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CDTX Patent

Compositions and methods for the treatment of viral infections include conjugates containing inhibitors of viral neuraminidase (e.g., zanamivir, peramivir, or analogs thereof) linked to an Fc monomer, an Fc domain, and Fc-binding peptide, an albumin protein, or albumin-binding peptide. In particular, conjugates can be used in the treatment of viral infections (e.g., influenza viral infections).

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Charles Gross

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0% found this document useful (0 votes)

132 views364 pages

CDTX Patent

Uploaded by

Charles Gross

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Compositions And Methods For The Treatment Of Viral Infections

DOCUMENT ID DATE PUBLISHED

US 11510992 B1 2022-11-29

INVENTOR INFORMATION
NAME CITY STATE ZIP CODE COUNTRY Borchardt;
Allen
San Diego CA N/A US Brady; San Diego
Thomas P. CA
N/A US Chen; Zhi- La Jolla CA N/A
Yong US Lam; Thanh San Diego
CA N/A US Tari; Leslie W. Solana Beach CA
N/A US

APPLICANT INFORMATION
NAME CITY STATE ZIP CODE COUNTRY AUTHORITY
Cidara San Diego CA N/A US N/A
Therapeutics,
TYPE
Inc.
assignee

ASSIGNEE INFORMATION
NAME CITY STATE ZIP CODE COUNTRY TYPE CODE
Cidara San Diego CA N/A US 02
Therapeutics,
Inc.
APPLICATION NO DATE FILED
17/236745 2021-04-21

DOMESTIC PRIORITY (CONTINUITY DATA)

continuation parent-doc US 17194093 20210305 ABANDONED child-doc US 17236745

continuation parent-doc US PCT/US2020/049772 20200908 PENDING child-doc US 17194093

us-provisional-application US 63062377 20200806

us-provisional-application US 63032488 20200529

us-provisional-application US 62988304 20200311

us-provisional-application US 62984705 20200303

us-provisional-application US 62970491 20200205

us-provisional-application US 62966500 20200127

us-provisional-application US 62959857 20200110

us-provisional-application US 62948143 20191213

us-provisional-application US 62941405 20191127

us-provisional-application US 62897036 20190906

US CLASS CURRENT:

1/1

CPC CURRENT
TYPE CPC DATE
CPCI C 07 D 309/28 2013-01-01
CPCI A 61 P 31/16 2018-01-01
CPCI A 61 K 47/6803 2017-08-01

KWIC Hits

APPLICANT GROUP
Cidara Therapeutics, Inc. San Diego CA US

ASSIGNEE NAME (USPAT, PGPUB)

Cidara Therapeutics, Inc.

Abstract

Compositions and methods for the treatment of viral infections include conjugates containing inhibitors
of viral neuraminidase (e.g., zanamivir, peramivir, or analogs thereof) linked to an Fc monomer, an Fc
domain, and Fc-binding peptide, an albumin protein, or albumin-binding peptide. In particular,
conjugates can be used in the treatment of viral infections (e.g., influenza viral infections).

Background/Summary

BACKGROUND

(1) The need for novel antiviral treatments for influenza is significant and especially critical in the
medical field. Influenza virus, the causative agent of influenza, or the flu, is responsible for three to five
million cases of severe illness annually, and approximately 500,000 deaths worldwide. While most
people recover completely from influenza in about one to two weeks, others develop life-threatening
complications, such as pneumonia. Thus, influenza can be deadly, especially for the young, old, or
chronically ill. People with weak or compromised immune systems, such as people with advanced HIV
infection or transplant patients, whose immune systems are medically suppressed to prevent
transplant organ rejection, are at greater risk for complications relating to influenza. Pregnant women
and young children are also at a high risk for complications.

(2) The development of antiviral treatments for influenza has been a continuing challenge. Several
influenza antiviral agents have been approved for use in the clinic, and these agents play important
roles in modulating disease severity and controlling pandemics while vaccines are prepared. However,
drug-resistant strains have emerged to the most commonly used inhibitors.

(3) Influenza Antiviral agents largely target proteins presented on the surface of the influenza virus
particle. The envelope of the influenza virus contains two immunodominant glycoproteins,
hemagglutinin and neuraminidase, that play key roles in viral infection and spread. Hemagglutinin
effects attachment of the virus to the host cell through its interaction with surface sialic acids, thereby
initiating entry.

(4) Neuraminidase is an exo-glycosidase enzyme that cleaves sialic acids (terminal neuraminic acid
residues) from glycan structures on the surface of infected host cells, releasing progeny viruses and
allowing the spread of the virus from the host cell to uninfected surrounding cells. Inhibition of
neuraminidase therefore serves as a pharmacological target for antiviral drugs. Viral neuraminidase
inhibitors used to reduce viral spread have been identified, including oseltamivir (Tamiflu™), zanamivir
(Relenza™), and peramivir (Rapivab™)

(5) However, influenza in transplant recipients remains characterized by prolonged viral shedding,
increasing the likelihood of developing drug resistant strains. New, more effective therapies for treating
influenza are needed.

SUMMARY

(6) The disclosure relates to conjugates, compositions, and methods for inhibiting viral growth, and
methods for the treatment of viral infections. In particular, such conjugates contain monomers or
dimers of a moiety that inhibits influenza virus neuraminidase (e.g., zanamivir, peramivir, or analogs
thereof) conjugated to Fc monomers, Fc domains, Fc-binding peptides, albumin proteins, or albumin
protein-binding peptides. The neuraminidase inhibitor (e.g., zanamivir, peramivir, or analogs thereof) in
the conjugates targets neuraminidase on the surface of the viral particle. The Fc monomers or Fc
domains in the conjugates bind to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb)
on immune cells, e.g., neutrophils, to activate phagocytosis and effector functions, such as antibody-
dependent cell-mediated cytotoxicity (ADCC), thus leading to the engulfment and destruction of viral
particles by immune cells and further enhancing the antiviral activity of the conjugates. The albumin or
albumin-binding peptide may extend the half-life of the conjugate, for example, by binding of albumin
to the recycling neonatal Fc receptor. Such compositions are useful in methods for the inhibition of
viral growth and in methods for the treatment of viral infections, such as those caused by an influenza
virus A, influenza virus B and influenza virus C.

(7) In a first aspect, the invention features a conjugate described by any one of formulas (D-I), (M-I),
(1), or (2):

(8) ##STR00001##

wherein each A.sub.1 and each A.sub.2 is independently selected from any one of formulas (A-I)-(A-
XII):

(9) ##STR00002##

wherein R.sub.1 is selected from —OH, —NH.sub.2, —NHC(═NH)NH.sub.2, and —

NHC(═NH)NHR.sub.6; R.sub.2 and R.sub.3 are each independently selected from —H, —OH, —F, —
Cl, and —Br; R.sub.4 is selected from —CO.sub.2H, —P(═O)(OH).sub.2, —SO.sub.3H; R.sub.5 is
selected from —COCH.sub.3, —COCF.sub.3, —SO.sub.2CH.sub.3; X is selected from —O— and —S
—; Y is selected from

(10) ##STR00003##

R.sub.6 is selected from

(11) ##STR00004## ##STR00005##

(12) R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15
aryl, and C2-C15 heteroaryl; R.sub.8 is selected from C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-
C15 heteroaryl;

(13) n is 1 or 2;

(14) each E comprises an Fc domain monomer, an albumin protein, an albumin protein-binding

peptide, or an Fc-binding peptide;

(15) L is a linker covalently attached to E and to each Y of each A.sub.1 or each A.sub.1 and A.sub.2;

(16) T is an integer from 1 to 20, and

(17) each squiggly line in formulas (D-I), (M-I), (1), or (2) indicates that L is covalently attached to each
E;

(18) or a pharmaceutically acceptable salt thereof.

(19) In some embodiments, n is 1 and each E includes an Fc domain monomer (e.g., an Fc domain
monomer having the sequence of any one of SEQ ID NOs: 1-138), an albumin protein (e.g., an
albumin protein having the sequence of any one of SEQ ID NOs: 139-141), an albumin protein-binding
peptide, or an Fc-binding peptide;

(20) In some embodiments, n is 2 and each E includes an Fc domain monomer (e.g., an Fc domain
monomer having the sequence of any one of SEQ ID NOs: 1-138), wherein the Fc domain monomers
dimerize to form and Fc domain;

(21) L is a linker covalently attached to each E and to each Y or each A.sub.1 and/or A.sub.2;

(22) T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
20), and each squiggly line in formulas (D-I), (M-I), (1), or (2) indicates that L is covalently attached
(e.g., by way of a covalent bond or linker) to each E; or a pharmaceutically acceptable salt thereof.
When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20),
each A.sub.1-L or each A.sub.1-L-A.sub.2 may be independently selected (e.g., independently
selected from any of the A.sub.1-L or A.sub.1-L-A.sub.2 structures described herein).

(23) In preferred embodiments of any of the aspects described herein, n is 2 and each E includes an
Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs:
1-138). In a conjugate having two Fc domain monomers (e.g., a conjugate of formula (1), formula (2),
formula (D-I) where n equals 2, or (M-I) where n equals 2), the Fc domain monomers dimerize to form
an Fc domain.

(24) In another aspect, the invention features a conjugate described by formula (D-I):

(25) ##STR00006##

wherein each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence
of any one of SEQ ID NOs: 1-138); L in each A.sub.1-L-A.sub.2 is a linker covalently attached to a
sulfur atom of a hinge cysteine in E and to each of A.sub.1 and A.sub.2; n is 1 or 2 (e.g., when n is 2,
the two Fc domain monomers dimerize to form and Fc domain); T is an integer from 1 to 20 (e.g., 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the squiggly line connected to the E
indicates that each A.sub.1-L-A.sub.2 is covalently attached (e.g., by way of a covalent bond or linker)
to a sulfur atom of a hinge cysteine in E, or a pharmaceutically acceptable salt thereof. When T is
greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each
A.sub.1-L-A.sub.2 may be independently selected (e.g., independently selected from any of the
A.sub.1-L-A.sub.2 structures described herein).

(26) In another aspect, the invention features a conjugate described by formula (D-I):

(27) ##STR00007##

wherein each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence
of any one of SEQ ID NOs: 1-138); L in each A.sub.1-L-A.sub.2 is a linker covalently attached to a
nitrogen atom of a surface exposed lysine in E and to each of A.sub.1 and A.sub.2; n is 1 or 2 (e.g.,
when n is 2, the two Fc domain monomers dimerize to form and Fc domain); T is an integer from 1 to
20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the squiggly line
connected to the E indicates that each A.sub.1-L-A.sub.2 is covalently attached (e.g., by way of a
covalent bond or linker) to the nitrogen atom of a surface exposed lysine in E, or a pharmaceutically
acceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20), each A.sub.1-L-A.sub.2 may be independently selected (e.g., independently
selected from any of the A.sub.1-L-A.sub.2 structures described herein). In some embodiments, each
of A.sub.1 and A.sub.2 may be independently selected from any one of formulas (A-I), (A-II), (A-VI), or
(A-VII). In other embodiments, each of A.sub.1 and A.sub.2 may be independently selected from
formula (A-I).

(28) In another aspect, the invention features a conjugate described by formula (M-I):
(29) ##STR00008##

wherein each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence
of any one of SEQ ID NOs: 1-138); L in each L-A.sub.1 is a linker covalently attached to a sulfur atom
of a hinge cysteine in E and to A.sub.1; n is 1 or 2 (e.g., when n is 2, the two Fc domain monomers
dimerize to form and Fc domain); T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, 14, 15, 16, 17, 18, 19, or 20); and the squiggly line connected to E indicates that each L-A.sub.1 is
covalently attached (e.g., by way of a covalent bond or linker) to the sulfur atom of the hinge cysteine
in E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A.sub.1 may be independently selected from
any structure described by formula (A-I)-(A-XII). In some embodiments, each A.sub.1 may be
independently selected from any one of formulas (A-I), (A-II), (A-VI), or (A-VII). In other embodiments,
each A.sub.1 may be independently selected from formula (A-I).

(30) In another aspect, the invention features a conjugate described by formula (M-I):

(31) ##STR00009##

wherein each E includes an Fc domain monomer (e.g., an Fc domain monomer having the sequence
of any one of SEQ ID NOs: 1-138); L in each L-A.sub.1 is a linker covalently attached to a nitrogen
atom of a surface exposed lysine in E and to A.sub.1; n is 1 or 2 (e.g., when n is 2, the two Fc domain
monomers dimerize to form and Fc domain); T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), the squiggly line connected to E indicates that each L-
A.sub.1 is covalently attached (e.g., by way of a covalent bond or linker) to the nitrogen atom of a
surface exposed lysine in E, or a pharmaceutically acceptable salt thereof. When T is greater than 1
(e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A.sub.1 may be
independently selected from any structure described by formula (A-I)-(A-XII). In some embodiments,
each A.sub.1 may be independently selected from any one of formulas (A-I), (A-II), (A-VI), or (A-VII).
In other embodiments, each A.sub.1 may be independently selected from formula (A-I).

(32) In one aspect, the disclosure features a conjugate described by formula (1):

(33) ##STR00010##

wherein each A.sub.1 and each A.sub.2 is independently selected from any one of formulas (A-I)-(A-
XII); each E comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of
any one of SEQ ID NOs: 1-138); L in each A.sub.1-L-A.sub.2 is a linker covalently attached to a sulfur
atom of a hinge cysteine in each E and to each of A.sub.1 and A.sub.2; T is an integer from 1 to 20
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the two squiggly lines
connected to the two Es indicate that each A.sub.1-L-A.sub.2 is covalently attached (e.g., by way of a
covalent bond or a linker) to a pair of sulfur atoms of two hinge cysteines in the two Es, or a
pharmaceutically acceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A.sub.1-L-A.sub.2 may be independently selected (e.g.,
independently selected from any of the A.sub.1-L-A.sub.2 structures described herein).

(34) In some embodiments, each of A.sub.1 and A.sub.2 may be independently selected from any one
of formulas (A-I), (A-II), (A-VI), or (A-VII). In other embodiments, each of A.sub.1 A.sub.2 may be
independently selected from formula (A-I).

(35) In another aspect, the disclosure features a conjugate described by formula (1):

(36) ##STR00011##

wherein each A.sub.1 and each A.sub.2 is independently selected from any one of formulas (A-I)-(A-
V); each E comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of
any one of SEQ ID NOs: 1-138); L in each A.sub.1-L-A.sub.2 is a linker covalently attached to a sulfur
atom of a hinge cysteine in each E and to each of A.sub.1 and A.sub.2; T is an integer from 1 to 20
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the two squiggly lines
connected to the two Es indicate that each A.sub.1-L-A.sub.2 is covalently attached (e.g., by way of a
covalent bond or a linker) to a pair of sulfur atoms of two hinge cysteines in the two Es, or a
pharmaceutically acceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A.sub.1-L-A.sub.2 may be independently selected (e.g.,
independently selected from any of the A.sub.1-L-A.sub.2 structures described herein).

(37) In another aspect, the disclosure features a conjugate described by formula (1):

(38) ##STR00012##

wherein each A.sub.1 and each A.sub.2 is independently selected from any one of formulas (A-VI)-(A-
IX); each E comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of
any one of SEQ ID NOs: 1-138); L in each A.sub.1-L-A.sub.2 is a linker covalently attached to a sulfur
atom of a hinge cysteine in each E and to each of A.sub.1 and A.sub.2; T is an integer from 1 to 20
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), and the two squiggly lines
connected to the two Es indicate that each A.sub.1-L-A.sub.2 is covalently attached (e.g., by way of a
covalent bond or a linker) to a pair of sulfur atoms of two hinge cysteines in the two Es, or a
pharmaceutically acceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A.sub.1-L-A.sub.2 may be independently selected (e.g.,
independently selected from any of the A.sub.1-L-A.sub.2 structures described herein).

(39) In another aspect, the invention features a conjugate described by formula (2):

(40) ##STR00013##

wherein each A.sub.1 is independently selected from any one of formulas (A-I)-(A-XII); each E
comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138); L in each L-A.sub.1 is a linker covalently attached to a sulfur atom in a hinge
cysteine in E and to A.sub.1; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20), and the two squiggly lines connected to the two sulfur atoms indicate
that each L-A.sub.1 is covalently (e.g., by way of a covalent bond or a linker) attached to a pair of
sulfur atoms of two hinge cysteines in the two Es, or a pharmaceutically acceptable salt thereof. When
T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each
A.sub.1 may be independently selected from any one of formulas (A-I)-(A-XII). In some embodiments,
each of A.sub.1 and A.sub.2 may be independently selected from any one of formulas (A-I), (A-II), (A-
VI), or (A-VII). In other embodiments, each of A.sub.1 A.sub.2 may be independently selected from
formula (A-I).

(41) In another aspect, the invention features a conjugate described by formula (2):

(42) ##STR00014##

wherein each A.sub.1 is independently selected from any one of formulas (A-I)-(A-V); each E
comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138); L in each L-A.sub.1 is a linker covalently attached to a sulfur atom in a hinge
cysteine in E and to A.sub.1; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20), and the two squiggly lines connected to the two sulfur atoms indicate
that each L-A.sub.1 is covalently attached (e.g., by way of a covalent bond or a linker) to a pair of
sulfur atoms of two hinge cysteines in the two Es, or a pharmaceutically acceptable salt thereof. When
T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each
A.sub.1 may be independently selected from any one of formulas (A-I)-(A-V).

(43) In another aspect, the invention features a conjugate described by formula (2):

(44) ##STR00015##

wherein each A.sub.1 is independently selected from any one of formulas (A-VI)-(A-IX); each E
comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138); L in each L-A.sub.1 is a linker covalently attached to a sulfur atom in a hinge
cysteine in E and to A.sub.1; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20), and the two squiggly lines connected to the two sulfur atoms indicate
that each L-A.sub.1 is covalently attached (e.g., by way of a covalent bond or a linker) to a pair of
sulfur atoms of two hinge cysteines in the two Es, or a pharmaceutically acceptable salt thereof. When
T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each
A.sub.1 may be independently selected from any one of formulas (A-VI)-(A-IX).

(45) In some embodiments of any of the foregoing embodiments, each E includes an Fc domain
monomer having the sequence of any one of SEQ ID NOs: 1-138.

(46) In some embodiments, at least one of the pair of sulfur atoms is the sulfur atom corresponding to
(e.g., the sulfur atom of) a hinge cysteine of SEQ ID NO: 10 or SEQ ID NO: 11, i.e., Cys10, Cys13,
Cys16, or Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11. In some embodiments, the pair of sulfur atoms
are the sulfur atoms corresponding to (e.g., the sulfur atoms of) Cys10 and Cys13 in SEQ ID NO: 10
or SEQ ID NO: 11, Cys10 and Cys16 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys 30 and Cys18 in SEQ
ID NO: 10 or SEQ ID NO: 11, Cys13 and Cys 36 in SEQ ID NO: 10 or SEQ ID NO: 11, Cys13 and Cys
38 in SEQ ID NO: 10 or SEQ ID NO: 11, and/or Cys 36 and Cys 38 in SEQ ID NO: 10 or SEQ ID NO:
11. In some embodiments, when T is 2, the pair of sulfur atoms are (e.g., the sulfur atoms
corresponding to) Cys10 and Cys13 in SEQ ID NO: 10 or SEQ ID NO: 11 or Cys 36 and Cys 38 in
SEQ ID NO: 10 or SEQ ID NO: 11.

(47) In some embodiments, the pair of sulfur atoms include one sulfur atom of a cysteine from each E,
i.e., L-A along with the sulfur atoms to which it is attached forms a bridge between two Fc domains
(e.g., two Fc domains comprising the sequence of SEQ ID NO: 10 or SEQ ID NO: 11). In some
embodiments, the pair of sulfur atoms are the sulfur atom corresponding to (e.g., the sulfur atom of)
Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the
sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, the
pair of sulfur atoms are the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID
NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of)
Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, the pair of sulfur
atoms are the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ
ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID
NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, the pair of sulfur atoms are the
sulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11
from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or
SEQ ID NO: 11 from another E.

(48) In some embodiments, when T is 2, the pairs of sulfur atoms are the sulfur atom corresponding to
(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E
and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID
NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID
NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, the pairs of sulfur
atoms are the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ
ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID
NO: 10 or SEQ ID NO: 11 from another E and the sulfur atom corresponding to (e.g., the sulfur atom
of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g.,
the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments,
when T is 2, the pairs of sulfur atoms are the sulfur atom corresponding to (e.g., the sulfur atom of)
Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the
sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and
the sulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11
from another E.
(49) In some embodiments, when T is 2, the pairs of sulfur atoms are the sulfur atom corresponding to
(e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E
and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID
NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID
NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is 2, the pairs of sulfur
atoms are the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ
ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID
NO: 10 or SEQ ID NO: 11 from another E and the sulfur atom corresponding to (e.g., the sulfur atom
of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g.,
the sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

(50) In some embodiments, when T is 2, the pairs of sulfur atoms are the sulfur atom corresponding to
(e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E
and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID
NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID
NO: 10 or SEQ ID NO: 11 from another E.

(51) In some embodiments, when T is 3, the pairs of sulfur atoms are the sulfur atom corresponding to
(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E;
the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11
from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or
SEQ ID NO: 11 from another E; and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16
of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur
atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is
3, the pairs of sulfur atoms are the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of
SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur
atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; the sulfur atom corresponding to
(e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E;
and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID
NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID
NO: 10 or SEQ ID NO: 11 from another E. In some embodiments, when T is 3, the pairs of sulfur
atoms are the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ
ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID
NO: 10 or SEQ ID NO: 11 from another E; the sulfur atom corresponding to (e.g., the sulfur atom of)
Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the
sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and
the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11
from another E. In some embodiments, when T is 3, the pairs of sulfur atoms are the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and
the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11
from another E; the sulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or
SEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of
SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and the sulfur atom corresponding to (e.g., the
sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E.

(52) In some embodiments, when T is 3, the pairs of sulfur atoms are the sulfur atom corresponding to
(e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E;
the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or SEQ ID NO: 11
from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 10 or
SEQ ID NO: 11 from another E; the sulfur atom corresponding to (e.g., the sulfur atom of) Cys16 of
SEQ ID NO: 10 or SEQ ID NO: 11 from one E and the sulfur atom corresponding to (e.g., the sulfur
atom of) Cys16 of SEQ ID NO: 10 or SEQ ID NO: 11 from another E; and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11 from one E and
the sulfur atom corresponding to (e.g., the sulfur atom of) Cys18 of SEQ ID NO: 10 or SEQ ID NO: 11
from another E.

(53) In some embodiments, the conjugate has the structure:

(54) ##STR00016##

wherein each of a, b, c, and d is, independently, 0 or 1 and wherein when a, b, c, or d is 0, the two
sulfur atoms form a disulfide bond.

(55) In some embodiments, a is 1 and b, c, and d are 0. In some embodiments, a and b are 1 and c
and d are 0. In some embodiments, a and c are 1 and b and d are 0. In some embodiments, a and d
are 1 and b and c are 0. In some embodiments, a, b, and c are 1 and d is 0. In some embodiments, a,
b, and d are 1 and c is 0. In some embodiments, a, c, and d are 1 and b is 0. In some embodiments, b
and c are 1 and a and d are 0. In some embodiments, b and d are 1 and a and c are 0. In some
embodiments, b, c, and d are 1 and a is 0. In some embodiments, c and d are 1 and a and b are 0. In
some embodiments, a, b, c, and d are 1.

(56) In some embodiments, each E comprises the sequence

MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 4).

(57) In some embodiments, each E comprises the sequence

MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVE
VH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FS CSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 33).

(58) In some embodiments, at least one of the pair of sulfur atoms is the sulfur atom corresponding to
(e.g., the sulfur atom of) a hinge cysteine of SEQ ID NO: 4 or SEQ ID NO: 33, i.e., Cys10 and/or
Cys13. In some embodiments, the pair of sulfur atoms are the sulfur atoms corresponding to (e.g., the
sulfur atoms of) Cys10 and Cys13 in SEQ ID NO: 4 or SEQ ID NO: 33.

(59) In some embodiments, the pair of sulfur atoms include one sulfur atom of a cysteine from each E,
i.e., L-A along with the sulfur atoms to which it is attached forms a bridge between two Fc domains
(e.g., two Fc domains comprising the sequence of SEQ ID NO: 4 or SEQ ID NO: 33). In some
embodiments, the pair of sulfur atoms are the sulfur atom corresponding to (e.g., the sulfur atom of)
Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from one E and the sulfur atom corresponding to (e.g., the
sulfur atom of) Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E. In some embodiments, the
pair of sulfur atoms are the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID
NO: 4 or SEQ ID NO: 33 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of)
Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E. In some embodiments, when T is 2, the
pairs of sulfur atoms are the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID
NO: 4 or SEQ ID NO: 33 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of)
Cys10 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E and the sulfur atom corresponding to (e.g.,
the sulfur atom of) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from one E and the sulfur atom
corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO: 4 or SEQ ID NO: 33 from another E.
(60) In some embodiments, the conjugate has the structure:

(61) ##STR00017##

wherein each of a and b is, independently, 0 or 1 and wherein when a or b is 0, the two sulfur atoms
form a disulfide bond. In some embodiments, a is 1 and b is 0. In some embodiments, a is 0 and b is
1. In some embodiments, a and b are 1.

(62) In some embodiments, at least one of the pair of sulfur atoms is the sulfur atom corresponding to
(e.g., the sulfur atom of) a hinge cysteine of SEQ ID NO: 8, i.e., Cys10 and/or Cys13. In some
embodiments, the pair of sulfur atoms are the sulfur atoms corresponding to (e.g., the sulfur atoms of)
Cys10 and Cys13 in SEQ ID NO: 8.

(63) In some embodiments, the pair of sulfur atoms include one sulfur atom of a cysteine from each E,
i.e., L-A along with the sulfur atoms to which it is attached forms a bridge between two Fc domains
(e.g., two Fc domains comprising the sequence of SEQ ID NO: 8). In some embodiments, the pair of
sulfur atoms are the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 8
from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID NO: 8
from another E. In some embodiments, the pair of sulfur atoms are the sulfur atom corresponding to
(e.g., the sulfur atom of) Cys13 of SEQ ID NO: 8 from one E and the sulfur atom corresponding to
(e.g., the sulfur atom of) Cys13 of SEQ ID NO: 8 from another E. In some embodiments, when T is 2,
the pairs of sulfur atoms are the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ
ID NO: 8 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys10 of SEQ ID
NO: 8 from another E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID
NO: 8 from one E and the sulfur atom corresponding to (e.g., the sulfur atom of) Cys13 of SEQ ID NO:
8 from another E.

(64) In some embodiments, the conjugate has the structure:

(65) ##STR00018##

(66) In some embodiments, the conjugate has the structure:

(67) ##STR00019##

(68) In some embodiments, the conjugate has the structure:

(69) ##STR00020##

(70) In some embodiments, the conjugate has the structure:

(71) ##STR00021##

wherein each of a and b is, independently, 0 or 1 and wherein when a or b is 0, the sulfur atoms is a
thiol.
(72) In some embodiments, a is 1 and b is 0. In some embodiments, a is 0 and b is 1. In some
embodiments, a and b are 1.

(73) In some embodiments of the previous three aspects, the nitrogen atom is the nitrogen of a
surface exposed lysine, e.g., the nitrogen atom corresponding to (e.g., the nitrogen atom of) Lys35,
Lys63, Lys77, Lys79, Lys106, Lys123, Lys129, Lys181, Lys203, Lys228, or Lys236 of SEQ ID NO: 10
or SEQ ID NO: 11. In some embodiments, the nitrogen atom is the nitrogen atom corresponding to
(e.g., the nitrogen atom of) Lys65, Lys79, Lys108, Lys230, and/or Lys238 of SEQ ID NO: 10 or SEQ ID
NO: 11.

(74) In some embodiments, the conjugate has the structure:

(75) ##STR00022##

wherein each of a, b, c, d, and e is, independently, 0 or 1 and wherein when a, b, c, d, or e is 0, the

two nitrogen atom is NH.sub.2. In some embodiments, a is 1 and b, c, d, and e are 0. In some
embodiments, b is 1 and a, c, d, and e are 0. In some embodiments, c is 1 and a, b, d, and e are 0. In
some embodiments, d is 1 and a, b, c, and e are 0. In some embodiments, e is 1 and a, b, c, and d are
0. In some embodiments, a and b are 1 and c, d, and e are 0. In some embodiments, a and c are 1
and b, d, and e are 0. In some embodiments, a and d are 1 and b, c, and e are 0. In some
embodiments, a and e are 1 and b, c, and d are 0. In some embodiments, b and c are 1 and a, d, and
e are 0. In some embodiments, b and d are 1 and a, c, and e are 0. In some embodiments, b and e
are 1 and a, c, and d are 0. In some embodiments, c and d are 1 and a, b, and e are 0. In some
embodiments, c and e are 1 and a, b, and d are 0. In some embodiments, d and e are 1 and a, b, and
c are 0. In some embodiments, a, b, and c are 1 and d and e are 0. In some embodiments, a, b, and d
are 1 and c and e are 0. In some embodiments, a, b, and e are 1 and c and d are 0. In some
embodiments, a, c, and d are 1 and b and e are 0. In some embodiments, a, c, and e are 1 and b and
d are 0. In some embodiments, a, d, and e are 1 and b and c are 0. In some embodiments, b, c, and d
are 1 and a and e are 0. In some embodiments, b, d, and e are 1 and a and c are 0. In some
embodiments, c, d, and e are 1 and a and b are 0.

(76) In some embodiments of any of the conjugates described herein, the conjugate forms a
homodimer including an Fc domain. In some embodiments of the conjugates described herein, E
homodimerizes with another E to form an Fc domain.

(77) In another aspect, the invention features a conjugate described by (D-I):

(78) ##STR00023##

wherein E includes an albumin protein (e.g., an albumin protein having the sequence of any one of
SEQ ID NOs: 139-141), an albumin protein-binding peptide, or an Fc-binding peptide; L in each
A.sub.1-L-A.sub.2 is a linker independently covalently attached to a sulfur atom of a surface exposed
cysteine or a nitrogen atom of a surface exposed lysine in E and to each of A.sub.1 and A.sub.2; n is
1; T is an integer from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or
20), and the squiggly line connected to the E indicates that each A.sub.1-L-A.sub.2 is independently
covalently attached to the sulfur atom of a solvent-exposed cysteine or the nitrogen atom of a solvent-
exposed lysine in E, or a pharmaceutically acceptable salt thereof. When T is greater than 1 (e.g., T is
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A.sub.1-L-A.sub.2 may be
independently selected (e.g., independently selected from any of the A.sub.1-L-A.sub.2 structures
described herein). In some embodiments, each of A.sub.1 A.sub.2 may be independently selected
from any one of formulas (A-I), (A-II), (A-VI), or (A-VII). In other embodiments, each of A.sub.1 A.sub.2
may be independently selected from formula (A-I).

(79) In a preferred embodiment of the above, x is 2.

(80) In another aspect, the invention features a conjugate described by formula (M-I):

(81) ##STR00024##
wherein E includes an albumin protein (e.g., an albumin protein having the sequence of any one of
SEQ ID NOs: 139-141), an albumin protein-binding peptide, or an Fc-binding peptide; L in each L-
A.sub.1 is a linker independently covalently attached to a sulfur atom of a surface exposed cysteine or
a nitrogen atom of a surface exposed lysine in E and to A.sub.1; n is 1; T is an integer from 1 to 20
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20); and the squiggly line
connected to E indicates that each L-A.sub.1 is independently covalently attached to the sulfur atom of
the solvent-exposed cysteine or the nitrogen atom of the solvent-exposed lysine in E, or a
pharmaceutically acceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A.sub.1 may be independently selected from any
structure described by formula (A-I)-(A-XII). In some embodiments, each A.sub.1 may be
independently selected from any one of formulas (A-I), (A-II), (A-VI), or (A-VII). In other embodiments,
each A.sub.1 may be independently selected from formula (A-I). In a preferred embodiment of the
above, x is 2.

(82) In some embodiments, each E includes an albumin protein having the sequence of any one of
SEQ ID NOs: 139-141.

(83) In some embodiments, T is 1 and L-A.sub.1 is covalently attached to the sulfur atom
corresponding to Cys34 of SEQ ID NO: 139.

(84) Intermediates of Table 1a may be conjugated to an Fc domain or Fc domain monomer (e.g., by

way of a linker) by any suitable methods known to those of skill in the art, including any of the methods
described or exemplified herein. In some embodiments, the conjugate (e.g., a conjugate described by
any one of formulas (1), (2), (D-I)-(D-XI), or (M-I)-(M-XI)) includes E, wherein E is an Fc domain
monomer or an Fc domain (e.g., an Fc domain monomer or an Fc domain, each Fc domain monomer
having, independently, the sequence of any one of SEQ ID NOs: 1-138). In preferred embodiments,
one or more nitrogen atoms of one or more surface exposed lysine residues of E or one or more sulfur
atoms of one or more surface exposed cysteines in E is covalently conjugated to a linker (e.g., a
PEG.sub.2-PEG.sub.20 linker). The linker conjugated to E may be functionalized such that it may
react to form a covalent bond with any of the Ints described herein (e.g., an Int of Table 1a). In
preferred embodiments, E is conjugated to a linker functionalized with an azido group and the Int (e.g.,
an Int of Table 1a) is functionalized with an alkyne group. Conjugation (e.g., by click chemistry) of the
linker-azido of E and linker-alkyne of the Int forms a conjugate of the invention, for example a
conjugate described by formula (5). In yet other embodiments, E is conjugated to a linker
functionalized with an alkyne group and the Int (e.g., an Int of Table 1a) is functionalized with an azido
group. Conjugation (e.g., by click chemistry; see, e.g., FIG. 103) of the linker-alkyne of E and linker-
azido of the Int forms a conjugate of the invention, for example a conjugate described by any one of
formulas (1), (2), (D-I)-(D-XI), or (M-I)-(M-XI).

(85) TABLE-US-00001 TABLE 1a Intermediates Intermediate Structure Int-1 Int-2 Int-3 Int-4 Int-5
Int-6 0 Int-7 Int-9 Int-12 Int-13 Int-14 Int-15 Int-16 Int-17 Int-18 Int-19 0 Int-20 Int-21 Int-22 Int-
23 Int-24 Int-25 Int-26 Int-27 Int-28 Int-30 0 Int-31 Int-34 Int-38 Int-39 Int-40 Int-42 Int-43 Int-44
Int-45 Int-46 0 Int-47 Int-48 Int-49 Int-50 Int-52 Int-53 Int-54 Int-55 Int-57 Int-58 0 Int-59 Int-60
Int-61 Int-62 Int-63 Int-64 Int-65 Int-67 Int-68 Int-69 0 Int-70 Int-71 Int-72 Int-73 Int-74 Int-75
Int-76 Int-77 Int-78 Int-79 0 Int-80 Int-81 Int-82 Int-83 Int-84 Int-85 Int-86 Int-87 Int-88 Int-89 00
Int-90 01 Int-91 02 Int-92 03

(86) In another aspect, the invention features conjugates of Table 1b. Each conjugate of Table 1b
corresponds to a conjugate of either formula (M-I) or formula (D-I), as indicated. Conjugates of Table
1b include conjugates formed by the covalent reaction of an Int of Table 1a with a linker which is in
turn conjugated to E (e.g., an Fc domain monomer, an albumin protein, an albumin protein-binding
peptide, or an Fc-binding peptide). In some embodiments, the reactive moiety of the Int (e.g., the
alkyne or azido group) reacts with a corresponding reactive group (e.g., an alkyne or azido group) of a
linker (represented by L′) covalently attached to E, such that an Int of Table 1a is covalently attached
to E. As represented in Table 1 b, L′ corresponds to the remainder of L as defined in (M-I) or (D-I)
(e.g., L′ is a linker that covalently joins the Int and E). For example, L′ may include a triazole (formed
by the click chemistry reaction between the Int and a linker conjugated to E) and a linker (e.g., a
PEG.sub.2-PEG.sub.20 linker) which in turn is conjugated to an amino acid side chain of E (see, e.g.,
FIG. 103).

(87) In some embodiments the conjugate of Table 1 b, n is 1 or 2. When n is 1, each E includes an Fc

domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-
138), an albumin protein (e.g., an albumin protein having a sequence of any one of SEQ ID NOs: 139-
141), an albumin protein-binding peptide, or an Fc-binding peptide. When n is 2, each E includes an
Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of SEQ ID NOs:
1-138), and the Fc domain monomers dimerize to form an Fc domain.

(88) In some embodiments of any conjugate of Table 1 b, T is an integer from 1 to 20 (e.g., 1, 2, 3, 4,

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). The disclosure also provides a population of
any of the conjugates of Table 2 wherein the average value of T is 1 to 20 (e.g., the average value of T
is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5,
5.5 to 7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5). In some embodiments, the average value of T is 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the average T is
1 to 10 (e.g., 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10). In certain
embodiments, the average T is 1 to 5 (e.g., 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3,
2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,
4.8, 4.9, or 5). In some embodiment, the average T is 5 to 10 (e.g., 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,
5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1,
8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10). In some
embodiments, the average T is 2.5 to 7.5 (e.g., 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,
3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1,
6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, or 7.5).

(89) The squiggly line in the conjugates of Table 1 b indicates that each L′-Int is covalently attached to
an amino acid side chain in E (e.g., the nitrogen atom of a surface exposed lysine or the sulfur atom of
a surface exposed cysteine in E), or a pharmaceutically acceptable salt thereof.

(90) TABLE-US-00002 TABLE 1b Conjugates Corresponding to Intermediates of Table 1a

Corresponding Intermediate Described by of Table 1a Formula Conjugate Structure Int-1 (D-I) 04 Int-2
(D-I) 05 Int-3 (D-I) 06 Int-4 (M-I) 07 Int-5 (M-I) 08 Int-6 (D-I) 09 Int-7 (D-I) 0 Int-9 (D-I) Int-12 (D-I) Int-
13 (M-I) Int-14 (M-I) Int-15 (D-I) Int-16 (M-I) Int-17 (D-I) Int-18 (D-I) Int-19 (D-I) Int-20 (D-I) 0 Int-21
(D-I) Int-22 (M-I) Int-23 (D-I) Int-24 (D-I) Int-25 (D-I) Int-26 (D-I) Int-27 (D-I) Int-28 (D-I) Int-30 (M-I)
Int-31 (D-I) 0 Int-34 (D-I) Int-38 (D-I) Int-39 (D-I) Int-40 (D-I) Int-42 (D-I) Int-43 (D-I) Int-44 (D-I)
Int-45 (D-I) Int-46 (D-I) Int-47 (D-I) 0 Int-48 (D-I) Int-49 (D-I) Int-50 (D-I) Int-52 (D-I) Int-53 (D-I) Int-
54 (D-I) Int-55 (D-I) Int-57 (D-I) Int-58 (D-I) Int-59 (D-I) 0 Int-60 (D-I) Int-61 (D-I) Int-62 (D-I) Int-63
(D-I) Int-64 (D-I) Int-65 (D-I) Int-67 (D-I) Int-68 (D-I) Int-69 (D-I) Int-70 (D-I) 0 Int-71 (D-I) Int-72 (D-
I) Int-73 (D-I) Int-74 (D-I) Int-75 (D-I) Int-76 (D-I) Int-77 (D-I) Int-78 (D-I) Int-79 (D-I) Int-80 (D-I) 0
Int-81 (D-I) Int-82 (D-I) Int-83 (D-I) Int-84 (D-I) Int-85 (D-I) Int-86 (D-I) Int-87 (D-I) Int-88 (D-I) Int-
89 (D-I) Int-90 (D-I) 0 Int-91 (M-I) Int-92 (M-I)

(91) In some embodiments, each E includes an Fc domain monomer having the sequence of any one
of SEQ ID NOs: 1-138. In other embodiments, each E includes an Fc domain monomer having a
sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 63 or SEQ ID NO: 64. In
other embodiments, each E includes an Fc domain monomer having the amino acid sequence of SEQ
ID NO: 63 or SEQ ID NO: 64. In other embodiments, each E includes an Fc domain monomer having
a sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 67 or SEQ ID NO: 68. In
other embodiments, each E includes an Fc domain monomer having the amino acid sequence of SEQ
ID NO: 67 or SEQ ID NO: 68. In other embodiments, each E includes an Fc domain monomer having
a sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 72 or SEQ ID NO: 73. In
other embodiments, each E includes an Fc domain monomer having the amino acid sequence of SEQ
ID NO: 72 or SEQ ID NO: 73. In other embodiments, each E includes an Fc domain monomer having
a sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 76 or SEQ ID NO: 77. In
other embodiments, each E includes an Fc domain monomer having the amino acid sequence of SEQ
ID NO: 76 or SEQ ID NO: 77. In other embodiments, each E includes an Fc domain monomer having
a sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 81 or SEQ ID NO: 82. In
other embodiments, each E includes an Fc domain monomer having the amino acid sequence of SEQ
ID NO: 81 or SEQ ID NO: 82. In other embodiments, each E includes an Fc domain monomer having
a sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 85 or SEQ ID NO: 86. In
other embodiments, each E includes an Fc domain monomer having the amino acid sequence of SEQ
ID NO: 85 or SEQ ID NO: 86.

(92) In another aspect, the invention features a conjugate including (i) a first moiety, A.sub.1; (ii) a
second moiety, A.sub.2; (iii) an Fc domain monomer or an Fc domain; and (iv) a linker covalently
attached to A.sub.1 and A.sub.2, and to the Fc domain monomer or the Fc domain; wherein each
A.sub.1 and each A.sub.2 is independently selected from any one of formulas (A-I)-(A-XII). In some
embodiments, each of A.sub.1 and A.sub.2 may be independently selected from any one of formulas
(A-I), (A-II), (A-VI), or (A-VII). In other embodiments, each of A.sub.1 and A.sub.2 may be
independently selected from formula (A-I). In a preferred embodiment of the above, x is 2.

(93) In another aspect, the invention features a conjugate including (i) a first moiety, Int; (ii) an Fc
domain monomer or an Fc domain; and (iv) a linker covalently attached to Int, and to the Fc domain
monomer or the Fc domain; wherein each Int is independently selected from any one of the
intermediates of Table 1a.

(94) In another aspect, the invention features a conjugate including (i) a first moiety, A.sub.1; (ii) a
second moiety, A.sub.2; (iii) an albumin protein, an albumin protein-binding peptide, or an Fc-binding
peptide; and (iv) a linker covalently attached to A.sub.1 and A.sub.2, and to the albumin protein, the
albumin protein-binding peptide, or the Fc-binding peptide; wherein each A.sub.1 and each A.sub.2 is
independently selected from any one of formulas (A-I)-(A-XII). In some embodiments, each of A.sub.1
and A.sub.2 may be independently selected from any one of formulas (A-I), (A-II), (A-VI), or (A-VII). In
other embodiments, each of A.sub.1 and A.sub.2 may be independently selected from formula (A-I). In
a preferred embodiment of the above, x is 2.

(95) In another aspect, the invention features a conjugate described by formula (D-I):

(96) ##STR00183##

wherein each A.sub.1 and each A.sub.2 is independently selected from any one of formulas (A-I)-(A-
XII); each E comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of
any one of SEQ ID NOs: 1-138), an albumin protein (e.g., an albumin protein having the sequence of
any one of SEQ ID NOs: 139-141), an albumin protein-binding peptide, or an Fc-binding peptide; n is 1
or 2; T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20); and L is a linker covalently attached to each of E, A.sub.1, and A.sub.2, or a pharmaceutically
acceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20), each A.sub.1-L-A.sub.2 may be independently selected (e.g., independently
selected from any of the A.sub.1-L-A.sub.2 structures described herein). In some embodiments, each
of A.sub.1 and A.sub.2 may be independently selected from any one of formulas (A-I), (A-II), (A-VI), or
(A-VII). In other embodiments, each of A.sub.1 and A.sub.2 may be independently selected from
formula (A-I).

(97) In another aspect, the invention features a conjugate described by formula (D-I):

(98) ##STR00184##

wherein each A.sub.1 and each A.sub.2 is independently selected from any one of formulas (A-I)-(A-
V); each E comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of
any one of SEQ ID NOs: 1-138), an albumin protein (e.g., an albumin protein having the sequence of
any one of SEQ ID NOs: 139-141), an albumin protein-binding peptide, or an Fc-binding peptide; n is 1
or 2; T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20); and L is a linker covalently attached to each of E, A.sub.1, and A.sub.2, or a pharmaceutically
acceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20), each A.sub.1-L-A.sub.2 may be independently selected (e.g., independently
selected from any of the A.sub.1-L-A.sub.2 structures described herein).

(99) In another aspect, the invention features a conjugate described by formula (D-I):

(100) ##STR00185##

wherein each A.sub.1 and each A.sub.2 is independently selected from any one of formulas (A-VI)-(A-
IX); each E comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of
any one of SEQ ID NOs: 1-138), an albumin protein (e.g., an albumin protein having the sequence of
any one of SEQ ID NOs: 139-141), an albumin protein-binding peptide, or an Fc-binding peptide; n is 1
or 2; T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20); and L is a linker covalently attached to each of E, A.sub.1, and A.sub.2, or a pharmaceutically
acceptable salt thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or 20), each A.sub.1-L-A.sub.2 may be independently selected (e.g., independently
selected from any of the A.sub.1-L-A.sub.2 structures described herein).

(101) In some embodiments, the conjugate is described by formula (D-I1):

(102) ##STR00186##

or a pharmaceutically acceptable salt thereof.

(103) In some embodiments, the conjugate is described by formula (D-II-1):

(104) ##STR00187##

or a pharmaceutically acceptable salt thereof.

(105) In some embodiments, the conjugate is described by formula (D-II-2):

(106) ##STR00188##

or a pharmaceutically acceptable salt thereof.

(107) In some embodiments, the conjugate is described by formula (D-II-3):

(108) ##STR00189##

wherein L′ is the remainder of L, and y.sub.1 and y.sub.2 are each independently an integer from 1-20
(e.g., y.sub.1 and y.sub.2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20), or a pharmaceutically acceptable salt thereof. In some embodiments, L′ is a nitrogen
atom.

(109) In some embodiments, the conjugate has the structure selected from:

(110) ##STR00190##

(111) In some embodiments, the conjugate is described by formula (D-II-4):

(112) ##STR00191##

or a pharmaceutically acceptable salt thereof.

(113) In some embodiments, the conjugate is described by formula (D-II-5):

(114) ##STR00192##
wherein L′ is the remainder of L, and y.sub.1 and y.sub.2 are each independently an integer from 1-20
(e.g., y.sub.1 and y.sub.2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20), or a pharmaceutically acceptable salt thereof. In some embodiments, L′ is a nitrogen
atom.

(115) In some embodiments, the conjugate has the structure selected from:

(116) ##STR00193##

or a pharmaceutically acceptable salt thereof.

(117) In some embodiments, the conjugate has the structure selected from:

(118) ##STR00194##

or a pharmaceutically acceptable salt thereof.

(119) In some embodiments, the conjugate is described by formula (D-II-6):

(120) ##STR00195##

wherein R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-
C15 aryl, and C2-C15 heteroaryl; or a pharmaceutically acceptable salt thereof. In some
embodiments, R.sub.7 is selected from C1-C20 alkyl (e.g., methyl, ethyl, propyl, or butyl).

(121) In some embodiments, the conjugate is described by formula (D-II-7):

(122) ##STR00196##

or a pharmaceutically acceptable salt thereof.

(123) In some embodiments, the conjugate is described by formula (D-II-8):

(124) ##STR00197##

(125) In some embodiments, the conjugate has the structure

(126) ##STR00198##

or a pharmaceutically acceptable salt thereof.

(127) In some embodiments, the conjugate has the structure

(128) ##STR00199##

or a pharmaceutically acceptable salt thereof.

(129) In some embodiments, the conjugate has the structure

(130) ##STR00200##

or a pharmaceutically acceptable salt thereof.

(131) In some embodiments, the conjugate has the structure

(132) ##STR00201##

or a pharmaceutically acceptable salt thereof.

(133) In some embodiments, the conjugate is described by formula (D-II-9):

(134) ##STR00202##

or a pharmaceutically acceptable salt thereof.

(135) In some embodiments, the conjugate is described by formula (D-II-10):

(136) ##STR00203##

(137) In some embodiments, the conjugate has the structure

(138) ##STR00204##

or a pharmaceutically acceptable salt thereof.

(139) In some embodiments, the conjugate has the structure of

(140) ##STR00205##

or a pharmaceutically acceptable salt thereof.

(141) In some embodiments, the conjugate is described by formula (D-III):

(142) ##STR00206##

or a pharmaceutically acceptable salt thereof.

(143) In some embodiments, the conjugate is described by formula (D-III-1):

(144) ##STR00207##

or a pharmaceutically acceptable salt thereof.

(145) In some embodiments, the conjugate is described by formula (D-III-2):

(146) ##STR00208##

or a pharmaceutically acceptable salt thereof.

(147) In some embodiments, the conjugate is described by formula (D-III-3):

(148) ##STR00209##

(149) In some embodiments, the conjugate is described by formula (D-III-4):

(150) ##STR00210##

or a pharmaceutically acceptable salt thereof.

(151) In some embodiments, the conjugate is described by formula (D-III-5):

(152) ##STR00211##

(153) In some embodiments, the conjugate is described by formula (D-III-6):

(154) ##STR00212##

or a pharmaceutically acceptable salt thereof.

(155) In some embodiments, the conjugate is described by formula (D-III-7):

(156) ##STR00213##

(157) In some embodiments, the conjugate is described by formula (D-III-8):

(158) ##STR00214##

or a pharmaceutically acceptable salt thereof.

(159) In some embodiments, the conjugate is described by formula (D-III-9):

(160) ##STR00215##

(161) In some embodiments, the conjugate is described by formula (D-IV):

(162) ##STR00216##

or a pharmaceutically acceptable salt thereof.

(163) In some embodiments, the conjugate is described by formula (D-IV-1):

(164) ##STR00217##

or a pharmaceutically acceptable salt thereof.

(165) In some embodiments, the conjugate is described by formula (D-IV-2):

(166) ##STR00218##
wherein L′ is the remainder of L, and y.sub.1 and y.sub.2 are each independently an integer from 1-20
(e.g., y.sub.1 and y.sub.2 are each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or 20), or a pharmaceutically acceptable salt thereof. In some embodiments, L′ is a nitrogen
atom.

(167) In some embodiments, the conjugate is described by formula (D-V):

(168) ##STR00219##

or a pharmaceutically acceptable salt thereof.

(169) In some embodiments, the conjugate is described by formula (D-V-1):

(170) ##STR00220##

or a pharmaceutically acceptable salt thereof.

(171) In some embodiments, the conjugate is described by formula (D-V-2):

(172) ##STR00221##

or a pharmaceutically acceptable salt thereof.

(173) In some embodiments, the conjugate is described by formula (D-V-3):

(174) ##STR00222##

(175) In some embodiments, the conjugate is described by formula (D-V-4):

(176) ##STR00223##

or a pharmaceutically acceptable salt thereof.

(177) In some embodiments, the conjugate is described by formula (D-V-5):

(178) ##STR00224##

(179) In some embodiments, the conjugate is described by formula (D-V-6):

(180) ##STR00225##

or a pharmaceutically acceptable salt thereof.

(181) In some embodiments, the conjugate is described by formula (D-V-7):

(182) ##STR00226##
or a pharmaceutically acceptable salt thereof.

(183) In some embodiments, the conjugate is described by formula (D-V-8):

(184) ##STR00227##

(185) In some embodiments, the conjugate is described by formula (D-V-9):

(186) ##STR00228##

or a pharmaceutically acceptable salt thereof.

(187) In some embodiments, the conjugate is described by formula (D-V-10):

(188) ##STR00229##

(189) In some embodiments, the conjugate is described by formula (D-VI):

(190) ##STR00230##

or a pharmaceutically acceptable salt thereof.

(191) In some embodiments, the conjugate is described by formula (D-VI-1):

(192) ##STR00231##

or a pharmaceutically acceptable salt thereof.

(193) In some embodiments, the conjugate is described by formula (D-VI-2):

(194) ##STR00232##

or a pharmaceutically acceptable salt thereof.

(195) In some embodiments, the conjugate is described by formula (D-VI-3):

(196) ##STR00233##

(197) In some embodiments, the conjugate is described by formula (D-VI-4):

(198) ##STR00234##
or a pharmaceutically acceptable salt thereof.

(199) In some embodiments, the conjugate is described by formula (D-VI-5):

(200) ##STR00235##

(201) In some embodiments, the conjugate is described by formula (D-VI-6):

(202) ##STR00236##

or a pharmaceutically acceptable salt thereof.

(203) In some embodiments, the conjugate is described by formula (D-VI-7):

(204) ##STR00237##

(205) In some embodiments, the conjugate is described by formula (D-VI-8):

(206) ##STR00238##

or a pharmaceutically acceptable salt thereof.

(207) In some embodiments, the conjugate is described by formula (D-VI-9):

(208) ##STR00239##

(209) In some embodiments, the conjugate is described by formula (D-VII):

(210) ##STR00240##

or a pharmaceutically acceptable salt thereof.

(211) In some embodiments of any of the aspects described herein, R.sub.1 is OH. In some
embodiments of any of the aspects described herein, R.sub.1 is NH.sub.2. In some embodiments of
any of the aspects described herein, R.sub.1 is —NHC(═NH)NH.sub.2.

(212) In some embodiments, the conjugate is described by formula (D-VIII):

(213) ##STR00241##

or a pharmaceutically acceptable salt thereof.

(214) In some embodiments, the conjugate is described by formula (D-VIII-1):

(215) ##STR00242##

or a pharmaceutically acceptable salt thereof.

(216) In some embodiments, the conjugate is described by formula (D-VIII-2):

(217) ##STR00243##

or a pharmaceutically acceptable salt thereof.

(218) In some embodiments, the conjugate is described by formula (D-VIII-3):

(219) ##STR00244##

(220) In some embodiments, the conjugate has the structure selected from

(221) ##STR00245##

or a pharmaceutically acceptable salt thereof.

(222) In some embodiments, the conjugate is described by formula (D-VIII-4):

(223) ##STR00246##

or a pharmaceutically acceptable salt thereof.

(224) In some embodiments, the conjugate is described by formula (D-VIII-5):

(225) ##STR00247##

(226) In some embodiments, the conjugate has the structure selected from:

(227) ##STR00248##

or a pharmaceutically acceptable salt thereof.

(228) In some embodiments, the conjugate is described by the structure

(229) ##STR00249##

or a pharmaceutically acceptable salt thereof.

(230) In some embodiments, the conjugate is described by formula (D-VIII-6):

(231) ##STR00250##

or a pharmaceutically acceptable salt thereof.

(232) In some embodiments, the conjugate is described by formula (D-VIII-7):

(233) ##STR00251##

(234) In some embodiments, the conjugate is described by formula (D-VIII-8):

(235) ##STR00252##

or a pharmaceutically acceptable salt thereof.

(236) In some embodiments, the conjugate is described by formula (D-VIII-9):

(237) ##STR00253##

(238) In some embodiments, the conjugate is described by formula (D-VIII-10):

(239) ##STR00254##

or a pharmaceutically acceptable salt thereof.

(240) In some embodiments, the conjugate is described by formula (D-VIII-11):

(241) ##STR00255##

(242) In some embodiments, the conjugate is described by formula (D-IX):

(243) ##STR00256##

or a pharmaceutically acceptable salt thereof.

(244) In some embodiments, the conjugate is described by formula (D-IX-1):

(245) ##STR00257##

or a pharmaceutically acceptable salt thereof.

(246) In some embodiments, the conjugate is described by formula (D-IX-2):

(247) ##STR00258##

or a pharmaceutically acceptable salt thereof.

(248) In some embodiments, the conjugate is described by formula (D-IX-3):

(249) ##STR00259##

or a pharmaceutically acceptable salt thereof.

(250) In some embodiments, the conjugate is described by formula (D-IX-4):

(251) ##STR00260##

or a pharmaceutically acceptable salt thereof.

(252) In some embodiments, the conjugate is described by formula (D-IX-5):

(253) ##STR00261##

or a pharmaceutically acceptable salt thereof.

(254) In some embodiments, the conjugate is described by formula (D-IX-6):

(255) ##STR00262##

or a pharmaceutically acceptable salt thereof.

(256) In some embodiments, the conjugate is described by formula (D-X):

(257) ##STR00263##

or a pharmaceutically acceptable salt thereof.

(258) In some embodiments, the conjugate is described by formula (D-X-1):

(259) ##STR00264##

or a pharmaceutically acceptable salt thereof.

(260) In some embodiments, the conjugate is described by formula (D-X-2):

(261) ##STR00265##

or a pharmaceutically acceptable salt thereof.

(262) In some embodiments, the conjugate is described by formula (D-X-3):

(263) ##STR00266##

or a pharmaceutically acceptable salt thereof.

(264) In some embodiments of any of the aspects described herein, L or L′ includes one or more
optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally
substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted
C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20
cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20
cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20
cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15
arylene, optionally substituted C2-C15 heteroarylene, O, S, NR.sup.i, P, carbonyl, thiocarbonyl,
sulfonyl, phosphate, phosphoryl, or imino, wherein R.sup.i is H, optionally substituted C1-C20 alkyl,
optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted
C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20
heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl,
optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl,
optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl,
optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl.

(265) In some embodiments of any of the aspects described herein, the backbone of L or L′ consists of
one or more optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene,
optionally substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally
substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted
C3-C20 cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-
C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-
C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-
C15 arylene, optionally substituted C2-C15 heteroarylene, O, S, NR.sup.i, P, carbonyl, thiocarbonyl,
sulfonyl, phosphate, phosphoryl, or imino, wherein R.sup.i is H, optionally substituted C1-C20 alkyl,
optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted
C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20
heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl,
optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl,
optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl,
optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl.

(266) In some embodiments of any of the aspects described herein, L or L′ is oxo substituted. In some
embodiments, the backbone of L or L′ comprises no more than 250 atoms. In some embodiments, L or
L′ is capable of forming an amide, a carbamate, a sulfonyl, or a urea linkage. In some embodiments L
or L′ is a bond. In some embodiments, L or L′ is an atom.

(267) In some embodiments of any of the aspects described herein, each L is described by formula
(D-L-I)

(268) ##STR00267##

wherein L.sup.A is described by formula G.sup.A1-(Z.sup.A1).sub.g1—(Y.sup.A1).sub.h1—

(Z.sup.A2).sub.i1—(Y.sup.A2).sub.j1—(Z.sup.A3).sub.k1—(Y.sup.A3).sub.l1—(Z.sup.A4).sub.m1—
(Y.sup.A4).sub.n1—(Z.sup.A5).sub.o1-G.sup.A2; L.sup.B is described by formula G.sup.B1-
(Z.sup.B1).sub.g2—(Y.sup.B1).sub.h2—(Z.sup.B2).sub.i2—(Y.sup.B2).sub.j2—(Z.sup.B3).sub.k2—
(Y.sup.B3).sub.l2—(Z.sup.B4).sub.m2—(Y.sup.B4).sub.n2—(Z.sup.B5).sub.o2-G.sup.B2; L.sup.C is
described by formula G.sup.C1-(Z.sup.C1).sub.g3—(Y.sup.C1).sub.h3—(Z.sup.C2).sub.i3—
(Y.sup.C2).sub.j3—(Z.sup.C3).sub.k3—(Y.sup.C3).sub.l3—(Z.sup.C4).sub.m3—(Y.sup.C4).sub.n3—
(Z.sup.C5).sub.o3-G.sup.C2; G.sup.A1 is a bond attached to Q; G.sup.A2 is a bond attached to
A.sub.1; G.sup.B1 is a bond attached to Q); G.sup.B2 is a bond attached to A.sub.2; G.sup.G1 is a
bond attached to Q; G.sup.C2 is a bond attached to E or a functional group capable of reacting with a
functional group conjugated to E (e.g., maleimide and cysteine, amine and activated carboxylic acid,
thiol and maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and
alkene and tetrazine); each of Z.sup.A1, Z.sup.A2, Z.sup.A3, Z.sup.A4, Z.sup.A5, Z.sup.B1, Z.sup.B2,
Z.sup.B3, Z.sup.B4, Z.sup.B5, Z.sup.C1, Z.sup.C2, Z.sup.C3, Z.sup.C4 and Z.sup.C5 is,
independently, optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene,
optionally substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally
substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted
C3-C20 cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-
C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-
C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-
C15 arylene, or optionally substituted C2-C15 heteroarylene; each of Y.sup.A1, Y.sup.A2, Y.sup.A3,
Y.sup.A4, Y.sup.B1, Y.sup.B2, Y.sup.B3, Y.sup.B4, Y.sup.C1, Y.sup.C2, Y.sup.C3 and Y.sup.C4 is,
independently, O, S, NR.sup.i, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino;
R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally
substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-
C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl,
optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally
substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally
substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted
C2-C15 heteroaryl; each of g1, h1, i1, j1, k1, l1, m1, n1, o1, g2, h2, i2, j2, k2, l2, m2, n2, o2, g3, h3, i3,
j3, k3, l3, m3, n3, and o3 is, independently, 0 or 1; Q is a nitrogen atom, optionally substituted C1-C20
alkylene, optionally substituted C1-C20 heteroalkylene, optionally substituted C2-C20 alkenylene,
optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally
substituted C2-C20 heteroalkynylene, optionally substituted C3-C20 cycloalkylene, optionally
substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally
substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally
substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene, or optionally
substituted C2-C15 heteroarylene.

(269) In some embodiments, L.sup.c may have two points of attachment to the Fc domain, Fc-binding
peptide, albumin protein, or albumin protein-binding peptide (e.g., two G.sup.C2) In some
embodiments of any of the aspects described herein, L includes a polyethylene glycol (PEG) linker. A
PEG linker includes a linker having the repeating unit structure (—CH.sub.2CH.sub.2O—).sub.n,
wherein n is an integer from 2 to 100. A polyethylene glycol linker may covalently join a neuraminidase
inhibitor and E (e.g., in a conjugate of any one of formulas (M-I)-(M-XI)). A polyethylene glycol linker
may covalently join a first neuraminidase inhibitor and a second neuraminidase inhibitor (e.g., in a
conjugate of any one of formulas (D-I)-(D-XI)). A polyethylene glycol linker may covalently join a
neuraminidase inhibitor dimer and E (e.g., in a conjugate of any one of formulas (D-I)-(D-XI)). A
polyethylene glycol linker may be selected any one of PEG.sub.2 to PEG.sub.100 (e.g., PEG.sub.2,
PEG.sub.3, PEG.sub.4, PEG.sub.5, PEG.sub.5-PEG.sub.10, PEG.sub.10-PEG.sub.20, PEG.sub.20-
PEG.sub.30, PEG.sub.30-PEG.sub.40, PEG.sub.50-PEG.sub.60, PEG.sub.60-PEG.sub.70,
PEG.sub.70-PEG.sub.80, PEG.sub.80-PEG.sub.90, PEG.sub.90-PEG.sub.10). In some
embodiments, L.sup.c includes a PEG linker, where L.sup.c is covalently attached to each of Q and E.

(270) In some embodiments, L is

(271) ##STR00268## ##STR00269## ##STR00270## ##STR00271## ##STR00272##

##STR00273## ##STR00274## ##STR00275## ##STR00276## ##STR00277## ##STR00278##

wherein z.sub.1 and z.sub.2 are each, independently, and integer from 1 to 20; and R.sub.9 is
selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15 aryl, and C2-C15
heteroaryl.

(272) In some embodiments, L is

(273) ##STR00279## ##STR00280## ##STR00281## ##STR00282## ##STR00283##

##STR00284## ##STR00285## ##STR00286## ##STR00287##

wherein R* is a bond or includes one or more of optionally substituted C1-C20 alkylene, optionally
substituted C1-C20 heteroalkylene, optionally substituted C2-C20 alkenylene, optionally substituted
C2-C20 heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20
heteroalkynylene, optionally substituted C3-C20 cycloalkylene, optionally substituted C3-C20
heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20
heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20
heterocycloalkynylene, optionally substituted C5-C15 arylene, optionally substituted C2-C15
heteroarylene, O, S, NR.sup.i, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, and imino, and wherein
R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally
substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-
C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl,
optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally
substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally
substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted
C2-C15 heteroaryl.

(274) In some embodiments, Y is:

(275) ##STR00288##

(—NH(C═O)O—) and L is:

(276) ##STR00289##

(277) In some embodiments, Y is:

(278) ##STR00290##

(—NH(C═O)O—) and L is:

(279) ##STR00291##

(280) In some embodiments, Y is:

(281) ##STR00292##

(—NH(C═O)O—) and L is:

(282) ##STR00293##

(283) In some embodiment, Y is:

(284) ##STR00294##

(—O—) and L is:

(285) ##STR00295##

(286) In another aspect, the invention features a conjugate described by formula (M-I):

(287) ##STR00296##

wherein each A.sub.1 is independently selected from any one of formulas (A-I)-(A-XII); each E
comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138), an albumin protein (e.g., an albumin protein having the sequence of any one of
SEQ ID NOs: 139-141), an albumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2; T is
an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20);
and L is a linker covalently attached to each of E and A.sub.1, or a pharmaceutically acceptable salt
thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20), each A.sub.1 may be independently selected from any one of formulas (A-I)-(A-XII). In some
embodiments, each A.sub.1 may be independently selected from any one of formulas (A-I), (A-II), (A-
VI), or (A-VII). In other embodiments, each A.sub.1 may be independently selected from formula (A-I).

(288) In another aspect, the invention features a conjugate described by formula (M-I):

(289) ##STR00297##

wherein each A.sub.1 is independently selected from any one of formulas (A-I)-(A-V); each E
comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138), an albumin protein (e.g., an albumin protein having the sequence of any one of
SEQ ID NOs: 139-141), an albumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2; T is
an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20);
and L is a linker covalently attached to each of E and A.sub.1, or a pharmaceutically acceptable salt
thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20), each A.sub.1 may be independently selected from any one of formulas (A-I)-(A-V).

(290) In another aspect, the invention features a conjugate described by formula (M-I):
(291) ##STR00298##

wherein each A.sub.1 is independently selected from any one of formulas (A-VI)-(A-IX); each E
comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138), an albumin protein (e.g., an albumin protein having the sequence of any one of
SEQ ID NOs: 139-141), an albumin protein-binding peptide, or an Fc-binding peptide; n is 1 or 2; T is
an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20);
and L is a linker covalently attached to each of E and A.sub.1, or a pharmaceutically acceptable salt
thereof. When T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
or 20), each A.sub.1 may be independently selected from any one of formulas (A-VI)-(A-IX).

(292) In some embodiments, the conjugate is described by formula (M-II):

(293) ##STR00299##

or a pharmaceutically acceptable salt thereof.

(294) In some embodiments, the conjugate is described by formula (M-II-1):

(295) ##STR00300##

or a pharmaceutically acceptable salt thereof.

(296) In some embodiments, the conjugate is described by formula (M-II-2):

(297) ##STR00301##

or a pharmaceutically acceptable salt thereof.

(298) In some embodiments, the conjugate is described by formula (M-II-3):

(299) ##STR00302##

wherein L′ is the remainder of L, and y.sub.1 is an integer from 1-20 (e.g., y.sub.1 is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.

(300) In some embodiments, the conjugate is described by formula (M-II-4):

(301) ##STR00303##

or a pharmaceutically acceptable salt thereof.

(302) In some embodiments, the conjugate is described by formula (M-II-5):

(303) ##STR00304##

(304) In some embodiments, the conjugate has the structure

(305) ##STR00305##

or a pharmaceutically acceptable salt thereof.

(306) In some embodiments, the conjugate is described by formula (M-II-6):

(307) ##STR00306##
wherein R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-
C15 aryl, and C2-C15 heteroaryl; or a pharmaceutically acceptable salt thereof. In some
embodiments, R.sub.7 is selected from C1-C20 alkyl (e.g., methyl, ethyl, propyl, or butyl).

(308) In some embodiments, the conjugate is described by formula (M-II-7):

(309) ##STR00307##

or a pharmaceutically acceptable salt thereof.

(310) In some embodiments, the conjugate is described by formula (M-II-8):

(311) ##STR00308##

(312) In some embodiments, the conjugate has the structure:

(313) ##STR00309##

(314) In some embodiments, the conjugate is described by formula (M-II-9):

(315) ##STR00310##

or a pharmaceutically acceptable salt thereof.

(316) In some embodiments, the conjugate is described by formula (M-II-10):

(317) ##STR00311##

(318) In some embodiments, the conjugate has the structure

(319) ##STR00312##

or a pharmaceutically acceptable salt thereof.

(320) In some embodiments, the conjugate is described by formula (M-III):

(321) ##STR00313##

or a pharmaceutically acceptable salt thereof.

(322) In some embodiments, the conjugate is described by formula (M-III-1):

(323) ##STR00314##

or a pharmaceutically acceptable salt thereof.

(324) In some embodiments, the conjugate is described by formula (M-III-2):

(325) ##STR00315##

or a pharmaceutically acceptable salt thereof.

(326) In some embodiments, the conjugate is described by formula (M-III-3):

(327) ##STR00316##

(328) In some embodiments, the conjugate is described by formula (M-III-4):

(329) ##STR00317##

or a pharmaceutically acceptable salt thereof.

(330) In some embodiments, the conjugate is described by formula (M-III-5):

(331) ##STR00318##

(332) In some embodiments, the conjugate is described by formula (M-III-6):

(333) ##STR00319##

or a pharmaceutically acceptable salt thereof.

(334) In some embodiments, the conjugate is described by formula (M-III-7):

(335) ##STR00320##

(336) In some embodiments, the conjugate is described by formula (M-III-8):

(337) ##STR00321##

or a pharmaceutically acceptable salt thereof.

(338) In some embodiments, the conjugate is described by formula (M-III-9):

(339) ##STR00322##

(340) In some embodiments, the conjugate is described by formula (M-IV):

(341) ##STR00323##

or a pharmaceutically acceptable salt thereof.

(342) In some embodiments, the conjugate is described by formula (M-IV-1):

(343) ##STR00324##

or a pharmaceutically acceptable salt thereof.

(344) In some embodiments, the conjugate is described by formula (M-IV-2):

(345) ##STR00325##
wherein L′ is the remainder of L, and y.sub.1 is an integer from 1-20 (e.g., y.sub.1 is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.

(346) In some embodiments, the conjugate is described by formula (M-V):

(347) ##STR00326##

or a pharmaceutically acceptable salt thereof.

(348) In some embodiments, the conjugate is described by formula (M-V-1):

(349) ##STR00327##

or a pharmaceutically acceptable salt thereof.

(350) In some embodiments, the conjugate is described by formula (M-V-2):

(351) ##STR00328##

or a pharmaceutically acceptable salt thereof.

(352) In some embodiments, the conjugate is described by formula (M-V-3):

(353) ##STR00329##

(354) In some embodiments, the conjugate is described by formula (M-V-4):

(355) ##STR00330##

or a pharmaceutically acceptable salt thereof.

(356) In some embodiments, the conjugate is described by formula (M-V-5):

(357) ##STR00331##

(358) In some embodiments, the conjugate is described by formula (M-V-6):

(359) ##STR00332##

or a pharmaceutically acceptable salt thereof.

(360) In some embodiments, the conjugate is described by formula (M-V-7):

(361) ##STR00333##

or a pharmaceutically acceptable salt thereof.

(362) In some embodiments, the conjugate is described by formula (M-V-8):

(363) ##STR00334##

(365) ##STR00335##

or a pharmaceutically acceptable salt thereof.

(366) In some embodiments, the conjugate is described by formula (M-V-10):

(367) ##STR00336##

(368) In some embodiments, the conjugate is described by formula (M-VI):

(369) ##STR00337##

or a pharmaceutically acceptable salt thereof.

(370) In some embodiments, the conjugate is described by formula (M-VI-1):

(371) ##STR00338##

or a pharmaceutically acceptable salt thereof.

(372) In some embodiments, the conjugate is described by formula (M-VI-2):

(373) ##STR00339##

or a pharmaceutically acceptable salt thereof.

(374) In some embodiments, the conjugate is described by formula (M-VI-3):

(375) ##STR00340##

(376) wherein L′ is the remainder of L, and y.sub.1 is an integer from 1-20 (e.g., y.sub.1 is 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.

(377) In some embodiments, the conjugate is described by formula (M-VI-4):

(378) ##STR00341##

or a pharmaceutically acceptable salt thereof.

(379) In some embodiments, the conjugate is described by formula (M-VI-5):

(380) ##STR00342##

(381) In some embodiments, the conjugate is described by formula (M-VI-6):

(382) ##STR00343##

or a pharmaceutically acceptable salt thereof.

(383) In some embodiments, the conjugate is described by formula (M-VI-7):

(384) ##STR00344##
wherein L′ is the remainder of L, and y.sub.1 is an integer from 1-20 (e.g., y.sub.1 is 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.

(385) In some embodiments, the conjugate is described by formula (M-VI-8):

(386) ##STR00345##

or a pharmaceutically acceptable salt thereof.

(387) In some embodiments, the conjugate is described by formula (M-VI-9):

(388) ##STR00346##

(389) In some embodiments, the conjugate is described by formula (M-VII):

(390) ##STR00347##

or a pharmaceutically acceptable salt thereof.

(391) In some embodiments of any of the aspects described herein, R.sub.1 is OH. In some
embodiments of any of the aspects described herein, R.sub.1 is NH.sub.2. In some embodiments of
any of the aspects described herein, R.sub.1 is —NHC(═NH)NH.sub.2.

(392) In some embodiments, the conjugate is described by formula (M-VIII):

(393) ##STR00348##

or a pharmaceutically acceptable salt thereof.

(394) In some embodiments, the conjugate is described by formula (M-VIII-1):

(395) ##STR00349##

or a pharmaceutically acceptable salt thereof.

(396) In some embodiments, the conjugate is described by (M-VIII-2):

(397) ##STR00350##

or a pharmaceutically acceptable salt thereof.

(398) In some embodiments, the conjugate is described by formula (M-VIII-3):

(399) ##STR00351##

(400) In some embodiments, the conjugate is described by formula (M-VIII-4):

(401) ##STR00352##

or a pharmaceutically acceptable salt thereof.

(402) In some embodiments, the conjugate is described by formula (M-VIII-5):

(403) ##STR00353##

wherein L′ is the remainder of L, and y.sub.1 is an integer from 1-20, or a pharmaceutically acceptable
salt thereof.

(404) In some embodiments, the conjugate has the structure of

(405) ##STR00354##

or a pharmaceutically acceptable salt thereof.

(406) In some embodiments, the conjugate is described by formula (M-VIII-6):

(407) ##STR00355##

or a pharmaceutically acceptable salt thereof.

(408) In some embodiments, the conjugate is described by formula (M-VIII-7):

(409) ##STR00356##

(410) In some embodiments, the conjugate is described by formula (M-VIII-8):

(411) ##STR00357##

or a pharmaceutically acceptable salt thereof.

(412) In some embodiments, the conjugate is described by formula (M-VIII-9):

(413) ##STR00358##

(414) In some embodiments, the conjugate is described by formula (M-VIII-10):

(415) ##STR00359##

or a pharmaceutically acceptable salt thereof.

(416) In some embodiments, the conjugate is described by formula (M-VIII-11):

(417) ##STR00360##

(418) In some embodiments, the conjugate is described by formula (M-IX):

(419) ##STR00361##

or a pharmaceutically acceptable salt thereof.

(420) In some embodiments, the conjugate is described by formula (M-IX-1):

(421) ##STR00362##
or a pharmaceutically acceptable salt thereof.

(422) In some embodiments, the conjugate is described by formula (M-IX-2):

(423) ##STR00363##

or a pharmaceutically acceptable salt thereof.

(424) In some embodiments, the conjugate is described by formula (M-IX-3):

(425) ##STR00364##

or a pharmaceutically acceptable salt thereof.

(426) In some embodiments, the conjugate is described by formula (M-IX-4):

(427) ##STR00365##

or a pharmaceutically acceptable salt thereof.

(428) In some embodiments, the conjugate is described by formula (M-IX-5):

(429) ##STR00366##

or a pharmaceutically acceptable salt thereof.

(430) In some embodiments, the conjugate is described by formula (M-IX-6):

(431) ##STR00367##

or a pharmaceutically acceptable salt thereof.

(432) In some embodiments, the conjugate is described by formula (M-X):

(433) ##STR00368##

or a pharmaceutically acceptable salt thereof.

(434) In some embodiments, the conjugate is described by formula (M-X-1):

(435) ##STR00369##

or a pharmaceutically acceptable salt thereof.

(436) In some embodiments, the conjugate is described by formula (M-X-2):

(437) ##STR00370##

or a pharmaceutically acceptable salt thereof.

(438) In some embodiments, the conjugate is described by formula (M-X-3):

(439) ##STR00371##

or a pharmaceutically acceptable salt thereof.

(440) In some embodiments of any of the aspects described herein, L or L′ comprises one or more
optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally
substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted
C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20
cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20
cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20
cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15
arylene, optionally substituted C2-C15 heteroarylene, O, S, NR.sup.i, P, carbonyl, thiocarbonyl,
sulfonyl, phosphate, phosphoryl, or imino, wherein R.sup.i is H, optionally substituted C1-C20 alkyl,
optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted
C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20
heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl,
optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl,
optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl,
optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl.

(441) In some embodiments of any of the aspects described herein, the backbone of L or L′ consists of
one or more optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene,
optionally substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally
substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted
C3-C20 cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-
C20 cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-
C20 cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-
C15 arylene, optionally substituted C2-C15 heteroarylene, O, S, NR.sup.i, P, carbonyl, thiocarbonyl,
sulfonyl, phosphate, phosphoryl, or imino, wherein R.sup.i is H, optionally substituted C1-C20 alkyl,
optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted
C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20
heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl,
optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl,
optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl,
optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl.

(442) In some embodiments of any of the aspects described herein, L or L′ is oxo substituted. In some
embodiments, the backbone of L or L′ comprises no more than 250 atoms. In some embodiments, L or
L′ is capable of forming an amide, a carbamate, a sulfonyl, or a urea linkage. In some embodiments, L
or L′ is a bond. In some embodiments, L or L′ is an atom. In some embodiments, L′ is a nitrogen atom.

(443) In some embodiments, each L is described by formula (M-L-I):

J.sup.1-(Q.sup.1).sub.g-(T.sup.1).sub.h-(Q.sup.2).sub.i-(T.sup.2).sub.j-(Q.sup.3).sub.k-(T.sup.3).sub.l-
(Q.sup.4).sub.m-(T.sup.4).sub.n-(Q.sup.5).sub.o-J.sup.2

wherein: J.sup.1 is a bond attached to A.sub.1; J.sup.2 is a bond attached to E or a functional group
capable of reacting with a functional group conjugated to E (e.g., maleimide and cysteine, amine and
activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine, isocyanate and
amine, azide and alkyne, and alkene and tetrazine); each of Q.sup.1, Q.sup.2, Q.sup.3, Q.sup.4 and
Q.sup.5 is, independently, optionally substituted C1-C20 alkylene, optionally substituted C1-C20
heteroalkylene, optionally substituted C2-C20 alkenylene, optionally substituted C2-C20
heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20
heteroalkynylene, optionally substituted C3-C20 cycloalkylene, optionally substituted C3-C20
heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20
heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20
heterocycloalkynylene, optionally substituted C5-C15 arylene, or optionally substituted C2-C15
heteroarylene; each of T.sup.1, T.sup.2, T.sup.3, T.sup.4 is, independently, O, S, NR.sup.i, P, carbonyl,
thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino; R.sup.i is H, optionally substituted C1-C20
alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally
substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20
heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl,
optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl,
optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl,
optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl; and each of g, h, i, j, k,
l, m, n, and o is, independently, 0 or 1; or a pharmaceutically acceptable salt thereof.

(444) In some embodiments, J.sup.2 may have two points of attachment to the Fc domain, Fc-binding
peptide, albumin protein, or albumin protein-binding peptide (e.g., two J.sup.2).

(445) In some embodiments, L is

(446) ##STR00372##

wherein d is an integer from 1 to 20 (e.g., d is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20).

(447) In some embodiments, L is

(448) ##STR00373## ##STR00374## ##STR00375##

wherein each of d and e is, independently, an integer from 1 to 26; or a pharmaceutically acceptable
salt thereof.

(449) In some embodiments of any of the aspects described herein, L includes a polyethylene glycol
(PEG) linker. A PEG linker includes a linker having the repeating unit structure (—CH.sub.2CH.sub.2O
—).sub.n, wherein n is an integer from 2 to 100. A polyethylene glycol linker may covalently join a
neuraminidase inhibitor and E (e.g., in a conjugate of any one of formulas (M-I)-(M-XI)). A
polyethylene glycol linker may covalently join a first neuraminidase inhibitor and a second
neuraminidase inhibitor (e.g., in a conjugate of any one of formulas (D-I)-(D-XI)). A polyethylene glycol
linker may covalently join a neuraminidase inhibitor dimer and E (e.g., in a conjugate of any one of
formulas (D-I)-(D-XI)). A polyethylene glycol linker may be selected any one of PEG.sub.2 to
PEG.sub.100 (e.g., PEG.sub.2, PEG.sub.3, PEG.sub.4, PEG.sub.5, PEG.sub.5-PEG.sub.10,
PEG.sub.10-PEG.sub.20, PEG.sub.20-PEG.sub.30, PEG.sub.30-PEG.sub.40, PEG.sub.50-
PEG.sub.60, PEG.sub.60-PEG.sub.70, PEG.sub.70-PEG.sub.80, PEG.sub.80-PEG.sub.90,
PEG.sub.90-PEG.sub.100). In some embodiments, L.sup.c includes a PEG linker, where L.sup.c is
covalently attached to each of Q and E.

(450) In some embodiments of any of the aspects described herein, R.sub.1 is —NHC(═NH)NH.sub.2.
In some embodiments of any of the aspects described herein, R.sub.2 is —F. In some embodiments of
any of the aspects described herein, R.sub.3 is —F. In some embodiments of any of the aspects
described herein, R.sub.4 is —CO.sub.2H. In some embodiments of any of the aspects described
herein, R.sub.5 is —COCH.sub.3.

(451) In some embodiments of any of the aspects described herein, L is covalently attached to the
nitrogen atom of a surface exposed lysine of E or L is covalently attached to the sulfur atom of a
surface exposed cysteine of E.

(452) In some embodiments of any of the aspects described herein, E is an Fc domain monomer. In
some embodiments, n is 2 and each E dimerizes to form an Fc domain.

(453) In some embodiments, n is 2, each E is an Fc domain monomer, each E dimerizes to form an Fc

domain, and the conjugate is described by formula (D-I-1):

(454) ##STR00376##

wherein J is an Fc domain; and T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.

(455) In some embodiments, the conjugate has the structure of

(456) ##STR00377##
or a pharmaceutically acceptable salt thereof.

(457) In some embodiments, n is 2, each E is an Fc domain monomer, each E dimerizes to form an Fc

domain, and the conjugate is described by formula (M-I-1):

(458) ##STR00378##

wherein J is an Fc domain; and T is an integer from 1 to 20 (e.g., T is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,

12, 13, 14, 15, 16, 17, 18, 19, or 20), or a pharmaceutically acceptable salt thereof.

(459) In some embodiments of any of the aspects described herein, E has the sequence of any one of
SEQ ID NOs: 1-138.

(460) In some embodiments of any of the aspects described herein, E is an albumin protein, an
albumin protein-binding peptide, or an Fc-binding peptide. In some embodiments, where E is an
albumin protein, an albumin protein-binding peptide, or an Fc-binding peptide, n is 1.

(461) In some embodiments, n is 1, E is an albumin protein, an albumin protein-binding peptide, or an

Fc-binding peptide and the conjugate is described by formula (D-I-2):

(462) ##STR00379##

wherein E is an albumin protein, an albumin protein-binding peptide, or Fc-binding peptide; and T is an

integer from 1 to 20, or a pharmaceutically acceptable salt thereof.

(463) In some embodiments, n is 1, E is an albumin protein, an albumin protein-binding peptide, or an

Fc-binding peptide, and the conjugate is described by formula (M-I-2):

(464) ##STR00380##

wherein E is an albumin protein, an albumin protein-binding peptide, or an Fc-binding peptide; and T is

an integer from 1 to 20, or a pharmaceutically acceptable salt thereof.

(465) In some embodiments of any of the aspects described herein, E is an albumin protein having the
sequence of any one of SEQ ID NOs: 139-141.

(466) In some embodiments of any of the aspects described herein, T is 1, 2, 3, 4, or 5.

(467) In another aspect, the invention provides a population of conjugates having the structure of any
of the conjugates described herein (e.g., a population of conjugates having the formula of any one of
formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)), wherein the average value of T is 1 to 20
(e.g., the average value of T is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to
4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to 7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5). In some embodiments, the
average value of T is about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,
11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20.

(468) In some embodiments of any of the aspects described herein, when T is greater than 1 (e.g., T
is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A.sub.1-L-A.sub.2 may be
independently selected (e.g., independently selected from any of the A.sub.1-L-A.sub.2 structures
described herein). In some embodiments, E may be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more
different A.sub.1-L-A.sub.2 moieties. In some embodiments, E is conjugated to a first A.sub.1-L-
A.sub.2 moiety, and a second A.sub.1-L-A.sub.2, moiety. In some embodiments, A.sub.1 and A.sub.2
of the first A.sub.1-L-A.sub.2 moiety are independently selected from any one of formulas (A-III)-(A-V),
and A.sub.1 and A.sub.2 of the second A.sub.1-L-A.sub.2 moiety are independently selected from any
one of formulas (A-I), (A-II), (A-VI), (A-VII), (A-VIII), and (A-IX).

(469) In some embodiments, each of the first A.sub.1-L-A.sub.2 moieties is conjugated specifically to a
lysine residue of E (e.g., the nitrogen atom of a surface exposed lysine residue of E), and each of the
second A.sub.1-L-A.sub.2 moieties is conjugated specifically to a cysteine residue of E (e.g., the sulfur
atom of a surface exposed cysteine residue of E). In some embodiments, each of the first A.sub.1-L-
A.sub.2 moieties is conjugated specifically to a cysteine residue of E (e.g., the sulfur atom of a surface
exposed cysteine residue of E), and each of the second A.sub.1-L-A.sub.2 moieties is conjugated
specifically to a lysine residue of E (e.g., the nitrogen atom of a surface exposed lysine residue of E).

(470) In some embodiments, the number of first A.sub.1-L-A.sub.2 moieties conjugated to E is an

integer from 1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the number of second
A.sub.1-L-A.sub.2 moieties conjugated to E is an integer from 1 to 10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

(471) In some embodiments of any of the aspects described herein, when T is greater than 1 (e.g., T
is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), each A.sub.1-L may be
independently selected (e.g., independently selected from any of the A.sub.1-L structures described
herein). In some embodiments, E may be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different
A.sub.1-L moieties. In some embodiments, E is conjugated to a first A.sub.1-L moiety, and a second
A.sub.1-L, moiety. In some embodiments, A.sub.1 of the first A.sub.1-L moiety is selected from any
one of formulas (A-III)-(A-V), and A.sub.1 of the second A.sub.1-L moiety is selected from any one of
formulas (A-I), (A-II), (A-VI), (A-VII), (A-VIII), or (A-IX).

(472) In some embodiments, each of the first A.sub.1-L moieties is conjugated specifically to a lysine
residue of E (e.g., the nitrogen atom of a surface exposed lysine residue of E), and each of the second
A.sub.1-L moieties is conjugated specifically to a cysteine residue of E (e.g., the sulfur atom of a
surface exposed cysteine residue of E). In some embodiments, each of the first A.sub.1-L moieties is
conjugated specifically to a cysteine residue of E (e.g., the sulfur atom of a surface exposed cysteine
residue of E), and each of the second A.sub.1-L moieties is conjugated specifically to a lysine residue
of E (e.g., the nitrogen atom of a surface exposed lysine residue of E).

(473) In some embodiments, the number of first A.sub.1-L moieties conjugated to E is an integer from
1 to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the number of second A.sub.1-L
moieties conjugated to E is an integer from 1 to 10 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10).

(474) In another aspect, the invention features a conjugate described by formula (D′-I):

(475) ##STR00381##

wherein each A.sub.1 is independently selected from any one of formulas (A-III)-(A-V); wherein each
A.sub.2 is independently selected from any one of formulas (A-I), (A-II), (A-VI), (A-VII), (A-VIII), and
(A-IX); each E comprises an Fc domain monomer (e.g., an Fc domain monomer having the sequence
of any one of SEQ ID NOs: 1-138), an albumin protein (e.g., an albumin protein having the sequence
of any one of SEQ ID NOs: 139-141), an albumin protein-binding peptide, or an Fc-binding peptide; n
is 1 or 2; Ti is an integer from 1 to 10 (e.g., T.sub.1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); L.sub.1 is a linker
covalently conjugated to E and to each A.sub.1; Ti is an integer from 1 to 10 (e.g., Ti is 1, 2, 3, 4, 5, 6,
7, 8, 9, 10); L.sub.2 is a linker covalently conjugated to E and each A.sub.2; T.sub.2 is an integer from
1 to 10 (e.g., T.sub.2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), or a pharmaceutically acceptable salt thereof.

(476) In some embodiments, each A.sub.1-L-A.sub.1 is conjugated specifically to a lysine residue of E

(e.g., the nitrogen atom of a surface exposed lysine residue of E), and each the A.sub.2-L-A.sub.2 is
conjugated specifically to a cysteine residue of E (e.g., the sulfur atom of a surface exposed cysteine
residue of E). In some embodiments, each A.sub.1-L-A.sub.1 moiety is conjugated specifically a
cysteine residue of E (e.g., the sulfur atom of a surface exposed cysteine residue of E), and each
A.sub.2-L-A.sub.2 moiety is conjugated specifically to a lysine residue of E (e.g., the nitrogen atom of
a surface exposed lysine residue of E).

(477) In another aspect, the invention features a conjugate described by formula (M′-I):

(478) ##STR00382##
wherein each A.sub.1 is independently selected from any one (M-IX) of formulas (A-III)-(A-V); wherein
each A.sub.2 is independently selected from any one of formulas (A-I), (A-II), (A-VI), (A-VII), (A-VIII),
and (A-IX); each E comprises an Fc domain monomer (e.g., an Fc domain monomer having the
sequence of any one of SEQ ID NOs: 1-138), an albumin protein (e.g., an albumin protein having the
sequence of any one of SEQ ID NOs: 139-141), an albumin protein-binding peptide, or an Fc-binding
peptide; n is 1 or 2; T.sub.1 is an integer from 1 to 10 (e.g., T.sub.1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);
L.sub.1 is a linker covalently conjugated to E and A.sub.1; T.sub.1 is an integer from 1 to 10 (e.g.,
T.sub.1 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); L.sub.2 is a linker covalently conjugated to E and A.sub.2;
T.sub.2 is an integer from 1 to 10 (e.g., T.sub.2 is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), or a pharmaceutically
acceptable salt thereof.

(479) In some embodiments, each A.sub.1-L is conjugated specifically to a lysine residue of E (e.g.,
the nitrogen atom of a surface exposed lysine residue of E), and each the A.sub.2-L is conjugated
specifically to a cysteine residue of E (e.g., the sulfur atom of a surface exposed cysteine residue of
E). In some embodiments, each A.sub.1-L moiety is conjugated specifically a cysteine residue of E
(e.g., the sulfur atom of a surface exposed cysteine residue of E), and each A.sub.2-L moiety is
conjugated specifically to a lysine residue of E (e.g., the nitrogen atom of a surface exposed lysine
residue of E).

(480) In another aspect, the invention provides a pharmaceutical composition comprising any of the
conjugates described herein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-
(M-XI), or (M′-I)), or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable
excipient.

(481) In another aspect, the invention provides a method for the treatment of a subject having a viral
infection or presumed to have a viral infection, the method comprising administering to the subject an
effective amount of any of the conjugates or compositions described herein (e.g., a conjugate of any
one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)).

(482) In another aspect, the invention provides a method for the prophylactic treatment of a viral
infection in a subject in need thereof, the method comprising administering to the subject an effective
amount of any of the conjugates or compositions described herein (e.g., a conjugate of any one of
formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)).

(483) In some embodiments, the viral infection is caused by influenza virus or parainfluenza virus. In
some embodiments, the viral infection is influenza virus A, B, or C, or parainfluenza virus.

(484) In some embodiments, the subject is immunocompromised.

(485) In some embodiments, the subject has been diagnosed with humoral immune deficiency, T cell
deficiency, neutropenia, asplenia, or complement deficiency.

(486) In some embodiments, the subject is being treated or is about to be treated with an
immunosuppressive therapy.

(487) In some embodiments, the subject has been diagnosed with a disease which causes
immunosuppression. In some embodiments, the disease is cancer or acquired immunodeficiency
syndrome. In some embodiments, the cancer is leukemia, lymphoma, or multiple myeloma.

(488) In some embodiments, the subject has undergone or is about to undergo hematopoietic stem
cell transplantation.

(489) In some embodiments, the subject has undergone or is about to undergo an organ transplant.

(490) In some embodiments, the subject has or is at risk of developing a secondary infection. In some
embodiments, the secondary infection is a bacterial infection (e.g., methicillin-resistant Staphylococcus
aureus (MRSA), Streptococcus pneumoniae, Pseudomonas aeruginosa, and/or Haemophilus
influenzae), a viral infection, or a fungal infection. In particular embodiments, the secondary infection is
MRSA. In certain embodiments, the secondary infection is S. pneumoniae. In some embodiments, the
secondary infection is a respiratory infection (e.g., an infection of the respiratory tract). In some
embodiments, the secondary infection is associated with (e.g., causes) pneumonia (e.g., bacterial or
viral pneumonia). In some embodiments, the subject has or is at risk of developing pneumonia.

(491) In another aspect, the disclosure features a method of preventing a secondary infection in a
subject diagnosed with an influenza infection, wherein the method includes administering to the
subject a conjugate or composition described herein. In some embodiments, the method includes
administering to the subject conjugate 45 or a pharmaceutical composition including conjugate 45.

(492) In some embodiments, administering a conjugate or composition of the present invention to a

subject diagnosed with an influenza infection decreases the likelihood of developing a secondary
infection, e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%,
500% or more (e.g., as compared to a subject suffering from influenza not treated with the conjugate
or composition). For example, administering a conjugate or composition of the present invention to a
subject diagnosed with an influenza infection decreases the likelihood of developing a secondary
bacterial infection (e.g., MRSA, Streptococcus pneumoniae, Pseudomonas aeruginosa, and/or
Haemophilus influenzae), e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%,
300%, 400%, 500% or more. In some embodiments, the conjugate or composition is administered
intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally,
intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally,
intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival,
intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, by
inhalation, by injection, or by infusion.

(493) In some embodiments, the subject is treated with a second therapeutic agent. In some
embodiments, the second therapeutic agent is an antiviral agent. In some embodiments, the antiviral
agent is selected from pimovidir, oseltamivir, zanamivir, peramivir, laninamivir, amantadine, or
rimantadine. In particular embodiments, the second therapeutic agent is pimovidir. In some
embodiments, the second therapeutic agent is a viral vaccine. In some embodiments, the viral vaccine
elicits an immune response in the subject against influenza virus A, B, or C, or parainfluenza virus.

(494) In some embodiments, the conjugate is administered in combination with an antiviral agent,
where the antiviral agent is baloxavir. In certain embodiments, the conjugate is described by formula
(D-II-6). In other embodiments, the conjugate is described by formula (D-II-7). In certain embodiments,
each E includes an Fc domain that has an amino acid sequence at least 95% identical to the
sequence of any one of SEQ ID NOs: 63-138. In particular embodiments, each E includes an Fc
domain that has an amino acid sequence at least 95% identical to the sequence of any one of SEQ ID
NO: 63, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID
NO: 76, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, or SEQ ID NO: 86. In
particular embodiments, each E includes an Fc domain that has the amino acid sequence of any one
of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73,
SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, or SEQ ID NO: 86.
In preferred embodiments, the conjugate is conjugate 45 (e.g., conjugate 45a or 45b) or conjugate 46.

(495) In certain embodiments, the conjugate and baloxavir are administered sequentially. In other
embodiments, the conjugate and baloxavir are administered simultaneously.

(496) In one aspect, the disclosure provides a method for treating or preventing a viral infection in
subject by administering to the subject: a) an effective amount of a conjugate or composition of any
one of claims 1-215; and b) a second therapeutic agent. In certain embodiments, the conjugate is
administered to the subject after the subject has a viral infection, is presumed to have a viral infection,
or has been exposed to a virus. In some embodiments, the conjugate is administered to the subject
prophylactically. In certain embodiments, the second therapeutic agent is administered to the subject
after the subject has a viral infection, is presumed to have a viral infection, or has been exposed to the
virus. In some embodiments, the second therapeutic agent is administered to the subject
prophylactically. In some embodiments, the second therapeutic agent is administered within 30 days,
within 14 days, within 7 days, within 2 days, or within 24 hours days of the conjugate. In particular
embodiments, the second therapeutic agent is administered within 2 days of the conjugate. In certain
embodiments, the second therapeutic agent is an antiviral agent (e.g., pimovidir, oseltamivir,
zanamivir, peramivir, laninamivir, amantadine, baloxavir marboxil, baloxavir acid, rimantadine, or a
pharmaceutically acceptable salt thereof). In particular embodiments, the antiviral agent is baloxavir
marboxil, baloxavir acid, or a pharmaceutically acceptable salt thereof. In certain embodiments, the
baloxavir marboxil is administered in an amount between 20 mg and 90 mg (e.g., between 25 mg and
50 mg, between 45 mg and 70 mg, or between 65 and 90 mg). In some embodiments, the baloxavir
marboxil is administered orally. In certain embodiments, the baloxavir marboxil is administered as a
single dose. In other embodiments, the baloxavir marboxil is administered as more than one dose. In
particular embodiments, the baloxavir marboxil is administered in an amount between 20 mg and 40
mg. In other embodiments, the baloxavir marboxil is administered in an amount between 30 and 80
mg. In certain embodiments, conjugate is described by formula (D-II-6). In other embodiments, the
conjugate is described by formula (D-II-7). In certain embodiments, each E has a sequence at least
95% identical to the sequence of any one of SEQ ID NOs: 63-138. In particular embodiments, each E
includes an Fc domain that has an amino acid sequence at least 95% identical to the sequence of any
one of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID
NO: 73, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, or SEQ ID
NO: 86. In particular embodiments, each E includes an Fc domain that has the amino acid sequence
of any one of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 72, SEQ
ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, or SEQ
ID NO: 86. In certain embodiments, the conjugate is conjugate 45 (e.g., conjugate 45a or conjugate
45b) or conjugate 46. In particular embodiments, the conjugate is administered intramuscularly,
intravenously, intradermally, intraarterially, intraperitoneally, intralesionally, intracranially,
intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally,
intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly,
mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, by inhalation, by injection, or
by infusion. In some embodiments, the conjugate is administered intravenously. In some
embodiments, the conjugate is administered intramuscularly. In some embodiments, the viral infection
is caused by an influenza virus or a parainfluenza virus. In certain embodiments, the virus is influenza
virus A, B, or C, or parainfluenza virus.

(497) In some embodiments, an Fc-domain-containing composition may be substituted for an Fc

domain and an FC-domain-monomer-containing composition may be substituted for an Fc domain
monomer in any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I) (e.g., any one of
formulas (1), (2), (3), (4), (5), (D-I), (D-I-1), (D-II-2), (D-II-3), (D-II-4), (D-II-5), (D-II-6), (D-II-7), (D-II-8),
(D-IV-9), (D-II-10), (D-III), (D-II-1), (D-II-2), (D-III-3), (D-III-4), (D-III-5), (D-I-6), (D-III-7), (D-II-8), (D-III-
9), (D-IV), (D-IV-1), (D-IV-2), (D-V), (D-V-1), (D-V-2), (D-V-3), (D-V-4), (D-V-5), (D-V-6), (D-V-7), (D-V-
8), (D-V-9), (D-V-10), (D-VI), (D-VI-1), (D-VI-2), (D-VI-3), (D-VI-4), (D-VI-5), (D-VI-6), (D-VI-7), (D-VI-
8), (D-VI-9), (D-VII), (D-VIII), (D-VIII-1), (D-VIII-2), (D-VIII-3), (D-VIII-4), (D-VIII-5), (D-VIII-6), (D-VIII-7),
(D-VIII-8), (D-VIII-9), (D-VIII-10), (D-VIII-11), (D-IX), (D-IX-1), (D-IX-2), (D-X-3), (D-IX-4), (D-IX-5), (D-
IX-6), (D-X), (D-X-1), (D-X-2), (D-X-3), (D-XI), (D-XI-1), (D′-I), (M-I), (M-II), (M-II-1), (M-II-2), (M-II-3),
(M-II-4), (M-II-5), (M-II-6), (M-II-7), (M-II-8), (M-II-9), (M-II-10), (M-III), (M-III-1), (M-III-2), (M-III-3), (M-
III-4), (M-III-5), (M-III-6), (M-III-7), (M-III-8), (M-III-9), (M-IV), (M-IV-1), (M-IV-2), (M-V), (M-V-1), (M-V-
2), (M-V-3), (M-V-4), (M-V-5), (M-V-6), (M-V-7), (M-V-8), (M-V-9), (M-V-10), (M-VI), (M-VI-1), (M-VI-2),
(M-VI-3), (M-VI-4), (M-VI-5), (M-VI-6), (M-VI-7), (M-VI-8), (M-VI-9), (M-VII), (M-VIII), (M-VIII-1), (M-VIII-
2), (M-VIII-3), (M-VIII-4), (M-VIII-5), (M-VIII-6), (M-VIII-7), (M-VIII-8), (M-VIII-9), (M-VIII-10), (M-VIII-11),
(M-IX), (M-IX-1), (M-IX-2), (M-IX-3), (M-IX-4), (M-IX-5), (M-IX-6), (M-X), (M-X-1), (M-X-2), (M-X-3), (M-
XI), (M-XI-1), or (M′-I)). In any of the formulas described herein (e.g., any one of formulas (1)-(5), (D-I)-
(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)), when n is 1, E is an Fc-domain-monomer-containing composition.
In any of the formulas described herein (e.g., any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-
XI), or (M′-I)), when n is 2, E is an Fc-domain-containing composition.

(498) In certain embodiments, the Fc-domain-containing composition is an antibody or an antibody

fragment. An antibody may include any form of immunoglobulin, heavy chain antibody, light chain
antibody, LRR-based antibody, or other protein scaffold with antibody-like properties, as well as any
other immunological binding moiety known in the art, including antibody fragments (e.g., a Fab, Fab′,
Fab′2, F(ab′)2, Fd, Fv, Feb, scFv, or SMIP). The subunit structures and three-dimensional
configurations of different classes of antibodies are known in the art. An antibody fragment may
include a binding moiety that includes a portion derived from or having significant homology to an
antibody, such as the antigen-determining region of an antibody. Exemplary antibody fragments
include Fab, Fab′, Fab′2, F(ab′)2, Fd, Fv, Feb, scFv, and SMIP.

(499) In particular embodiments, the antibody or antibody fragment is a human, mouse, camelid (e.g.,
llama, alpaca, or camel), goat, sheep, rabbit, chicken, guinea pig, hamster, horse, or rat antibody or
antibody fragment. In specific embodiments, the antibody is an IgG, IgA, IgD, IgE, IgM, or intrabody. In
certain embodiments, the antibody fragment includes an scFv, sdAb, dAb, Fab, Fab′, Fab′2, F(ab)2,
Fd, Fv, Feb, or SMIP.

(500) In some embodiments, the Fc-domain-containing composition (e.g., an antibody or antibody

fragment) confers binding specificity to a one or more targets (e.g., an antigen).

(501) In some embodiments, the one or more targets (e.g., an antigen) bound by the Fc-domain-
containing composition (e.g., an antibody or antibody fragment) is a viral (e.g., influenza) protein such
as neuraminidase or hemagglutinin. In some embodiments, the antibody or antibody fragment
recognizes a viral surface antigen. In some embodiments, the antibody or antibody fragment targets
hemagglutinin. Hemagglutinin-targeting antibodies include monoclonal antibodies, such as CR6261,
CR8020, MED18852, MHAA4549A, and VIS410. In some embodiments, the antibody or antibody
fragment is a broadly neutralizing antibody or antibody fragment targeting influenza hemagglutinin
(e.g., an antibody or antibody fragment described in Wu et al., J. Mol. Biol. 429:2694-2709 (2017)). In
some embodiments the antibody or antibody fragment targets a viral matrix protein (e.g., matrix 2
protein). TCN032 is a matrix 2 protein targeting monoclonal antibody.

(502) In some embodiments, the Fc-domain-containing composition (e.g., an antibody or antibody

fragment) includes one or more single-domain antibodies (sdAbs). In some embodiments the Fc-
domain-containing composition is an antibody or antibody fragment including a sdAb with influenza A
reactivity, such as a sdAb that binds to hemagglutinin of influenza A (e.g., SD38 or SD38, described in
Laursen et al. Science. 362:598-602 (2018)). In some embodiments the Fc-domain-containing
composition is an antibody or antibody fragment including a sdAb with influenza B reactivity, such as a
sdAb that binds to hemagglutinin of influenza B (e.g., SD83 or SD84, described in Laursen et al.
Science. 362:598-802 (2018)).

(503) In some embodiments the Fc-domain-containing composition is a multidomain antibody (MDAb)

or multidomain antibody fragment including 2 or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) sdAbs.
In some embodiments, the MDAb or fragment thereof includes one or more sdABs that binds to
hemagglutinin of influenza A, and one or more sdAbs that binds to hemagglutinin of influenza B. In
some embodiments, the MDAb is JNJ-7445 (also known as MD3806), which is described in Laursen
et al. Science. 362:598-802 (2018). In brief, JNJ-7445 is an MDAb that includes two sdAbs that bind to
hemagglutinin of influenza A (SD36 and SD38) and two sdAbs that bind to hemagglutinin of influenza
B (SD83 and SD84), which are linked to an Fc domain (IgG1). The sdAbs were produced by
immunizing llamas with influenza vaccine and H7 and H2 recombinant hemagglutinin.

(504) In another aspect, the invention includes a conjugate described by any one of formulas (1)-(5),
(D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I) (e.g., any one of formulas (1), (2), (3), (4), (5), (D-I), (D-II), (D-
II-1), (D-II-2), (D-II-3), (D-I1-4), (D-II-5), (D-II-6), (D-II-7), (D-II-8), (D-II-9), (D-II-10), (D-III), (D-III-1), (D-
III-2), (D-III-3), (D-III-4), (D-III-5), (D-III-6), (D-III-7), (D-III-8), (D-III-9), (D-IV), (D-IV-1), (D-IV-2), (D-V),
(D-V-1), (D-V-2), (D-V-3), (D-V-4), (D-V-5), (D-V-6), (D-V-7), (D-V-8), (D-V-9), (D-V-10), (D-VI), (D-VI-
1), (D-VI-2), (D-VI-3), (D-VI-4), (D-VI-5), (D-VI-6), (D-VI-7), (D-VI-8), (D-VI-9), (D-VII), (D-VIII), (D-VIII-
1), (D-VIII-2), (D-VIII-3), (D-VIII-4), (D-VIII-5), (D-VIII-6), (D-VIII-7), (D-VIII-8), (D-VIII-9), (D-VIII-10),
(D-VIII-11), (D-IX), (D-IX-1), (D-IX-2), (D-IX-3), (D-IX-4), (D-IX-5), (D-IX-6), (D-X), (D-X-1), (D-X-2), (D-
X-3), (D-XI), (D-XI-1), (D′-1), (M-I), (M-V), (M-V-1), (M-II-2), (M-V-3), (M-V-4), (M-V-5), (M-V-6), (M-V-
7), (M-V-8), (M-M-II-10), (M-III), (M-III-1), (M-III-2), (M-III-3), (M-III-4), (M-III-5), (M-III-6), (M-III-7), (M-
III-8), (M-III-9), (M-IV), (M-IV-1), (M-IV-2), (M-V), (M-V-1), (M-V-2), (M-V-3), (M-V-4), (M-V-5), (M-V-6),
(M-V-7), (M-V-8), (M-V-9), (M-V-10), (M-VI), (M-VI-1), (M-VI-2), (M-VI-3), (M-VI-4), (M-VI-5), (M-VI-6),
(M-VI-7), (M-VI-8), (M-VI-9), (M-VII), (M-VIII), (M-VIII-1), (M-VIII-2), (M-VIII-3), (M-VIII-4), (M-VIII-5),
(M-VIII-6), (M-VII-7), (M-VIII-8), (M-VIII-9), (M-VIII-10), (M-VIII-11), (M-IX), (M-IX-1), (M-IX-2), (M-IX-3),
(M-IX-4), (M-IX-5), (M-IX-6), (M-X), (M-X-1), (M-X-2), (M-X-3), or (M′-I)), where E is an antibody or
antibody fragment. In preferred embodiments, where E is an antibody or antibody fragment, n is 1. In
some embodiments, the antibody or antibody fragment includes any antibody or antibody fragment
described herein, such as a monoclonal antibody that binds to viral hemagglutinin (e.g., CR6261,
CR8020, MED18852, MHAA4549A, or VIS410); a broadly neutralizing antibody or antibody fragment
targeting viral hemagglutinin (e.g., antibodies or antibody fragments described in Wu et al., J. Mol.
Biol. 429:2694-2709 (2017)); a sdAb targeting viral hemagglutinin (e.g., SD36, SD38, SD83, or SD84);
or a MDAb or fragment thereof targeting viral hemagglutinin (e.g., JNJ-7445).

(505) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 1. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 1.

(506) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 2. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 2.

(507) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 3. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 3.

(508) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 4. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 4.

(509) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 5. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 5.

(510) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 6. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 6.

(511) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 7. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 7.

(512) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 8. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 8.

(513) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 9. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 9.

(514) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 10. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 10.

(515) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 11. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 11.

(516) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 12. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 12.

(517) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 13. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 13.

(518) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 14. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 14.

(519) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 15. In some embodiments. E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 15.

(520) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 16. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 16.

(521) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 17. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 17.

(522) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 18. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 18.

(523) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 19. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 19.

(524) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 20. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 20.

(525) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 21. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 21.
(526) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 22. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 22.

(527) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 23. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 23.

(528) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 24. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 24.

(529) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 25. In some embodiments. E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 25.

(530) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 26. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 26.

(531) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 27. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 27.

(532) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 28. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 28.

(533) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 29. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 29.

(534) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 30. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 30.

(535) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 31. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 31.

(536) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 32. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 32.

(537) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 33. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 33.

(538) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 34. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 34.

(539) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 35. In some embodiments. E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 35.

(540) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 36. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 36.

(541) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 37. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 37.

(542) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 38. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 38.

(543) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 39. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 39.

(544) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 40. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 40.

(545) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 41. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 41.

(546) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 42. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 42.

(547) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 43. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 43.

(548) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 44. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 45.
(549) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 46. In some embodiments. E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 46.

(550) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 47. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 47.

(551) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 48. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 48.

(552) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 49. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 49.

(553) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 50. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 50.

(554) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 51. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 51.

(555) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 52. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 52.

(556) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 53. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 53.

(557) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 54. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 54.

(558) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 55. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 55.

(559) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 56. In some embodiments. E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 56.

(560) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 57. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 57.

(561) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 58. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 58.

(562) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 59. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 59.

(563) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 60. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 60.

(564) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 61. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 61.

(565) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 62. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 62.

(566) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 63. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 63.

(567) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 64. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 64.

(568) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 65. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 65.

(569) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 66. In some embodiments. E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 66.

(570) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 67. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 67.

(571) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 68. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 68.
(572) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 69. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 69.

(573) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 70. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 70.

(574) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 71. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 71.

(575) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 72. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 72.

(576) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 73. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 73.

(577) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 74. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 74.

(578) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 75. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 75.

(579) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 76. In some embodiments. E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 76.

(580) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 77. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 77.

(581) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 78. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 78.

(582) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 79. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 79.

(583) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 80. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 80.

(584) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 81. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 81.

(585) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 82. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 82.

(586) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 83. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 83.

(587) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 84. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 84.

(588) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 85. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 85.

(589) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 86. In some embodiments. E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 86.

(590) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 87. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 87.

(591) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 88. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 88.

(592) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 89. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 89.

(593) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 90. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 90.

(594) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 91. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 91.
(595) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 92. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 92.

(596) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 93. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%. 85%, 90%, 95%. 96%, 97%, 98%. 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 93.

(597) In some embodiments of any of the aspects described herein. E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 94. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 94.

(598) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 95. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 95.

(599) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 96. In some embodiments. E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 96.

(600) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 97. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 97.

(601) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 98. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 98.

(602) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 99. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 99.

(603) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 100. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 100.

(604) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 101. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 101.

(605) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 102. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 102.

(606) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 103. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 103.

(607) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 104. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 104.

(608) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 105. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 105.

(609) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 106. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 106.

(610) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 107. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 107.

(611) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 108. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 108.

(612) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 109. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 109.

(613) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 110. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 110.

(614) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 111. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 111.

(615) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 112. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 112.

(616) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 113. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 113.

(617) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 114. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 114.
(618) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 115. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 115.

(619) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 116. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 116.

(620) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 117. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 117.

(621) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 118. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 118.

(622) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 119. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 119.

(623) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 120. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 120.

(624) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 121. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 121.

(625) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 122. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 122.

(626) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 123. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 123.

(627) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 124. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 124.

(628) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 125. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 125.

(629) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 126. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 126.

(630) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 127. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 127.

(631) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 128. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 128.

(632) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 129. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 129.

(633) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 130. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 130.

(634) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 131. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 131.

(635) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 132. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 132.

(636) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 133. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 133.

(637) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 134. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 134.

(638) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 135. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 135.

(639) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 136. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 136.

(640) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 137. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 137.
(641) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 138. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 138.

(642) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 139. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 139.

(643) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 140. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 140.

(644) In some embodiments of any of the aspects described herein, E (e.g., each E) includes the
amino acid sequence of SEQ ID NO: 145. In some embodiments, E includes an amino acid sequence
that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of SEQ ID NO: 141.

(645) In some embodiments of any of the aspects described herein, wherein E includes an Fc domain
monomer, the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138) includes a triple mutation corresponding to M252Y/S254T/T256E (YTE). As used
herein, an amino acid “corresponding to” a particular amino acid residue (e.g., of a particular SEQ ID
NO.) should be understood to include any amino acid residue that one of skill in the art would
understand to align to the particular residue (e.g., of the particular sequence). For example, any one of
SEQ ID NOs: 1-138 may be mutated to include a YTE mutation.

(646) In some embodiments of any of the aspects described herein, wherein E includes an Fc domain
monomer, the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138) includes a double mutant corresponding to M428L/N434S (LS). As used herein,
an amino acid “corresponding to” a particular amino acid residue (e.g., of a particular SEQ ID NO.)
should be understood to include any amino acid residue that one of skill in the art would understand to
align to the particular residue (e.g., of the particular sequence). For example, any one of SEQ ID NOs:
1-138 may be mutated to include a LS mutation.

(647) In some embodiments of any of the aspects described herein, wherein E includes an Fc domain
monomer, the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138) includes a mutant corresponding to N434H. As used herein, an amino acid
“corresponding to” a particular amino acid residue (e.g., of a particular SEQ ID NO.) should be
understood to include any amino acid residue that one of skill in the art would understand to align to
the particular residue (e.g., of the particular sequence). For example, any one of SEQ ID NOs: 1-138
may be mutated to include an N434H mutation.

(648) In some embodiments of any of the aspects described herein, wherein E includes an Fc domain
monomer, the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138) includes a mutant corresponding to C220S. As used herein, an amino acid
“corresponding to” a particular amino acid residue (e.g., of a particular SEQ ID NO.) should be
understood to include any amino acid residue that one of skill in the art would understand to align to
the particular residue (e.g., of the particular sequence). For example, any one of SEQ ID NOs: 1-138
may be mutated to include a C220S mutation.

(649) In some embodiments of any of the aspects described herein, the Fc domain monomer (e.g., the
Fc domain monomer having the sequence of any one of SEQ ID NOs: 1-95) includes a triple mutation
corresponding to V309D/Q311H/N434S (DHS). As used herein, an amino acid “corresponding to” a
particular amino acid residue (e.g., of a particular SEQ ID NO.) should be understood to include any
amino acid residue that one of skill in the art would understand to align to the particular residue (e.g.,
of the particular sequence). For example, any one of SEQ ID NOs: 1-95 may be mutated to include a
DHS mutation.

(650) In some embodiments of any of the aspects described herein, wherein E includes an Fc domain
monomer, the Fc domain monomer (e.g., the Fc domain monomer having the sequence of any one of
SEQ ID NOs: 1-138) is a fragment of the Fc domain monomer (e.g., a fragment of at least 25 (e.g., 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,
48, 49, 50 or more), at least 50 (e.g., 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,
68, 69, 70, 71, 72, 73, 74, 75 or more), at least 75 (e.g., 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more) consecutive amino acids in length from
SEQ ID NOs: 1-138.

(651) In some embodiments of any of the aspects described herein (e.g., a conjugate of any one of
formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-II)), one or more nitrogen atoms of one or more
surface exposed lysine residues of E or one or more sulfur atoms of one or more surface exposed
cysteines in E is covalently conjugated to a linker (e.g., a PEG.sub.2-PEG.sub.20 linker). The linker
conjugated to E may be functionalized such that it may reacts to form a covalent bond with the L of
any A.sub.1-L or any A.sub.2-L-A.sub.1 described herein. In preferred embodiments, E is conjugated
to a linker functionalized with an azido group and the L of A.sub.1-L or any A.sub.2-L-A.sub.1 is
functionalized with an alkyne group. Conjugation (e.g., by click chemistry) of the linker-azido of E and
linker-alkyne of A.sub.1-L or A.sub.2-L-A.sub.1 forms a conjugate of the invention, for example a
conjugate described by any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-1). In yet
other embodiments, E is conjugated to a linker functionalized with an alkyne group and L of any
A.sub.1-L or of any A.sub.2-L-A.sub.1 is functionalized with an azido group. Conjugation (e.g., by click
chemistry, see, e.g., FIG. 103) of the linker-alkyne of E and linker-azido of A.sub.1-L or A.sub.2-L-
A.sub.1 forms a conjugate of the invention, for example a conjugate described by formula (1)-(5), (D-
I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I).

(652) In some embodiments of any of the aspects described herein, the squiggly line of any one of
formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I) may represent a covalent bond between E
and the L of A.sub.1-L or A.sub.2-L-A.sub.1.

(653) In some embodiments of any of the aspects described herein, the squiggly line of any one of
formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I) may represent that one or more amino acid
side chains of E (e.g., one or more nitrogen atoms of one or more surface exposed lysine residues of
E or one or more sulfur atoms of one or more surface exposed cysteines in E) have been conjugated
to a linker (e.g., a PEG.sub.2-PEG.sub.20 linker) wherein the linker has been functionalized with a
reactive moiety, such that the reactive moiety forms a covalent bond with the L of any A.sub.1-L or any
A.sub.2-L-A.sub.1 described herein (e.g., by click chemistry between an azido functionalized linker
and an alkyne functionalized linker, as described above; see, e.g., FIG. 103).

(654) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-I):

(655) ##STR00383##

In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-I):
R.sub.1 is —NHC(═NH)NH.sub.2, R.sub.4 is —CO.sub.2H, R.sub.5 is —COCH.sub.3, and/or X is —
O—. In preferred embodiments, A.sub.1 and/or A.sub.2 have the structure of zanamivir described by:

(656) ##STR00384##

(657) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-II):

(658) ##STR00385##
(659) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
II): R.sub.1 is —NHC(═NH)NH.sub.2, R.sub.2 is H or F, R.sub.3 is H or F, R.sub.4 is —CO.sub.2H,
R.sub.5 is —COCH.sub.3, and/or X is —O—. In preferred embodiments, A.sub.1 and/or A.sub.2 have
the structure described by:

(660) ##STR00386##

(661) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-III):

(662) ##STR00387##

(663) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
III): R.sub.1 is —NHC(═NH)NH.sub.2, R.sub.4 is —CO.sub.2H, and/or R.sub.5 is —COCH.sub.3. In
preferred embodiments, A.sub.1 and/or A.sub.2 have the structure of peramivir described by:

(664) ##STR00388##

(665) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-IV):

(666) ##STR00389##

(667) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
IV): R.sub.1 is —NHC(═NH)NH.sub.2, R.sub.4 is —CO.sub.2H, and/or R.sub.5 is —COCH.sub.3. In
preferred embodiments, A.sub.1 and/or A.sub.2 have the structure described by:

(668) ##STR00390##

(669) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-V):

(670) ##STR00391##

(671) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
V): R.sub.1 is —NHC(═NH)NH.sub.2, R.sub.4 is —CO.sub.2H, and/or R.sub.5 is —COCH.sub.3. In
preferred embodiments, A.sub.1 and/or A.sub.2 have the structure described by:

(672) ##STR00392##

(673) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-VI):

(674) ##STR00393##

(675) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
VI): R.sub.1 is —NHC(═NH)NH.sub.2, R.sub.4 is —CO.sub.2H, R.sub.5 is —COCH.sub.3, and/or X is
—O—. In preferred embodiments, A.sub.1 and/or A.sub.2 have the structure of zanamivir described
by:

(676) ##STR00394##

(677) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-VII):

(678) ##STR00395##

(679) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
VII): R.sub.1 is —NHC(═NH)NH.sub.2, R.sub.2 is H or F, R.sub.3 is H or F, R.sub.4 is —CO.sub.2H,
R.sub.5 is —COCH.sub.3, and/or X is —O—. In preferred embodiments, A.sub.1 and/or A.sub.2 have
the structure described by:

(680) ##STR00396##

(681) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-VIII):

(682) ##STR00397##

(683) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
VIII): R.sub.1 is —NHC(═NH)NH.sub.2, R.sub.5 is —COCH.sub.3, and/or X is —O—. In preferred
embodiments, A.sub.1 and/or A.sub.2 have the structure described by:

(684) ##STR00398##

(685) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-IX):

(686) ##STR00399##

(687) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
IX): R.sub.1 is —NHC(═NH)NH.sub.2, R.sub.2 is H or F, R.sub.3 is H or F, R.sub.5 is —COCH.sub.3,
and/or X is —O—. In preferred embodiments, A.sub.1 and/or A.sub.2 have the structure described by:

(688) ##STR00400##

(689) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-X):

(690) ##STR00401##

(691) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
X): R.sub.1 is —NHC(═NH)NH.sub.2, R.sub.3 is H, R.sub.5 is —COCH.sub.3, and/or X is —O—. In
preferred embodiments, A.sub.1 and/or A.sub.2 have the structure of sulfozanamivir described by:

(692) ##STR00402##

(693) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-XI):

(694) ##STR00403##

(695) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
XI): R.sub.4 is —CO.sub.2H, and/or R.sub.5 is —COCH.sub.3. In preferred embodiments, the alkene
is (E), (Z), or a racemic mixture of (E)/(Z). In preferred embodiments, A.sub.1 and/or A.sub.2 have the
structure of A-315675 (Abbott) described by:

(696) ##STR00404##

(697) In some embodiments of any of the aspects described herein, A.sub.1 and/or A.sub.2 have the
structure described by (A-XII):

(698) ##STR00405##

(699) In preferred embodiments, wherein A.sub.1 and/or A.sub.2 have the structure described by (A-
XII): R.sub.4 is —CO.sub.2H. In preferred embodiments, A.sub.1 and/or A.sub.2 have the structure of
A-315675 (Abbott) described by:
(700) ##STR00406##

(701) In some embodiments, the conjugate is conjugate 1, or any regioisomer thereof, and the drug-to-
antibody ratio (DAR) (e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2,
1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,
3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,
6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2,
8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some
embodiments the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between
6.0 and 8.0, or between 8.0 and 10.0. In another aspect the invention provides a population of
conjugates, each conjugate having the structure of conjugate 1, wherein the average DAR (e.g., T) of
the population of conjugates is 1 to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to
4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to 7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(702) In some embodiments, the conjugate is conjugate 2, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 2, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(703) In some embodiments, the conjugate is conjugate 3, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 3, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(704) In some embodiments, the conjugate is conjugate 4, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 4, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(705) In some embodiments, the conjugate is conjugate 5, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 5, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(706) In some embodiments, the conjugate is conjugate 6, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 6, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(707) In some embodiments, the conjugate is conjugate 7, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 7, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(708) In some embodiments, the conjugate is conjugate 8, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 8, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(709) In some embodiments, the conjugate is conjugate 9, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 9, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(710) In some embodiments, the conjugate is conjugate 10, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 10, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(711) In some embodiments, the conjugate is conjugate 11, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 11, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(712) In some embodiments, the conjugate is conjugate 12, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 12, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(713) In some embodiments, the conjugate is conjugate 13, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 13, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(714) In some embodiments, the conjugate is conjugate 14, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 14, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.
(715) In some embodiments, the conjugate is conjugate 15, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 15, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(716) In some embodiments, the conjugate is conjugate 16, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 16, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(717) In some embodiments, the conjugate is conjugate 17, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 17, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(718) In some embodiments, the conjugate is conjugate 18, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 18, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(719) In some embodiments, the conjugate is conjugate 19, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 19, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(720) In some embodiments, the conjugate is conjugate 20, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 20, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(721) In some embodiments, the conjugate is conjugate 21, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 21, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(722) In some embodiments, the conjugate is conjugate 22, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 22, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(723) In some embodiments, the conjugate is conjugate 23, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 23, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(724) In some embodiments, the conjugate is conjugate 24, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 24, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(725) In some embodiments, the conjugate is conjugate 25, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 25, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(726) In some embodiments, the conjugate is conjugate 26, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 26, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(727) In some embodiments, the conjugate is conjugate 27, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 27, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(728) In some embodiments, the conjugate is conjugate 28, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 28, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(729) In some embodiments, the conjugate is conjugate 29, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 29, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(730) In some embodiments, the conjugate is conjugate 30, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 30, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(731) In some embodiments, the conjugate is conjugate 31, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 31, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(732) In some embodiments, the conjugate is conjugate 32, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 32, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(733) In some embodiments, the conjugate is conjugate 33, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 33 wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.
(734) In some embodiments, the conjugate is conjugate 34, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 34, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(735) In some embodiments, the conjugate is conjugate 35, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 35, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(736) In some embodiments, the conjugate is conjugate 36, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 36, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(737) In some embodiments, the conjugate is conjugate 37, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 37, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(738) In some embodiments, the conjugate is conjugate 38, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 38, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(739) In some embodiments, the conjugate is conjugate 39, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 39, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(740) In some embodiments, the conjugate is conjugate 40, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 40, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(741) In some embodiments, the conjugate is conjugate 41, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 41, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(742) In some embodiments, the conjugate is conjugate 42, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 42, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(743) In some embodiments, the conjugate is conjugate 43, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 43, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(744) In some embodiments, the conjugate is conjugate 44, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 44, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(745) In some embodiments, the conjugate is conjugate 45 (e.g., conjugate 45a or conjugate 45b), or
any regioisomer thereof, and the DAR (e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8,
0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2,
3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5,
5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0,
7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In
some embodiments the DAR is between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0,
between 6.0 and 8.0, or between 8.0 and 10.0. In another aspect the invention provides a population
of conjugates, each conjugate having the structure of conjugate 45 (e.g., conjugate 45a or conjugate
45b), wherein the average DAR (e.g., T) of the population of conjugates is 1 to 2, 1 to 3, 1 to 4, 1 to 5,
5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to 7.5, 6.5 to 8.5, 7.5 to 9.5,
or 8.5 to 10.5.

(746) In some embodiments, the conjugate is conjugate 46, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 46, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(747) In some embodiments, the conjugate is conjugate 47, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 47, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(748) In some embodiments, the conjugate is conjugate 48, or any regioisomer thereof, and the DAR
(e.g., T) is between 0.5 and 10.0, e.g., about 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7,
1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1,
4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,
6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7,
8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, or 10.0. In some embodiments the DAR is
between 0.5 and 2.0, between 2.0 and 4.0, between 4.0 and 6.0, between 6.0 and 8.0, or between 8.0
and 10.0. In another aspect the invention provides a population of conjugates, each conjugate having
the structure of conjugate 48, wherein the average DAR (e.g., T) of the population of conjugates is 1 to
2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5.

(749) In some embodiments of any of the aspects described herein, the invention features a conjugate
described by any one of formulas (D-I), (M-I), (1), or (2): wherein each A.sub.1 and each A.sub.2 is
independently selected from any one of formulas (A-XIII):

(750) ##STR00407##

wherein R.sub.1 is selected from —OH, —NH.sub.2, —NHC(═NH)NH.sub.2, and —

NHC(═NH)NHR.sub.6; R.sub.4 is selected from —CO.sub.2H, —P(═O)(OH).sub.2, —SO.sub.3H;
R.sub.5 is selected from —COCH.sub.3, —COCF.sub.3, —SO.sub.2CH.sub.3; X is selected from —O
— and —S—; Y is selected from

(751) ##STR00408## ##STR00409##

(752) R.sub.6 is selected from

(753) ##STR00410## ##STR00411## ##STR00412##

(754) R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15
aryl, and C2-C15 heteroaryl;

(755) R.sub.8 is selected from C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl;

(756) R.sub.9 is selected from —H, a halogen (e.g., Cl or F), —OR.sub.10, —NHC(═O)R.sub.7,
optionally substituted C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15 aryl, and C2-
C15 heteroaryl;

(757) R.sub.10 is selected from C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15
aryl, and C2-C15 heteroaryl;

(758) n is 1 or 2;

(759) each E comprises an Fc domain monomer, an albumin protein, an albumin protein-binding

peptide, or an Fc-binding peptide;

(760) L is a linker covalently attached to E and to each Y of each A.sub.1 or each A.sub.1 and
A.sub.2;

(761) T is an integer from 1 to 20, and

(762) each squiggly line in formulas (D-I), (M-I), (1), or (2) indicates that L is covalently attached to
each E;

(763) or a pharmaceutically acceptable salt thereof.

(764) In some embodiments, each A.sub.1 and each A.sub.2 is described by formula (A-XIII-1):

(765) ##STR00413##
(766) In some embodiments, each A.sub.1 and each A.sub.2 is independently selected from any one
of formulas (A-XIII-1a)-(A-XIII-1d):

(767) ##STR00414##

(768) In some embodiments, the conjugate is described by formula (D-XI):

(769) ##STR00415##

or a pharmaceutically acceptable salt thereof.

(770) In some embodiments, the conjugate is described by formula (D-XI-1):

(771) ##STR00416##

wherein R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-
C15 aryl, and C2-C15 heteroaryl; or a pharmaceutically acceptable salt thereof.

(772) In some embodiments, the conjugate is described by formula (M-XI):

(773) ##STR00417##

or a pharmaceutically acceptable salt thereof.

(774) In some embodiments, the conjugate is described by formula (M-XI-1):

(775) ##STR00418##

wherein R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-
C15 aryl, and C2-C15 heteroaryl; or a pharmaceutically acceptable salt thereof.

Definitions

(776) To facilitate the understanding of this invention, a number of terms are defined below. Terms
defined herein have meanings as commonly understood by a person of ordinary skill in the areas
relevant to the present invention. Terms such as “a”, “an,” and “the” are not intended to refer to only a
singular entity, but include the general class of which a specific example may be used for illustration.
The terminology herein is used to describe specific embodiments of the invention, but their usage
does not delimit the invention, except as outlined in the claims.

(777) The term “neuraminidase inhibitor” or ““viral neuraminidase inhibitor,” as used herein, refers to
compounds that decreases the activity of the enzyme influenza virus neuraminidase (e.g., from
influenza virus A, B, or C). A neuraminidase inhibitor may be identified by methods known to those of
skill in the art, for example, by reduction of viral replication in an influenza viral plaque reduction assay,
e.g., at concentrations less than 20 μM (e.g., less than 10 μM, 5 μM, 2 μM, 1 μM, 500 nM or 100 nM).
Viral neuraminidase inhibitors known to those of skill in the art include zanamivir, sulfozanamivir,
peramivir, and A-315675 (Abbott) (see, for example, Hadházi et al. A sulfozanamivir analogue has
potent anti-influenza virus activity. ChemMedChem Comm. 13:785-789 (2018) and In vitro
characterization of A-315675, a highly potent inhibitor of A and B strain of influenza virus
neuraminidases and influenza virus replication. Antimicrobial Agents and Chemotherapy 46(4):1014-
1021 (2002)). Viral neuraminidase inhibitors of the invention include zanamivir, sulfozanamivir,
peramivir, A-315675 and analogs thereof, such as the viral neuraminidase inhibitors of formulas (A-I)-
(A-XIII):

(778) ##STR00419## ##STR00420## ##STR00421## ##STR00422##

wherein R.sub.1 is selected from —OH, —NH.sub.2, —NHC(═NH)NH.sub.2, and —

(779) ##STR00423## ##STR00424##

(780) R.sub.6 is selected from

(781) ##STR00425## ##STR00426## ##STR00427##

R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15 aryl,
and C2-C15 heteroaryl; and R.sub.8 is selected from C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-
C15 heteroaryl; R.sub.9 is selected from —H, a halogen (e.g., Cl, F, or Br), —OR.sub.10, —
NHC(═O)R.sub.7, optionally substituted C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl;
C5-C15 aryl, and C2-C15 heteroaryl; and R.sub.10 is selected from C1-C20 alkyl, C3-C20 cycloalkyl,
C3-C20 heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl.

(782) The term “inhibits neuraminidase activity,” as used herein refers to an IC.sub.50 of less than or
equal to 1,000 nM, for example, as measured in accordance with the neuraminidase inhibition assay in
Example 2 herein. Specifically, the IC.sub.50 represents the concentration of the influenza virus
neuraminidase inhibitor that is required for 50% inhibition in vitro. In some aspects, an IC.sub.50 of
less than or equal to 100 nM or less than or equal to 10 nM in accordance with neuraminidase
inhibition assay is indicative of a compound inhibiting neuraminidase activity.

(783) By “viral infection” is meant the pathogenic growth of a virus (e.g., the influenza virus) in a host
organism (e.g., a human subject). A viral infection can be any situation in which the presence of a viral
population(s) is damaging to a host body. Thus, a subject is “suffering” from a viral infection when an
excessive amount of a viral population is present in or on the subject's body, or when the presence of
a viral population(s) is damaging the cells or other tissue of the subject.

(784) As used herein, the term “Fc domain monomer” refers to a polypeptide chain that includes at
least a hinge domain and second and third antibody constant domains (C.sub.H2 and C.sub.H3) or
functional fragments thereof (e.g., fragments that that capable of (i) dimerizing with another Fc domain
monomer to form an Fc domain, and (ii) binding to an Fc receptor. The Fc domain monomer can be
any immunoglobulin antibody isotype, including IgG, IgE, IgM, IgA, or IgD (e.g., IgG). Additionally, the
Fc domain monomer can be an IgG subtype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4) (e.g., IgG1). An
Fc domain monomer does not include any portion of an immunoglobulin that is capable of acting as an
antigen-recognition region, e.g., a variable domain or a complementarity determining region (CDR). Fc
domain monomers in the conjugates as described herein can contain one or more changes from a
wild-type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 amino acid substitutions, additions,
or deletions) that alter the interaction between an Fc domain and an Fc receptor. Examples of suitable
changes are known in the art. In certain embodiments, a human Fc domain monomer (e.g., an IgG
heavy chain, such as IgG1) comprises a region that extends from any of Asn208, Glu216, Asp221,
Lys222, or Cys226 to the carboxyl-terminus of the heavy chain at Lys447. C-terminal Lys447 of the Fc
region may or may not be present, without affecting the structure or stability of the Fc region. C-
terminal Lys 447 may be proteolytically cleaved upon expression of the polypeptide. In some
embodiments of any of the Fc domain monomers described herein, C-terminal Lys 447 is optionally
present or absent. The disclosure specifically contemplates any of SEQ ID NOs: 1-4, 11, 16, 19, 20,
32-37, 48-53, and 60-68 that do not include the C-terminal Lys corresponding to Lys447. The N-
terminal N (Asn) of the Fc region (e.g., of any one of SEQ ID NOs: 60-77) may or may not be present,
without affecting the structure of stability of the Fc region. N-terminal Asn may be deamidated upon
expression of the polypeptide. In some embodiments of any of the Fc domain monomers described
herein, N-terminal Asn is optionally present or absent. The disclosure specifically contemplates any of
SEQ ID NOs: 60-77 that do not include the N-terminal Asn. Unless otherwise specified herein,
numbering of amino acid residues in the IgG or Fc domain monomer is according to the EU numbering
system for antibodies, also called the Kabat EU index, as described, for example, in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of
Health, Bethesda, Md., 1991.

(785) As used herein, the term “Fc domain” refers to a dimer of two Fc domain monomers that is
capable of binding an Fc receptor. In the wild-type Fc domain, the two Fc domain monomers dimerize
by the interaction between the two C.sub.H3 antibody constant domains, in some embodiments, one
or more disulfide bonds form between the hinge domains of the two dimerizing Fc domain monomers.
The term “covalently attached” refers to two parts of a conjugate that are linked to each other by a
covalent bond formed between two atoms in the two parts of the conjugate.

(786) As used herein, the term “Fc-binding peptide” refers to refers to a polypeptide having an amino
acid sequence of 5 to 50 (e.g., 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or 10 to 20)
amino acid residues that has affinity for and functions to bind an Fc domain, such as any of the Fc
domain described herein. An Fc-binding peptide can be of different origins, e.g., synthetic, human,
mouse, or rat. Fc-binding peptides of the invention include Fc-binding peptides which have been
engineered to include one or more (e.g., two, three, four, or five) solvent-exposed cysteine or lysine
residues, which may provide a site for conjugation to a compound of the invention (e.g., conjugation to
a neuraminidase inhibitor monomer or dimer, including by way of a linker). Most preferably, the Fc-
binding peptide will contain a single solvent-exposed cysteine or lysine, thus enabling site-specific
conjugation of a compound of the invention. Fc-binding peptides may include only naturally occurring
amino acid residues, or may include one or more non-naturally occurring amino acid residues. Where
included, a non-naturally occurring amino acid residue (e.g., the side chain of a non-naturally occurring
amino acid residue) may be used as the point of attachment for a compound of the invention (e.g., a
neuraminidase inhibitor monomer or dimer, including by way of a linker). Fc-binding peptides of the
invention may be linear or cyclic. Fc-binding peptides of the invention include any Fc-binding peptides
known to one of skill in the art.

(787) As used here, the term “albumin protein” refers to a polypeptide comprising an amino acid
sequence corresponding to a naturally-occurring albumin protein (e.g., human serum albumin) or a
variant thereof, such as an engineered variant of a naturally-occurring albumin protein. Variants of
albumin proteins include polymorphisms, fragments such as domains and sub-domains, and fusion
proteins (e.g., an albumin protein having a C-terminal or N-terminal fusion, such as a polypeptide
linker). Preferably the albumin protein has the amino acid sequence of human serum albumin (HSA) or
a variant or fragment thereof, most preferably a functional variant or fragment thereof. Albumin
proteins of the invention include proteins having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, or 99% identity to any one of SEQ ID NOs: 139-141. Albumin proteins of
the invention include albumin proteins which have been engineered to include one or more (e.g., two,
three, four, or five) solvent-exposed cysteine or lysine residues, which may provide a site for
conjugation to a compound of the invention (e.g., conjugation to a neuraminidase inhibitor monomer or
dimer, including by way of a linker). Most preferably, the albumin protein will contain a single solvent-
exposed cysteine or lysine, thus enabling site-specific conjugation of a compound of the invention.
Albumin proteins may include only naturally occurring amino acid residues, or may include one or
more non-naturally occurring amino acid residues. Where included, a non-naturally occurring amino
acid residue (e.g., the side chain of a non-naturally occurring amino acid residue) may be used as the
point of attachment for a compound of the invention (e.g., a neuraminidase inhibitor monomer or
dimer, including by way of a linker).

(788) As used herein, the term “albumin protein-binding peptide” refers to a polypeptide having an
amino acid sequence of 5 to 50 (e.g., 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or
10 to 20) amino acid residues that has affinity for and functions to bind an albumin protein, such as
any of the albumin proteins described herein. Preferably, the albumin protein-binding peptide binds to
a naturally-occurring serum albumin, most preferably human serum albumin. An albumin protein-
binding peptide can be of different origins, e.g., synthetic, human, mouse, or rat. Albumin protein-
binding peptides of the invention include albumin protein-binding peptides which have been
engineered to include one or more (e.g., two, three, four, or five) solvent-exposed cysteine or lysine
residues, which may provide a site for conjugation to a compound of the invention (e.g., conjugation to
a neuraminidase inhibitor monomer or dimer, including by way of a linker). Most preferably, the
albumin protein-binding peptide will contain a single solvent-exposed cysteine or lysine, thus enabling
site-specific conjugation of a compound of the invention. Albumin protein-binding peptides may include
only naturally occurring amino acid residues, or may include one or more non-naturally occurring
amino acid residues. Where included, a non-naturally occurring amino acid residue (e.g., the side
chain of a non-naturally occurring amino acid residue) may be used as the point of attachment for a
compound of the invention (e.g., a neuraminidase inhibitor monomer or dimer, including by way of a
linker). Albumin protein-binding peptides of the invention may be linear or cyclic. Albumin protein-
binding peptide of the invention include any albumin protein-binding peptides known to one of skill in
the art, examples of which, are provided herein. Further exemplary albumin protein-binding peptides
are provided in U.S. Patent Application No. 2005/0287153, which is incorporated herein by reference
in its entirety.

(789) As used-herein, a “surface exposed amino acid” or “solvent-exposed amino acid,” such as a
surface exposed cysteine or a surface exposed lysine refers to an amino acid that is accessible to the
solvent surrounding the protein. A surface exposed amino acid may be a naturally-occurring or an
engineered variant (e.g., a substitution or insertion) of the protein. In some embodiments, a surface
exposed amino acid is an amino acid that when substituted does not substantially change the three-
dimensional structure of the protein.

(790) The terms “linker,” “L,” and “L′,” as used herein, refer to a covalent linkage or connection
between two or more components in a conjugate (e.g., between two neuraminidase inhibitors in a
conjugate described herein, between a neuraminidase inhibitor and an Fc domain or albumin protein
in a conjugate described herein, and between a dimer of two neuraminidase inhibitors and an Fc
domain or an albumin protein in a conjugate described herein). In some embodiments, a conjugate
described herein may contain a linker that has a trivalent structure (e.g., a trivalent linker). A trivalent
linker has three arms, in which each arm is covalently linked to a component of the conjugate (e.g., a
first arm conjugated to a first neuraminidase inhibitor, a second arm conjugated to a second
neuraminidase inhibitor, and a third arm conjugated to an Fc domain or an albumin protein).

(791) Molecules that may be used as linkers include at least two functional groups, which may be the
same or different, e.g., two carboxylic acid groups, two amine groups, two sulfonic acid groups, a
carboxylic acid group and a maleimide group, a carboxylic acid group and an alkyne group, a
carboxylic acid group and an amine group, a carboxylic acid group and a sulfonic acid group, an
amine group and a maleimide group, an amine group and an alkyne group, or an amine group and a
sulfonic acid group. The first functional group may form a covalent linkage with a first component in the
conjugate and the second functional group may form a covalent linkage with the second component in
the conjugate. In some embodiments of a trivalent linker, two arms of a linker may contain two
dicarboxylic acids, in which the first carboxylic acid may form a covalent linkage with the first
neuraminidase inhibitor in the conjugate and the second carboxylic acid may form a covalent linkage
with the second neuraminidase inhibitor in the conjugate, and the third arm of the linker may for a
covalent linkage with an Fc domain or albumin protein in the conjugate. Examples of dicarboxylic acids
are described further herein. In some embodiments, a molecule containing one or more maleimide
groups may be used as a linker, in which the maleimide group may form a carbon-sulfur linkage with a
cysteine in a component (e.g., an Fc domain or an albumin protein) in the conjugate. In some
embodiments, a molecule containing one or more alkyne groups may be used as a linker, in which the
alkyne group may form a 1,2,3-triazole linkage with an azide in a component (e.g., an Fc domain or an
albumin protein) in the conjugate. In some embodiments, a molecule containing one or more azide
groups may be used as a linker, in which the azide group may form a 1,2,3-triazole linkage with an
alkyne in a component (e.g., an Fc domain or an albumin protein) in the conjugate. In some
embodiments, a molecule containing one or more bis-sulfone groups may be used as a linker, in which
the bis-sulfone group may form a linkage with an amine group a component (e.g., an Fc domain or an
albumin protein) in the conjugate. In some embodiments, a molecule containing one or more sulfonic
acid groups may be used as a linker, in which the sulfonic acid group may form a sulfonamide linkage
with a component in the conjugate. In some embodiments, a molecule containing one or more
isocyanate groups may be used as a linker, in which the isocyanate group may form a urea linkage
with a component in the conjugate. In some embodiments, a molecule containing one or more
haloalkyl groups may be used as a linker, in which the haloalkyl group may form a covalent linkage,
e.g., C—N and C—O linkages, with a component in the conjugate.

(792) In some embodiments, a linker provides space, rigidity, and/or flexibility between the two or more
components. In some embodiments, a linker may be a bond, e.g., a covalent bond. The term “bond”
refers to a chemical bond, e.g., an amide bond, a disulfide bond, a C—O bond, a C—N bond, a N—N
bond, a C—S bond, or any kind of bond created from a chemical reaction, e.g., chemical conjugation.
In some embodiments, a linker includes no more than 250 atoms. In some embodiments, a linker
includes no more than 250 non-hydrogen atoms. In some embodiments, the backbone of a linker
includes no more than 250 atoms. The “backbone” of a linker refers to the atoms in the linker that
together form the shortest path from one part of a conjugate to another part of the conjugate (e.g., the
shortest path linking a first neuraminidase inhibitor and a second neuraminidase inhibitor). The atoms
in the backbone of the linker are directly involved in linking one part of a conjugate to another part of
the conjugate (e.g., linking a first neuraminidase inhibitor and a second neuraminidase inhibitor). For
examples, hydrogen atoms attached to carbons in the backbone of the linker are not considered as
directly involved in linking one part of the conjugate to another part of the conjugate.

(793) In some embodiments, a linker may comprise a synthetic group derived from, e.g., a synthetic
polymer (e.g., a polyethylene glycol (PEG) polymer). In some embodiments, a linker may comprise
one or more amino acid residues, such as D- or L-amino acid residues. In some embodiments, a linker
may be a residue of an amino acid sequence (e.g., a 1-25 amino acid, 1-10 amino acid, 1-9 amino
acid, 1-8 amino acid, 1-7 amino acid, 1-6 amino acid, 1-5 amino acid, 1-4 amino acid, 1-3 amino acid,
1-2 amino acid, or 1 amino acid sequence). In some embodiments, a linker may comprise one or
more, e.g., 1-100, 1-50, 1-25, 1-10, 1-5, or 1-3, optionally substituted alkylene, optionally substituted
heteroalkylene (e.g., a PEG unit), optionally substituted alkenylene, optionally substituted
heteroalkenylene, optionally substituted alkynylene, optionally substituted heteroalkynylene, optionally
substituted cycloalkylene, optionally substituted heterocycloalkylene, optionally substituted
cycloalkenylene, optionally substituted heterocycloalkenylene, optionally substituted cycloalkynylene,
optionally substituted heterocycloalkynylene, optionally substituted arylene, optionally substituted
heteroarylene (e.g., pyridine), O, S, NR.sup.i (R.sup.i is H, optionally substituted alkyl, optionally
substituted heteroalkyl, optionally substituted alkenyl, optionally substituted heteroalkenyl, optionally
substituted alkynyl, optionally substituted heteroalkynyl, optionally substituted cycloalkyl, optionally
substituted heterocycloalkyl, optionally substituted cycloalkenyl, optionally substituted
heterocycloalkenyl, optionally substituted cycloalkynyl, optionally substituted heterocycloalkynyl,
optionally substituted aryl, or optionally substituted heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl,
phosphate, phosphoryl, or imino. For example, a linker may comprise one or more optionally
substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene (e.g., a PEG unit),
optionally substituted C2-C20 alkenylene (e.g., C2 alkenylene), optionally substituted C2-C20
heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20
heteroalkynylene, optionally substituted C3-C20 cycloalkylene (e.g., cyclopropylene, cyclobutylene),
optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene,
optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene,
optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15 arylene (e.g., C6
arylene), optionally substituted C2-C15 heteroarylene (e.g., imidazole, pyridine), O, S, NR.sup.i
(R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally
substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-
C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl,
optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally
substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally
substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted
C2-C15 heteroaryl), P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino.

(794) The terms “alkyl,” “alkenyl,” and “alkynyl,” as used herein, include straight-chain and branched-
chain monovalent substituents, as well as combinations of these, containing only C and H when
unsubstituted. When the alkyl group includes at least one carbon-carbon double bond or carbon-
carbon triple bond, the alkyl group can be referred to as an “alkenyl” or “alkynyl” group respectively.
The monovalency of an alkyl, alkenyl, or alkynyl group does not include the optional substituents on
the alkyl, alkenyl, or alkynyl group. For example, if an alkyl, alkenyl, or alkynyl group is attached to a
compound, monovalency of the alkyl, alkenyl, or alkynyl group refers to its attachment to the
compound and does not include any additional substituents that may be present on the alkyl, alkenyl,
or alkynyl group. In some embodiments, the alkyl or heteroalkyl group may contain, e.g., 1-20. 1-18, 1-
16, 1-14, 1-12, 1-10, 1-8, 1-6, 1-4, or 1-2 carbon atoms (e.g., C1-C20, C1-C18, C1-C16, C1-C14, C1-
C12, C1-C10, C1-C8, C1-C6, C1-C4, or C1-C2). In some embodiments, the alkenyl, heteroalkenyl,
alkynyl, or heteroalkynyl group may contain, e.g., 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, or 2-4
carbon atoms (e.g., C2-C20, C2-C18, C2-C16, C2-C14, C2-C12, C2-C10, C2-C8, C2-C6, or C2-C4).
Examples include, but are not limited to, methyl, ethyl, isobutyl, sec-butyl, tert-butyl, 2-propenyl, and 3-
butynyl.

(795) The term “cycloalkyl,” as used herein, represents a monovalent saturated or unsaturated non-
aromatic cyclic alkyl group. A cycloalkyl may have, e.g., three to twenty carbons (e.g., a C3-C7, C3-
C8, C3-C9, C3-C10, C3-C11, C3-C12, C3-C14, C3-C16, C3-C18, or C3-C20 cycloalkyl). Examples of
cycloalkyls include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and
cycloheptyl. When the cycloalkyl group includes at least one carbon-carbon double bond, the
cycloalkyl group can be referred to as a “cycloalkenyl” group. A cycloalkenyl may have, e.g., four to
twenty carbons (e.g., a C4-C7, C4-C8, C4-C9, C4-C10, C4-C11, C4-C12, C4-C14, C4-C16, C4-C18,
or C4-C20 cycloalkenyl). Exemplary cycloalkenyl groups include, but are not limited to, cyclopentenyl,
cyclohexenyl, and cycloheptenyl. When the cycloalkyl group includes at least one carbon-carbon triple
bond, the cycloalkyl group can be referred to as a “cycloalkynyl” group. A cycloalkynyl may have, e.g.,
eight to twenty carbons (e.g., a C8-C9, C8-C10, C8-C11, C8-C12, C8-C14, C8-C16, C8-C18, or C8-
C20 cycloalkynyl). The term “cycloalkyl” also includes a cyclic compound having a bridged multicyclic
structure in which one or more carbons bridges two non-adjacent members of a monocyclic ring, e.g.,
bicyclo[2.2.1.]heptyl and adamantane. The term “cycloalkyl” also includes bicyclic, tricyclic, and
tetracyclic fused ring structures, e.g., decalin and spiro cyclic compounds.

(796) The term “aryl,” as used herein, refers to any monocyclic or fused ring bicyclic or tricyclic system
which has the characteristics of aromaticity in terms of electron distribution throughout the ring system,
e.g., phenyl, naphthyl, or phenanthrene. In some embodiments, a ring system contains 5-15 ring
member atoms or 5-10 ring member atoms. An aryl group may have, e.g., five to fifteen carbons (e.g.,
a C5-C6, C5-C7, C5-C8, C5-C9, C5-C10, C5-C11, C5-C12, C5-C13, C5-C14, or C5-C15 aryl). The
term “heteroaryl” also refers to such monocyclic or fused bicyclic ring systems containing one or more,
e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms selected from O, S and N. A heteroaryl group may have, e.g.,
two to fifteen carbons (e.g., a C2-C3, C2-C4, C2-C5, C2-C6, C2-C7, C2-C8, C2-C9. C2-C10, C2-C11,
C2-C12, C2-C13, C2-C14, or C2-C15 heteroaryl). The inclusion of a heteroatom permits inclusion of
5-membered rings to be considered aromatic as well as 6-membered rings. Thus, typical heteroaryl
systems include, e.g., pyridyl, pyrimidyl, indolyl, benzimidazolyl, benzotriazolyl, isoquinolyl, quinolyl,
benzothiazolyl, benzofuranyl, thienyl, furyl, pyrrolyl, thiazolyl, oxazolyl, isoxazolyl, benzoxazolyl,
benzoisoxazolyl, and imidazolyl. Because tautomers are possible, a group such as phthalimido is also
considered heteroaryl. In some embodiments, the aryl or heteroaryl group is a 5- or 6-membered
aromatic rings system optionally containing 1-2 nitrogen atoms. In some embodiments, the aryl or
heteroaryl group is an optionally substituted phenyl, pyridyl, indolyl, pyrimidyl, pyridazinyl,
benzothiazolyl, benzimidazolyl, pyrazolyl, imidazolyl, isoxazolyl, thiazolyl, or imidazopyridinyl. In some
embodiments, the aryl group is phenyl. In some embodiments, an aryl group may be optionally
substituted with a substituent such an aryl substituent, e.g., biphenyl.

(797) The term “alkaryl,” refers to an aryl group that is connected to an alkylene, alkenylene, or
alkynylene group. In general, if a compound is attached to an alkaryl group, the alkylene, alkenylene,
or alkynylene portion of the alkaryl is attached to the compound. In some embodiments, an alkaryl is
C6-C35 alkaryl (e.g., C6-C16, C6-C14, C6-C12, C6-C10, C6-C9, C6-C8, C7, or C6 alkaryl), in which
the number of carbons indicates the total number of carbons in both the aryl portion and the alkylene,
alkenylene, or alkynylene portion of the alkaryl. Examples of alkaryls include, but are not limited to,
(C1-C8)alkylene(C6-C12)aryl, (C2-C8)alkenylene(C6-C12)aryl, or (C2-C8)alkynylene(C6-C12)aryl. In
some embodiments, an alkaryl is benzyl or phenethyl. In a heteroalkaryl, one or more heteroatoms
selected from N, O, and S may be present in the alkylene, alkenylene, or alkynylene portion of the
alkaryl group and/or may be present in the aryl portion of the alkaryl group. In an optionally substituted
alkaryl, the substituent may be present on the alkylene, alkenylene, or alkynylene portion of the alkaryl
group and/or may be present on the aryl portion of the alkaryl group.

(798) The term “amino,” as used herein, represents —N(R.sup.x).sub.2 or —N.sup.+(R.sup.x).sub.3,

where each R.sup.x is, independently, H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R.sup.x
combine to form a heterocycloalkyl. In some embodiment, the amino group is —NH.sub.2.

(799) The term “alkamino,” as used herein, refers to an amino group, described herein, that is
attached to an alkylene (e.g., C1-C5 alkylene), alkenylene (e.g., C2-C5 alkenylene), or alkynylene
group (e.g., C2-C5 alkenylene). In general, if a compound is attached to an alkamino group, the
alkylene, alkenylene, or alkynylene portion of the alkamino is attached to the compound. The amino
portion of an alkamino refers to —N(R.sup.x).sub.2 or —N.sup.+(R.sup.x).sub.3, where each R.sup.x
is, independently, H, alkyl, alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R.sup.x combine to form a
heterocycloalkyl. In some embodiment, the amino portion of an alkamino is —NH.sub.2. An example
of an alkamino group is C1-C5 alkamino, e.g., C2 alkamino (e.g., CH.sub.2CH.sub.2NH.sub.2 or
CH.sub.2CH.sub.2N(CH.sub.3).sub.2). In a heteroalkamino group, one or more, e.g., 1-4, 1-3, 1, 2, 3,
or 4, heteroatoms selected from N, O, and S may be present in the alkylene, alkenylene, or alkynylene
portion of the heteroalkamino group. In some embodiments, an alkamino group may be optionally
substituted. In a substituted alkamino group, the substituent may be present on the alkylene,
alkenylene, or alkynylene portion of the alkamino group and/or may be present on the amino portion of
the alkamino group. The term “alkamide,” as used herein, refers to an amide group that is attached to
an alkylene (e.g., C1-C5 alkylene), alkenylene (e.g., C2-C5 alkenylene), or alkynylene (e.g., C2-C5
alkenylene) group. In general, if a compound is attached to an alkamide group, the alkylene,
alkenylene, or alkynylene portion of the alkamide is attached to the compound. The amide portion of
an alkamide refers to —C(O)—N(R.sup.x).sub.2, where each R.sup.x is, independently, H, alkyl,
alkenyl, alkynyl, aryl, alkaryl, cycloalkyl, or two R.sup.x combine to form a heterocycloalkyl. In some
embodiment, the amide portion of an alkamide is —C(O)NH.sub.2. An alkamide group may be —
(CH.sub.2).sub.2—C(O)NH.sub.2 or —CH.sub.2—C(O)NH.sub.2. In a heteroalkamide group, one or
more, e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms selected from N, O, and S may be present in the
alkylene, alkenylene, or alkynylene portion of the heteroalkamide group. In some embodiments, an
alkamide group may be optionally substituted. In a substituted alkamide group, the substituent may be
present on the alkylene, alkenylene, or alkynylene portion of the alkamide group and/or may be
present on the amide portion of the alkamide group.

(800) The terms “alkylene,” “alkenylene,” and “alkynylene,” as used herein, refer to divalent groups
having a specified size. In some embodiments, an alkylene may contain, e.g., 1-20, 1-18, 1-16, 1-14,
1-12, 1-10, 1-8, 1-6, 1-4, or 1-2 carbon atoms (e.g., C1-C20, C1-C18, C1-C16, C1-C14, C1-C12, C1-
C10, C1-C8, C1-C6, C1-C4, or C1-C2). In some embodiments, an alkenylene or alkynylene may
contain, e.g., 2-20, 2-18, 2-16, 2-14, 2-12, 2-10, 2-8, 2-6, or 2-4 carbon atoms (e.g., C2-C20, C2-C18,
C2-C16, C2-C14, C2-C12, C2-C10, C2-C8, C2-C6, or C2-C4). Alkylene, alkenylene, and/or alkynylene
includes straight-chain and branched-chain forms, as well as combinations of these. The divalency of
an alkylene, alkenylene, or alkynylene group does not include the optional substituents on the
alkylene, alkenylene, or alkynylene group. For example, two neuraminidase inhibitors may be attached
to each other by way of a linker that includes alkylene, alkenylene, and/or alkynylene, or combinations
thereof. Each of the alkylene, alkenylene, and/or alkynylene groups in the linker is considered divalent
with respect to the two attachments on either end of alkylene, alkenylene, and/or alkynylene group.
For example, if a linker includes -(optionally substituted alkylene)-(optionally substituted alkenylene)-
(optionally substituted alkylene)-, the alkenylene is considered divalent with respect to its attachments
to the two alkylenes at the ends of the linker. The optional substituents on the alkenylene are not
included in the divalency of the alkenylene. The divalent nature of an alkylene, alkenylene, or
alkynylene group (e.g., an alkylene, alkenylene, or alkynylene group in a linker) refers to both of the
ends of the group and does not include optional substituents that may be present in an alkylene,
alkenylene, or alkynylene group. Because they are divalent, they can link together multiple (e.g., two)
parts of a conjugate, e.g., a first neuraminidase inhibitor and a second neuraminidase inhibitor.
Alkylene, alkenylene, and/or alkynylene groups can be substituted by the groups typically suitable as
substituents for alkyl, alkenyl and alkynyl groups as set forth herein. For example, C═O is a C1
alkylene that is substituted by an oxo (═O). For example, —HCR—C═C— may be considered as an
optionally substituted alkynylene and is considered a divalent group even though it has an optional
substituent, R. Heteroalkylene, heteroalkenylene, and/or heteroalkynylene groups refer to alkylene,
alkenylene, and/or alkynylene groups including one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms,
e.g., N, O, and S. For example, a polyethylene glycol (PEG) polymer or a PEG unit —
(CH.sub.2).sub.2—O— in a PEG polymer is considered a heteroalkylene containing one or more
oxygen atoms.

(801) As used herein, a “combination therapy” or “administered in combination” means that a

conjugate described herein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-
(M-XI), or (M′-I)) and one (or more) different agents or treatments are administered to a subject as part
of a defined treatment regimen for a viral infection. The treatment regimen defines the doses and
periodicity of administration of each agent such that the effects of the separate agents on the subject
overlap. In some embodiments, the delivery of the conjugate and the one or more agents is
simultaneous or concurrent and the conjugate and the one or more agents may be co-formulated. In
some embodiments, the conjugate and the one or more agents are not co-formulated and are
administered in a sequential manner as part of a prescribed regimen. In some embodiments,
administration of the conjugate and the one or more agents or treatments in combination is such that
the reduction in a symptom, or other parameter related to the viral infection, is greater than what would
be observed with one agent or treatment delivered alone or in the absence of the other. The effect of
the conjugate and the one or more agents can be partially additive, wholly additive, or greater than
additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic
agent can be by any appropriate route including, but not limited to, oral routes, intravenous routes,
intramuscular routes, and direct absorption through mucous membrane tissues. The therapeutic
agents can be administered by the same route or by different routes. For example, a conjugate
described herein may be administered by intravenous injection while a second therapeutic agent of the
combination may be administered orally.

(802) The term “cycloalkylene,” as used herein, refers to a divalent cyclic group linking together two
parts of a compound. For example, one carbon within the cycloalkylene group may be linked to one
part of the compound, while another carbon within the cycloalkylene group may be linked to another
part of the compound. A cycloalkylene group may include saturated or unsaturated non-aromatic cyclic
groups. A cycloalkylene may have, e.g., three to twenty carbons in the cyclic portion of the
cycloalkylene (e.g., a C3-C7, C3-C8, C3-C9, C3-C10, C3-C11, C3-C12, C3-C14, C3-C16, C3-C18, or
C3-C20 cycloalkylene). When the cycloalkylene group includes at least one carbon-carbon double
bond, the cycloalkylene group can be referred to as a “cycloalkenylene” group. A cycloalkenylene may
have, e.g., four to twenty carbons in the cyclic portion of the cycloalkenylene (e.g., a C4-C7, C4-C8,
C4-C9. C4-C10, C4-C11, C4-C12, C4-C14, C4-C16, C4-C18, or C4-C20 cycloalkenylene). When the
cycloalkylene group includes at least one carbon-carbon triple bond, the cycloalkylene group can be
referred to as a “cycloalkynylene” group. A cycloalkynylene may have, e.g., four to twenty carbons in
the cyclic portion of the cycloalkynylene (e.g., a C4-C7, C4-C8, C4-C9. C4-C10, C4-C11, C4-C12, C4-
C14, C4-C16, C4-C18, or C8-C20 cycloalkynylene). A cycloalkylene group can be substituted by the
groups typically suitable as substituents for alkyl, alkenyl and alkynyl groups as set forth herein.
Heterocycloalkylene refers to a cycloalkylene group including one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4,
heteroatoms, e.g., N, O, and S. Examples of cycloalkylenes include, but are not limited to,
cyclopropylene and cyclobutylene. A tetrahydrofuran may be considered as a heterocycloalkylene.

(803) The term “arylene,” as used herein, refers to a multivalent (e.g., divalent or trivalent) aryl group
linking together multiple (e.g., two or three) parts of a compound. For example, one carbon within the
arylene group may be linked to one part of the compound, while another carbon within the arylene
group may be linked to another part of the compound. An arylene may have, e.g., five to fifteen
carbons in the aryl portion of the arylene (e.g., a C5-C6, C5-C7, C5-C8, C5-C9. C5-C10, C5-C11, C5-
C12, C5-C13, C5-C14, or C5-C15 arylene). An arylene group can be substituted by the groups
typically suitable as substituents for alkyl, alkenyl and alkynyl groups as set forth herein. Heteroarylene
refers to an aromatic group including one or more, e.g., 1-4, 1-3, 1, 2, 3, or 4, heteroatoms, e.g., N, O,
and S. A heteroarylene group may have, e.g., two to fifteen carbons (e.g., a C2-C3, C2-C4, C2-C5,
C2-C6, C2-C7, C2-C8, C2-C9. C2-C10, C2-C11, C2-C12, C2-C13, C2-C14, or C2-C15 heteroarylene).
(804) The term “optionally substituted,” as used herein, refers to having 0, 1, or more substituents,
such as 0-25, 0-20, 0-10 or 0-5 substituents. Substituents include, but are not limited to, alkyl, alkenyl,
alkynyl, aryl, alkaryl, acyl, heteroaryl, heteroalkyl, heteroalkenyl, heteroalkynyl, heteroalkaryl, halogen,
oxo, cyano, nitro, amino, alkamino, hydroxy, alkoxy, alkanoyl, carbonyl, carbamoyl, guanidinyl, ureido,
amidinyl, any of the groups or moieties described above, and hetero versions of any of the groups or
moieties described above. Substituents include, but are not limited to, F, Cl, methyl, phenyl, benzyl,
OR, NR.sub.2, SR, SOR, SO.sub.2R, OCOR, NRCOR, NRCONR.sub.2, NRCOOR, OCONR.sub.2,
RCO, COOR, alkyl-OOCR, SO.sub.3R, CONR.sub.2, SO.sub.2NR.sub.2, NRSO.sub.2NR.sub.2, CN,
CF.sub.3, OCF.sub.3, SiR.sub.3, and NO.sub.2, wherein each R is, independently, H, alkyl, alkenyl,
aryl, heteroalkyl, heteroalkenyl, or heteroaryl, and wherein two of the optional substituents on the
same or adjacent atoms can be joined to form a fused, optionally substituted aromatic or nonaromatic,
saturated or unsaturated ring which contains 3-8 members, or two of the optional substituents on the
same atom can be joined to form an optionally substituted aromatic or nonaromatic, saturated or
unsaturated ring which contains 3-8 members.

(805) An optionally substituted group or moiety refers to a group or moiety (e.g., any one of the groups
or moieties described above) in which one of the atoms (e.g., a hydrogen atom) is optionally replaced
with another substituent. For example, an optionally substituted alkyl may be an optionally substituted
methyl, in which a hydrogen atom of the methyl group is replaced by, e.g., OH. As another example, a
substituent on a heteroalkyl or its divalent counterpart, heteroalkylene, may replace a hydrogen on a
carbon or a hydrogen on a heteroatom such as N. For example, the hydrogen atom in the group —R—
NH—R— may be substituted with an alkamide substituent, e.g., —R—
N[(CH.sub.2C(O)N(CH.sub.3).sub.2]—R. Generally, an optional substituent is a noninterfering
substituent. A “noninterfering substituent” refers to a substituent that leaves the ability of the
conjugates described herein (e.g., conjugates of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-
(M-XI), or (M′-I)) to either bind to viral neuraminidase or to inhibit the proliferation of influenza virus.
Thus, in some embodiments, the substituent may alter the degree of such activity. However, as long
as the conjugate retains the ability to bind to viral neuraminidase or to inhibitor viral proliferation, the
substituent will be classified as “noninterfering.” For example, the noninterfering substituent would
leave the ability of the compound to provide antiviral efficacy based on an IC50 value of 10 μM or less
in a viral plaque reduction assay, such as in Example 2 based on an IC50 value against influenza virus
neuraminidase of less than 500 nM. Thus, the substituent may alter the degree of inhibition based on
plaque reduction or influenza virus neuraminidase inhibition. However, as long as the compounds
herein such as compounds of formulas (A-I), (A-II), (A-III), (A-IV), (A-V), (A-VI), (A-VII), (A-VIII), (A-IX),
(A-X), (A-XI), (A-XII), and (A-XIII) retain the ability to inhibit influenza virus neuraminidase activity, the
substituent will be classified as “noninterfering.” A number of assays for determining viral plaque
reduction or the ability of any compound to inhibit influenza virus neuraminidase are available in the
art, and some are exemplified in the Examples below.

(806) The term “hetero,” when used to describe a chemical group or moiety, refers to having at least
one heteroatom that is not a carbon or a hydrogen, e.g., N, O, and S. Any one of the groups or
moieties described above may be referred to as hetero if it contains at least one heteroatom. For
example, a heterocycloalkyl, heterocycloalkenyl, or heterocycloalkynyl group refers to a cycloalkyl,
cycloalkenyl, or cycloalkynyl group that has one or more heteroatoms independently selected from,
e.g., N, O, and S. An example of a heterocycloalkenyl group is a maleimido. For example, a heteroaryl
group refers to an aromatic group that has one or more heteroatoms independently selected from,
e.g., N, O, and S. One or more heteroatoms may also be included in a substituent that replaced a
hydrogen atom in a group or moiety as described herein. For example, in an optionally substituted
heteroaryl group, if one of the hydrogen atoms in the heteroaryl group is replaced with a substituent
(e.g., methyl), the substituent may also contain one or more heteroatoms (e.g., methanol).

(807) The term “acyl,” as used herein, refers to a group having the structure:

(808) ##STR00428##

wherein R.sup.z is an optionally substituted alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,

cycloalkynyl, aryl, alkaryl, alkamino, heteroalkyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl,
heterocycloalkenyl, heterocycloalkynyl, heteroaryl, heteroalkaryl, or heteroalkamino.

(809) The term “halo” or “halogen,” as used herein, refers to any halogen atom, e.g., F, Cl, Br, or I. Any
one of the groups or moieties described herein may be referred to as a “halo moiety” if it contains at
least one halogen atom, such as haloalkyl.

(810) The term “hydroxyl,” as used herein, represents an —OH group.

(811) The term “oxo,” as used herein, refers to a substituent having the structure ═O, where there is a
double bond between an atom and an oxygen atom.

(812) The term “carbonyl,” as used herein, refers to a group having the structure:

(813) ##STR00429##

(814) The term “thiocarbonyl,” as used herein, refers to a group having the structure:

(815) ##STR00430##

(816) The term “phosphate,” as used herein, represents the group having the structure:

(817) ##STR00431##

(818) The term “phosphoryl,” as used herein, represents the group having the structure:

(819) ##STR00432##

(820) ##STR00433##

(821) The term “sulfonyl,” as used herein, represents the group having the structure:

(822) ##STR00434##

(823) The term “imino,” as used herein, represents the group having the structure:

(824) ##STR00435##

wherein R is an optional substituent.

(825) The term “N-protecting group,” as used herein, represents those groups intended to protect an
amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting
groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 5th Edition (John Wiley &
Sons, New York, 2014), which is incorporated herein by reference. N-protecting groups include, e.g.,
acyl, aryloyl, and carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-
chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-
chlorobutyryl, benzoyl, carboxybenzyl (CBz), 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and
chiral auxiliaries such as protected or unprotected D, L or D, L-amino acid residues such as alanine,
leucine, phenylalanine; sulfonyl-containing groups such as benzenesulfonyl and p-toluenesulfonyl;
carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-
methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-
bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyl oxycarbonyl, 2,4-
dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,
3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-
dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl, t-butyloxycarbonyl (BOC),
diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl,
allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-
9-methoxycarbonyl (Fmoc), cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl,
and phenylthiocarbonyl; alkaryl groups such as benzyl, triphenylmethyl, and benzyloxymethyl; and silyl
groups such as trimethylsilyl.

(826) The term “amino acid,” as used herein, means naturally occurring amino acids and non-naturally
occurring amino acids.

(827) The term “naturally occurring amino acids,” as used herein, means amino acids including Ala,
Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

(828) The term “non-naturally occurring amino acid,” as used herein, means an alpha amino acid that
is not naturally produced or found in a mammal. Examples of non-naturally occurring amino acids
include D-amino acids; an amino acid having an acetylaminomethyl group attached to a sulfur atom of
a cysteine; a pegylated amino acid; the omega amino acids of the formula
NH.sub.2(CH.sub.2).sub.nCOOH where n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-
butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine; oxymethionine; phenylglycine;
citrulline; methionine sulfoxide; cysteic acid; ornithine; diaminobutyric acid; 3-aminoalanine; 3-hydroxy-
D-proline; 2,4-diaminobutyric acid; 2-aminopentanoic acid; 2-aminooctanoic acid, 2-carboxy
piperazine; piperazine-2-carboxylic acid, 2-amino-4-phenylbutanoic acid; 3-(2-naphthyl)alanine, and
hydroxyproline. Other amino acids are α-aminobutyric acid, α-amino-α-methylbutyrate,
aminocyclopropane-carboxylate, aminoisobutyric acid, aminonorbornyl-carboxylate, L-
cyclohexylalanine, cyclopentylalanine, L-N-methylleucine, L-N-methylmethionine, L-N-methylnorvaline,
L-N-methylphenylalanine, L-N-methylproline, L-N-methylserine, L-N-methyltryptophan, D-ornithine, L-
N-methylethylglycine, L-norleucine, α-methyl-aminoisobutyrate, α-methylcyclohexylalanine, D-α-
methylalanine, D-α-methylarginine, D-α-methylasparagine, D-α-methylaspartate, D-α-methylcysteine,
D-α-methylglutamine, D-α-methylhistidine, D-α-methylisoleucine, D-α-methylleucine, D-α-methyllysine,
D-α-methylmethionine, D-α-methylornithine, D-α-methylphenylalanine, D-α-methylproline, D-α-
methylserine, D-N-methylserine, D-α-methylthreonine, D-α-methyltryptophan, D-α-methyltyrosine, D-α-
methylvaline, D-N-methylalanine, D-N-methylarginine, D-N-methylasparagine, D-N-methylaspartate,
D-N-methylcysteine, D-N-methylglutamine, D-N-methylglutamate, D-N-methylhistidine, D-N-
methylisoleucine, D-N-methylleucine, D-N-methyllysine, N-methylcyclohexylalanine, D-N-
methylornithine, N-methylglycine, N-methylaminoisobutyrate, N-(1-methylpropyl)glycine, N-(2-
methylpropyl)glycine, D-N-methyltryptophan, D-N-methyltyrosine, D-N-methylvaline, γ-aminobutyric
acid, L-t-butylglycine, L-ethylglycine, L-homophenylalanine, L-α-methylarginine, L-α-methylaspartate,
L-α-methylcysteine, L-α-methylglutamine, L-α-methylhistidine, L-α-methylisoleucine, L-α-
methylleucine, L-α-methylmethionine, L-α-methylnorvaline, L-α-methylphenylalanine, L-α-
methylserine, L-α-methyltryptophan, L-α-methylvaline, N-(N-(2,2-diphenylethyl)
carbamylmethylglycine, 1-carboxy-1-(2,2-diphenyl-ethylamino) cyclopropane, 4-hydroxyproline,
ornithine, 2-aminobenzoyl (anthraniloyl), D-cyclohexylalanine, 4-phenyl-phenylalanine, L-citrulline, α-
cyclohexylglycine, L-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, L-thiazolidine-4-carboxylic acid,
L-homotyrosine, L-2-furylalanine, L-histidine (3-methyl), N-(3-guanidinopropyl)glycine, O-methyl-L-
tyrosine, O-glycan-serine, meta-tyrosine, nor-tyrosine, L-N,N′,N″-trimethyllysine, homolysine,
norlysine, N-glycan asparagine, 7-hydroxy-1,2,3,4-tetrahydro-4-fluorophenylalanine, 4-
methylphenylalanine, bis-(2-picolyl)amine, pentafluorophenylalanine, indoline-2-carboxylic acid, 2-
aminobenzoic acid, 3-amino-2-naphthoic acid, asymmetric dimethylarginine, L-tetrahydroisoquinoline-
1-carboxylic acid, D-tetrahydroisoquinoline-1-carboxylic acid, 1-amino-cyclohexane acetic acid, D/L-
allylglycine, 4-aminobenzoic acid, 1-amino-cyclobutane carboxylic acid, 2 or 3 or 4-aminocyclohexane
carboxylic acid, 1-amino-1-cyclopentane carboxylic acid, 1-aminoindane-1-carboxylic acid, 4-amino-
pyrrolidine-2-carboxylic acid, 2-aminotetraline-2-carboxylic acid, azetidine-3-carboxylic acid, 4-benzyl-
pyrolidine-2-carboxylic acid, tert-butylglycine, b-(benzothiazolyl-2-yl)-alanine, b-cyclopropyl alanine,
5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, (2R,4S)4-hydroxypiperidine-2-carboxylic acid, (2S,4S)
and (2S,4R)-4-(2-naphthylmethoxy)-pyrolidine-2-carboxylic acid, (2S,4S) and (2S,4R)4-phenoxy-
pyrrolidine-2-carboxylic acid, (2R,5S) and (2S,5R)-5-phenyl-pyrrolidine-2-carboxylic acid, (2S,4S)-4-
amino-1-benzoyl-pyrrolidine-2-carboxylic acid, t-butylalanine, (2S,5R)-5-phenyl-pyrrolidine-2-
carboxylic acid, 1-aminomethyl-cyclohexane-acetic acid, 3,5-bis-(2-amino)ethoxy-benzoic acid, 3,5-
diamino-benzoic acid, 2-methylamino-benzoic acid, N-methylanthranylic acid, L-N-methylalanine, L-N-
methylarginine, L-N-methylasparagine, L-N-methylaspartic acid, L-N-methylcysteine, L-N-
methylglutamine, L-N-methylglutamic acid, L-N-methylhistidine, L-N-methylisoleucine, L-N-
methyllysine, L-N-methylnorleucine, L-N-methylornithine, L-N-methylthreonine, L-N-methyltyrosine, L-
N-methylvaline, L-N-methyl-t-butylglycine, L-norvaline, α-methyl-γ-aminobutyrate, 4,4′-biphenylalanine,
α-methylcylcopentylalanine, α-methyl-α-napthylalanine, α-methylpenicillamine, N-(4-
aminobutyl)glycine, N-(2-aminoethyl)glycine, N-(3-aminopropyl)glycine, N-amino-α-methylbutyrate, α-
napthylalanine, N-benzylglycine, N-(2-carbamylethyl)glycine, N-(carbamylmethyl)glycine, N-(2-
carboxyethyl)glycine, N-(carboxymethyl)glycine, N-cyclobutylglycine, N-cyclodecylglycine, N-
cycloheptylglycine, N-cyclohexylglycine, N-cyclodecylglycine, N-cylcododecylglycine, N-
cyclooctylglycine, N-cyclopropylglycine, N-cycloundecylglycine, N-(2,2-diphenylethyl)glycine, N-(3,3-
diphenylpropyl)glycine, N-(3-guanidinopropyl)glycine, N-(1-hydroxyethyl)glycine, N-
(hydroxyethyl))glycine, N-(imidazolylethyl))glycine, N-(3-indolylyethyl)glycine, N-methyl-γ-
aminobutyrate, D-N-methylmethionine, N-methylcyclopentylalanine, D-N-methylphenylalanine, D-N-
methylproline, D-N-methylthreonine, N-(1-methylethyl)glycine, N-methyl-napthylalanine, N-
methylpenicillamine, N-(p-hydroxyphenyl)glycine, N-(thiomethyl)glycine, penicillamine, L-α-
methylalanine, L-α-methylasparagine, L-α-methyl-t-butylglycine, L-methylethylglycine, L-α-
methylglutamate, L-α-methylhomophenylalanine, N-(2-methylthioethyl)glycine, L-α-methyllysine, L-α-
methylnorleucine, L-α-methylornithine, L-α-methylproline, L-α-methylthreonine, L-α-methyltyrosine, L-
N-methyl-homophenylalanine, N-(N-(3,3-diphenylpropyl) carbamylmethylglycine, L-pyroglutamic acid,
D-pyroglutamic acid, O-methyl-L-serine, O-methyl-L-homoserine, 5-hydroxylysine, α-
carboxyglutamate, phenylglycine, L-pipecolic acid (homoproline), L-homoleucine, L-lysine (dimethyl),
L-2-naphthylalanine, L-dimethyldopa or L-dimethoxy-phenylalanine, L-3-pyridylalanine, L-histidine
(benzoyloxymethyl), N-cycloheptylglycine, L-diphenylalanine, O-methyl-L-homotyrosine, L-β-
homolysine, O-glycan-threoine, Ortho-tyrosine, L-N,N′-dimethyllysine, L-homoarginine, neotryptophan,
3-benzothienylalanine, isoquinoline-3-carboxylic acid, diaminopropionic acid, homocysteine, 3,4-
dimethoxyphenylalanine, 4-chlorophenylalanine, L-1,2,3,4-tetrahydronorharman-3-carboxylic acid,
adamantylalanine, symmetrical dimethylarginine, 3-carboxythiomorpholine, D-1,2,3,4-
tetrahydronorharman-3-carboxylic acid, 3-aminobenzoic acid, 3-amino-1-carboxymethyl-pyridin-2-one,
1-amino-1-cyclohexane carboxylic acid, 2-aminocyclopentane carboxylic acid, 1-amino-1-
cyclopropane carboxylic acid, 2-aminoindane-2-carboxylic acid, 4-amino-tetrahydrothiopyran-4-
carboxylic acid, azetidine-2-carboxylic acid, b-(benzothiazol-2-yl)-alanine, neopentylglycine, 2-
carboxymethyl piperidine, b-cyclobutyl alanine, allylglycine, diaminopropionic acid, homo-cyclohexyl
alanine, (2S,4R)-4-hydroxypiperidine-2-carboxylic acid, octahydroindole-2-carboxylic acid, (2S,4R)
and (2S,4R)-4-(2-naphthyl), pyrrolidine-2-carboxylic acid, nipecotic acid, (2S,4R) and (2S,4S)-4-(4-
phenylbenzyl) pyrrolidine-2-carboxylic acid, (3S)-1-pyrrolidine-3-carboxylic acid, (2S,4S)-4-
tritylmercapto-pyrrolidine-2-carboxylic acid, (2S,4S)-4-mercaptoproline, t-butylglycine, N,N-bis(3-
aminopropyl)glycine, 1-amino-cyclohexane-1-carboxylic acid, N-mercaptoethylglycine, and
selenocysteine. In some embodiments, amino acid residues may be charged or polar. Charged amino
acids include alanine, lysine, aspartic acid, or glutamic acid, or non-naturally occurring analogs
thereof. Polar amino acids include glutamine, asparagine, histidine, serine, threonine, tyrosine,
methionine, or tryptophan, or non-naturally occurring analogs thereof. It is specifically contemplated
that in some embodiments, a terminal amino group in the amino acid may be an amido group or a
carbamate group.

(829) As used herein, the term “percent (%) identity” refers to the percentage of amino acid residues of
a candidate sequence, e.g., an Fc-IgG, or fragment thereof, that are identical to the amino acid
residues of a reference sequence after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent identity (i.e., gaps can be introduced in one or both of the candidate
and reference sequences for optimal alignment and non-homologous sequences can be disregarded
for comparison purposes). Alignment for purposes of determining percent identity can be achieved in
various ways that are within the skill in the art, for instance, using publicly available computer software
such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine
appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal
alignment over the full length of the sequences being compared. In some embodiments, the percent
amino acid sequence identity of a given candidate sequence to, with, or against a given reference
sequence (which can alternatively be phrased as a given candidate sequence that has or includes a
certain percent amino acid sequence identity to, with, or against a given reference sequence) is
calculated as follows:
100×(fraction of A/B)

where A is the number of amino acid residues scored as identical in the alignment of the candidate
sequence and the reference sequence, and where B is the total number of amino acid residues in the
reference sequence. In some embodiments where the length of the candidate sequence does not
equal to the length of the reference sequence, the percent amino acid sequence identity of the
candidate sequence to the reference sequence would not equal to the percent amino acid sequence
identity of the reference sequence to the candidate sequence.

(830) Two polynucleotide or polypeptide sequences are said to be “identical” if the sequence of
nucleotides or amino acids in the two sequences is the same when aligned for maximum
correspondence as described above. Comparisons between two sequences are typically performed by
comparing the sequences over a comparison window to identify and compare local regions of
sequence similarity. A “comparison window” as used herein, refers to a segment of at least about 15
contiguous positions, about 20 contiguous positions, about 25 contiguous positions, or more (e.g.,
about 30 to about 75 contiguous positions, or about 40 to about 50 contiguous positions), in which a
sequence may be compared to a reference sequence of the same number of contiguous positions
after the two sequences are optimally aligned.

(831) The term “treating” or “to treat,” as used herein, refers to a therapeutic treatment of a viral
infection (e.g., a viral infection such as and influenza infection) in a subject. In some embodiments, a
therapeutic treatment may slow the progression of the viral infection, improve the subject's outcome,
and/or eliminate the infection. In some embodiments, a therapeutic treatment of a viral infection in a
subject may alleviate or ameliorate of one or more symptoms or conditions associated with the viral
infection, diminish the extent of the viral, stabilize (i.e., not worsening) the state of the viral infection,
prevent the spread of the viral infection, and/or delay or slow the progress of the viral infection, as
compare the state and/or the condition of the viral infection in the absence of the therapeutic
treatment.

(832) The term “average value of T,” as used herein, refers to the mean number of monomers of
neuraminidase inhibitor or dimers of neuraminidase inhibitors conjugated to an Fc domain or an
albumin protein within a population of conjugates. In some embodiments, within a population of
conjugates, the average number of monomers of neuraminidase inhibitor or dimers of neuraminidase
inhibitors conjugated to an Fc domain monomer may be from 1 to 20 (e.g., the average value of T is 1
to 2, 1 to 3, 1 to 4, 1 to 5, 5 to 10, 10 to 15, 15 to 20, 1.5 to 3.5, 2.5 to 4.5, 3.5 to 5.5, 4.5 to 6.5, 5.5 to
7.5, 6.5 to 8.5, 7.5 to 9.5, or 8.5 to 10.5). In some embodiments, the average value of T is 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

(833) The term “subject,” as used herein, can be a human, non-human primate, or other mammal,
such as but not limited to dog, cat, horse, cow, pig, turkey, goat, fish, monkey, chicken, rat, mouse, and
sheep. The term “therapeutically effective amount,” as used herein, refers to an amount, e.g.,
pharmaceutical dose, effective in inducing a desired effect in a subject or in treating a subject having a
condition or disorder described herein (e.g., a viral infection, such as an influenza infection). It is also
to be understood herein that a “therapeutically effective amount” may be interpreted as an amount
giving a desired therapeutic and/or preventative effect, taken in one or more doses or in any dosage or
route, and/or taken alone or in combination with other therapeutic agents (e.g., an antiviral agent
described herein). For example, in the context of administering a conjugate described herein (e.g., a
conjugate of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) that is used for the
treatment of a viral infection, an effective amount of a conjugate is, for example, an amount sufficient
to prevent, slow down, or reverse the progression of the viral infection as compared to the response
obtained without administration of the conjugate.

(834) As used herein, the term “pharmaceutical composition” refers to a medicinal or pharmaceutical
formulation that contains at least one active ingredient (e.g., a conjugate of any one of formulas (1)-
(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) as well as one or more excipients and diluents to enable
the active ingredient suitable for the method of administration. The pharmaceutical composition of the
present disclosure includes pharmaceutically acceptable components that are compatible with a
conjugate described herein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-
(M-XI), or (M′-I)).

(835) As used herein, the term “pharmaceutically acceptable carrier” refers to an excipient or diluent in
a pharmaceutical composition. For example, a pharmaceutically acceptable carrier may be a vehicle
capable of suspending or dissolving the active conjugate (e.g., a conjugate of any one of formulas (1)-
(5), (D-I)-(D-XI), or (M-I)-(M-VI)). The pharmaceutically acceptable carrier must be compatible with the
other ingredients of the formulation and not deleterious to the recipient. In the present disclosure, the
pharmaceutically acceptable carrier must provide adequate pharmaceutical stability to a conjugate
described herein. The nature of the carrier differs with the mode of administration. For example, for
oral administration, a solid carrier is preferred; for intravenous administration, an aqueous solution
carrier (e.g., WFI, and/or a buffered solution) is generally used.

(836) The term “pharmaceutically acceptable salt,” as used herein, represents salts of the conjugates
described herein (e.g., conjugates of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or
(M′-I)) that are, within the scope of sound medical judgment, suitable for use in methods described
herein without undue toxicity, irritation, and/or allergic response. Pharmaceutically acceptable salts are
well known in the art. For example, pharmaceutically acceptable salts are described in:
Pharmaceutical Salts: Properties, Selection, and Use (Eds. P. H. Stahl and C. G. Wermuth), Wiley-
VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the
conjugates described herein or separately by reacting the free base group with a suitable organic acid.

(837) The term “drug-to-antibody ratio” or “DAR” refers to the average number of small molecule drug
moieties (e.g., the average number of small molecule drug monomers or dimers) conjugated to an
antibody, Fc domain, or albumin protein described herein. In some embodiments described herein, the
DAR is represented by “T” (e.g., in formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)). As used
herein, each monomer moiety (e.g., each zanamivir or peramivir monomer) or each dimer moiety (e.g.,
each zanamivir dimer or peramivir dimer) conjugated to the Fc domain, antibody, or albumin protein
corresponds to a DAR value of 1.0 (e.g., a “T” value of 1.0). For example, an Fc domain conjugated to
4 zanamivir monomers would have a DAR of 4.0 (e.g., a “T” of 4.0). An Fc domain conjugated to 4
zanamivir dimers (e.g., 8 total zanamivir molecules) would also have a DAR of 4.0 (e.g., a “T” of 4.0).
DAR may also be computed as the average DAR for a population of molecules, such as a population
of Fc domains, antibodies, or albumin proteins. DAR values may affect the efficacy, potency,
pharmacokinetics, or toxicity of the drug.

(838) The term “secondary infection,” as used herein, refers to an infection that occurs in a subject
during or after another (referred to as primary) infection in that subject (e.g., during or after a primary
influenza infection). A secondary infections may be caused by the primary infection or may be caused
by treatment of the primary infection. In some cases, primary infections alter the immune system
making the subject more susceptible to a secondary infection. In some cases, treatment of the primary
infection makes the subject more susceptible to a secondary infection. For example, the influenza
virus has been associated with secondary infections (e.g., increased risk of developing a secondary
infection), such as bacterial secondary infections, for example of the respiratory tract. Secondary
infections associated with influenza infection increase the morbidity and mortality of influenza.
Secondary infections include co-infections. The terms “secondary infection” and “co-infection” are
used interchangeably herein.

(839) The term “about,” as used herein, indicates a deviation of up to ±5%. For example, about 10%
refers to from 9.5% to 10.5%.

(840) Any values provided in a range of values include both the upper and lower bounds, and any
values contained within the upper and lower bounds.

(841) The term “(1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-)”, as used herein, represents the
formulas of any one of (1), (2), (3), (4), (5), (D-I), (D-II), (D-II-1), (D-II-2), (D-I1-3), (D-I1-4), (D-I1-5), (D-
II-6), (D-I1-7), (D-II-8), (D-II-9), (D-II-10), (D-III), (D-III-1), (D-III-2), (D-III-3), (D-III-4), (D-III-5), (D-III-6),
(D-II-7), (D-III-8), (D-III-9), (D-IV), (D-IV-1), (D-IV-2), (D-V), (D-V-1), (D-V-2), (D-V-3), (D-V-4), (D-V-5),
(D-V-6), (D-V-7), (D-V-8), (D-V-9), (D-V-10), (D-VI), (D-VI-1), (D-VI-2), (D-VI-3), (D-VI-4), (D-VI-5), (D-
VI-6), (D-VI-7), (D-VI-8), (D-VI-9), (D-VII), (D-VIII), (D-VIII-1), (D-VIII-2), (D-VIII-3), (D-VIII-4), (D-VIII-
5), (D-VIII-6), (D-VIII-7), (D-VII-8), (D-VIII-9), (D-VIII-10), (D-VIII-11), (D-IX), (D-IX-1), (D-IX-2), (D-IX-
3), (D-IX-4), (D-IX-5), (D-IX-6), (D-X), (D-X-1), (D-X-2), (D-X-3), (D-XI), (D-XI-1), (D′-1), (M-I), (M-II),
(M-II-1), (M-II-2), (M-II-3), (M-II-4), (M-II-5), (M-II-6), (M-II-7), (M-II-8), (M-II-9), (M-I1-10), (M-III), (M-III-
1), (M-III-2), (M-III-3), (M-III-4), (M-III-5), (M-III-6), (M-III-7), (M-III-8), (M-III-9), (M-IV), (M-IV-1), (M-IV-
2), (M-V), (M-V-1), (M-V-2), (M-V-3), (M-V-4), (M-V-5), (M-V-6), (M-V-7), (M-V-8), (M-V-9), (M-V-10),
(M-VI), (M-VI-1), (M-VI-2), (M-VI-3), (M-V1-4), (M-VI-5), (M-VI-6), (M-VI-7), (M-VI-8), (M-VI-9), (M-VI-
10), (M-VIII-2), (M-VIII-1), (M-VIII-2), (M-VIII-3), (M-VIII-4), (M-VIII-5), (M-VIII-6), (M-VIII-7), (M-VIII-8),
(M-VIII-9), (M-VIII-10), (M-VIII-11), (M-IX), (M-IX-1), (M-IX-2), (M-IX-3), (M-IX-4), (M-IX-5), (M-IX-6),
(M-X), (M-X-1), (M-X-2), (M-X-3), (M-XI), (M-XI-1), or (M′-I)).

(842) Other features and advantages of the conjugates described herein will be apparent from the
following Detailed Description and the claims.

Description

DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is an image depicting exemplary methods of conjugating a neuraminidase inhibitor

monomer or dimer, e.g., by way of a linker, to an Fc domain monomer, an Fc domain, an Fc-binding
peptide, an albumin protein, or an albumin protein-binding peptide.

(2) FIG. 2 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain
formed from Fc domain monomers having the sequence of SEQ ID NO: 1.

(3) FIG. 3 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain
formed from Fc domain monomers having the sequence of SEQ ID NO: 3.

(4) FIG. 4 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain
formed from Fc domain monomers having the sequence of SEQ ID NO: 5.

(5) FIG. 5 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain
formed from Fc domain monomers having the sequence of SEQ ID NO: 7.

(6) FIG. 6 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain
formed from Fc domain monomers having the sequence of SEQ ID NO: 9.

(7) FIG. 7 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain
formed from Fc domain monomers having the sequence of SEQ ID NO: 12.

(8) FIG. 8 shows non-reducing and reducing SDS-PAGE and a schematic illustration of an Fc domain
formed from Fc domain monomers having the sequence of SEQ ID NO: 14.

(9) FIG. 9 shows a non-reducing SDS-PAGE of Conjugate 1.

(10) FIG. 10 shows a non-reducing SDS-PAGE of Conjugate 2.

(11) FIG. 11 shows a non-reducing SDS-PAGE of Conjugate 3.

(12) FIG. 12 shows a non-reducing SDS-PAGE of Conjugate 4.

(13) FIG. 13 shows a non-reducing SDS-PAGE of Conjugate 5.

(14) FIG. 14 shows a non-reducing SDS-PAGE of Conjugate 6.

(15) FIG. 15 is a graph showing the IC50 values from an H1N1 neuraminidase inhibition assay for Int-
2 and Conjugate 1.
(16) FIG. 16 is a graph showing the IC50 values from an H1N1 neuraminidase inhibition assay for
Conjugates 1-6.

(17) FIG. 17 is a graph showing the IC50 values from an H3N2 neuraminidase inhibition assay for
Conjugates 1-6.

(18) FIGS. 18A-18C are a series of graphs showing the cell by viability of A549 cells treated with
Conjugate 3 (FIG. 18A), Conjugate 4 (FIG. 18B), or Conjugate 6 (FIG. 18C).

(19) FIGS. 19A-19E are a series of graphs showing the ability of Conjugate 3 to inhibit the growth of
pathogenic influenza viral strains A/WSN/33 H1N1 (FIG. 19A), A/Wyoming/3/03 H3N2 (FIG. 19B),
A/California/04/09 H1N1 pdm (FIG. 19C), A/Vietnam/1203/04 H5N1 HALo (FIG. 19D), or B/Lee/40
Victoria (FIG. 19E) in human epithelial cells.

(20) FIGS. 20A-20E are a series of graphs showing the ability of Conjugate 4 to inhibit the growth of
pathogenic influenza viral strains A/WSN/33 H1N1 (FIG. 20A), A/Wyoming/3/03 H3N2 (FIG. 20B),
A/California/04/09 H1N1 pdm (FIG. 20C), A/Vietnam/1203/04 H5N1 HALo (FIG. 20D), or B/Lee/40
Victoria (FIG. 20E) in human epithelial cells.

(21) FIGS. 21A-21E are a series of graphs showing the ability of Conjugate 6 to inhibit the growth of
pathogenic influenza viral strains A/WSN/33 H1N1 (FIG. 21A), A/Wyoming/3/03 H3N2 (FIG. 21B),
A/California/04/09 H1N1 pdm (FIG. 21C), A/Vietnam/1203/04 H5N1 HALo (FIG. 21D), or B/Lee/40
Victoria (FIG. 21E) in human epithelial cells.

(22) FIGS. 22A-22E are a series of graphs showing the ability of Conjugate 6 to inhibit the growth of
pathogenic influenza viral strains A/WSN/33 H1N1 (FIG. 22A), A/Wyoming/3/03 H3N2 (FIG. 22B),
A/California/04/09 H1N1 pdm (FIG. 22C), B/Lee/40 Victoria (FIG. 22D), or A/Vietnam/1203/04 (FIG.
22E) in human epithelial cells, compared to Oseltamivir.

(23) FIG. 23 is a graph showing the relative mouse serum concentration of Conjugate 6 compared to
Fc (hIgG1) alone.

(24) FIG. 24 is a graph showing the effect of Conjugate 6 on mouse weight in a lethal mouse influenza
model. The study was performed as described in Example 29.

(25) FIG. 25 is a graph showing the effect of Conjugate 6 on survival in a lethal mouse influenza
model. The study was performed as described in Example 29.

(26) FIG. 26 is a graph showing the effect of Conjugate 6 on mouse weight in a lethal mouse influenza
model. The study was performed as described in Example 30.

(27) FIG. 27 is a graph showing the effect of Conjugate 6 on survival in a lethal mouse influenza
model. The study was performed as described in Example 30.

(28) FIG. 28 is an image depicting a method of conjugating a neuraminidase inhibitor monomer or

dimer, e.g., by way of a linker, to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an
albumin protein, or an albumin protein-binding peptide by oxime conjugation to an amino acid residue,
e.g., a nitrogen atom of a surface exposed lysine.

(29) FIG. 29 is an image depicting a method of conjugating a neuraminidase inhibitor monomer or

dimer, e.g., by way of a linker, to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an
albumin protein, or an albumin protein-binding peptide by thioether conjugation to an amino acid
residue, e.g., a nitrogen atom of a surface exposed lysine.

(30) FIG. 30 is an image depicting a method of conjugating a neuraminidase inhibitor monomer or

dimer, e.g., by way of a linker, to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an
albumin protein, or an albumin protein-binding peptide by rebridged cysteine conjugation, e.g.,
rebridged cysteine conjugation to a pair of sulfur atoms of two hinge cysteines in an Fc domain
monomer or Fc domain.

(31) FIGS. 31A-31F are a series of graphs showing the survival of mice treated with Conjugate 6 in a
lethal mouse influenza model. Mice were treated with either Oseltamivir (Tamiflu™) control, 20 mg/kg,
2× daily, starting 8 hours post-infection (FIG. 31A); Conjugate 6, 50 mg/kg, 1 dose 28 days prior to
infection (FIG. 31B); Conjugate 6, 10 mg/kg, 1 dose 28 days prior to infection (FIG. 31C); Conjugate 6,
5 mg/kg, 1 dose 28 days prior to infection (FIG. 31D); Conjugate 6, 2.5 mg/kg, 1 dose 28 days prior to
infection (FIG. 31E); or Conjugate 6, 1.25 mg/kg, 1 dose 28 days prior to infection (FIG. 31F). This
study was performed as described in Example 33.

(32) FIGS. 32A-32F is a series of graphs showing that Conjugate 6 extends treatment window as
compared to Oseltamivir (Tamiflu™) as determined by survival in a lethal mouse influenza model. Mice
were treated with either vehicle (PBS), Fc only 10 mpk, or Conjugate 6, 4 hours prior to infection (FIG.
32A); Conjugate 6 or Oseltamivir (Tamiflu™), 8 hours post-infection (FIG. 32B); Conjugate 6 or
Oseltamivir (Tamiflu™), 24 hours post-infection (FIG. 32C); Conjugate 6 or Oseltamivir (Tamiflu™), 48
hours post-infection (FIG. 32D); Conjugate 6 or Oseltamivir (Tamiflu™), 72 hours post-infection (FIG.
32E); or Conjugate 6 or Oseltamivir (Tamiflu™), 96 hours post-infection (FIG. 32F). The study was
performed as described in Example 34.

(33) FIG. 33 is a graph showing no significant effect of body weight gain were observed following
administration of Conjugate 6 in a 14 day rat dose-range finder toxicity study. The study was
performed as described in Example 35.

(34) FIGS. 34A-34D is a series of graphs showing that Conjugate 6 extends treatment window as
compared to Oseltamivir (Tamiflu™) as determined by survival in a lethal mouse influenza model. Mice
were treated with either Oseltamivir (Tamiflu™) control, 20 mg/kg, 2× daily, starting 8 hours post-
infection (FIG. 34A); Conjugate 6, 10 mg/kg, 1 dose 4 hours prior to infection (FIG. 34B); Conjugate 6,
2 mg/kg, 1 dose 4 hours prior to infection (FIG. 34C); or Conjugate 6, 0.4 mg/kg, 1 dose 4 days prior to
infection (FIG. 34D). The study was performed as described in Example 37.

(35) FIG. 35 is a graph showing the effect of Conjugate 6 on mouse weight in a lethal mouse influenza
model. The study was performed as described in Example 37.

(36) FIG. 36 is a graph showing a 7-day rat pharmacokinetic study following IV administration of
Conjugate 6. The study was performed as described in Example 38.

(37) FIG. 37 is a graph showing a 14-day rat pharmacokinetic study following IV administration of
Conjugate 6. The study was performed as described in Example 39.

(38) FIG. 38 is a graph showing a 28-day rat pharmacokinetic study comparing IV and SC
administration of Conjugate 6. The study was performed as described in Example 40.

(39) FIG. 39 is a graph showing a 28-day non-human primate pharmacokinetic study following IV
administration of Conjugate 6. The study was performed as described in Example 41.

(40) FIG. 40 is a graph showing a mouse lung distribution pharmacokinetic study following IV
administration of Conjugate 6. The study was performed as described in Example 42.

(41) FIG. 41 is a graph showing a 5-day mouse pharmacokinetic study comparing IV, SC and IM
administration of Conjugate 6. The study was performed as described in Example 43.

(42) FIG. 42 shows a non-reducing SDS-PAGE of Conjugate 8.

(43) FIG. 43 shows the structure of Conjugate 6.

(44) FIG. 44 is a graph showing a 24 hour in vitro mouse plasma stability study comparing Conjugate
6 incubated at 37° C. for 24 hr compared to a control and neat compound.
(45) FIG. 45 is a graph showing a 24 hour in vitro human plasma stability study comparing Conjugate
6 incubated at 37° C. for 24 hr compared to a control and neat compound.

(46) FIG. 46 is a graph showing a 24 hour mouse liver microsomal stability study comparing Conjugate
6 incubated at 37° C. for 24 hr in mouse liver microsomal cells and heat killed mouse liver microsomal
cells as a control.

(47) FIG. 47 is a graph showing a 24 hour human liver microsomal stability study comparing
Conjugate 6 incubated at 37° C. for 24 hr in human liver microsomal cells and heat killed mouse liver
microsomal cells as a control.

(48) FIG. 48 is a graph showing the % body weight change in mice over 15 days post viral challenge.
The study was performed as described in Example 66.

(49) FIG. 49 is a graph showing the % survival in mice over 15 days post viral challenge. The study
was performed as described in Example 66.

(50) FIG. 50 is a graph showing the binding of Conjugate 6 and Conjugate 12 compared to hIgG1 Fc
(WT) and hIgG1 Fc (N297A) to Fcγ receptor IIIA. The study was performed as described in Example
67.

(51) FIG. 51 is a graph showing the binding of Conjugate 6 and Conjugate to Fcγ receptor IIIA. The
study was performed as described in Example 67.

(52) FIG. 52 is a graph showing a 7-day non-human primate toxicokinetic study following IV
administration of Conjugate 6. The study was performed as described in Example 68.

(53) FIG. 53 is a graph showing a 28-day non-human primate pharmacokinetic study comparing IV
and SC administration of Conjugate 6. The study was performed as described in Example 68.

(54) FIG. 54 shows a non-reducing SDS-PAGE of Conjugate 12.

(55) FIG. 55 shows a non-reducing SDS-PAGE of Conjugate 13 having different drug-to-antibody

(DAR) ratio values (Conjugate 13a, Conjugate 13b, Conjugate 13c, Conjugate 13d, Conjugate 13e,
Conjugate 13f, and Conjugate 13g).

(56) FIG. 56 is a graph showing the % survival in immune compromised mice over 35 days post viral
challenge. The 0.3 mg/kg conjugate 6 treatment group remained at 100% survival but is slightly offset
in the graph for clarity. The study was performed as described in Example 95 FIG. 57 is a graph
showing the % body weight change in immune compromised mice over 35 days post viral challenge.
The study was performed as described in Example 95.

(57) FIG. 58 is a graph showing administration of conjugate 6 results in dose-dependent viral

clearance in the lungs of a mouse model infected with Influenza A (H1N1) and that this viral clearance
is greater than PBS control, Fc-only control, or Oseltamivir control. This study was performed as
described in Example 96.

(58) FIG. 59 is a graph showing that administration of conjugate 6 results in a dose-dependent

reduction in inflammatory cytokines in the lungs in a mouse model infected with Influenza A (H1N1).
This study was performed as described in Example 97.

(59) FIG. 60 is a graph showing the pharmacokinetics of conjugate 6 in BALB/c SCID

(immunocompromised) mice and CD-1 mice (immunocompetent). This study was performed as
described in Example 98.

(60) FIG. 61 is an image depicting conjugate 33.

(61) FIGS. 62A-62B are graphs showing the body weight change (%) in BALB/c mice challenged
intranasally with 3× the LD.sub.95 of mouse adapted influenza A.PR/8/1934 (H1N1). This study was
performed as described in Example 133.

(62) FIG. 63A is a graph showing the viral burden on day 4 post infection. This study was performed
as described in Example 133.

(63) FIG. 63B is a graph showing the log reduction in viral burden on day 4 post infection. This study
was performed as described in Example 133.

(64) FIG. 64A is a graph showing administration of conjugate 33 results in dose-dependent reduction
of viral burden in a mouse model infected with Influenza A (H1N1) compared with PBS control or Fc-
only control. This study was performed as described in Example 133.

(65) FIG. 64B is a graph showing the log reduction in viral burden on day 4 post infection. This study
was performed as described in Example 133.

(66) FIGS. 65A-65E are a series bar graphs showing administration of conjugate 33 results in dose-
dependent fold-reduction in cytokine levels for TNF-α (FIG. 65A), IL-6 (FIG. 65B), INF-γ (FIG. 65C),
MCP-1 (FIG. 65D), and MIP-1α (FIG. 65E). This study was performed as described in Example 133.

(67) FIG. 66 is a chromatograph showing the stability of Int-80 compared to Int-4 on day 7 of
incubating at 37° C. and 60° C. This study was performed as describe in Example 142.

(68) FIG. 67 is a graph showing serial passage of A/CA/09 pdm in the presence of Conjugate 6,
oseltamivir, baloxavir or PBS control in A549 cells to evaluate the potential for development of drug
resistant mutant viral strains under selective pressure with viral inhibitors. This study was performed
as described in Example 147.

(69) FIG. 68 is a graph showing serial passage of A/WSN/1933 in the presence of Conjugate 6,
Conjugate 33, oseltamivir, baloxavir or PBS control in MDCK cells to evaluate the potential for
development of drug resistant mutant viral strains under selective pressure with viral inhibitors. This
study was performed as described in Example 147.

(70) FIG. 69 is a graph showing an MOI-dependent increase in ADCC by conjugate 6 against

influenza A H1N1. This study was performed as described in Example 152.

(71) FIG. 70 is a graph showing a dose-dependent increase in ADCC by conjugate 6 against influenza
A/PR/8/1934 (H1N1) at an MOI of 1. This study was performed as described in Example 152.

(72) FIGS. 71A-71C are graphs showing an MOI-dependent increase in conjugate 33 against
influenza A/PR/8/1934 (H1N1, FIG. 71A), influenza A/CA/07/2009 (H1N1)pdm (FIG. 71B) and
influenza A/HK/1/1968 (H3N2, FIG. 71C). This study was performed as described in Example 152.

(73) FIG. 72 is a graph showing an MOI-dependent increase in ADCC by conjugate 33 against

influenza B/Malaysia/2506/2004 (Victoria). This study was performed as described in Example 152.

(74) FIGS. 73A-73B are graphs showing a dose-dependent increase in ADCC by conjugate 33 against
influenza A/PR/8/1934 at an MOI of 1 (FIG. 73A) and an MOI of 10 (FIG. 73B). This study was
performed as described in Example 152.

(75) FIG. 74 is a graph showing an MOI-dependent increase in ADCC by a control full-length

monoclonal antibody, Gedivumab (Genentech), against influenza A/PR/8/1934 (H1N1). This study was
performed as described in Example 152.

(76) FIGS. 75A-75B are graphs showing a dose-dependent increase in ADCC by a control full-length
monoclonal antibody, Gedivumab (Genentech), against influenza A/PR/8/1934 at an MOI of 1 (FIG.
75A) and an MOI of 10 (FIG. 75B). This study was performed as described in Example 152.
(77) FIG. 76 is a graph showing an MOI-dependent increase in ADCP by conjugate 6 against influenza
A H1N1. This study was performed as described in Example 153.

(78) FIGS. 77A-77B are graphs showing a dose-dependent increase in ADCP by conjugate 6 against
influenza A/PR/8/1934 (H1N1) at an MOI of 1 (FIG. 77A) and an MOI of 10 (FIG. 77B). This study was
performed as described in Example 153.

(79) FIGS. 78A-78C are graphs showing an MOI-dependent increase in conjugate 33 against
influenza A/PR/8/1934 (H1N1, FIG. 78A), influenza A/CA/07/2009 (H1N1)pdm (FIG. 78B) and
influenza A/HK/1/1968 (H3N2, FIG. 78C). This study was performed as described in Example 153.

(80) FIG. 79 is a graph showing an MOI-dependent increase in ADCP by conjugate 33 against

influenza B/Malaysia/2506/2004 (Victoria). This study was performed as described in Example 153.

(81) FIGS. 80A-80B are graphs showing a dose-dependent increase in ADCP by conjugate 33 against
influenza A/PR/8/1934 at an MOI of 1 (FIG. 80A) and an MOI of 10 (FIG. 80B). This study was
performed as described in Example 153.

(82) FIG. 81 is a graph showing an MOI-dependent increase in ADCP by a control full-length

monoclonal antibody, Gedivumab (Genentech), against influenza A/PR/8/1934 (H1N1). This study was
performed as described in Example 153.

(83) FIGS. 82A-82B are graphs showing a dose-dependent increase in ADCP by a control full-length
monoclonal antibody, Gedivumab (Genentech), against influenza A/PR/8/1934 at an MOI of 1 (FIG.
82A) and an MOI of 10 (FIG. 82B). This study was performed as described in Example 153.

(84) FIG. 83 is a graph showing serial passage of A/Ca/07/2009 (H1N1)pdm in the presence of
Conjugate 45b, oseltamivir, baloxavir, or PBS control in MDCK cells to evaluate the potential for
development of drug resistant mutant viral strains under selective pressure with viral inhibitors. This
study was performed as described in Example 165.

(85) FIG. 84 is a graph showing plasma levels of conjugate 45b in a mouse PK study comparing IV,
SC, and IM administration. Plasma concentration levels were determined by NA capture. This study
was performed as described in Example 173.

(86) FIG. 85 is a graph showing plasma levels of conjugate 45b in a mouse PK study comparing IV,
SV, and IM administration. Plasma concentration levels were determined by hIgG Fc capture. This
study was performed as described in Example 173.

(87) FIG. 86 is a graph showing dose linearity in plasma levels of conjugate 45b by NA capture. This
study was performed as described in Example 174.

(88) FIG. 87 is a graph showing dose linearity in plasma levels of conjugate 45b by hIgG capture. This
study was performed as described in Example 174.

(89) FIG. 88 is a graph showing the plasma concentration of conjugate 45b at 50 mg/kg and 5 mg/kg
SC dosing in a rat PK study. Plasma concentrations were determined by ELISA using a neuraminidase
coated plate. This study was performed as described in Example 175.

(90) FIG. 89 is a graph showing the plasma concentrations of conjugate 45b comparing IV and SV
administration of conjugate 45b at 5 mg/kg. Plasma concentrations were determined by ELISA using a
neuraminidase coated place. This study was performed as described in Example 175.

(91) FIG. 90 is a graph showing the plasma concentrations of conjugate 45b in a non-human primate,
cynomolgus monkeys. Plasma concentrations were determined by ELISA using a neuraminidase
coated plate. This study was performed as described in Example 176.
(92) FIG. 91 is a graph showing the plasma concentrations of conjugate 45b in non-human primate,
cynomolgus monkeys. Plasma concentrations were determined by hIgG Fc capture. This study was
performed as described in Example 176.

(93) FIG. 92 is a graph showing the plasma concentrations of conjugate 45b from day 1 to day 14 in
non-human primate, cynomolgus monkeys. Plasma concentrations were determined by
neuraminidase (NA) capture and Fc capture. This study was performed as described in Example 176.

(94) FIG. 93 is a graph showing the plasma levels of conjugate 45b (3 mpk IV) in ferret PK studies
determined by neuraminidase (NA) capture and Fc capture. This study was performed as described in
Example 183.

(95) FIG. 94 is a graph showing nasal wash levels of conjugate 45b in ferret PK studies determined by
H1N1 neuraminidase (NA) capture. This study was performed as described in Example 186.

(96) FIG. 95 is a graph showing nasal wash levels of conjugate 45b in ferret PK studies determined by
hIgG Fc capture. This study was performed as described in Example 186.

(97) FIG. 96 is a graph showing the comparison of clinical observations following administration of
conjugate 45b (0.3 mg/kg, single) as compared to oseltamivir (10 mg/kg, bid×5) or vehicle. This study
was performed as described in Example 182.

(98) FIG. 97 is a graph showing plasma levels of conjugate 45b (2 mpk IV) compared to conjugate 46
(2 mpk IV) in non-human primate PK studies determined by Fc capture. This study was performed as
described in Example 189.

(99) FIG. 98 is a graph showing plasma concentration levels of conjugate 45b compared to epithelial
lining fluid (ELF) levels of conjugate 45b in mice. This study was performed as described in Example
191.

(100) FIG. 99 is a graph showing the plasma levels of conjugate 45b in SCID mouse PK studies
determined by H1N1 Fc capture. This study was performed as described in Example 192.

(101) FIG. 100 is a graph showing plasma levels of conjugate 45b in SCID mouse PK studies
determined by neuraminidase (NA) capture. This study was performed as described in Example 192.

(102) FIG. 101 is a graph showing the plasma levels of conjugate 45b compared to conjugate 46 in
mouse PK studies determined by neuraminidase (NA) capture. This study was performed as
described in Example 193.

(103) FIG. 102 is an image depicting conjugate 45 and conjugate 46.

(104) FIG. 103 is an image depicting an exemplary click chemistry conjugation of an azido-
functionalized Fc domain monomer or Fc domain with a pre-conjugation intermediate (Int).

(105) FIG. 104 is an image depicting the experimental procedure for secondary bacterial infection
model with methicillin-resistant Staphylococcus aureus (MRSA) as described in Example 200.

(106) FIGS. 105A-105B are graphs showing the percent survival (FIG. 105A) and the CFU/g lung
(FIG. 105B) of secondary infection mouse model after treatment with conjugate 45b. This study was
performed as described in Example 200.

(107) FIG. 106 is an image depicting the experimental procedure for secondary bacterial infection
model with Streptococcus pneumoniae as described in Example 200.

(108) FIG. 107 is a graph depicting the percent survival of mice in a secondary infection model of S.
pneumoniae after treatment with conjugate 45b. This study was performed as described in Example
200.
(109) FIG. 108A-108B are graphs showing the survival of mice (FIG. 108A) and mean body weight
(FIG. 108B) following treatment with conjugate 45a on a single occasion, 7 days before virus
challenge, with influenza A/Vietnam/1203/2004 (H5N1). Following challenge infection and treatment
with conjugate 45a, 100% protection was observed for the 10 mg/kg dose, 80% protection for the 0.3
and 3.0 mg/kg doses, and 70% protection for the 1 mg/kg dose (FIG. 108A). 30% survival in the
placebo group is unusual, so the 1 mg/kg dose did not provide significant protection. In addition, all
doses provided significant protection from weight loss (FIG. 108B), (*P<0.05, **P<0.01, ****P<0.0001).
This study was performed as described in Example 201.

(110) FIG. 109A-109B are graphs showing the survival of mice (FIG. 109A) and mean body weight
(FIG. 109B) following treatment with conjugate 45a on a single occasion, 4 hours post-virus challenge
with influenza A/Vietnam/1203/2004 (H5N1). Following challenge infection and treatment with
Conjugate 45a, 100% protection was observed for the 1, 3, and 10 mg/kg doses, and 70% protection
for the 0.3 mg/kg dose (FIG. 109A). 30% survival in the placebo group is unusual, so the 0.3 mg/kg
dose did not provide significant protection. In addition, all doses provided significant protection from
weight loss (FIG. 109B), (*P<0.05, **P<0.01, ****P<0.0001). This study was performed as described in
Example 201.

(111) FIG. 110A is an image depicting a crystal structure presentation of tetrameric zanamivir (shown
in ball and stick model) complexed to A/California/04/2009 H1N1 neuraminidase (NA) (PDB code
3T15). The linear distance between adjacent NA active sites within the tetramer are shown. The C7
position on zanamivir that was chosen for attachment to the Fc carrier (via an NHS ester) is
highlighted. C7 on zanamivir is solvent exposed and unencumbered sterically, allowing for conjugation
to C7 with minimal impact on binding affinity. This study was performed as described in Example 203.

(112) FIG. 110B is an image of a semi-transparent molecular surface representation of the structure of
full length hIgG1 (PDB code 1 HZH) redacted to show the only the Fc construct boundaries used in
conjugate 45b. Positions of lysines that were preferentially conjugated in conjugate 45b are shown in
green. Conjugate 45b is a multivalent conjugate of zanamivir dimers (Int-83, structure shown on right)
stably conjugated to lysine on the N-terminal extended hIgG1 Fc with an average drug-antibody ratio
(DAR) of 4.5:1. The spatial disposition of conjugated lysine of the Fc domain surface and the length
and flexibility of the PEG linkers result in Int.83 dimer constellations on conjugate 45b where sufficient
separation (>90 A) exists between zanamivir dimers to allow simultaneous engagement of multiple
active-sites within an NA tetramer, between neighboring NA tetramers on the same virus particle, or
between NA tetramers on different virus particles by a single conjugate 45b molecule. This study was
performed as described in Example 203.

(113) FIGS. 111A-111J are graphs showing conjugate 45b binding to Fcγ receptors and triggering
ADCC. FIGS. 111A-111D show conjugate 45b binds to human Fcγ RI (FIG. 111A), RIIa (FIG. 111B),
RIIb (FIG. 111C), and RIIIa (FIG. 111D), as determined by ELISA. FIGS. 111E-111H show conjugate
45b binds to murine Fcγ RI (FIG. 111E), RIII (FIG. 111F), RIIb (FIG. 111G), and RIV (FIG. 111H), as
determined by ELISA. FIGS. 111I-111J show conjugate 45b induces antibody-dependent cellular
toxicity in dose—(FIG. 111I) and MOI—(FIG. 111J) dependency, using reporter cells. This study was
performed as described in Example 203.

(114) FIGS. 112A-112M are graphs showing the efficacy of conjugate 45b against lethal challenge with
influenza in Balb/C mice. FIG. 112A is a graph showing dose-response of conjugate 45b ranging from
0.01-1 mg/kg dosed SC at 2 h post-infection against lethal challenge with influenza A/PR/8/1934
(H1N1) in survival. FIGS. 112B-112H are graphs showing the efficacy of minimal protective dose of
conjugate 45b administered SC against influenza A/CA/07/2009 (H1N1)pdm (FIG. 112B),
A/WSN/1933 (H1N1) (FIG. 112C), A/CA/12/2012 (H1N1)pdm09 (FIG. 112D), A/Texas/23/2012
(H1N1)pdm09 H275Y (FIG. 112E), A/Hong Kong/1/1968 (H3N2) (FIG. 112F), B/Florida/4/2006
(Yamagata) (FIG. 112G), and B/Malaysia/2506/2004 (Victoria) (FIG. 112H). FIG. 112I is a graph
showing the efficacy of conjugate 45 against BSL-3 strain A/Vietnam/1203/2005 (H5N1). FIG. 112J is
a bar graph showing viral burden in the lung on day 4 post-infection. FIGS. 112K-112L are graphs
depicting the lung cytokine levels of IL-6 (FIG. 112K) and MCP-1 (FIG. 112L). FIG. 112M is a graph
showing the minimal protective dose of conjugate 45b administered SC against influenza A/PR/8/1934
(H1N1). This study was performed as described in Example 203.

(115) FIGS. 113A-113J are graphs showing the efficacy of conjugate 45b against lethal challenge with
influenza in Balb/C mice by percent body weight change. FIG. 113A is a graph showing dose-
response of conjugate 45b ranging from 0.01-1 mg/kg dosed SC at 2 h post-infection against lethal
challenge with influenza A/PR/8/1934 (H1N1). FIGS. 113B-113H show percent body weight change
following minimal protective dose of conjugate 45b administered SC against influenza A/CA/07/2009
(H1N1)pdm (FIG. 113B), A/WSN/1933 (H1N1) (FIG. 113C), A/CA/12/2012 (H1N1)pdm09 (FIG. 113D),
A/Texas/23/2012 (H1N1)pdm09 H275Y (FIG. 113E), A/Hong Kong/1/1968 (H3N2) (FIG. 113F),
B/Florida/4/2006 (Yamagata) (FIG. 113G), and B/Malaysia/2506/2004 (Victoria) (FIG. 113H).

(116) FIG. 1131 is a graph showing the efficacy of conjugate 45b against BSL-3 strain
A/Vietnam/1203/2005 (H5N1). FIG. 113J is a graph showing the body weight change when the
minimal protective dose of conjugate 45b is administered SC against influenza A/PR/8/1934 (H1N1).
This study was performed as described in Example 203.

(117) FIGS. 114A-114B is a pair of graphs showing the efficacy of oseltamivir at a human equivalent
dose at 5 mg/kg (BID x 5 days) or 10× human equivalent dose at 50 mg/kg (BID x 5 days) in lethal
mouse model of A/PR/8/1934 (H1N1) in survival (FIG. 114A) and body weight change (FIG. 114B).

(118) FIGS. 115A-115C are bar graphs showing lung cytokine levels for NF-α (FIG. 115A), KC (FIG.
115B), MIP-1α (FIG. 115C) on day 4 post-infection with lethal challenge of A/PR/8/1934 (H1N1).

(119) FIGS. 116A-116B are graphs depicting the efficacy of conjugate 45b following a single dose
administered SC, IM, or IV against influenza A/CA/07/2009 (H1N1)pdm in percent survival (FIG. 111A)
and percent body weight change (FIG. 116B).

(120) FIGS. 116C-116D are graphs depicting the plasma level of conjugate 45b following a single dose
administered SC, IM, or IV in mice over 168 h determined by NA capture (FIG. 116C) or Fc capture
(FIG. 116D).

(121) FIGS. 117A-117C are graphs showing comparable FcRn binding pattern as hIgG1 Fc or full-
length IgG1 isotype control to human (FIG. 117A), cynomolgus monkey (FIG. 117B), and mouse (FIG.
117C) with stronger binding at pH 5.8 and reduced binding at pH 7.4.

(122) FIGS. 118A-118F are graphs depicting the long duration of action of conjugate 45b. FIG. 118A is
a graph showing the plasma levels of conjugate 45b dose response from 1-100 mg/kg after SC
administration in mice. FIG. 118B is a graph depicting the levels of conjugate 45b after SC dosing in
plasma, ELF, and lung of mice. FIGS. 118C-F show the protection provided by conjugate 45b dosed
28 days priorto lethal challenge against influenza A/CA/07/2009 (H1N1)pdm (FIG. 118C), A/HK/1/1968
(H3N2) (FIG. 118D), B/Malaysia/2506/2004 (Victoria) (FIG. 118E), and B/Florida/4/2006 (Yamagata)
(FIG. 118F) in percent survival.

(123) FIG. 119 is a graph depicting survival curve of lower conjugate 45a dose groups. This study was
performed as described in Example 209.

(124) FIG. 120 is a graph depicting Kaplan-Meier survival curve of controls and conjugate 45a at 0.3
and 0.03 mg/kg. This study was performed as described in Example 210.

(125) FIG. 121 is a graph depicting Kaplan-Meier survival curve of controls and conjugate 45a (protein
A column purified) and *conjugate 45a (protein A column flow-through) at 0.1 mg/kg. This study was
performed as described in Example 215.

(126) FIG. 122 is a graph depicting Kaplan-Meier survival curve of controls and conjugates 45a and 46
at 0.1 mg/kg. This study was performed as described in Example 216.

DETAILED DESCRIPTION
(127) The disclosure features conjugates, compositions, and methods for the treatment of viral
infections (e.g., influenza viral infections). The conjugates disclosed herein include monomers or
dimers of viral neuraminidase inhibitors (e.g., zanamivir, peramivir, or analogs thereof) conjugated to
Fc monomers, Fc domains, Fc-binding peptides, albumin proteins, or albumin protein-binding
peptides. The neuraminidase inhibitor (e.g., zanamivir, peramivir, or analogs thereof) in the conjugates
targets neuraminidase on the surface of the viral particle. The Fc monomers or Fc domains in the
conjugates bind to FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune
cells, e.g., neutrophils, to activate phagocytosis and effector functions, such as antibody-dependent
cell-mediated cytotoxicity (ADCC), thus leading to the engulfment and destruction of viral particles by
immune cells and further enhancing the antiviral activity of the conjugates. The albumin or albumin-
binding peptide may extend the half-life of the conjugate, for example, by binding of albumin to the
recycling neonatal Fc receptor. Such compositions are useful in methods for the inhibition of viral
growth and in methods for the treatment of viral infections, such as those caused by an influenza virus
A, influenza virus B and influenza virus C.

(128) I. Viral Infections

(129) The compounds and pharmaceutical compositions described herein (e.g., a conjugate of any
one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) can be used to treat a viral infection
(e.g., an influenza viral infection, such as influenza A, B, C, or parainfluenza).

(130) Viral infection refers to the pathogenic growth of a virus (e.g., the influenza virus) in a host
organism (e.g., a human subject). A viral infection can be any situation in which the presence of a viral
population(s) is damaging to a host body. Thus, a subject is suffering from a viral infection when an
excessive amount of a viral population is present in or on the subject's body, or when the presence of
a viral population(s) is damaging the cells or other tissue of the subject.

(131) Influenza, commonly known as “the flu”, is an infectious disease caused by an influenza virus.
Symptoms can be mild to severe. The most common symptoms include: a high fever, runny nose, sore
throat, muscle pains, headache, coughing, and feeling tired. These symptoms typically begin two days
after exposure to the virus and most last less than a week. The cough, however, may last for more
than two weeks. In children, there may be nausea and vomiting, but these are less common in adults.
Complications of influenza may include viral pneumonia, secondary bacterial pneumonia, sinus
infections, and worsening of previous health problems such as asthma or heart failure. Severe
complications may occur in subjects having weakened immune systems, such as the young, the old,
those with illnesses that weaken the immune system, and those undergoing therapy treatment
resulting in a weakening of the immune system.

(132) Subjects infected with influenza are also at increased risk of developing secondary infections
(e.g., secondary bacterial, viral, or fungal infections), in particular, bacterial infections such as
methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, Pseudomonas
aeruginosa, and/or Haemophilus influenzae. Bacterial secondary infections further increase morbidity
and mortality of influenza infection.

(133) Three types of influenza viruses affect human subjects, namely Type A, Type B, and Type C.
Usually, the virus is spread through the air from coughs or sneezes. This is believed to occur mostly
over relatively short distances. It can also be spread by touching surfaces contaminated by the virus
and then touching the mouth or eyes. A person may be infectious to others both before and during the
time they are showing symptoms. The infection may be confirmed by testing the throat, sputum, or
nose for the virus. A number of rapid tests are available; however, people may still have the infection if
the results are negative. A type of polymerase chain reaction that detects the virus's RNA may be
used to diagnose influenza infection.

(134) II. Conjugates of the Disclosure

(135) Provided herein are synthetic conjugates useful in the treatment of viral infections (e.g.,
influenza infections). The conjugates disclosed herein include an Fc domain or an albumin protein
conjugated to one or more monomers neuraminidase inhibitors or one or more dimers of two
neuraminidase inhibitors (e.g., neuraminidase inhibitors selected from zanamivir, sulfozanamivir,
peramivir, A-315675, or analogs thereof). The dimers of two neuraminidase inhibitors include a
neuraminidase inhibitor (e.g., a first neuraminidase inhibitor of formula (A-I), (A-II), (A-III), (A-IV), (A-V),
(A-VI), (A-VII), (A-VIII), (A-IX), (A-X), (A-XI), (A-XII), or (A-XIII)) and a second neuraminidase inhibitor
(e.g., a second neuraminidase inhibitor of formula (A-I), (A-II), (A-III), (A-IV), (A-V), (A-VI), (A-VII), (A-
VIII), (A-IX), (A-X), (A-XI), (A-XII), or (A-XIII)). The first and second neuraminidase inhibitors are linked
to each other by way of a linker. Without being bound by theory, in some aspects, conjugates
described herein bind to the surface of a viral particle (e.g., bind to viral neuraminidase enzyme on the
surface on an influenza viral particle) through the interactions between the neuraminidase inhibitor
moieties in the conjugates and proteins on the surface of the viral particle. The neuraminidase inhibitor
disrupts neuraminidase, an envelope glycoprotein that cleaves sialic acids, i.e., terminal neuraminic
acid residues, from glycan structures on the surface of infected host cells, releasing progeny viruses
and allowing the spread of the virus from the host cell to uninfected surrounding cells.

(136) Conjugates of the invention include neuraminidase inhibitor monomers and dimers conjugated to
an Fc domain, Fc monomer, or Fc-binding peptide. The Fc domain in the conjugates described herein
binds to the FcγRs (e.g., FcRn, FcγRI, FcγRIIa, FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells. The
binding of the Fc domain in the conjugates described herein to the FcγRs on immune cells activates
phagocytosis and effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC),
thus leading to the engulfment and destruction of viral particles by immune cells and further enhancing
the antiviral activity of the conjugates.

(137) Conjugates of the invention include neuraminidase inhibitor monomers and dimers conjugated to
an albumin protein or an albumin protein-binding peptide. The albumin protein or albumin protein-
binding peptide may extend the half-life of the conjugate, for example, by binding of albumin to the
recycling neonatal Fc receptor.

(138) Conjugates provided herein are described by any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-
I)-(M-XI), or (M′-I). In some embodiments, the conjugates described herein include one or more
monomers of neuraminidase inhibitors conjugated to an Fc domain or an albumin protein. In some
embodiments, the conjugates described herein include one or more dimers of neuraminidase inhibitors
conjugated to an Fc domain or an albumin protein. In some embodiments, when n is 2, E (an Fc
domain monomer) dimerizes to form an Fc domain.

(139) Conjugates described herein may be synthesized using available chemical synthesis techniques
in the art. In cases where a functional group is not available for conjugation, a molecule may be
derivatized using conventional chemical synthesis techniques that are well known in the art. In some
embodiments, the conjugates described herein contain one or more chiral centers. The conjugates
include each of the isolated stereoisomeric forms as well as mixtures of stereoisomers in varying
degrees of chiral purity, including racemic mixtures. It also encompasses the various diastereomers,
enantiomers, and tautomers that can be formed.

(140) Neuraminidase inhibitors

(141) A component of the conjugates described herein is an influenza virus neuraminidase inhibitor
moiety. An influenza virus neuraminidase inhibitor disrupts neuraminidase, an envelope glycoprotein
that cleaves sialic acids, i.e., terminal neuraminic acid residues, from glycan structures on the surface
of infected host cells, releasing progeny viruses and allowing the spread of the virus from the host cell
to uninfected surrounding cells. Examples of an influenza virus neuraminidase inhibitor include
zanamivir (Relenza), sulfozanamivir, A-315675 and peramivir. In addition, derivatives of zanamivir,
sulfozanamivir, A-315675 and peramivir, such as those found in the literature, have neuraminidase
inhibitor activity and are useful as neuraminidase inhibitor moieties of the compounds herein (see, for
example, Hadházi et al. A sulfozanamivir analogue has potent anti-influenza virus activity.
ChemMedChem Comm. 13:785-789 (2018) and In vitro characterization of A-315675, a highly potent
inhibitor of A and B strain of influenza virus neuraminidases and influenza virus replication.
Antimicrobial Agents and Chemotherapy 46(4):1014-1021 (2002)).
(142) Conjugates described herein are separated into two types: (1) one or more dimers of
neuraminidase inhibitors conjugated to an Fc domain or an albumin protein and (2) one or more
monomers of neuraminidase inhibitors conjugated to an Fc domain or an albumin protein. The dimers
of neuraminidase inhibitors are linked to each other by way of a linker, such as the linkers described
herein.

(143) Viral neuraminidase inhibitors of the invention include zanamivir, sulfozanamivir, A-315675,
peramivir, and analogs thereof, such as the viral neuraminidase inhibitors of formulas (A-I)-(A-XIII):

(144) ##STR00436## ##STR00437## ##STR00438##

wherein R.sub.1 is selected from —OH, —NH.sub.2, —NHC(═NH)NH.sub.2, and —

NHC(═NH)NHR.sub.6; R.sub.2 and R.sub.3 are each independently selected from —H, —OH, —F, —
Cl, and —Br; R.sub.4 is selected from —CO.sub.2H, —P(═O)(OH).sub.2, —SO.sub.3H; Rs is
selected from —COCH.sub.3, —COCF.sub.3, —SO.sub.2CH.sub.3; X is selected from —O— and —S
—; Y is selected from

(145) ##STR00439##

(146) R.sub.6 is selected from

(147) ##STR00440## ##STR00441## ##STR00442##

(148) R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15
aryl, and C2-C15 heteroaryl; R.sub.8 is selected from C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-
C15 heteroaryl; R.sub.9 is selected from —H, a halogen (e.g., C1, F, or Br), —OR.sub.10, —
NHC(═O)R.sub.7, optionally substituted C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl;
C5-C15 aryl, and C2-C15 heteroaryl; and R.sub.10 is selected from C1-C20 alkyl, C3-C20 cycloalkyl,
C3-C20 heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl. Most preferably the viral
neuraminidase inhibitor of formula (A-I), (A-II), (A-III), (A-IV), (A-V), (A-VI), (A-VII), (A-VIII), (A-IX), (A-
X), (A-XI), (A-XII), or (A-XIII) is covalently attached to the conjugate through Y.

(149) Preferably the viral neuraminidase inhibitor is selected from zanamivir, sulfozanamivir, peramivir,
or A-315675:

(150) ##STR00443##

Conjugates of Dimers of Neuraminidase Inhibitors Linked to an Fc Domain or an Albumin Protein

(151) The conjugates described herein include an Fc domain, and Fc monomer, an Fc-binding
peptide, and albumin protein, or an albumin protein-binding peptide covalently linked to one or more
dimers of neuraminidase inhibitors. The dimers of two neuraminidase inhibitors include a first
neuraminidase inhibitor (e.g., a first viral neuraminidase inhibitor of formulas (A-I)-(A-XIII)) and a
second neuraminidase inhibitor (e.g., a second viral neuraminidase inhibitor of formulas (A-I)-(A-XIII)).
The first and second neuraminidase inhibitors are linked to each other by way of a linker, such as a
linker described herein. In some embodiments of the dimers of neuraminidase inhibitors, the first and
second neuraminidase inhibitors are the same. In some embodiments, the first and second
neuraminidase inhibitors are different.

(152) Dimers of neuraminidase inhibitors include homo-dimers of zanamivir or analogs thereof (e.g.,
(A-I), (A-II), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X), or (A-XIII). For example, neuraminidase inhibitor
dimers of the invention include dimers having the structure A.sub.1-L-A.sub.2, wherein each A.sub.1
and each A.sub.2 is selected from (A-I), (A-II), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X), and (A-XIII).

(153) Dimers of neuraminidase inhibitors include homo-dimers of peramivir or analogs thereof (e.g.,
(A-III), (A-IV), or (A-V)). For example, neuraminidase inhibitor dimers of the invention include dimers
having the structure A.sub.1-L-A.sub.2, wherein each A.sub.1 and each A.sub.2 is selected from (A-
III), (A-IV), and (A-V).
(154) Dimers of neuraminidase inhibitors include hetero-dimers including zanamivir or analogs thereof
and peramivir of analogs thereof (e.g., (A-I)-(A-XIII)). For example, neuraminidase inhibitor dimers of
the invention include dimers having the structure A.sub.1-L-A.sub.2, wherein each A.sub.1 is selected
from (A-I), (A-II), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X), and (A-XIII), and each A.sub.2 is selected from
(A-III), (A-IV), and (A-V).

(155) In some embodiments, when T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20), each A.sub.1-L-A.sub.2 may be independently selected (e.g.,
independently selected from any of the A.sub.1-L-A.sub.2 structures described herein). In some
embodiments, E may be conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different A.sub.1-L-A.sub.2
moieties. In some embodiments, E is conjugated to a first A.sub.1-L-A.sub.2 moiety, and a second
A.sub.1-L-A.sub.2, moiety. In some embodiments, A.sub.1 and A.sub.2 of the first A.sub.1-L-A.sub.2
moiety are independently selected from any one of formulas (A-III)-(A-V):

(156) ##STR00444##

and A.sub.1 and A.sub.2 of the second A.sub.1-L-A.sub.2 moiety are independently selected from any
one of formulas (A-I), (A-II), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X), or (A-XIII):

(157) ##STR00445## ##STR00446##

(158) In some embodiments, the first A.sub.1-L-A.sub.2 moiety is conjugated specifically to lysine
residues of E (e.g., the nitrogen atoms of surface exposed lysine residues of E), and the second
A.sub.1-L-A.sub.2 moiety is conjugated specifically to cysteine residues of E (e.g., the sulfur atoms of
surface exposed cysteine residues of E). In some embodiments, the first A.sub.1-L-A.sub.2 moiety is
conjugated specifically to cysteine residues of E (e.g., the sulfur atoms of surface exposed cysteine
residues of E), and the second A.sub.1-L-A.sub.2 moiety is conjugated specifically to lysine residues
of E (e.g., the nitrogen atoms of surface exposed lysine residues of E).

(159) In some embodiments, the disclosure provides a conjugate, or a pharmaceutically acceptable

salt thereof, described by the formulae below:

(160) ##STR00447## ##STR00448## ##STR00449## ##STR00450## ##STR00451##

##STR00452## ##STR00453## ##STR00454## ##STR00455## ##STR00456## ##STR00457##
##STR00458## ##STR00459## ##STR00460## ##STR00461## ##STR00462## ##STR00463##
##STR00464## ##STR00465##

or a pharmaceutically acceptable salt thereof.

(161) In the conjugates described herein, the squiggly line connected to E indicates that one or more
(e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) dimers of neuraminidase
inhibitors may be attached to an Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein,
or albumin protein-binding peptide. In some embodiments, when n is 1, one or more (e.g., 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10) dimers of neuraminidase inhibitors may be attached to an Fc domain monomer, Fc
domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide. In some
embodiments, when n is 2, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20) dimers of neuraminidase inhibitors may be attached to an Fc domain. The squiggly line in
the conjugates described herein is not to be construed as a single bond between one or more dimers
of neuraminidase inhibitors and an atom in the Fc domain or albumin protein. In some embodiments,
when T is 1, one dimer of neuraminidase inhibitors may be attached to an atom in the Fc domain
monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide. In some
embodiments, when T is 2, two dimers of neuraminidase inhibitors may be attached to an atom in the
Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding
peptide.

(162) As described further herein, a linker in a conjugate described herein (e.g., L or L′) may be a
branched structure. As described further herein, a linker in a conjugate described herein (e.g., L or L′)
may be a multivalent structure, e.g., a divalent or trivalent structure having two or three arms,
respectively. In some embodiments when the linker has three arms, two of the arms may be attached
to the first and second neuraminidase inhibitors and the third arm may be attached to the Fc domain
monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide.

(163) In conjugates having an Fc domain covalently linked to one or more dimers of neuraminidase
inhibitors, as represented by the formulae above, when n is 2, two Fc domain monomers (each Fc
domain monomer is represented by E) dimerize to form an Fc domain.

(164) Conjugates of Monomers of Neuraminidase Inhibitors Linked to an Fc Domain or an Albumin

Protein

(165) In some embodiments, the conjugates described herein include an Fc domain monomer, Fc
domain, Fc-binding peptide, albumin protein, or albumin protein-binding peptide covalently linked to
one or more monomers of neuraminidase inhibitors. Conjugates of an Fc domain monomer or albumin
protein and one or more monomers of neuraminidase inhibitors may be formed by linking the Fc
domain or albumin protein to each of the monomers of neuraminidase inhibitors through a linker, such
as any of the linkers described herein.

(166) In the conjugates having an Fc domain or albumin protein covalently linked to one or more
monomers of neuraminidase inhibitors described herein, the squiggly line connected to E indicates
that one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) monomers
of neuraminidase inhibitors may be attached to an Fc domain monomer, Fc domain, Fc-binding
peptide, albumin protein, or albumin protein-binding peptide. In some embodiments, when n is 1, one
or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) monomers of neuraminidase inhibitors may be attached to
an Fc domain monomer or an albumin protein. In some embodiments, when n is 2, one or more (e.g.,
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) monomers of neuraminidase
inhibitors may be attached to an Fc domain. The squiggly line in the conjugates described herein is not
to be construed as a single bond between one or more monomers of neuraminidase inhibitors and an
atom in the Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or albumin protein-
binding peptide. In some embodiments, when T is 1, one monomer of neuraminidase inhibitor may be
attached to an atom in the Fc domain monomer, Fc domain, Fc-binding peptide, albumin protein, or
albumin protein-binding peptide. In some embodiments, when T is 2, two monomers of neuraminidase
inhibitors may be attached to an atom in the Fc domain monomer, Fc domain, Fc-binding peptide,
albumin protein, or albumin protein-binding peptide.

(167) In some embodiments, when T is greater than 1 (e.g., T is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20), each A.sub.1-L may be independently selected (e.g., independently
selected from any of the A.sub.1-L structures described herein). In some embodiments, E may be
conjugated to 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different A.sub.1-L moieties. In some embodiments, E is
conjugated to a first A.sub.1-L moiety, and a second A.sub.1-L, moiety. In some embodiments, A.sub.1
of the first A.sub.1-L moiety is selected from any one of formulas (A-III)-(A-V):

(168) ##STR00466##

and A.sub.1 of the second A.sub.1-L moiety is selected from any one of formulas (A-I), (A-II), (A-VI),
(A-VII), (A-VIII), (A-IX), (A-X), or (A-XIII):

(169) ##STR00467## ##STR00468##

(170) In some embodiments, the first A.sub.1-L moiety is conjugated specifically to lysine residues of
E (e.g., the nitrogen atoms of surface exposed lysine residues of E), and the second A.sub.1-L moiety
is conjugated specifically to cysteine residues of E (e.g., the sulfur atoms of surface exposed cysteine
residues of E). In some embodiments, the first A.sub.1-L moiety is conjugated specifically to cysteine
residues of E (e.g., the sulfur atoms of surface exposed cysteine residues of E), and the second
A.sub.1-L moiety is conjugated specifically to lysine residues of E (e.g., the nitrogen atoms of surface
exposed lysine residues of E).
(171) As described further herein, a linker in a conjugate having an Fc domain monomer, Fc domain,
Fc-binding peptide, albumin protein, or albumin protein-binding peptide covalently linked to one or
more monomers of neuraminidase inhibitors described herein (e.g., L or L′) may be a divalent structure
having two arms. One arm in a divalent linker may be attached to the monomer of neuraminidase
inhibitor and the other arm may be attached to the Fc domain monomer, Fc domain, Fc-binding
peptide, albumin protein, or albumin protein-binding peptide.

(172) In some embodiments, a conjugate containing an Fc domain monomer, Fc domain, Fc-binding

peptide, albumin protein, or albumin protein-binding peptide covalently linked to one or more
monomers of neuraminidase inhibitors provided herein is described by any one of formulae below:

(173) ##STR00469## ##STR00470## ##STR00471## ##STR00472## ##STR00473##

##STR00474## ##STR00475## ##STR00476## ##STR00477## ##STR00478## ##STR00479##
##STR00480## ##STR00481## ##STR00482## ##STR00483## ##STR00484## ##STR00485##
##STR00486## ##STR00487## ##STR00488## ##STR00489##

or a pharmaceutically acceptable salt thereof.

(174) In conjugates having an Fc domain covalently linked to one or more monomers of

neuraminidase inhibitors, as represented by the formulae above, when n is 2, two Fc domain
monomers (each Fc domain monomer is represented by E) dimerize to form an Fc domain.

(175) Regioisomers of Conjugates Including Zanamivir or Analogs Thereof

(176) Conjugates (e.g., monomer or dimer conjugates as described in detail herein) may be produced
as a mixture or regioisomers. A particular regioisomer or mixture of regioisomers may be preferred for
reasons such as ease of synthesis, thermostability, oxidative stability, pharmacokinetics (e.g.,
metabolic stability or bioavailability), effector binding, or therapeutic efficacy.

(177) In some embodiments, a conjugate of the invention includes zanamivir or an analog thereof
(e.g., any of (A-I), (A-II), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X), or (A-XIII)). Zanamivir or an analog
thereof may be conjugated to an Fc domain or an albumin protein (e.g., by way of a linker) through, for
example, the C7 position (see, e.g., (A-I), (A-II), (A-X), or (A-XIII)) or through the C9 position (see, e.g.,
(A-VI) or (A-VII)):

(178) ##STR00490## ##STR00491##

(179) The present disclosure includes a population of monomeric conjugates (e.g., a population of
conjugates of formula (M-I)) wherein the population of conjugates includes any of the monomeric
conjugates described herein and one or more of its corresponding regioisomers. For example, a
population of conjugates may include (1) zanamivir or an analog thereof conjugated (e.g., by way of a
linker) at the C7 position to an Fc domain or an albumin protein, and (2) zanamivir or an analog
thereof conjugated (e.g., by way of a linker) at the C9 position to an Fc domain or an albumin protein.
The population of monomeric conjugates may have a specified ratio of C7-linked conjugate to C9-
linked conjugate. For example, the population of conjugates may have substantially 100% C-7 linked
conjugate and substantially 0% C-9 linked conjugate. The population of conjugates may have about
95% C-7 linked conjugate and about 5% C-9 linked conjugate. The population of conjugates may have
about 90% C-7 linked conjugate and about 10% C-9 linked conjugate. The population of conjugates
may have about 85% C-7 linked conjugate and about 15% C-9 linked conjugate. The population of
conjugates may have about 80% C-7 linked conjugate and about 20% C-9 linked conjugate. The
population of conjugates may have about 75% C-7 linked conjugate and about 25% C-9 linked
conjugate. The population of conjugates may have about 70% C-7 linked conjugate and about 30% C-
9 linked conjugate. The population of conjugates may have about 65% C-7 linked conjugate and about
35% C-9 linked conjugate. The population of conjugates may have about 60% C-7 linked conjugate
and about 40% C-9 linked conjugate. The population of conjugates may have about 55% C-7 linked
conjugate and about 45% C-9 linked conjugate. The population of conjugates may have about 50% C-
7 linked conjugate and about 50% C-9 linked conjugate. The population of conjugates may have about
45% C-7 linked conjugate and about 55% C-9 linked conjugate. The population of conjugates may
have about 40% C-7 linked conjugate and about 60% C-9 linked conjugate. The population of
conjugates may have about 35% C-7 linked conjugate and about 65% C-9 linked conjugate. The
population of conjugates may have about 30% C-7 linked conjugate and about 70% C-9 linked
conjugate. The population of conjugates may have about 25% C-7 linked conjugate and about 75% C-
9 linked conjugate. The population of conjugates may have about 20% C-7 linked conjugate and about
80% C-9 linked conjugate. The population of conjugates may have about 15% C-7 linked conjugate
and about 85% C-9 linked conjugate. The population of conjugates may have about 10% C-7 linked
conjugate and about 90% C-9 linked conjugate. The population of conjugates may have substantially
0% C-7 linked conjugate and substantially 100% C-9 linked conjugate. The population of conjugates
may have greater than 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70%, 60%, 65%, 60%,
55%, or 50% C7-linked conjugate. The population of conjugates may have less than 50%, 40%, 30%,
25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1% C9-linked conjugate.

(180) The present disclosure also includes a population of dimeric conjugates (e.g., a population of
conjugates of formula (D-I)) wherein the population of conjugates includes any of the dimeric
conjugates described herein and one or more of its corresponding regioisomers. For example, a
population of conjugates may include a (1) a C7-C7 dimer (e.g., both zanamivir or analog thereof
moieties of the dimer are conjugated (e.g., by way of a linker) at their respective C7 positions to an Fc
domain or an albumin protein), (2) a C9-C9 dimer (e.g., both zanamivir or analog thereof moieties of
the dimer are conjugated (e.g., by way of a linker) at their respective C9 positions to an Fc domain or
an albumin protein), and/or (3) a C7-C7 dimer (e.g., one zanamivir or analog thereof moiety is
conjugated (e.g., by way of a linker) to and Fc domain or an albumin protein through its C7 position
and the other zanamivir or analog thereof moiety is conjugated (e.g., by way of a linker) to an Fc
domain or an albumin protein through its C9 position).

(181) The population of dimeric conjugates may have a specified ratio of C7-C7 linked conjugate to
C7-C9 linked conjugate to C9-C9 linked conjugate. For example, the population of conjugates may
have substantially 100% C7-C7 linked conjugate, and substantially 0% C7-C9 or C9-C9 linked
conjugate. The population of conjugates may have substantially 100% C9-C9 linked conjugate, and
substantially 0% C7-C7 or C7-C9 linked conjugate. The population of conjugates may have
substantially 100% C7-C9 linked conjugate, and substantially 0% C7-C7 or C9-C9 linked conjugate.

(182) The population of conjugates may have greater than 99%, 98%, 97%, 96%, 95%, 90%, 85%,
80%, 75%, 70%, 60%, 65%, 60%, 55%, or 50% C7-C7 linked conjugate.

(183) The population of conjugates may have less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%,
4%, 3%, 2%, or 1% C9-C9 linked conjugate.

(184) The population of conjugates may have less than 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%,
4%, 3%, 2%, or 1% C7-C9 linked conjugate.

(185) For any of the above-described populations of regioisomers, A.sub.1 and/or A.sub.2 (e.g., of (M-
I) or (D-I)) may be selected from zanamivir or any of the zanamivir analogs described herein (e.g., any
of (A-I), (A-II), (A-VI), (A-VII), (A-VIII), (A-IX), (A-X), or (A-XIII)). In particular, the C7-linked zanamivir or
analogs thereof is described by (A-I), (A-II), (A-X), and (A-XIII), and C9-linked zanamivir or analogs
thereof is described by (A-VI) or (A-VII). Exemplary methods for preparing regioisomers, e.g., C7, C9,
C7-C7, C7-C9, and C9-C9 linked regioisomers, are described in Examples 100-103, 123 and 124. In
some instances, it may be preferable to have 95% or more, 96% or more, 97% or more, 98% or more,
99% or more, or substantially 100% C7-linked monomer conjugates or C7-C7 linked dimer conjugates.
In these instances, it may be preferable to prepare the intermediate with a method that forms
substantially C7 linked monomer or C7-C7 linked dimer intermediates, such as the methods
described, for example, in Examples 103 and 123. The method of Example 103 is exemplary of
methods used to achieve primarily the C7 or C7-C7 linked intermediate and may be used to prepare
any intermediate described herein. Zanamivir analogs having a modification (e.g., a substituent other
than OH) at position C9 (e.g., zanamivir analogs described by (A-XIII)) may increase the ratio of C7-
linked zanamivir to C9-linked zanamivir by preventing the migration from C7-linked zanamivir to C9-
linked zanamivir. Exemplary C9-modified zanamivir analogs are described herein (see, e.g.,
conjugates described by D-XI or M-XI, for example Conjugate 47 (Int-91) or Conjugate 48 (Int-92).

(186) In preferred embodiments, the conjugate is a conjugate of any one of formulas (D-I)-(D-XI), (D′-
I), (M-I)-(M-XI), or (M′-I), wherein A.sub.1 and/or A.sub.2 are described by formula (A-I), (A-II), (A-X),
or (A-XIII) and Y is

(187) ##STR00492##

wherein R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-
C15 aryl, and C2-C15 heteroaryl. In preferred embodiments, A.sub.1 and/or A.sub.2 are described by
formula (A-I) (e.g., zanamivir). In preferred embodiments, R.sub.7 is C1-C20 alkyl (e.g., —CH.sub.3,
—CH.sub.2CH.sub.3, —CH.sub.2CH.sub.2CH.sub.3). Such conjugates have been shown to exhibit
increased stability of the C7-linkage, resulting in less C7 to C9 migration (see, e.g., conjugates
described by D-II-6 or D-II-7, such as Conjugate 45 (Int-83) or Conjugate 46 (Int-83)). The resulting
product is therefore expected to be more homogenous and exhibit increased efficacy. The preferred
conjugate is more homogenous, has an increased proportion (e.g., substantially pure, such as greater
than 95%, 96%, 97%, 98%, or 99% pure) C7-linked zanamivir, and retains efficacy against influenza.

III. Fc Domain Monomers and Fc Domains

(188) An Fc domain monomer includes a hinge domain, a C.sub.H2 antibody constant domain, and a
C.sub.H3 antibody constant domain. The Fc domain monomer can be of immunoglobulin antibody
isotype IgG, IgE, IgM, IgA, or IgD. The Fc domain monomer can also be of any immunoglobulin
antibody isotype (e.g., IgG1, IgG2a, IgG2b, IgG3, or IgG4). The Fc domain monomer can be of any
immunoglobulin antibody allotype (e.g., IGHG1*01 (i.e., G1m(za)), IGHG1*07 (i.e., G1m(zax)),
IGHG1*04 (i.e., G1m(zav)), IGHG1*03 (G1m(f)), IGHG1*08 (i.e., G1m(fa)), IGHG2*01, IGHG2*06,
IGHG2*02, IGHG3*01, IGHG3*05, IGHG3*10, IGHG3*04, IGHG3*09, IGHG3*11, IGHG3*12,
IGHG3*06, IGHG3*07, IGHG3*08, IGHG3*13, IGHG3*03, IGHG3*14, IGHG3*15, IGHG3*16,
IGHG3*17, IGHG3*18, IGHG3*19, IGHG2*04, IGHG4*01, IGHG4*03, or IGHG4*02) (as described in,
for example, in Vidarsson et al. IgG subclasses and allotypes: from structure to effector function.
Frontiers in Immunology. 5(520):1-17 (2014)). The Fc domain monomer can also be of any species,
e.g., human, murine, or mouse. A dimer of Fc domain monomers is an Fc domain that can bind to an
Fc receptor, which is a receptor located on the surface of leukocytes.

(189) In some embodiments, an Fc domain monomer in the conjugates described herein may contain
one or more amino acid substitutions, additions, and/or deletion relative to an Fc domain monomer
having a sequence of any one of SEQ ID NOs: 1-138. In some embodiments, an Asn in an Fc domain
monomer in the conjugates as described herein may be replaced by Ala in order to prevent N-linked
glycosylation (see, e.g., SEQ ID NOs: 12-15, where Asn to Ala substitution is labeled with *). In some
embodiments, an Fc domain monomer in the conjugates described herein may also containing
additional Cys additions (see, e.g., SEQ ID NOs: 9, 10, and 11, where Cys additions are labeled with
*).

(190) In some embodiments, an Fc domain monomer in the conjugates as described herein includes
an additional moiety, e.g., an albumin-binding peptide, a purification peptide (e.g., a hexa-histidine
peptide (HHHHHH (SEQ ID NO: 146)), or a signal sequence (e.g., IL2 signal sequence
MYRMQLLSCIALSLALVTNS (SEQ ID NO: 147)) attached to the N- or C-terminus of the Fc domain
monomer. In some embodiments, an Fc domain monomer in the conjugate does not contain any type
of antibody variable region, e.g., V.sub.H, V.sub.L, a complementarity determining region (CDR), or a
hypervariable region (HVR).

(191) In some embodiments, an Fc domain monomer in the conjugates as described herein may have
a sequence that is at least 95% identical (e.g., 97%, 99%, or 99.5% identical) to the sequence of any
one of SEQ ID NOs: 1-138 shown below. In some embodiments, an Fc domain monomer in the
conjugates as described herein may have a sequence of any one of SEQ ID NOs: 1-138 shown below.
(192) TABLE-US-00003 SEQ ID NO: 1: murine Fc-IgG2a with IL2 signal sequence
at the N-terminus (bold)
MYRMQLLSCIALSLALVTNSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVD
VS
EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER
TIS
KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY
FM YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 2: mature
murine Fc-IgG2a
PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVH
TA
QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPE
EE
MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSY
SC SVVHEGLHNHHTTKSFSRTPGK SEQ ID NO: 3: human Fc-IgG1 with IL2 signal
sequence at the N-terminus (bold) and N-terminal MVRS amino acid residues
added (underlined)
MYRMQLLSCIALSLALVTNSMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 4: mature
human Fc-IgG1 with N-terminal MVRS amino acid residues added (underlined)
MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FS CSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 5: murine Fc-IgG2a with IL2 signal
sequence (bold) at the N-terminus and hexa-histidine peptide (italicized) at the C-
terminus
MYRMQLLSCIALSLALVTNSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVD
VS
EDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER
TIS
KPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSY
FM YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHH SEQ ID NO: 6:
mature murine Fc-IgG2a with hexa-histidine peptide (italicized) at the C-terminus
PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVH
TA
QTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPE
EE
MTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSY
SC SVVHEGLHNHHTTKSFSRTPGKHHHHHH SEQ ID NO: 7: human Fc-IgG1 with IL2
signal sequence (bold) at the N-terminus, N-terminal MVRS amino acid residues
added (underlined), and hexa-histidine peptide (italicized) at the C-terminus
MYRMQLLSCIALSLALVTNSMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 8:
mature human Fc-IgG1 with hexa-histidine peptide (italicized) at the C-terminus and
N-terminal MVRS amino acid residues added (underlined)
MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FS CSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 9: human Fc-IgG1 with IL2
signal sequence (bold) at the N-terminus, N-terminal MVRS amino acid residues
added (underlined), two additional cysteines in the hinge region (*), and hexa-
histidine peptide (italicized) at the C-terminus
MYRMQLLSCIALSLALVTNSMVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPE
VT
CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK
AL
PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID
NO: 10: mature human Fc-IgG1 with N-terminal MVRS amino acid residues added
(underlined), two additional cysteines in the hinge region (*), and hexa-histidine
peptide (italicized) at the C-terminus
MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
W QQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 11: mature human
Fc-IgG1 with N-terminal MVRS amino acid residues added (underlined) and two
additional cysteines in the hinge region (*)
MVRSDKTHTCPPCPPC*KC*PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW
YV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP
QV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR
W QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 12: murine Fc-IgG2a with
IL2 signal sequence (bold) at the N-terminus, Asn to Ala substitution (*), and hexa-
histidine peptide (italicized) at the C-terminus
MYRMQLLSCIALSLALVTNSPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVD
VS
EDDPDVQISWFVNNVEVHTAQTQTHREDYA*STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIER
TI
SKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGS
YF MYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGKHHHHHH SEQ ID NO: 13:
mature murine Fc-IgG2a with Asn to Ala substitution (*) and hexa-histidine peptide
(italicized) at the C-terminus
PRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVH
TA
QTQTHREDYA*STLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPP
EE
EMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNS
YS CSVVHEGLHNHHTTKSFSRTPGKHHHHHH SEQ ID NO: 14: human Fc-IgG1 with
IL2 signal sequence (bold) at the N-terminus, N-terminal MVRS amino acid
residues added (underlined), Asn to Ala substitution (*), and hexa-histidine peptide
(italicized) at the C-terminus
MYRMQLLSCIALSLALVTNSMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYA*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EK
TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO:
15: mature human Fc-IgG1 with Asn to Ala substitution (*), N-terminal MVRS
amino acid residues added (underlined), and hexa-histidine peptide (italicized) at the
C-terminus
MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE
VH
NAKTKPREEQYA*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
S
REEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
V FSCSVMHEALHNHYTQKSLSLSPGKHHHHHH SEQ ID NO: 16: human IgG1 Fc with
Human Serum Albumin Signal Sequence (bold) at the N-terminus and N-terminal
ISAMVRS amino acid residues added (underlined)
MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 17: human
IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N-terminus, N-
terminal ISAMVRS amino acid residues added (underlined), C-terminal G45 linker
(italicized), and C-terminal c-Myc tag (underlined, italicized)
MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ
ID NO: 18: mature human IgG1 Fc with N-terminal ISAMVRS amino acid
residues added (underlined), C-terminal G45 linker (italicized), and C-terminal c-Myc
tag (underlined, italicized)
ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
N VFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 19: human
IgG1 Fc with Human Serum Albumin Signal Sequence (bold), N-terminal ISAMVRS
amino acid residues added (underlined), and lysine to serine modification (*) to
prevent lysine conjugation at this site
MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS*DTLMISRTPEVTCVV
VD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EK
TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
GS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 20:
mature human IgG1 Fc with N-terminal ISAMVRS amino acid residues added
(underlined) and lysine to serine modification (*) to prevent lysine conjugation at
this site
ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS*DTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD
GV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
LP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
G NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 21: human IgG1 Fc with
Human Serum Albumin Signal Sequence (bold) at the N-terminus, N-terminal
ISAMVRS amino acid residues added (underlined), lysine to serine modification (*)
to prevent lysine conjugation at this site, C-terminal G4S linker (italicized), and C-
terminal C-Myc tag (underlined, italicized)
MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS(*)DTLMISRTPEVTCV
VV
DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA
PI
EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS
D GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ
ID NO: 22: mature human IgG1 Fc with N-terminal ISAMVRS amino acid
residues added (underlined), lysine to serine modification (*) to prevent lysine
conjugation at this site, C- terminal G4S linker (italicized), and C-terminal C-Myc
tag (underlined, italicized)
ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPS(*)DTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DG
VEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
Q GNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 23: human
IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N-terminus, N-
terminal ISAMVRS amino acid residues added (underlined), Asn to Ala substitution
(*), C-terminal G4S linker (italicized), and C-terminal C-myc tag (underlined, italicized)
MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP
IE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
G SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ
ID NO: 24: mature human IgG1 Fc with N-terminal ISAMVRS amino acid
residues added (underlined), Asn to Ala substitution (*), C-terminal G4S linker
(italicized), and C-terminal C-myc tag (underlined, italicized)
ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VE
VHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
Q GNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 25: human
IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N-terminus, N-
terminal ISAMVRS amino acid residues added (underlined), H310A (*) and H435A
(*) mutations to impede FcRn binding, C-terminal G4S (italicized), and C-terminal C-
myc tag (underlined, italicized)
MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLA(*)QDWLNGKEYKCKVSNKALPAP
IE
KTISKA(*)KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD
SD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNAYTQKSLSLSPGGGGGSEQKLISEEDL
SEQ ID NO: 26: mature human IgG1 Fc with Human Serum Albumin Signal
Sequence (bold) at the N- terminus, N-terminal ISAMVRS amino acid residues
added (underlined), with H310A (*) and H435A (*) mutations to impede FcRn
binding, C-terminal G4S (italicized), and C-terminal C-myc tag (underlined, italicized)
ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VE
VHNAKTKPREEQYNSTYRVVSVLTVLA(*)QDWLNGKEYKCKVSNKALPAPIEKTISKA(*)KGQPREPQV
Y
TLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW
Q QGNVFSCSVMHEALHNAYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 27: human
IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N-terminus, N-
terminal ISAMVRS amino acid residues added (underlined), C-terminal G4S linker
(italicized), and C-terminal mutated (lysine to phenylalanine, bold) C-myc tag
(underlined, italicized)
MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KT
ISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG
SF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS SEQ ID NO: 28:
mature human IgG1 Fc with N-terminal ISAMVRS amino acid residues added
(underlined), C-terminal G4S linker (italicized), and C-terminal mutated (lysine to
phenylalanine, bold) C-myc tag (underlined, italicized)
ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VE
VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PP
SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
N VFSCSVMHEALHNHYTQKSLSLSPGGGGGS SEQ ID NO: 29: human IgG1 Fc with
Human Serum Albumin Signal Sequence (bold) at the N-terminus, N-terminal
ISAMVRS amino acid residues added (underlined), Asn to Ala substitution (*), C-
terminal G4S linker (italicized), and C-terminal mutated (lysine to phenylalanine, bold)
C-myc tag (underlined, italicized)
MKWVTFISLLFLFSSAYSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP
IE
KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD
G SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQFLISEEDL SEQ
ID NO: 30: mature human IgG1 Fc with N-terminal MVRS amino acid residues
added (underlined), Asn to Ala substitution (*), C-terminal G4S linker (italicized),
and C-terminal mutated (lysine to phenylalanine, bold) C-myc tag (underlined, italicized)
ISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG
VE
VHNAKTKPREEQYA(*)STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVY
TL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ
Q GNVFSCSVMHEALHNHYTQKSLSLSPGGGGGS SEQ ID NO: 31: human IgG1 Fc
with Human Serum Albumin Signal Sequence (bold) at the N-terminus, allotype
G1m(fa) (bold italics), C-terminal G4S linker (italicized), and C-terminal mutated
(lysine to phenylalanine, bold) C-myc tag (underlined)
MKWVTFISLLFLFSSAYSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
E
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
G QPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQFLISEEDL SEQ ID NO: 32:
human IgG1 Fc with Human Serum Albumin Signal Sequence (bold) at the N-
terminus, allotype G1m(fa) (bold italics)
MKWVTFISLLFLFSSAYSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
E
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
G QPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 33: mature human
IgG1 Fc with a YTE triple mutation (bold and underlined) with N-terminal MVRS
amino acid residues added (underlined)
MVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVE
VH
NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP
SR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FS CSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 34: human IgG1 Fc with Human
Serum Albumin Signal Sequence (bold) at the N-terminus, contains residues EPKSS
comprising the full hinge region on the N-terminus of mature human IgG1 Fc
(underlined), Cys to Ser substitution (#), allotype G1m(fa) (bold italics)
MKWVTFISLLFLFSSAYSEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DV
SHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE
KT ISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 35: human
IgG1 Fc with murine IgG signal sequence (bold) at the N-terminus, with removal
of EPKSSD hinge residues from the N-terminus of the mature human IgG1 Fc,
allotype G1m(fa) (bold italics)
MGWSCIILFLVATATGVHSKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
E
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
G QPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 36: mature human
IgG1 Fc with a YTE triple mutation (bold and underlined), with removal of
EPKSSD hinge residues from the N-terminus of the mature human IgG1 Fc,
allotype G1m(fa) (bold italics)
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT
KP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
S VMHEALHNHYTQKSLSLSPGK SEQ ID NO: 37: mature human IgG1 Fc with an
LS double mutation (bold and underlined), with removal of EPKSSD hinge residues
from the N-terminus of the mature human IgG1 Fc, allotype G1m(fa) (bold italics)
KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK
TK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
S VLHEALHSHYTQKSLSLSPGK SEQ ID NO: 38: mature human IgG1 Fc with
Human Serum Albumin Signal Sequence (bold) at the N- terminus, a YTE triple
mutation (bold and underlined), allotype G1m(fa) (bold italics), C-terminal G4S linker
(italicized), and C-terminal C-myc tag (underlined)
MKWVTFISLLFLFSSAYSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDP
EV
KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK
GQ PREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID NO: 39:
mature human Fc IgG1, wherein X.sub.1 is Met or Tyr, X.sub.2 is Ser or Thr,
X.sub.3 is Thr or Glu, X.sub.4 is Asp or Glu, and X.sub.5 is Leu or Met,
X.sub.6 is Met or Leu, and X.sub.7 is Asn or Ser
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX.sub.1IX.sub.2RX.sub.3PEVTCVVVDVSHEDPEVKFN
WYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX.
sub.4E
X.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQG
NVFSC SVX.sub.6HEALHX.sub.7HYTQKSLSLSPG SEQ ID NO: 40: mature human Fc
IgG1 wherein X.sub.4 is Asp or Glu, and X.sub.5 is Leu or Met
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX.s
ub.4EX.sub.5
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SV MHEALHNHYTQKSLSLSPG SEQ ID NO: 41: mature human Fc IgG1 with a
YTE triple mutation (bold and underlined), and wherein X.sub.4 is Asp or Glu,
and X.sub.5 is Leu or Met
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX.su
b.4EX.sub.5T
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
V MHEALHNHYTQKSLSLSPG SEQ ID NO: 42: mature human Fc IgG1 with a YTE
triple mutation (bold and underlined), allotype G1m(fa) (bold italics)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
E TK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
M HEALHNHYTQKSLSLSPG SEQ ID NO: 43: mature human Fc IgG1 with a YTE
triple mutation (bold and underlined), allotype G1m(f) (bold italics)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
E TK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
M HEALHNHYTQKSLSLSPG SEQ ID NO: 44: mature human Fc IgG1 with a LS
double mutation (bold and underlined), and wherein X.sub.4 is Asp or Glu, and
X.sub.5 is Leu or Met
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KT
KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX.s
ub.4EX.sub.5
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC
SV LHEALHSHYTQKSLSLSPG SEQ ID NO: 45: mature human Fc IgG1 with a LS
double mutation (bold and underlined), allotype G1m(fa) (bold italics)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
ET
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VL HEALHSHYTQKSLSLSPG SEQ ID NO: 46: mature human Fc IgG1 with a LS
double mutation (bold and underlined), allotype G1m(f) (bold italics)
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNA
KT KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
ET
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS
VL HEALHSHYTQKSLSLSPG SEQ ID NO: 47: mature human Fc IgG1 with mouse
heavy chain MIgG Vh signal sequence (bold), Cys to Ser substitution (#), and
wherein X.sub.1 is Met or Tyr, X.sub.2 is Ser or Thr, X.sub.3 is Thr or Glu,
X.sub.4 is Asp or Glu, and X.sub.5 is Leu or Met, X.sub.6 is Met or Leu,
and X.sub.7 is Asn or Ser
MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKP
KD
TLX.sub.1IX.sub.2RX.sub.3PEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNG
KEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDI
AVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVX.sub.6HEALHX.sub.7HYTQKSLSLS
PG SEQ ID NO: 48: mature human IgG1 Fc with mouse heavy chain MIgG Vh
signal sequence (bold), Cys to Ser substitution (#), allotype G1m(fa) (bold italics)
MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKP
KD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID
NO: 49: mature human IgG1 Fc with mouse heavy chain MIgG Vh signal
sequence (bold), Cys to Ser substitution (#), allotype G1m(f) (bold italics)
MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKP
KD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID
NO: 50: mature human IgG1 Fc with mouse heavy chain MIgG Vh signal
sequence (bold), Cys to Ser substitution (#), M428L, N434S mutations
(Bold/Underlined), allotype G1m(fa) (bold italics)
MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKP
KD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID
NO: 51: mature human IgG1 Fc with mouse heavy chain MIgG Vh signal
sequence (bold), Cys to Ser substitution (#), M428L, N434S mutations
(Bold/Underlined), allotype G1m(f) (bold italics)
MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKP
KD
TLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID
NO: 52: mature human IgG1 Fc with mouse heavy chain MIgG Vh signal
sequence (bold), Cys to Ser substitution (#), YTE triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKP
KD
TLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID
NO: 53: mature human IgG1 Fc with mouse heavy chain MIgG Vh signal
sequence (bold), Cys to Ser substitution (#), YTE triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
MGWSCIILFLVATATGVHSNVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKP
KD
TLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNG
KE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID
NO: 54: mature human IgG1 Fc with mouse heavy chain MIgG Vh signal
sequence (bold), N- terminal ISAMVRS amino acid residues added (italicized),
M428L, N434S mutations (bold/underlined), G45 linker (italicized), and C-terminal C-myc-
tag (underlined), allotype G1m(f) (bold italics)
MGWSCIILFLVATATGVHSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EK TISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID
NO: 55: mature human IgG1 Fc with mouse heavy chain MIgG Vh signal
sequence (bold), N- terminal ISAMVRS amino acid residues added (italicized),
M428L, N434S mutations (bold/underlined), G4S linker (italicized), C-terminal C-myc-tag
(underlined), allotype G1m(fa) (bold italics)
MGWSCIILFLVATATGVHSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EK TISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID
NO: 56: mature human IgG1 Fc with mouse heavy chain MIgG Vh signal
sequence (bold), N- terminal ISAMVRS amino acid residues added (italicized), YTE
triple mutant (bold/underlined), G4S linker (italicized), and C-terminal C-myc-tag
(underlined), allotype G1m(f) (bold italics)
MGWSCIILFLVATATGVHSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVV
D
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EK TISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID
NO: 57: mature human IgG1 Fc with mouse heavy chain MIgG Vh signal
sequence (bold), N- terminal ISAMVRS amino acid residues added (italicized), YTE
triple mutant (bold/underlined), G4S linker (italicized), C-terminal C-myc-tag (underlined),
allotype G1m(fa) (bold italics)
MGWSCIILFLVATATGVHSISAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVV
D
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EK TISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSEQKLISEEDL SEQ ID
NO: 58: mature human IgG1 with mouse heavy chain MIgG1 signal sequence
(bold), Cys to Ser substitution (#), C-terminal G4S (italics), and C-terminal IgA
peptide (underline), allotype G1m(fa) (bold italics)
MGWSCIILFLVATATGVHSEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EK TISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSQRNPRLRLIRRHPTLRIPPI
SEQ ID NO: 59: mature human IgG1 with mouse heavy chain MIgG1 signal
sequence (bold), Cys to Ser substitution (#), M428L, N4345 mutations
(bold/underlined), C-terminal G4S (italics), and C- terminal IgA peptide (underline),
allotype G1m(fa) (bold italics)
MGWSCIILFLVATATGVHSEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VD
VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI
EK TISKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGGGGGSQRNPRLRLIRRHPTLRIPPI
SEQ ID NO: 60: mature human Fc IgG1, Z.sub.1 is Cys or Ser, and wherein
X.sub.1 is Met or Tyr, X.sub.2 is Ser or Thr, X.sub.3 is Thr or Glu, X.sub.4 is
Asp or Glu, and X.sub.5 is Leu or Met, X.sub.6 is Met or Leu, and X.sub.7
is Asn or Ser
NVNHKPSNTKVDKKVEPKSZ.sub.1DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX.sub.1IX.sub.2RX.
sub.3PEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS
KAKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVX.sub.6HEALHX.sub.7HYTQKSLSLSPGK SEQ
ID NO: 61: mature human Fc IgG1, Cys to Ser substitution (#), and wherein
X.sub.1 is Met or Tyr, X.sub.2 is Ser or Thr, X.sub.3 is Thr or Glu, X.sub.4 is
Asp or Glu, and X.sub.5 is Leu or Met, X.sub.6 is Met or Leu, and X.sub.7
is Asn or Ser
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX.sub.1IX.sub.2RX.su
b.3PEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
TI
SKAKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVX.sub.6HEALHX.sub.7HYTQKSLSLSPGK SEQ
ID NO: 62: mature human IgG1 Fc, Cys to Ser substitution (#), X.sub.4 is
Asp or Glu, and X.sub.5 is Leu or Met
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS
KAKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO:
63: mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold
italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 64: mature
human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 65: mature
human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations
(Bold/Underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID NO: 66: mature
human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations
(Bold/Underlined), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID NO: 67: mature
human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 68: mature
human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 69: mature
human Fc IgG1, Z.sub.1 is Cys or Ser, and wherein X.sub.1 is Met or Tyr,
X.sub.2 is Ser or Thr, X.sub.3 is Thr or Glu, X.sub.4 is Asp or Glu, and
X.sub.5 is Leu or Met, X.sub.6 is Met or Leu, and X.sub.7 is Asn or Ser
NVNHKPSNTKVDKKVEPKSZ.sub.1DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX.sub.1IX.sub.2RX.
sub.3PEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS
KAKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVX.sub.6HEALHX.sub.7HYTQKSLSLSPG SEQ ID
NO: 70: mature human Fc IgG1, Cys to Ser substitution (#), and wherein
X.sub.1 is Met or Tyr, X.sub.2 is Ser or Thr, X.sub.3 is Thr or Glu, X.sub.4 is
Asp or Glu, and X.sub.5 is Leu or Met, X.sub.6 is Met or Leu, and X.sub.7
is Asn or Ser
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX.sub.1IX.sub.2RX.su
b.3PEVTCVVVDVS
HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
TI
SKAKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVX.sub.6HEALHX.sub.7HYTQKSLSLSPG SEQ
ID NO: 71: mature human IgG1 Fc, Cys to Ser substitution (#), X.sub.4 is
Asp or Glu, and X.sub.5 is Leu or Met
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS
KAKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 72:
mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 73: mature
human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 74: mature
human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations
(Bold/Underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG SEQ ID NO: 75: mature
human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations
(Bold/Underlined), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG SEQ ID NO: 76: mature
human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 77: mature
human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 78: mature
human Fc IgG1, Z.sub.1 is Cys or Ser, and wherein X.sub.1 is Met or Tyr,
X.sub.2 is Ser or Thr, X.sub.3 is Thr or Glu, X.sub.4 is Asp or Glu, and
X.sub.5 is Leu or Met, X.sub.6 is Met or Leu, and X.sub.7 is Asn or Ser
VNHKPSNTKVDKKVEPKSZ.sub.1DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX.sub.1IX.sub.2RX.s
ub.3PEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK
AKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFL YSKLTVDKSRWQQGNVFSCSVX.sub.6HEALHX.sub.7HYTQKSLSLSPGK SEQ ID
NO: 79: mature human Fc IgG1, Cys to Ser substitution (#), and wherein
X.sub.1 is Met or Tyr, X.sub.2 is Ser or Thr, X.sub.3 is Thr or Glu, X.sub.4 is
Asp or Glu, and X.sub.5 is Leu or Met, X.sub.6 is Met or Leu, and X.sub.7
is Asn or Ser
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX.sub.1IX.sub.2RX.sub.
3PEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS
KAKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVX.sub.6HEALHX.sub.7HYTQKSLSLSPGK SEQ
ID NO: 80: mature human IgG1 Fc, Cys to Ser substitution (#), X.sub.4 is
Asp or Glu, and X.sub.5 is Leu or Met
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK
AKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 81:
mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 82: mature
human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 83: mature
human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations
(Bold/Underlined), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID NO: 84: mature
human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations
(Bold/Underlined), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPGK SEQ ID NO: 85: mature
human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 86: mature
human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 87: mature
human Fc IgG1, Z.sub.1 is Cys or Ser, and wherein X.sub.1 is Met or Tyr,
X.sub.2 is Ser or Thr, X.sub.3 is Thr or Glu, X.sub.4 is Asp or Glu, and
X.sub.5 is Leu or Met, X.sub.6 is Met or Leu, and X.sub.7 is Asn or Ser
VNHKPSNTKVDKKVEPKSZ.sub.1DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX.sub.1IX.sub.2RX.s
ub.3PEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK
AKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFL YSKLTVDKSRWQQGNVFSCSVX.sub.6HEALHX.sub.7HYTQKSLSLSPG SEQ ID
NO: 88: mature human Fc IgG1, Cys to Ser substitution (#), and wherein
X.sub.1 is Met or Tyr, X.sub.2 is Ser or Thr, X.sub.3 is Thr or Glu, X.sub.4 is
Asp or Glu, and X.sub.5 is Leu or Met, X.sub.6 is Met or Leu, and X.sub.7
is Asn or Ser
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX.sub.1IX.sub.2RX.sub.
3PEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT
IS
KAKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVX.sub.6HEALHX.sub.7HYTQKSLSLSPG SEQ ID
NO: 89: mature human IgG1 Fc, Cys to Ser substitution (#), X.sub.4 is Asp or
Glu, and X.sub.5 is Leu or Met
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK
AKGQPREPQVYTLPPSRX.sub.4EX.sub.5TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 90:
mature human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 91: mature
human IgG1 Fc, Cys to Ser substitution (#), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 92: mature
human IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations
(Bold/Underlined), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG SEQ ID NO: 93: mature human
IgG1 Fc, Cys to Ser substitution (#), M428L, N434S mutations (Bold/Underlined),
allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVLHEALHSHYTQKSLSLSPG SEQ ID NO: 94: mature human
IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and underlined),
allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 95: mature
human IgG1 Fc, Cys to Ser substitution (#), YTE triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG SEQ ID NO: 96: mature
human Fc IgG1, J.sub.1 is Asn or absent, J.sub.2 is Lys or absent, Z.sub.1 is
Cys or Ser, and wherein X.sub.1 is Met or Tyr, X.sub.2 is Ser or Thr, X.sub.3
is Thr or Glu, X.sub.4 is Asn or Ala, X.sub.5 is Leu or Asp, X.sub.6 is Gln
or His, X.sub.7 is Asp or Glu, and X.sub.8 is Leu or Met, X.sub.9 is Met or
Leu, and X.sub.10 is Asn or Ser
J.sub.1VNHKPSNTKVDKKVEPKSZ.sub.1DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLX.sub.1IX.su
b.2RX.sub.3PEVTCVVVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYX.sub.4STYRVVSVLTVX.sub.5HX.sub.6DWLNGKEYKCK
VSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVX.sub.9HEALHX.sub.10HYTQKSLSLSPGJ.sub.2
SEQ ID NO: 97: mature human Fc IgG1, Cys to Ser substitution (#), J.sub.1
is Asn or absent, J.sub.2 is Lys or absent, and wherein X.sub.4 is Asn or Ala,
X.sub.5 is Leu or Asp, X.sub.6 is Gln or His, X.sub.7 is Asp or Glu, and
X.sub.8 is Leu or Met, and X.sub.10 is Asn or Ser
J.sub.1VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYX.sub.4STYRVVSVLTVX.sub.5HX.sub.6DWLNGKEYKCK
VSNKALPAPIEKTI
SKAKGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHX.sub.10HYTQKSLSLSPGJ.sub.2 SEQ
ID NO: 98: mature human Fc IgG1, Cys to Ser substitution (#), DHS triple
mutation (bold and underlined), J.sub.1 is Asn or absent, J.sub.2 is Lys or
absent, wherein X.sub.4 is Asn or Ala, X.sub.7 is Asp or Glu, and X.sub.8 is
Leu or Met
J.sub.1VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV
VVDVSH
EDPEVKFNWYVDGVEVHNAKTKPREEQYX.sub.4STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPA
PIEKTIS
KAKGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGJ.sub.2 SEQ ID
NO: 99: mature human Fc IgG1, Cys to Ser substitution (#), DHS triple
mutation (bold and underlined), wherein X.sub.4 is Asn or Ala, X.sub.7 is Asp or
Glu, and X.sub.8 is Leu or Met
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYX.sub.4STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPA
PIEKTIS
KAKGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO:
100: mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation
(bold and underlined), wherein X.sub.7 is Asp or Glu and X.sub.8 is Leu or Met
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKT
IS
KAKGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO:
101: mature human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation
(bold and underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 102: mature
human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 103: mature
human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 104: mature
human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 105: mature
human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 106: mature
human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKT
IS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 107: mature
human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPG SEQ ID NO: 108: mature
human Fc IgG1, Cys to Ser substitution (#), DHS triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPG SEQ ID NO: 109: mature
human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS
triple mutation (bold and underlined), wherein X.sub.7 is Asp or Glu and X.sub.8
is Leu or Met
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIE
KTI
SKAKGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP
PVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO:
110: mature human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution
(*), DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIE
KTI SKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 111: mature
human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS
triple mutation (bold and underlined), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIE
KTI SKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 112: mature
human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS
triple mutation (bold and underlined), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEK
TIS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 113: mature
human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS
triple mutation (bold and underlined), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEK
TIS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 114: mature
human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS
triple mutation (bold and underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIE
KTI SKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 115: mature
human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS
triple mutation (bold and underlined), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV
SH
EDPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIE
KTI SKAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 116: mature
human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS
triple mutation (bold and underlined), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEK
TIS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPG SEQ ID NO: 117: mature
human Fc IgG1, Cys to Ser substitution (#), Asn to Ala substitution (*), DHS
triple mutation (bold and underlined), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSS(#)DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HE
DPEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEK
TIS KAKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPG SEQ ID NO: 118: mature
human Fc IgG1, J.sub.1 is Asn or absent, J.sub.2 is Lys or absent, and
wherein X.sub.4 is Asn or Ala, X.sub.5 is Leu or Asp, X.sub.6 is Gln or His,
X.sub.7 is Asp or Glu, and X.sub.8 is Leu or Met, and X.sub.10 is Asn or Ser
J.sub.1VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYX.sub.4STYRVVSVLTVX.sub.5HX.sub.6DWLNGKEYKCKV
SNKALPAPIEKTIS
KAKGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHX.sub.10HYTQKSLSLSPGJ.sub.2 SEQ
ID NO: 119: mature human Fc IgG1, DHS triple mutation (bold and underlined),
J.sub.1 is Asn or absent, J.sub.2 is Lys or absent, and wherein X.sub.4 is Asn
or Ala, X.sub.7 is Asp or Glu, and X.sub.8 is Leu or Met
J.sub.1VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV
DVSHE
DPEVKFNWYVDGVEVHNAKTKPREEQYX.sub.4STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPI
EKTISK
AKGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGJ.sub.2 SEQ ID NO:
120: mature human Fc IgG1, DHS triple mutation (bold and underlined), wherein
X.sub.4 is Asn or Ala, and X.sub.7 is Asp or Glu, and X.sub.8 is Leu or Met
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYX.sub.4STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIE
KTISKA
KGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 121:
mature human Fc IgG1, DHS triple mutation (bold and underlined), wherein
X.sub.7 is Asp or Glu and X.sub.8 is Leu or Met
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTIS
KA
KGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL
DSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 122:
mature human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa)
(bold italics)
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTIS
KA KGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 123: mature
human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold
italics)
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTIS
KA KGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 124: mature
human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold
italics)
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTISK
AK GQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 125: mature human
Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTISK
AK GQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 126: mature human
Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTIS
KA KGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 127: mature
human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold
italics)
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTIS
KA KGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 128: mature
human Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(fa) (bold
italics)
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTISK
AK GQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPG SEQ ID NO: 129: mature human
Fc IgG1, DHS triple mutation (bold and underlined), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DP
EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTISK
AK GQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPG SEQ ID NO: 130: mature human
Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined),
wherein X.sub.7 is Asp or Glu and X.sub.8 is Leu or Met
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTI
SK
AKGQPREPQVYTLPPSRX.sub.7EX.sub.8TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV
LDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 131:
mature human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 132: mature
human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 133: mature
human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DP
EVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTIS
KA KGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 134: mature
human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DP
EVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTIS
KA KGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 135: mature
human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 136: mature
human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and
underlined), allotype G1m(f) (bold italics)
NVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSH
ED
PEVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTI
SK AKGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPGK SEQ ID NO: 137: mature
human Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and
underlined), allotype G1m(fa) (bold italics)
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DP
EVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTIS
KA KGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPG SEQ ID NO: 138: mature human
Fc IgG1, Asn to Ala substitution (*), DHS triple mutation (bold and underlined),
allotype G1m(f) (bold italics)
VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DP
EVKFNWYVDGVEVHNAKTKPREEQYA(*)STYRVVSVLTVDHHDWLNGKEYKCKVSNKALPAPIEKTIS
KA KGQPREPQVYTLPPSR E
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHSHYTQKSLSLSPG

(193) As defined herein, an Fc domain includes two Fc domain monomers that are dimerized by the
interaction between the C.sub.H3 antibody constant domains, as well as one or more disulfide bonds
that form between the hinge domains of the two dimerizing Fc domain monomers. An Fc domain forms
the minimum structure that binds to an Fc receptor, e.g., Fc-gamma receptors (i.e., Fcγ receptors
(FcγR)), Fc-alpha receptors (i.e., Fcα receptors (FcαR)), Fc-epsilon receptors (i.e., Fcs receptors
(FcεR)), and/or the neonatal Fc receptor (FcRn). In some embodiments, an Fc domain of the present
invention binds to an Fcγ receptor (e.g., FcRn, FcγRI (CD64), FcγRIIa (CD32), FcγRIIb (CD32),
FcγRIIIa (CD16a), FcγRIIIb (CD16b)), and/or FcγRIV and/or the neonatal Fc receptor (FcRn).

(194) In some embodiments, the Fc domain monomer or Fc domain of the invention is an

aglycosylated Fc domain monomer or Fc domain (e.g., an Fc domain monomer or and Fc domain that
maintains engagement to an Fc receptor (e.g., FcRn). For example, the Fc domain is an aglycosylated
IgG1 variants that maintains engagement to an Fc receptor (e.g., an IgG1 having an amino acid
substitution at N297 and/or T299 of the glycosylation motif). Exemplary aglycosylated Fc domains and
methods for making aglycosylated Fc domains are known in the art, for example, as described in
Sazinsky S. L. et al., Aglycosylated immunoglobulin G1 variants productively engage activating Fc
receptors, PNAS, 2008, 105(51):20167-20172, which is incorporated herein in its entirety.

(195) In some embodiments, the Fc domain or Fc domain monomer of the invention is engineered to
enhance binding to the neonatal Fc receptor (FcRn). For example, the Fc domain may include the
triple mutation corresponding to M252Y/S254T/T256E (YTE) (e.g., an IgG1, such as a human or
humanized IgG1 having a YTE mutation, for example SEQ ID NO: 33, SEQ ID NO: 36, SEQ ID NO:
38, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 52, SEQ ID NO:
53, SEQ ID NO: 56, or SEQ ID NO: 57). The Fc domain may include the double mutant corresponding
to M428L/N434S (LS) (e.g., an IgG1, such as a human or humanized IgG1 having an LS mutation,
such as SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 44, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID
NO: 50, SEQ ID NO: 51, SEQ ID NO: 54, SEQ ID NO: 55, or SEQ ID NO: 59). The Fc domain may
include the single mutant corresponding to N434H (e.g., an IgG1, such as a human or humanized
IgG1 having an N434H mutation). The Fc domain may include the single mutant corresponding to
C220S (e.g., an IgG1, such as a human or humanized IgG1 having a C220S mutation, such as SEQ
ID NO: 34, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID
NO: 52, SEQ ID NO: 53, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID
NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID
NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID
NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID
NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID
NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 92, SEQ ID
NO: 93, SEQ ID NO: 94, and SEQ ID NO: 95). The Fc domain may include a quadruple mutant
corresponding to C220S/L309D/Q311 H/N434S (CDHS) (e.g., an IgG1, such as a human or
humanized IgG1 having a DHS mutation, such as SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98,
SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO:
104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID
NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115,
SEQ ID NO: 116, and SEQ ID NO: 117). The Fc domain may include a triple mutant corresponding to
L309D/Q311 H/N434S (DHS) (e.g., an IgG1, such as a human or humanized IgG1 having a DHS
mutation, such as SEQ ID NO: 118, SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO:
122, SEQ ID NO: 123, SEQ ID NO: 124, SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID
NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, SEQ ID NO: 131, SEQ ID NO: 132, SEQ ID NO: 133,
SEQ ID NO: 134, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, and SEQ ID NO: 138). The Fc
domain may include a combination of one or more of the above-described mutations that enhance
binding to the FcRn. Enhanced binding to the FcRn may increase the half-life Fc domain-containing
conjugate. For example, incorporation of one or more amino acid mutations that increase binding to
the FcRn (e.g., a YTE mutation, an LS mutation, or an N434H mutation) may increase the half-life of
the conjugate by 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%. 100%, 200%, 300%,
400%, 500% or more relative to a conjugate having the corresponding Fc domain without the mutation
that enhances FcRn binding. Exemplary Fc domains with enhanced binding to the FcRN and methods
for making Fc domains having enhanced binding to the FcRN are known in the art, for example, as
described in Maeda, A. et al., Identification of human IgG1 variant with enhanced FcRn binding and
without increased binding to rheumatoid factor autoantibody, MABS, 2017, 9(5):844-853, which is
incorporated herein in its entirety.

(196) As used herein, an amino acid “corresponding to” a particular amino acid residue (e.g., of a
particular SEQ ID NO.) should be understood to include any amino acid residue that one of skill in the
art would understand to align to the particular residue (e.g., of the particular sequence). For example,
any one of SEQ ID NOs: 1-138 may be mutated to include a YTE mutation, an LS mutation, and/or an
N434H mutation by mutating the “corresponding residues” of the amino acid sequence.

(197) As used herein, a sulfur atom “corresponding to” a particular cysteine residue of a particular
SEQ ID NO. should be understood to include the sulfur atom of any cysteine residue that one of skill in
the art would understand to align to the particular cysteine of the particular sequence. The protein
sequence alignment of human IgG1 (UniProtKB: P01857; SEQ ID NO: 142), human IgG2 (UniProtKB:
P01859; SEQ ID NO: 143), human IgG3 (UniProtKB: P01860; SEQ ID NO: 144), and human IgG4
(UniProtKB: P01861; SEQ ID NO: 145) is provided below (aligned with Clustal Omega Multiple
Pairwise Alignment). The alignment indicates cysteine residues (e.g., sulfur atoms of cysteine
residues) that “correspond to” one another (in boxes and indicated by the .Math. symbol). One of skill
in the art would readily be able to perform such an alignment with any IgG variant of the invention to
determine the sulfur atom of a cysteine that corresponds to any sulfur atom of a particular cysteine of
a particular SEQ ID NO. described herein (e.g., any one of SEQ ID NOs: 1-138). For example, one of
skill in the art would readily be able to determine that Cys10 of SEQ ID NO: 10 (the first cysteine of the
conserved CPPC motif of the hinge region of the Fc domain) corresponds to, for example, Cys109 of
IgG1, Cys106 of IgG2, Cys156 of IgG3, Cys29 of SEQ ID NO: 1, Cys9 of SEQ ID NO: 2, Cys30 of
SEQ ID NO: 3, or Cys10 of SEQ ID NO: 10. In some embodiments, the Fc domain or Fc domain
monomer of the invention has the sequence of any one of SEQ ID NOs: 39-138 may further include
additional amino acids at the N-terminus (Xaa)x and/or additional amino acids at the C-terminus
(Xaa)z, wherein Xaa is any amino acid and x and z are a whole number greater than or equal to zero,
generally less than 100, preferably less than 10 and more preferably 0, 1, 2, 3, 4, or 5. In some
embodiments, the additional amino acids are least 70% (e.g., 70%, 75%, 80%, 85%, 90%, 95%, 96%,
97%, 98%, 99%, or 100%) identical to one or more consecutive amino acids of SEQ ID NO: 81. For
example, the additional amino acids may be a single amino acid on the C-terminus corresponding to
Lys330 of IgG1 (SEQ ID NO: 119).

(198) As used herein, a nitrogen atom “corresponding to” a particular lysine residue of a particular
SEQ ID NO. should be understood to include the nitrogen atom of any lysine residue that one of skill in
the art would understand to align to the particular lysine of the particular sequence. The protein
sequence alignment of human IgG1 (UniProtKB: P01857; SEQ ID NO: 142), human IgG2 (UniProtKB:
P01859; SEQ ID NO: 143), human IgG3 (UniProtKB: P01860; SEQ ID NO: 144), and human IgG4
(UniProtKB: P01861; SEQ ID NO: 145) is provided below (aligned with Clustal Omega Multiple
Pairwise Alignment). The alignment indicates lysine residues (e.g., nitrogen atoms of lysine residues)
that “correspond to” one another (in boxes and indicated by the * symbol). One of skill in the art would
readily be able to perform such an alignment with any IgG variant of the invention to determine the
nitrogen atom of a lysine that corresponds to any nitrogen atom of a particular lysine of a particular
SEQ ID NO. described herein (e.g., any one of SEQ ID NOs: 1-138). For example, one of skill in the
art would readily be able to determine that Lys35 of SEQ ID NO: 10 corresponds to, for example,
Lys129 of IgG1, Lys126 of IgG2, Lys176 of IgG3, Lys51 of SEQ ID NO: 1, Lys31 of SEQ ID NO: 2,
Lys50 of SEQ ID NO: 3, or Lys30 of SEQ ID NO: 10.

(199) Protein sequence alignment of IgG1 (SEQ ID NO: 142), IgG2 (SEQ ID NO: 143), IgG3 (SEQ ID
NO:

(200) TABLE-US-00004

Activation of Immune Cells

(201) Fc-gamma receptors (FcγRs) bind the Fc portion of immunoglobulin G (IgG) and play important
roles in immune activation and regulation. For example, the IgG Fc domains in immune complexes
(ICs) engage FcγRs with high avidity, thus triggering signaling cascades that regulate immune cell
activation. The human FcγR family contains several activating receptors (FcγRI, FcγRIIa, FcγRIIc,
FcγRIIIa, and FcγRIIIb) and one inhibitory receptor (FcγRIIb). FcγR signaling is mediated by
intracellular domains that contain immune tyrosine activating motifs (ITAMs) for activating FcγRs and
immune tyrosine inhibitory motifs (ITIM) for inhibitory receptor FcγRIIb. In some embodiments, FcγR
binding by Fc domains results in ITAM phosphorylation by Src family kinases; this activates Syk family
kinases and induces downstream signaling networks, which include PI3K and Ras pathways.

(202) In the conjugates described herein, the portion of the conjugates including monomers or dimers
of neuraminidase inhibitors bind to and inhibits viral neuraminidase leading to inhibition of viral
replication, while the Fc domain portion of the conjugates bind to FcγRs (e.g., FcRn, FcγRI, FcγRIIa,
FcγRIIc, FcγRIIIa, and FcγRIIIb) on immune cells and activate phagocytosis and effector functions,
such as antibody-dependent cell-mediated cytotoxicity (ADCC), thus leading to the engulfment and
destruction of viral particles by immune cells and further enhancing the antiviral activity of the
conjugates. Examples of immune cells that may be activated by the conjugates described herein
include, but are not limited to, macrophages, neutrophils, eosinophils, basophils, lymphocytes,
follicular dendritic cells, natural killer cells, and mast cells.

(203) Tissue Distribution

(204) After a therapeutic enters the systemic circulation, it is distributed to the body's tissues.
Distribution is generally uneven because of different in blood perfusion, tissue binding, regional pH,
and permeability of cell membranes. The entry rate of a drug into a tissue depends on the rate of
blood flow to the tissue, tissue mass, and partition characteristics between blood and tissue.
Distribution equilibrium (when the entry and exit rates are the same) between blood and tissue is
reached more rapidly in richly vascularized areas, unless diffusion across cell membranes is the rate-
limiting step. The size, shape, charge, target binding, FcRn and target binding mechanisms, route of
administration, and formulation affect tissue distribution.

(205) In some instances, the conjugates described herein may be optimized to distribute to lung
tissue. In some instances, the conjugates have a concentration ratio of distribution in epithelial lining
fluid of at least 30% the concentration of the conjugates in plasma within 2 hours after administration.
In certain embodiments, ratio of the concentration is at least 45% within 2 hours after administration. In
some embodiments, the ratio of concentration is at least 55% within 2 hours after administration. In
particular, the ratio of concentration is at least 60% within 2 hours after administration. As shown in
Example 190 and FIG. 98, by 2 hours post injection, a conjugate having an Fc domain (SEQ ID NO:
73) ELF levels are surprisingly ˜60% of plasma exposure levels as measured by AUC across the rest
of the time course indicating nearly immediate partitioning of the conjugate from plasma to the ELF in
the lung. This demonstrates that an Fc containing conjugate rapidly distributes to lung, and maintains
high concentrations in lung relative to levels in plasma.

(206) IV. Albumin Proteins and Albumin Protein-Binding Peptides

(207) Albumin Proteins

(208) An albumin protein of the invention may be a naturally-occurring albumin or a variant thereof,
such as an engineered variant of a naturally-occurring albumin protein. Variants include
polymorphisms, fragments such as domains and sub-domains, and fusion proteins. An albumin protein
may include the sequence of an albumin protein obtained from any source. Preferably the source is
mammalian, such as human or bovine. Most preferably, the albumin protein is human serum albumin
(HSA), or a variant thereof. Human serum albumins include any albumin protein having an amino acid
sequence naturally occurring in humans, and variants thereof. An albumin protein coding sequence is
obtainable by methods know to those of skill in the art for isolating and sequencing cDNA
corresponding to human genes. An albumin protein of the invention may include the amino acid
sequence of human serum albumin (HSA), provided in SEQ ID NO: 139 or SEQ ID NO: 140, or the
amino acid sequence of mouse serum albumin (MSA), provided in SEQ ID NO: 141, or a variant or
fragment thereof, preferably a functional variant or fragment thereof. A fragment or variant may or may
not be functional, or may retain the function of albumin to some degree. For example, a fragment or
variant may retain the ability to bind to an albumin receptor, such as HSA or MSA, by at least 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 105% of the ability of the parent albumin (e.g.,
the parent albumin from which the fragment or variant is derived). Relative binding ability may be
determined by methods known in the art, such as by surface plasmon resonance.

(209) The albumin protein may be a naturally-occurring polymorphic variant of an albumin protein,
such as human serum albumin. Generally, variants or fragments of human serum albumin will have at
least 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, or 70%, and preferably 80%, 90%, 95%, 100%, or
105% or more of human serum albumin or mouse serum albumin's ligand binding activity.

(210) The albumin protein may include the amino acid sequence of bovine serum albumin. Bovine
serum albumin proteins include any albumin having an amino acid sequence naturally occurring in
cows, for example, as described by Swissprot accession number P02769, and variants thereof as
defined herein. Bovine serum albumin proteins also include fragments of full-length bovine serum
albumin or variants thereof, as defined herein.

(211) The albumin protein may comprise the sequence of an albumin derived from one of serum
albumin from dog (e.g., Swissprot accession number P49822-1), pig (e.g., Swissprot accession
number P08835-1), goat (e.g., Sigma product no. A2514 or A4164), cat (e.g., Swissprot accession
number P49064-1), chicken (e.g., Swissprot accession number P19121-1), ovalbumin (e.g., chicken
ovalbumin) (e.g., Swissprot accession number P01012-1), turkey ovalbumin (e.g., Swissprot
accession number O73860-1), donkey (e.g., Swissprot accession number Q5XLE4-1), guinea pig
(e.g., Swissprot accession number Q6WDN9-1), hamster (e.g., as described in DeMarco et al.
International Journal for Parasitology 37(11): 1201-1208 (2007)), horse (e.g., Swissprot accession
number P35747-1), rhesus monkey (e.g., Swissprot accession number Q28522-1), mouse (e.g.,
Swissprot accession number P07724-1), pigeon (e.g., as defined by Khan et al. Int. J. Biol. Macromol.
30(3-4), 171-8 (2002)), rabbit (e.g., Swissprot accession number P49065-1), rat (e.g., Swissprot
accession number P02770-1) or sheep (e.g., Swissprot accession number P14639-1), and includes
variants and fragments thereof as defined herein. Many naturally-occurring mutant forms of albumin
are known to those skilled in the art. Naturally-occurring mutant forms of albumin are described in, for
example, Peters, et al. All About Albumin: Biochemistry, Genetics and Medical Applications, Academic
Press, Inc., San Diego, Calif., p. 170-181 (1996).

(212) Albumin proteins of the invention include variants of naturally-occurring albumin proteins. A
variant albumin refers to an albumin protein having at least one amino acid mutation, such as an
amino acid mutation generated by an insertion, deletion, or substitution, either conservative or non-
conservative, provided that such changes result in an albumin protein for which at least one basic
property has not been significantly altered (e.g., has not been altered by more than 5%, 10%, 15%,
20%, 25%, 30%, 35%, or 40%). Exemplary properties which may define the activity of an albumin
protein include binding activity (e.g., including binding specificity or affinity to bilirubin, or a fatty acid
such as a long-chain fatty acid), osmolarity, or behavior in a certain pH-range.

(213) Typically an albumin protein variant will have at least 40%, at least 50%, at least 60%, and
preferably at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or at least 99% amino acid sequence identity with a naturally-occurring albumin protein, such as
the albumin protein of any one of SEQ ID NOs: 139-141.

(214) Methods for the production and purification of recombinant human albumins are well-established
(Sleep et al. Biotechnology, 8(1):42-6 (1990)), and include the production of recombinant human
albumin for pharmaceutical applications (Bosse et al. J Clin Pharmacol 45(1):57-67 (2005)). The three-
dimensional structure of HSA has been elucidated by X-ray crystallography (Carter et al. Science.
244(4909): 1195-8(1998)); Sugio et al. Protein Eng. 12(6):439-46 (1999)). The HSA polypeptide chain
has 35 cysteine residues, which form 17 disulfide bonds, and one unpaired (e.g., free) cysteine at
position 34 of the mature protein. Cys-34 of HSA has been used for conjugation of molecules to
albumin (Leger et al. Bioorg Med Chem Lett 14(17):4395-8 (2004); Thibaudeau et al. Bioconjug Chem
16(4):1000-8 (2005)), and provides a site for site-specific conjugation.

(215) SEQ ID NO: 139 (Human serum albumin (HSA), variant 1)

DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENCDKSLHTL
F
GDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKK
YL
YEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFG
ER
AFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKEC
C
EKPLLEKSHCLAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLL
RL
AKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQ
VS
TPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRP
CF
SALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKC
C KADDKETCFAEEGKKLVAASQAALGL

SEQ ID NO: 140 (Human serum albumin (HSA), variant 2)

RGVFRRDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAENC
D
KSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAFHDN
EE
TFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCA
SL
QKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSI
SS
KLKECCEKPLLEKSHCLAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDY
SV
VLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYT
KK
VPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESL
VN
RRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFA
AF VEKCCKADDKETCFAEEGKKLVAASQAALGL

SEQ ID NO: 141 (Mouse serum albumin (MSA))

RGVFRREAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANC
DK
SLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENP
TTF
MGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSS
M
QKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKYMCENQATI
SS
KLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDY
SV
SLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYT
QK
APQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLV
ER
RPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQF
L DTCCKAADKDTCFSTEGPNLVTRCKDALA

Conjugation of Albumin Proteins

(216) An albumin protein of the invention may be conjugated to (e.g., by way of a covalent bond) to
any compound of the invention (e.g., by way of the linker portion of a neuraminidase inhibitor
monomer or dimer). The albumin protein may be conjugated to any compound of the invention by any
method well-known to those of skill in the art for producing small-molecule-protein conjugates. This
may include covalent conjugation to a solvent-exposed amino acid, such as a solvent exposed
cysteine or lysine. For example, human serum albumin may be conjugated to a compound of the
invention by covalent linkage to the sulfur atom corresponding to Cys34 of SEQ ID NO: 139 or Cys40
of SEQ ID NO: 140.

(217) An albumin protein of the invention may be conjugated to any compound of the invention by way
of an amino acid located within 10 amino acid residues of the C-terminal or N-terminal end of the
albumin protein. An albumin protein may include a C-terminal or N-terminal polypeptide fusion of 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 or more amino acid. The C-terminal or N-terminal polypeptide fusion
may include one or more solvent-exposed cysteine or lysine residues, which may be used for covalent
conjugation of a compound of the invention (e.g., conjugation to a neuraminidase inhibitor monomer or
dimer, including by way of a linker).

(218) Albumin proteins of the invention include any albumin protein which has been engineered to
include one or more solvent-exposed cysteine or lysine residues, which may provide a site for
conjugation to a compound of the invention (e.g., conjugation to a neuraminidase inhibitor monomer or
dimer, including by way of a linker). Most preferably, the albumin protein will contain a single solvent-
exposed cysteine or lysine, thus enabling site-specific conjugation of a compound of the invention.
Exemplary methods for the production of engineered variants of albumin proteins that include one or
more conjugation-competent cysteine residues are provided in U.S. Patent Application No.
2017/0081389, which is incorporated herein by reference in its entirety. Briefly, preferred albumin
protein variants are those comprising a single, solvent-exposed, unpaired (e.g., free) cysteine residue,
thus enabling site-specific conjugation of a linker to the cysteine residue.

(219) Albumin proteins which have been engineered to enable chemical conjugation to a solvent-
exposed, unpaired cysteine residue include the following albumin protein variants:

(220) (a) an albumin protein having a substitution of a non-cysteine amino acid residue with a cysteine
at an amino acid residue corresponding to any of L585, D1, A2, D562, A364, A504, E505, T79, E86,
D129, D549, A581, D121, E82, S270, Q397, and A578 of SEQ ID NO: 139;

(221) (b) an albumin protein having an insertion of a cysteine at a position adjacent the N- or C-
terminal side of an amino acid residue corresponding to any of L585, D1, A2, D562, A364, A504,
E505, T79, E86, D129, D549, A581, D121, E82, S270, Q397, and A578 of SEQ ID NO: 139;

(222) (c) an albumin protein engineered to have an unpaired cysteine having a free thiol group at a
residue corresponding to any of C369, C361, C91, C177, C567, C316, C75, C169, C124, or C558 of
SEQ ID NO: 96, and which may or may not be generated by deletion or substitution of a residue
corresponding to C360, C316, C75, C168, C558, C361, C91, C124, C169, or C567 of SEQ ID NO:
139; and/or

(223) (d) addition of a cysteine to the N- or C-terminus of an albumin protein.

(224) In some embodiments of the invention, the net result of the substitution, deletion, addition, or
insertion events of (a), (b), (c) and/or (d) is that the number of conjugation competent cysteine
residues of the polypeptide sequence is increased relative to the parent albumin sequence. In some
embodiments of the invention, the net result of the substitution, deletion, addition, or insertion events
of (a), (b), (c) and/or (d) is that the number of conjugation competent-cysteine residues of the
polypeptide sequence is one, thus enabling site-specific conjugation.

(225) Preferred albumin protein variants also include albumin proteins having a single solvent-exposed
lysine residue, thus enabling site-specific conjugation of a linker to the lysine residue. Such variants
may be generated by engineering an albumin protein, including any of the methods previously
described (e.g., insertion, deletion, substitution, or C-terminal or N-terminal fusion).

(226) Albumin Protein-Binding Peptides

(227) Conjugation of a biologically-active compound to an albumin protein-binding peptide can alter

the pharmacodynamics of the biologically-active compound, including the alteration of tissue uptake,
penetration, and diffusion. In a preferred embodiment, conjugation of an albumin protein-binding
peptide to a compound of the invention (e.g., a neuraminidase inhibitor monomer or dimer, by way of a
linker) increases the efficacy or decreases the toxicity of the compound, as compared to the
compound alone.

(228) Albumin protein-binding peptides of the invention include any polypeptide having an amino acid
sequence of 5 to 50 (e.g., 5 to 40, 5 to 30, 5 to 20, 5 to 15, 5 to 10, 10 to 50, 10 to 30, or 10 to 20)
amino acid residues that has affinity for and functions to bind an albumin protein, such as any of the
albumin proteins described herein. Preferably, the albumin protein-binding peptide binds to a naturally
occurring serum albumin, most preferably human serum albumin. An albumin protein-binding peptide
can be of different origins, e.g., synthetic, human, mouse, or rat. Albumin protein-binding peptides of
the invention include albumin protein-binding peptides which have been engineered to include one or
more (e.g., two, three, four, or five) solvent-exposed cysteine or lysine residues, which may provide a
site for conjugation to a compound of the invention (e.g., conjugation to a neuraminidase inhibitor
monomer or dimer, including by way of a linker). Most preferably, the albumin protein-binding peptide
will contain a single solvent-exposed cysteine or lysine, thus enabling site-specific conjugation of a
compound of the invention. Albumin protein-binding peptides may include only naturally occurring
amino acid residues, or may include one or more non-naturally occurring amino acid residues. Where
included, a non-naturally occurring amino acid residue (e.g., the side chain of a non-naturally occurring
amino acid residue) may be used as the point of attachment for a compound of the invention (e.g., a
neuraminidase inhibitor monomer or dimer, including by way of a linker). Albumin protein-binding
peptides of the invention may be linear or cyclic. Albumin protein-binding peptides of the invention
include any albumin protein-binding peptides known to one of skill in the art, examples of which, are
provided herein.

(229) Albumin protein-binding peptide, and conjugates including an albumin protein-binding peptide,
preferably bind an albumin protein (e.g., human serum albumin) with an affinity characterized by a
dissociation constant, Kd, that is less than about 100 μM, preferably less than about 100 nM, and most
preferably do not substantially bind other plasma proteins. Specific examples of such compounds are
linear or cyclic peptides, preferably between about 10 and 20 amino acid residues in length, optionally
modified at the N-terminus or C-terminus or both.

(230) Albumin protein-binding peptides include linear and cyclic peptides comprising the following
general formulae, wherein Xaa is any amino acid:

Xaa-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Xaa-Xaa-Phe-Cys-Xaa-Asp-Trp-Pro-Xaa-Xaa-Xaa-Ser-
Cys SEQ ID NO: 148

Val-Cys-Tyr-Xaa-Xaa-Xaa-Ile-Cys-Phe SEQ ID NO: 149

Cys-Tyr-Xaa-Pro-Gly-Xaa-Cys SEQ ID NO: 150

Asp-Xaa-Cys-Leu-Pro-Xaa-Trp-Gly-Cys-Leu-Trp SEQ ID NO: 151

Trp-Cys-Asp-Xaa-Xaa-Leu-Xaa-Ala-Xaa-Asp-Leu-Cys SEQ ID NO: 152

Asp-Leu-Val-Xaa-Leu-Gly-Leu-Glu-Cys-Trp SEQ ID NO: 153

(231) Albumin protein-binding peptides of the invention further include any of the following peptide
sequences, which may be linear or cyclic:

DLCLRDWGCLW SEQ ID NO: 154

DICLPRWGCLW SEQ ID NO: 155

MEDICLPRWGCLWGD SEQ ID NO: 156

QRLMEDICLPRWGCLWEDDE SEQ ID NO: 157

QGLIGDICLPRWGCLWGRSV SEQ ID NO: 158

QGLIGDICLPRWGCLWGRSVK SEQ ID NO: 159

EDICLPRWGCLWEDD SEQ ID NO: 160

RLMEDICLPRWGCLWEDD SEQ ID NO: 161

MEDICLPRWGCLWEDD SEQ ID NO: 162

MEDICLPRWGCLWED SEQ ID NO: 163

RLMEDICLARWGCLWEDD SEQ ID NO: 164

EVRSFCTRWPAEKSCKPLRG SEQ ID NO: 165

RAPESFVCYWETICFERSEQ SEQ ID NO: 166

EMCYFPGICWM SEQ ID NO: 167

(232) Albumin protein-binding peptides of SEQ ID NOs: 154-167 may further include additional amino
acids at the N-terminus (Xaa)x and/or additional amino acids at the C-terminus (Xaa)z, wherein Xaa is
any amino acid and x and z are a whole number greater or equal to zero, generally less than 100,
preferably less than 10 and more preferably 0, 1, 2, 3, 4 or 5.

(233) Further exemplary albumin protein-binding peptides are provided in U.S. Patent Application No.
2005/0287153, which is incorporated herein by reference in its entirety.

(234) Conjugation of Albumin Protein-Binding Peptides

(235) An albumin protein-binding peptide of the invention may be conjugated to (e.g., by way of a
covalent bond) to any compound of the invention (e.g., by way of the linker portion of a neuraminidase
inhibitor monomer or dimer). The albumin protein-binding peptide may be conjugated to any
compound of the invention by any method known to those of skill in the art for producing peptide-small
molecule conjugates. This may include covalent conjugation to the side chain group of an amino acid
residue, such as a cysteine, a lysine, or a non-natural amino acid. Alternately, covalent conjugation
may occur at the C-terminus (e.g., to the C-terminal carboxylic acid, or to the side chain group of the
C-terminal residue) or at the N-terminus (e.g., to the N-terminal amino group, or to the side chain
group of the N-terminal amino acid).

(236) V. Linkers

(237) A linker refers to a linkage or connection between two or more components in a conjugate
described herein (e.g., between two neuraminidase inhibitors in a conjugate described herein,
between a neuraminidase inhibitor and an Fc domain or an albumin protein in a conjugate described
herein, and between a dimer of two neuraminidase inhibitors and an Fc domain or an albumin protein
in a conjugate described herein).

(238) Linkers in Conjugates Having an Fc Domain or an Albumin Protein Covalently Linked to Dimers
of Neuraminidase Inhibitors

(239) In a conjugate containing an Fc domain monomer, an Fc domain, an Fc-binding peptide, an

albumin protein, or an albumin protein-binding peptide covalently linked to one or more dimers of
neuraminidase inhibitors as described herein, a linker in the conjugate (e.g., L or L′) may be a
branched structure. As described further herein, a linker in a conjugate described herein (e.g., L or L)
may be a multivalent structure, e.g., a divalent or trivalent structure having two or three arms,
respectively. In some embodiments when the linker has three arms, two of the arms may be attached
to the first and second neuraminidase inhibitors and the third arm may be attached to the Fc domain
monomer, and Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding
peptide. In some embodiments when the linker has two arms, one arm may be attached to an Fc
domain or an albumin protein and the other arm may be attached to one of the two neuraminidase
inhibitors. In other embodiments, a linker with two arms may be used to attach the two neuraminidase
inhibitors on a conjugate containing an Fc domain or albumin protein covalently linked to one or more
dimers of neuraminidase inhibitors.

(240) In some embodiments, a linker in a conjugate having an Fc domain or an albumin protein

covalently linked to one or more dimers of neuraminidase inhibitors is described by formula (D-L-I):

(241) ##STR00500##

wherein L.sup.A is described by formula G.sup.A1-(Z.sup.A1).sub.g1—(Y.sup.A1).sub.h1—

(242) In some embodiments, L.sup.c may have two points of attachment to the Fc domain (e.g., two
G.sup.C2) In some embodiments, L includes a polyethylene glycol (PEG) linker. A PEG linker includes
a linker having the repeating unit structure (—CH.sub.2CH.sub.2O—).sub.n, where n is an integer
from 2 to 100. A polyethylene glycol linker may covalently join a neuraminidase inhibitor and E (e.g., in
a conjugate of any one of formulas (M-I)-(M-XI)). A polyethylene glycol linker may covalently join a first
neuraminidase inhibitor and a second neuraminidase inhibitor (e.g., in a conjugate of any one of
formulas (D-I)-(D-XI)). A polyethylene glycol linker may covalently join a neuraminidase inhibitor dimer
and E (e.g., in a conjugate of any one of formulas (D-I)-(D-XI)). A polyethylene glycol linker may be
selected any one of PEG.sub.2 to PEG.sub.100 (e.g., PEG.sub.2, PEG.sub.3, PEG.sub.4, PEG.sub.5,
PEG.sub.5-PEG.sub.10, PEG.sub.10-PEG.sub.20, PEG.sub.20-PEG.sub.30, PEG.sub.30-
PEG.sub.40, PEG.sub.50-PEG.sub.60, PEG.sub.60-PEG.sub.70, PEG.sub.70-PEG.sub.80,
PEG.sub.80-PEG.sub.90, PEG.sub.90-PEG.sub.100). In some embodiments, L.sup.c includes a PEG
linker, where L.sup.c is covalently attached to each of Q and E.

(243) Linkers of formula (D-L-I) that may be used in conjugates described herein include, but are not
limited to

(244) ##STR00501## ##STR00502## ##STR00503## ##STR00504## ##STR00505##

##STR00506## ##STR00507## ##STR00508## ##STR00509## ##STR00510## ##STR00511##

(245) Linkers of the formula (D-L-I) may also include any of

(246) ##STR00512## ##STR00513## ##STR00514## ##STR00515## ##STR00516##
##STR00517## ##STR00518## ##STR00519##

Linkers in Conjugates Having an Fc Domain or an Albumin Protein Covalently Linked to Monomers of

Neuraminidase Inhibitors

(247) In a conjugate containing an Fc domain monomer, an Fc domain, an Fc-binding peptide, an

albumin protein, or an albumin protein-binding peptide covalently linked to one or more monomers of
neuraminidase inhibitors as described herein, a linker in the conjugate (e.g., L, or L′) may be a divalent
structure having two arms. One arm in a divalent linker may be attached to the monomer of
neuraminidase inhibitor and the other arm may be attached to the Fc domain monomer, and Fc
domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide. In some
embodiments, the one or more monomers of neuraminidase inhibitors in the conjugates described
herein may each be, independently, connected to an atom in the Fc domain monomer, and Fc domain,
an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide.

(248) In some embodiments, a linker is described by formula (M-L-I):

J.sup.1-(Q.sup.1).sub.g-(T.sup.1).sub.h-(Q.sup.2).sub.i-(T.sup.2).sub.j-(Q.sup.3).sub.k-(T.sup.3).sub.l-
(Q.sup.4).sub.m-(T.sup.4).sub.n-(Q.sup.5).sub.o-J.sup.2

wherein J.sup.1 is a bond attached to a neuraminidase inhibitor; J.sup.2 is a bond attached to an Fc

domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-
binding peptide, or a functional group capable of reacting with a functional group conjugated to an Fc
domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-
binding peptide (e.g., maleimide and cysteine, amine and activated carboxylic acid, thiol and
maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene
and tetrazine); each of Q.sup.1, Q.sup.2, Q.sup.3, Q.sup.4 and Q.sup.5 is, independently, optionally
substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally substituted C2-
C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20
alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20
cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20
cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20
cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15
arylene, or optionally substituted C2-C15 heteroarylene; each of T.sup.1, T.sup.2, T.sup.3, T.sup.4 is,
independently, O, S, NR.sup.i, P, carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino;
R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally
substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-
C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl,
optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally
substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally
substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted
C2-C15 heteroaryl; and each of g, h, i, j, k, l, m, n, and o is, independently, 0 or 1.

(249) In some embodiments, J.sub.2 may have two points of attachment to the Fc domain monomer,
and Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide (e.g.,
two J.sup.2).

(250) Linkers of formula (M-L-I) that may be used in conjugates described herein include, but are not
limited to,

(251) ##STR00520##

(252) ##STR00521##

wherein d is an integer from 1 to 20 (e.g., d is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20).
(253) Linkers of formula (M-L-I) that may be used in conjugates described herein include, but are not
limited to,

(254) ##STR00522## ##STR00523## ##STR00524##

wherein each of d and e is, independently, an integer from 1 to 26.

Linking Groups

(255) In some embodiments, a linker provides space, rigidity, and/or flexibility between the
neuraminidase inhibitors and the Fc domain monomer, and Fc domain, an Fc-binding peptide, an
albumin protein, or an albumin protein-binding peptide in the conjugates described here or between
two neuraminidase inhibitors in the conjugates described herein. In some embodiments, a linker may
be a bond, e.g., a covalent bond, e.g., an amide bond, a disulfide bond, a C—O bond, a C—N bond, a
N—N bond, a C—S bond, or any kind of bond created from a chemical reaction, e.g., chemical
conjugation. In some embodiments, a linker (L or L′ as shown in any one of formulas (1)-(5), (D-I)-(D-
XI), (D′-I), (M-I)-(M-XI), or (M′-I)) includes no more than 250 atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12,
1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-
90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220,
1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120,
110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9,
8, 7, 6, 5, 4, 3, 2, or 1 atom(s)). In some embodiments, a linker (L or L) includes no more than 250
non-hydrogen atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40,
1-45, 1-50, 1-55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-
150, 1-160, 1-170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 non-hydrogen atom(s);
250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70,
65, 60, 55, 50, 45, 40, 35, 30, 28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 non-
hydrogen atom(s)). In some embodiments, the backbone of a linker (L or L) includes no more than 250
atoms (e.g., 1-2, 1-4, 1-6, 1-8, 1-10, 1-12, 1-14, 1-16, 1-18, 1-20, 1-25, 1-30, 1-35, 1-40, 1-45, 1-50, 1-
55, 1-60, 1-65, 1-70, 1-75, 1-80, 1-85, 1-90, 1-95, 1-100, 1-110, 1-120, 1-130, 1-140, 1-150, 1-160, 1-
170, 1-180, 1-190, 1-200, 1-210, 1-220, 1-230, 1-240, or 1-250 atom(s); 250, 240, 230, 220, 210, 200,
190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30,
28, 26, 24, 22, 20, 18, 16, 14, 12, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 atom(s)). The “backbone” of a linker
refers to the atoms in the linker that together form the shortest path from one part of the conjugate to
another part of the conjugate. The atoms in the backbone of the linker are directly involved in linking
one part of the conjugate to another part of the conjugate. For examples, hydrogen atoms attached to
carbons in the backbone of the linker are not considered as directly involved in linking one part of the
conjugate to another part of the conjugate.

(256) Molecules that may be used to make linkers (L or L′) include at least two functional groups, e.g.,
two carboxylic acid groups. In some embodiments of a trivalent linker, two arms of a linker may contain
two dicarboxylic acids, in which the first carboxylic acid may form a covalent linkage with the first
neuraminidase inhibitor in the conjugate and the second carboxylic acid may form a covalent linkage
with the second neuraminidase inhibitor in the conjugate, and the third arm of the linker may for a
covalent linkage (e.g., a C—O bond) with an Fc domain monomer, an Fc domain, an Fc-binding
peptide, an albumin protein, or an albumin protein-binding peptide in the conjugate. In some
embodiments of a divalent linker, the divalent linker may contain two carboxylic acids, in which the first
carboxylic acid may form a covalent linkage with one component (e.g., a neuraminidase inhibitor) in
the conjugate and the second carboxylic acid may form a covalent linkage (e.g., a C—S bond or a C—
N bond) with another component (e.g., an Fc domain monomer, an Fc domain, an Fc-binding peptide,
an albumin protein, or an albumin protein-binding peptide) in the conjugate.

(257) In some embodiments, dicarboxylic acid molecules may be used as linkers (e.g., a dicarboxylic
acid linker). For example, in a conjugate containing an Fc domain monomer, an Fc domain, an Fc-
binding peptide, an albumin protein, or an albumin protein-binding peptide covalently linked to one or
more dimers of neuraminidase inhibitors, the first carboxylic acid in a dicarboxylic acid molecule may
form a covalent linkage with a hydroxyl or amine group of the first neuraminidase inhibitor and the
second carboxylic acid may form a covalent linkage with a hydroxyl or amine group of the second
neuraminidase inhibitor.

(258) Examples of dicarboxylic acids molecules that may be used to form linkers include, but are not
limited to,

(259) ##STR00525## ##STR00526## ##STR00527##

wherein n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20).

(260) Other examples of dicarboxylic acids molecules that may be used to form linkers include, but are
not limited to,

(261) ##STR00528## ##STR00529## ##STR00530##

(262) In some embodiments, dicarboxylic acid molecules, such as the ones described herein, may be
further functionalized to contain one or more additional functional groups. Dicarboxylic acids may be
further functionalized, for example, to provide an attachment point to an Fc domain monomer, an Fc
domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide (e.g., by way
of a linker, such as a PEG linker).

(263) In some embodiments, when the neuraminidase inhibitor is attached to Fc domain monomer, an
Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide, the
linking group may comprise a moiety comprising a carboxylic acid moiety and an amino moiety that
are spaced by from 1 to 25 atoms. Examples of such linking groups include, but are not limited to,

(264) ##STR00531##

wherein n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20).

(265) In some embodiments, a linking group may include a moiety including a carboxylic acid moiety
and an amino moiety, such as the ones described herein, may be further functionalized to contain one
or more additional functional groups. Such linking groups may be further functionalized, for example,
to provide an attachment point to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an
albumin protein, or an albumin protein-binding peptide (e.g., by way of a linker, such as a PEG linker).

(266) In some embodiments, when the neuraminidase inhibitor is attached to Fc domain monomer, an
Fc domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide, the
linking group may comprise a moiety comprising two or amino moieties (e.g., a diamino moiety) that
are spaced by from 1 to 25 atoms. Examples of such linking groups include, but are not limited to,

(267) ##STR00532## ##STR00533##

wherein n is an integer from 1 to 20 (e.g., n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, or 20).

(268) In some embodiments, a linking group may include a diamino moiety, such as the ones
described herein, may be further functionalized to contain one or more additional functional groups.
Such diamino linking groups may be further functionalized, for example, to provide an attachment
point to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein, or an
albumin protein-binding peptide (e.g., by way of a linker, such as a PEG linker).

(269) In some embodiments, a molecule containing an azide group may be used to form a linker, in
which the azide group may undergo cycloaddition with an alkyne to form a 1,2,3-triazole linkage. In
some embodiments, a molecule containing an alkyne group may be used to form a linker, in which the
alkyne group may undergo cycloaddition with an azide to form a 1,2,3-triazole linkage. In some
embodiments, a molecule containing a maleimide group may be used to form a linker, in which the
maleimide group may react with a cysteine to form a C—S linkage. In some embodiments, a molecule
containing one or more sulfonic acid groups may be used to form a linker, in which the sulfonic acid
group may form a sulfonamide linkage with the linking nitrogen in a neuraminidase inhibitor. In some
embodiments, a molecule containing one or more isocyanate groups may be used to form a linker, in
which the isocyanate group may form a urea linkage with the linking nitrogen in a neuraminidase
inhibitor. In some embodiments, a molecule containing one or more haloalkyl groups may be used to
form a linker, in which the haloalkyl group may form a covalent linkage, e.g., C—N and C—O linkages,
with a neuraminidase inhibitor.

(270) In some embodiments, a linker (L or L′) may comprise a synthetic group derived from, e.g., a
synthetic polymer (e.g., a polyethylene glycol (PEG) polymer). In some embodiments, a linker may
comprise one or more amino acid residues. In some embodiments, a linker may be an amino acid
sequence (e.g., a 1-25 amino acid, 1-10 amino acid, 1-9 amino acid, 1-8 amino acid, 1-7 amino acid,
1-6 amino acid, 1-5 amino acid, 1-4 amino acid, 1-3 amino acid, 1-2 amino acid, or 1 amino acid
sequence). In some embodiments, a linker (L or L′) may include one or more optionally substituted C1-
C20 alkylene, optionally substituted C1-C20 heteroalkylene (e.g., a PEG unit), optionally substituted
C2-C20 alkenylene (e.g., C2 alkenylene), optionally substituted C2-C20 heteroalkenylene, optionally
substituted C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted
C3-C20 cycloalkylene (e.g., cyclopropylene, cyclobutylene), optionally substituted C3-C20
heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20
heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20
heterocycloalkynylene, optionally substituted C5-C15 arylene (e.g., C6 arylene), optionally substituted
C2-C15 heteroarylene (e.g., imidazole, pyridine), O, S, NR.sup.i (R.sup.i is H, optionally substituted
C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl,
optionally substituted C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally
substituted C2-C20 heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-
C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20
heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20
heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl), P,
carbonyl, thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino.

(271) Conjugation Chemistries

(272) Neuraminidase inhibitor monomer or dimers (e.g., in a conjugate of any one of formulas (1)-(5),
(D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) may be conjugated to an Fc domain monomer, an Fc
domain, an Fc-binding peptide, an albumin protein, or an albumin protein-binding peptide, e.g., by way
of a linker, by any standard conjugation chemistries known to those of skill in the art. The following
conjugation chemistries are specifically contemplated, e.g., for conjugation of a PEG linker (e.g., a
functionalized PEG linker) to an Fc domain monomer, an Fc domain, an Fc-binding peptide, an
albumin protein, or an albumin protein-binding peptide.

(273) Covalent conjugation of two or more components in a conjugate using a linker may be
accomplished using well-known organic chemical synthesis techniques and methods. Complementary
functional groups on two components may react with each other to form a covalent bond. Examples of
complementary reactive functional groups include, but are not limited to, e.g., maleimide and cysteine,
amine and activated carboxylic acid, thiol and maleimide, activated sulfonic acid and amine,
isocyanate and amine, azide and alkyne, and alkene and tetrazine. Site-specific conjugation to a
polypeptide (e.g., an Fc domain monomer, an Fc domain, an Fc-binding peptide, an albumin protein,
or an albumin protein-binding peptide) may be accomplished using techniques known in the art.
Exemplary techniques for site-specific conjugation of a small molecule to an Fc domain are provided in
Agarwall. P., et al. Bioconjugate Chem. 26:176-192 (2015).

(274) Other examples of functional groups capable of reacting with amino groups include, e.g.,
alkylating and acylating agents. Representative alkylating agents include: (i) an α-haloacetyl group,
e.g., XCH.sub.2CO— (where X═Br, Cl, or I); (ii) a N-maleimide group, which may react with amino
groups either through a Michael type reaction or through acylation by addition to the ring carbonyl
group; (iii) an aryl halide, e.g., a nitrohaloaromatic group; (iv) an alkyl halide; (v) an aldehyde or ketone
capable of Schiff's base formation with amino groups; (vi) an epoxide, e.g., an epichlorohydrin and a
bisoxirane, which may react with amino, sulfhydryl, or phenolic hydroxyl groups; (vii) a chlorine-
containing of s-triazine, which is reactive towards nucleophiles such as amino, sufhydryl, and hydroxyl
groups; (viii) an aziridine, which is reactive towards nucleophiles such as amino groups by ring
opening; (ix) a squaric acid diethyl ester; and (x) an α-haloalkyl ether.

(275) Examples of amino-reactive acylating groups include, e.g., (i) an isocyanate and an
isothiocyanate; (ii) a sulfonyl chloride; (iii) an acid halide; (iv) an active ester, e.g., a nitrophenylester or
N-hydroxysuccinimidyl ester, or derivatives thereof (e.g., azido-PEG.sub.2-PEG.sub.40-NHS ester);
(v) an acid anhydride, e.g., a mixed, symmetrical, or N-carboxyanhydride; (vi) an acylazide; and (vii)
an imidoester. Aldehydes and ketones may be reacted with amines to form Schiff's bases, which may
be stabilized through reductive amination.

(276) It will be appreciated that certain functional groups may be converted to other functional groups
prior to reaction, for example, to confer additional reactivity or selectivity. Examples of methods useful
for this purpose include conversion of amines to carboxyls using reagents such as dicarboxylic
anhydrides; conversion of amines to thiols using reagents such as N-acetylhomocysteine thiolactone,
S-acetylmercaptosuccinic anhydride, 2-iminothiolane, or thiol-containing succinimidyl derivatives;
conversion of thiols to carboxyls using reagents such as a -haloacetates; conversion of thiols to
amines using reagents such as ethylenimine or 2-bromoethylamine; conversion of carboxyls to amines
using reagents such as carbodiimides followed by diamines; and conversion of alcohols to thiols using
reagents such as tosyl chloride followed by transesterification with thioacetate and hydrolysis to the
thiol with sodium acetate.

(277) In some embodiments, a linker of the invention (e.g., L or L′, such as L.sup.c of D-L-I), is
conjugated (e.g., by any of the methods described herein) to E (e.g., an Fc domain or albumin
protein). In preferred embodiments of the invention, the linker is conjugated by way of: (a) a thiourea
linkage (i.e., —NH(C═S)NH—) to a lysine of E; (b) a carbamate linkage (i.e., —NH(C═O)—O) to a
lysine of E; (c) an amine linkage by reductive amination (i.e., —NHCH.sub.2) between a lysine and E;
(d) an amide (i.e., —NH—(C═O)CH.sub.2) to a lysine of E; (e) a cysteine-maleimide conjugate
between a maleimide of the linker to a cysteine of E; (f) an amine linkage by reductive amination (i.e.,
—NHCH.sub.2) between the linker and a carbohydrate of E (e.g., a glycosyl group of an Fc domain
monomer or an Fc domain); (g) a rebridged cysteine conjugate, wherein the linker is conjugated to two
cysteines of E; (h) an oxime linkage between the linker and a carbohydrate of E (e.g., a glycosyl group
of an Fc domain monomer or an Fc domain); (i) an oxime linkage between the linker and an amino
acid residue of E; (j) an azido linkage between the linker and E; (k) direct acylation of a linker to E; or
(I) a thioether linkage between the linker and E.

(278) In some embodiments, a linker is conjugated to E, wherein the linkage includes the structure —
NH(C═NH)X—, wherein X is O, HN, or a bond. In some embodiments, a linker is conjugated to E,
wherein the linkage between the remainder of the linker and E includes the structure —NH(C═O)NH
—. In some embodiments, a linker (e.g., an active ester, e.g., a nitrophenylester or N-
hydroxysuccinimidyl ester, or derivatives thereof (e.g., a functionalized PEG linker (e.g., azido-
PEG.sub.2-PEG.sub.40-NHS ester), is conjugated to E, with a T of (e.g., DAR) of between 0.5 and
10.0, e.g., 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5,
2.6, 2.7, 2.8, 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9,
5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,
7.3, 7.4, 7.5, 7.6, 7.7, 7.8.0, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5,
9.6, 9.7, 9.8, 9.9, or 10.0. In these instances, the E-(PEG.sub.2-PEG.sub.40)-azide can react with an
Int having a terminal alkyne linker (e.g., L, or L′, such as L.sup.c of D-L-I) through click conjugation.
During click conjugation, the copper-catalyzed reaction of the an azide (e.g., the Fc-(PEG.sub.2-
PEG.sub.40)-azide) with the alkyne (e.g., the Int having a terminal alkyne linker (e.g., L or L′, such as
L.sup.c of D-L-I) forming a 5-membered heteroatom ring. In some embodiments, the linker conjugated
to E is a terminal alkyne and is conjugated to an Int having a terminal azide. Exemplary preparations
of preparations of E-(PEG.sub.2-PEG.sub.40)-azide are described in Examples 7, 8, 61, 84, 88, and
124. Exemplary conjugates prepared through click conjugation are depicted in FIGS. 43, 61, and 102.
The click chemistry conjugation procedure is depicted in FIG. 103. One of skill in the art would readily
understand the final product from a click chemistry conjugation.

(279) Exemplary linking strategies (e.g., methods for linking a monomer or a dimer of a neuraminidase
inhibitor to E, such as, by way of a linker) are further depicted in FIGS. 1, 28, 29, 30, 43, and 61.

(280) VI. Combination Therapies

(281) Antiviral Agents

(282) In some embodiments, one or more antiviral agents may be administered in combination (e.g.,
administered substantially simultaneously (e.g., in the same pharmaceutical composition or in
separate pharmaceutical compositions) or administered separately at different times) with a conjugate
described herein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or
(M′-I)). The antiviral agent may be administered substantially simultaneously (e.g., in the same
pharmaceutical composition or in separate pharmaceutical compositions) as the conjugates, or may
be administered prior to or following the conjugates (e.g., within a period of 1 day, 2, days, 5, days, 1
week, 2 weeks, 3 weeks, 1 month, 2 months, 6 months, or 12 months, or more). In some
embodiments, the conjugate is administered by injection (e.g., intramuscularly, intradermally,
intranasally, or subcutaneously), and the antiviral agent is administered orally. Most preferably, the
conjugate is administered intravenously, and the antiviral agent is administered orally.

(283) In some embodiments, the conjugate is administered prophylactically (e.g., prior to the subject
coming into contact with the virus) and the antiviral agent is administered after the subject has a viral
infection, is presumed to have a viral infection, or has been exposed to the virus. In some
embodiments, the conjugate and the antiviral agent are both administered after the subject has a viral
infection, is presumed to have a viral infection, or has been exposed to the virus. In some
embodiments, the conjugate and the antiviral agent are both administered prophylactically.

(284) The conjugate and the antiviral agent may be formulated in the same pharmaceutical
composition or in separate pharmaceutical compositions. In preferred embodiments, the conjugate
and the antiviral agent is formulated in separate pharmaceutical compositions (e.g., formulated for
different routes of administration). In some embodiments, the conjugate and the antiviral agent are
administered simultaneously (e.g., at substantially the same time, such as within 5 minutes, 30
minutes, 1-6 hours, 1-12 hours, or 1 day) or sequentially (e.g., at different times, such as more than 1
day apart). Provided the antiviral agent and the conjugate are administered sequentially, the antiviral
agent is administered 1-50 (e.g., 1-15, 10-25, 20-35, 30-45, or 35-50) times after the administration of
the conjugate (e.g., administrations 1 day, 2, days, 5, days, 1 week, 2 weeks, 3 weeks, 1 month, 2
months, 6 months, or 12 months, or more after the conjugate).

(285) In some instances, an antiviral agent is administered to a subject in need thereof one or more
times (e.g., 1-10 times or more; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times) daily, weekly, monthly, biannually,
annually, or as medically necessary after the administration of a conjugate described herein (e.g., a
conjugate of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)).

(286) In some embodiments, the antiviral agent is an antiviral agent for the treatment of influenza
virus. For example, the antiviral agent may be an M2 ion channel blocker, a neuraminidase inhibitor
(e.g., a long-acting neuraminidase inhibitor), a polymerase inhibitor, a hemagglutinin inhibitor, a fusion
protein inhibitor, a COX-2 inhibitor, or a PPAR agonist. The antiviral agent may target either the virus
or the host subject. The antiviral agent for the treatment of influenza virus used in combination with a
conjugate described herein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-
(M-XI), or (M′-I)) may be selected from pimovidir, oseltamivir, zanamivir, peramivir, Ianinamivir, CS-
8958, amantadine, rimantadine, cyanovirin-N, a cap-dependent endonuclease inhibitor (e.g., baloxavir
marboxil), a polymerase inhibitor (e.g., T-705), a PB2 inhibitor (e.g., JNJ-63623872), a conjugated
sialidase (e.g., DAS181), a thiazolide (e.g., nitazoxanide), a COX inhibitor, a PPAR agonist, a
hemagglutinin-targeting antibody (e.g., a monoclonal antibody such as CR6261, CR8020, MED18852,
MHAA4549A, or VIS410), or an siRNA targeting a host or viral gene, or prodrugs thereof, or
pharmaceutically acceptable salts thereof.

(287) Preferably, the antiviral agent is directed to a different therapeutic target than the conjugate, for
example an M2 ion channel blocker, a polymerase inhibitor, a hemagglutinin inhibitor, a viral replication
inhibitor (e.g., a cap-dependent endonuclease inhibitor), a fusion protein inhibitor, a COX-2 inhibitor, or
a PPAR agonist. Most preferably, the antiviral agent is a cap-dependent endonuclease inhibitor (e.g.,
baloxavir marboxil). In some embodiments, the antiviral agent is administered in combination with a
conjugate described by formula (D-II-6). In some embodiments, the antiviral agent is administered in
combination with a conjugate described by formula (D-II-7). In preferred embodiments, an antiviral
agent (e.g., baloxavir marboxil) is administered in combination with a conjugate described by formula
(D-II-6). Most preferably, an antiviral agent (e.g., baloxavir marboxil) is administered in combination
with a conjugate described formula (D-II-7). More preferably, the conjugate is conjugate 45 or
conjugate 46.

(288) Baloxavir

(289) In some embodiments, Baloxavir marboxil (BXM, prodrug form) or baloxavir acid (BXA, active
form) or any salt thereof (Omoto et al. Scientific Reports. 8:9633, 2018; Japic CTI-153090; Japic CTI-
163417; each of which are incorporated herein by reference in their entirety) may be administered in
combination (e.g., administered substantially simultaneously (e.g., in the same pharmaceutical
composition or in separate pharmaceutical compositions) or administered separately at different times)
with a conjugate described herein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I),
(M-I)-(M-XI), or (M′-I)).

(290) ##STR00534##

(291) In some embodiments, Baloxavir marboxil, Baloxavir acid, or salt thereof is administered in a
dosage ranging from about 0.1 mg to about 3000 mg, preferably about 0.1 mg to about 1000 mg, most
preferable about 10 mg to about 100 mg (e.g., about 10 mg, about 20 mg, about 30 mg, about 40 mg,
about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, or about 100 mg) per adult a day,
if necessary, by division. In some embodiments, the Baloxavir marboxil, Baloxavir acid, or salt thereof
is administered at a decreased dose or frequency compared to standard of care when administered in
combination with the conjugate. The conjugate may be administered at a dose described herein.

(292) In some embodiments, Baloxavir marboxil, Baloxavir acid, or a salt thereof is administered more
frequently than the conjugate. For example, the conjugate may be administered once every 12
months, 6 months, 3 months, 2 months, 1 month, every 3 weeks, every 2 weeks, or weekly. The
Baloxavir marboxil, Baloxavir acid, or salt thereof may be administered three times daily, twice daily,
once daily, once every 2-6 days, once weekly, or once every two weeks. In some embodiments,
Baloxavir marboxil, Baloxavir acid, or salts thereof, are administered one or more times (e.g., 1-10
times or more; 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more) after (e.g., within 6 months, 3 months, 2
months, 1 month, 3 weeks, 2 weeks, or 1 week) the administration of a conjugate described herein.

(293) In some embodiments, Baloxavir marboxil, Baloxavir acid, or a salt thereof is administered orally.

(294) In some embodiments, Baloxavir marboxil, Baloxavir acid, or a salt thereof is administered, e.g.,
orally, in a dosage ranging from about 0.01 mg to about 1000 mg, preferably about 0.05 mg to about
500 mg, per day. Dosage forms and strengths for Baloxavir marboxil (XOFLUZA™) are well known,
with a single 40 mg oral dose for adults 40 to <80 kg and a single 80 mg oral dose for adults ≥80 kg.
Dosage forms and strengths for Baloxavir marboxil (XOFLUZA™) for pediatric subjects (e.g., subjects
≥12 years old and ≥40 kg) is well known, with a single 40 mg oral dose for pediatric subjects 40 to <80
kg and a single 80 mg oral dose for pediatric subjects >80 kg.

(295) When administered in combination with a conjugate of the present invention, the efficacy of
baloxavir (e.g., baloxavir marboxil, baloxavir acid, or a salt thereof) may be enhanced, e.g., by a
synergistic interaction of the baloxavir and the conjugate. This may permit the administration of
baloxavir at a reduced dose (e.g., relative to the present clinical standard of care) without any loss of
efficacy. This has the advantage of decreasding adverse events associate with administration of
baloxavir. In some embodiments, Baloxavir marboxil, Baloxavir acid, or a salt thereof is administered
in a reduced or subclinical dose (e.g., administered at a dose lower than without a conjugate described
herein and/or lower than the present clinical standard of care (e.g., a dose lower than 40 mg oral dose
(e.g., a dose ranging from 0.01 mg to 40 mg (e.g., 0.5 mg, 1.0 mg, 1.5 mg, 2.0 mg, 2.5 mg, 5.0 mg,
7.5 mg, 10 mg, 15 mg, 18 mg, 20 mg, 23 mg, 25 mg, 30 mg, 35 mg, or 38 mg oral dose))). Baloxavir
marboxil (XOFLUZA™) may be provided in any amount sufficient to treat an influenza viral infection in
a subject having previously been administered any conjugate described herein.

(296) Antiviral Vaccines

(297) In some embodiments, any one of conjugates described herein (e.g., a conjugate of any one of
formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) is administered in combination with an
antiviral vaccine (e.g., a composition that elicits an immune response in a subject directed against a
virus). The antiviral vaccine may be administered substantially simultaneously (e.g., in the same
pharmaceutical composition or in separate pharmaceutical compositions) as the conjugates, or may
be administered prior to or following the conjugates (e.g., within a period of 1 day, 2, days, 5, days, 1
week, 2 weeks, 3 weeks, 1 month, 2 months, 6 months, or 12 months, or more).

(298) In some embodiments the viral vaccine comprises an immunogen that elicits an immune
response in the subject against influenza virus A, B, C, or parainfluenza virus. In some embodiments
the immunogen is an inactivated virus (e.g., the vaccine is a trivalent influenza vaccine that contains
purified and inactivated material influenza virus A, B, C, or parainfluenza virus or any combination
thereof). In some embodiments the vaccine is given as an intramuscular injection. In some
embodiments, the vaccine is a live virus vaccine that contains live viruses that have been attenuated
(weakened). In some embodiments the vaccine is administered as a nasal spray.

(299) VII. Methods

(300) Methods described herein include, e.g., methods of protecting against or treating a viral infection
(e.g., an influenza viral infection) in a subject and methods of preventing, stabilizing, or inhibiting the
growth of viral particles. A method of treating a viral infection (e.g., an influenza viral infection) in a
subject includes administering to the subject a conjugate described herein (e.g., a conjugate of any
one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) or a pharmaceutical composition
thereof. In some embodiments, the viral infection is cause by the influenza virus (e.g., influenza virus
A, B, C, or parainfluenza virus). In some embodiments, the viral infection is caused by a resistant
strain of virus. A method of preventing, stabilizing, or inhibiting the growth of viral particles or
preventing the replication and spread of the virus includes contacting the virus or a site susceptible to
viral growth with a conjugate described herein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-
(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) or a pharmaceutical composition thereof.

(301) The disclosure also provides a method of protecting against or treating a viral infection (e.g., an
influenza viral infection) in a subject having or at risk of developing a secondary infection (e.g., a
secondary bacterial infection, a secondary viral infection, or a secondary fungal infection), wherein the
method includes administering to the subject a conjugate or composition described herein. The
disclosure further provides a method of preventing a secondary infection in a subject diagnosed with
an influenza infection, wherein the method includes administering to the subject a conjugate or
composition described herein. In some embodiments, the secondary infection is a bacterial infection
(e.g., methicillin-resistant Staphylococcus aureus (MRSA), Streptococcus pneumoniae, Pseudomonas
aeruginosa, and/or Haemophilus influenzae), a viral infection, or a fungal infection. In particular
embodiments, the secondary infection is MRSA. In certain embodiments, the secondary infection is S.
pneumoniae. In some embodiments, the secondary infection is a respiratory infection (e.g., an
infection of the respiratory tract). In some embodiments, the secondary infection is associated with
(e.g., causes) pneumonia (e.g., bacterial or viral pneumonia). In some embodiments, the subject has
or is at risk of developing pneumonia.
(302) Moreover, methods described herein also include methods of protecting against or treating viral
infection in a subject by administering to the subject a conjugate described herein (e.g., a conjugate of
any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)). In some embodiments, the
method further includes administering to the subject an antiviral agent or an antiviral vaccine.

(303) Methods described herein also include methods of protecting against or treating a viral infection
in a subject by administering to said subject (1) a conjugate described herein (e.g., a conjugate of any
one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) and (2) an antiviral agent or an
antiviral vaccine. Methods described herein also include methods of preventing, stabilizing, or
inhibiting the growth of viral particles or preventing the replication or spread of a virus, by contacting
the virus or a site susceptible to viral growth with (1) a conjugate described herein (e.g., a conjugate of
any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) and (2) an antiviral agent or an
antiviral vaccine.

(304) In some embodiments, the conjugate described herein (e.g., a conjugate of any one of formulas
(1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) is administered first, followed by administering of the
antiviral agent or antiviral vaccine alone. In some embodiments, the antiviral agent or antiviral vaccine
is administered first, followed by administering of the conjugate described herein alone. In some
embodiments, the conjugate described herein and the antiviral agent or antiviral vaccine are
administered substantially simultaneously (e.g., in the same pharmaceutical composition or in
separate pharmaceutical compositions). In some embodiments, the conjugate described herein or the
antiviral agent or antiviral vaccine is administered first, followed by administering of the conjugate
described herein and the antiviral agent or antiviral vaccine substantially simultaneously (e.g., in the
same pharmaceutical composition or in separate pharmaceutical compositions). In some
embodiments, the conjugate described herein and the antiviral agent or antiviral vaccine are
administered first substantially simultaneously (e.g., in the same pharmaceutical composition or in
separate pharmaceutical compositions), followed by administering of the conjugate described herein
or the antiviral agent or antiviral vaccine alone. In some embodiments, when a conjugate described
herein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) and
an antiviral agent or antiviral vaccine are administered together (e.g., substantially simultaneously in
the same or separate pharmaceutical compositions, or separately in the same treatment regimen),
inhibition of viral replication of each of the conjugate and the antiviral agent or antiviral vaccine may be
greater (e.g., occur at a lower concentration) than inhibition of viral replication of each of the conjugate
and the antiviral agent or antiviral vaccine when each is used alone in a treatment regimen.

(305) VII. Pharmaceutical Compositions and Preparations

(306) A conjugate described herein may be formulated in a pharmaceutical composition for use in the
methods described herein. In some embodiments, a conjugate described herein may be formulated in
a pharmaceutical composition alone. In some embodiments, a conjugate described herein may be
formulated in combination with an antiviral agent or antiviral vaccine in a pharmaceutical composition.
In some embodiments, the pharmaceutical composition includes a conjugate described herein (e.g., a
conjugate described by any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) and
pharmaceutically acceptable carriers and excipients.

(307) Acceptable carriers and excipients in the pharmaceutical compositions are nontoxic to recipients
at the dosages and concentrations employed. Acceptable carriers and excipients may include buffers
such as phosphate, citrate, HEPES, and TAE, antioxidants such as ascorbic acid and methionine,
preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride,
resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, dextran, and
immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acid residues such as
glycine, glutamine, histidine, and lysine, and carbohydrates such as glucose, mannose, sucrose, and
sorbitol.

(308) Examples of other excipients include, but are not limited to, antiadherents, binders, coatings,
compression aids, disintegrants, dyes, emollients, emulsifiers, fillers (diluents), film formers or
coatings, flavors, fragrances, glidants (flow enhancers), lubricants, sorbents, suspensing or dispersing
agents, or sweeteners. Exemplary excipients include, but are not limited to: butylated hydroxytoluene
(BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked
polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl
cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine,
methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, povidone,
pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl
cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid,
sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

(309) The conjugates herein may have ionizable groups so as to be capable of preparation as
pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or
organic acids or the salts may, in the case of acidic forms of the conjugates herein be prepared from
inorganic or organic bases. Frequently, the conjugates are prepared or used as pharmaceutically
acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases.
Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric,
sulphuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and
potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines, and the like
for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art.

(310) Representative acid addition salts include, but are not limited to, acetate, adipate, alginate,
ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate,
camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,
fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide,
hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate,
malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,
oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate,
propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and
valerate salts. Representative alkali or alkaline earth metal salts include, but are not limited to, sodium,
lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium,
and amine cations, including, but not limited to ammonium, tetramethylammonium,
tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

(311) Depending on the route of administration and the dosage, a conjugate herein or a
pharmaceutical composition thereof used in the methods described herein will be formulated into
suitable pharmaceutical compositions to permit facile delivery. A conjugate (e.g., a conjugate of any
one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) or a pharmaceutical composition
thereof may be formulated to be administered intramuscularly, intravenously (e.g., as a sterile solution
and in a solvent system suitable for intravenous use), intradermally, intraarterially, intraperitoneally,
intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally,
intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously,
subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally
(e.g., a tablet, capsule, caplet, gelcap, or syrup), topically (e.g., as a cream, gel, lotion, or ointment),
locally, by inhalation, by injection, or by infusion (e.g., continuous infusion, localized perfusion bathing
target cells directly, catheter, lavage, in cremes, or lipid compositions). Depending on the route of
administration, a conjugate herein or a pharmaceutical composition thereof may be in the form of, e.g.,
tablets, capsules, pills, powders, granulates, suspensions, emulsions, solutions, gels including
hydrogels, pastes, ointments, creams, plasters, drenches, osmotic delivery devices, suppositories,
enemas, injectables, implants, sprays, preparations suitable for iontophoretic delivery, or aerosols. The
compositions may be formulated according to conventional pharmaceutical practice.

(312) A conjugate described herein may be formulated in a variety of ways that are known in the art.
For use as treatment of human and animal subjects, a conjugate described herein can be formulated
as pharmaceutical or veterinary compositions. Depending on the subject (e.g., a human) to be treated,
the mode of administration, and the type of treatment desired, e.g., prophylaxis or therapy, a conjugate
described herein is formulated in ways consonant with these parameters. A summary of such
techniques is found in Remington: The Science and Practice of Pharmacy, 22nd Edition, Lippincott
Williams & Wilkins (2012); and Encyclopedia of Pharmaceutical Technology, 4th Edition, J. Swarbrick
and J. C. Boylan, Marcel Dekker, New York (2013), each of which is incorporated herein by reference.

(313) Formulations may be prepared in a manner suitable for systemic administration or topical or
local administration. Systemic formulations include those designed for injection (e.g., intramuscular,
intravenous or subcutaneous injection) or may be prepared for transdermal, transmucosal, or oral
administration. The formulation will generally include a diluent as well as, in some cases, adjuvants,
buffers, and preservatives. The conjugates can be administered also in liposomal compositions or as
microemulsions. Systemic administration may also include relatively noninvasive methods such as the
use of suppositories, transdermal patches, transmucosal delivery and intranasal administration. Oral
administration is also suitable for conjugates herein. Suitable forms include syrups, capsules, and
tablets, as is understood in the art.

(314) The pharmaceutical compositions can be administered parenterally in the form of an injectable
formulation. Pharmaceutical compositions for injection can be formulated using a sterile solution or
any pharmaceutically acceptable liquid as a vehicle. Formulations may be prepared as solid forms
suitable for solution or suspension in liquid prior to injection or as emulsions. Pharmaceutically
acceptable vehicles include, but are not limited to, sterile water, physiological saline, and cell culture
media (e.g., Dulbecco's Modified Eagle Medium (DMEM), α-Modified Eagles Medium (α-MEM), F-12
medium). Such injectable compositions may also contain amounts of nontoxic auxiliary substances
such as wetting or emulsifying agents, pH buffering agents, such as sodium acetate and sorbitan
monolaurate. Formulation methods are known in the art, see e.g., Pharmaceutical Preformulation and
Formulation, 2nd Edition, M. Gibson, Taylor & Francis Group, CRC Press (2009).

(315) The pharmaceutical compositions can be prepared in the form of an oral formulation.
Formulations for oral use include tablets containing the active ingredient(s) in a mixture with non-toxic
pharmaceutically acceptable excipients. These excipients may be, for example, inert diluents or fillers
(e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch,
calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium
phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline
cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding
agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch,
pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate,
carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose,
polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g.,
magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc).
Formulations for oral use may also be provided as chewable tablets, or as hard gelatin capsules
wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose,
microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules
wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid
paraffin, or olive oil. Powders, granulates, and pellets may be prepared using the ingredients
mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed
apparatus or a spray drying equipment.

(316) Other pharmaceutically acceptable excipients for oral formulations include, but are not limited to,
colorants, flavoring agents, plasticizers, humectants, and buffering agents. Formulations for oral use
may also be provided as chewable tablets, or as hard gelatin capsules wherein the active ingredient is
mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium
carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is
mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders,
granulates, and pellets may be prepared using the ingredients mentioned above under tablets and
capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying
equipment.

(317) Dissolution or diffusion controlled release of a conjugate described herein (e.g., a conjugate of
any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) or a pharmaceutical composition
thereof can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of
the conjugate, or by incorporating the conjugate into an appropriate matrix. A controlled release
coating may include one or more of the coating substances mentioned above and/or, e.g., shellac,
beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl
distearate, glycerol palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate
butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate,
methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydrogels, 1,3 butylene glycol, ethylene
glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix
material may also include, e.g., hydrated methylcellulose, carnauba wax and stearyl alcohol, carbopol
934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride,
polyethylene, and/or halogenated fluorocarbon.

(318) The pharmaceutical composition may be formed in a unit dose form as needed. The amount of
active component, e.g., a conjugate described herein (e.g., a conjugate of any one of formulas (1)-(5),
(D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)), included in the pharmaceutical compositions are such that a
suitable dose within the designated range is provided (e.g., a dose within the range of 0.01-100 mg/kg
of body weight).

(319) IX. Routes of Administration and Dosages

(320) In any of the methods described herein, conjugates herein may be administered by any
appropriate route for treating or protecting against a viral infection (e.g., an influenza infection), or for
preventing, stabilizing, or inhibiting the proliferation or spread of a virus (e.g., an influenza virus).
Conjugates described herein may be administered to humans, domestic pets, livestock, or other
animals with a pharmaceutically acceptable diluent, carrier, or excipient. In some embodiments,
administering comprises administration of any of the conjugates described herein (e.g., conjugates of
any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) or compositions intramuscularly,
intravenously (e.g., as a sterile solution and in a solvent system suitable for intravenous use),
intradermally, intraarterially, intraperitoneally, intralesionally, intracranially, intraarticularly,
intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally,
topically, intratumorally, peritoneally, subcutaneously, subconjunctival, intravesicularlly, mucosally,
intrapericardially, intraumbilically, intraocularally, orally (e.g., a tablet, capsule, caplet, gelcap, or
syrup), topically (e.g., as a cream, gel, lotion, or ointment), locally, by inhalation, by injection, or by
infusion (e.g., continuous infusion, localized perfusion bathing target cells directly, catheter, lavage, in
cremes, or lipid compositions). In some embodiments, if an antiviral agent is also administered in
addition to a conjugate described herein, the antiviral agent or a pharmaceutical composition thereof
may also be administered in any of the routes of administration described herein.

(321) The dosage of a conjugate described herein (e.g., a conjugate of any one of formulas (1)-(5), (D-
I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) or pharmaceutical compositions thereof depends on factors
including the route of administration, the disease to be treated (e.g., the extent and/or condition of the
viral infection), and physical characteristics, e.g., age, weight, general health, of the subject. Typically,
the amount of the conjugate or the pharmaceutical composition thereof contained within a single dose
may be an amount that effectively prevents, delays, or treats the viral infection without inducing
significant toxicity. A pharmaceutical composition may include a dosage of a conjugate described
herein ranging from 0.01 to 500 mg/kg (e.g., 0.01, 0.1, 0.2, 0.3, 0.4, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 100, 150, 200, 250, 300, 350, 400, 450, or 500 mg/kg) and, in a more specific
embodiment, about 0.1 to about 30 mg/kg and, in a more specific embodiment, about 1 to about 30
mg/kg. In some embodiments, when a conjugate described herein (e.g., a conjugate of any one of
formulas (1)-(5), (D-I)-(D-XI), (D′-I), (M-I)-(M-XI), or (M′-I)) and an antiviral agent or antiviral vaccine
are administered in combination (e.g., substantially simultaneously in the same or separate
pharmaceutical compositions, or separately in the same treatment regimen), the dosage needed of the
conjugate described herein may be lower than the dosage needed of the conjugate if the conjugate
was used alone in a treatment regimen.

(322) A conjugate described herein (e.g., a conjugate of any one of formulas (1)-(5), (D-I)-(D-XI), (D′-I),
(M-I)-(M-XI), or (M′-I)) or a pharmaceutical composition thereof may be administered to a subject in
need thereof, for example, one or more times (e.g., 1-10 times or more; 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
times) daily, weekly, monthly, biannually, annually, or as medically necessary. Dosages may be
provided in either a single or multiple dosage regimens. The timing between administrations may
decrease as the medical condition improves or increase as the health of the patient declines. The
dosage and frequency of administration may be adapted by the physician in accordance with
conventional factors such as the extent of the infection and different parameters of the subject.

EXAMPLES

(323) The following examples are put forth so as to provide those of ordinary skill in the art with a
description of how the compositions and methods described herein may be used, made, and
evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the
scope of what the inventors regard as their invention.

Example 1: Preparation of Fc Constructs

(324) Reverse translations of the amino acids comprising the protein constructs (SEQ ID NOs: 1, 3, 5,
7, 9, 12, and 14) were synthesized by solid-phase synthesis. The oligonucleotide templates were
cloned into pcDNA3.1 (Life Technologies, Carlsbad, Calif., USA) at the cloning sites BamHI and XhoI
(New England Biolabs, Ipswich, Mass., USA) and included signal sequences derived from the human
Interleukin-2 or human albumin. The pcDNA3.1 plasmids were transformed into Top10 E. coli cells
(LifeTech). DNA was amplified, extracted, and purified using the PURELINK® HiPure Plasmid Filter
Maxiprep Kit (LifeTech). The plasmid DNA is delivered, using the ExpiFectamine™ 293 Transfection
Kit (LifeTech), into HEK-293 cells per the manufacturer's protocol. Cells were centrifuged, filtered, and
the supernatants were purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA).
Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each
molecule into the gel, and staining using instant Blue staining. Each gel included a molecular weight
ladder with the indicated molecular weight standards (FIGS. 2-8). Reduced and non-reduced lanes are
denoted by “R” and “NR”. FIGS. 2-8 show non-reducing and reducing SDS-PAGE of an Fc domain
formed from Fc domain monomers having the sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 12, and 14,
respectively.

Example 2. Synthesis of Zanamivir Intermediate

(325) ##STR00535##

(326) Methyl 5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-

non-2-enonate (4.56 g, 10.0 mmol) was dissolved in anhydrous THF (15 mL), and the solution was
cooled to approximately 13° C. Triphenylphosphine (2.89 g, 11 mmol) was added in portions over 20
minutes. The resulting mixture was stirred at approximately 13° C. to room temperature for 2 hours,
then a solution of LiOH (24 mg, 1 mmol) in water (1.5 mL) was added dropwise. After stirring for 28
hours, the reaction mixture was added with N,N′-bis-boc-1-guanylpyrazole (3.26 g, 10.5 mmol) and 4-
dimethylaminopyridine (244.4 mg, 2 mmol). The reaction was stirred for 1.5 days. It was then diluted
with a 1:1 mixture of ethyl acetate: hexanes (100 mL) and extracted with water (30 mL). The aqueous
layer was back-extracted with ethyl acetate (30 mL). The combined organic layers were concentrated
by rotary evaporation. The residue was purified through C18 reversed phase column chromatography
(150 g, 25 to 70% acetonitrile and water). The collected fractions were concentrated by rotary
evaporation at room temperature. A cloudy aqueous solution resulted and the majority of the product
deposited on the flask as a gel. The solution was then extracted with ethyl acetate (150 mL). The
organic layer was used to re-dissolve the gel material. It was then dried over Na.sub.2SO.sub.4,
concentrated by rotary evaporation, and further dried under high vacuum to afford the title compound
as a white foam. Yield 6.24 g, 92.8%. Ion found by LCMS: [M+H].sup.+=673.2.

(327) Step b.

(328) ##STR00536##

(329) A solution of the product from step-a (6.24 g, 9.28 mmol) in anhydrous MeOH (20 mL) was
cooled in an ice-water bath and a 0.5 M solution of sodium methoxide in MeOH (26 mL, 13 mmol) was
slowly added. The reaction was stirred for 1 hour, then its pH was carefully adjusted to 7 to 7.5 by
dropwise addition of a 4 N solution of HCl in dioxane (3 mL). The solvent was removed by rotary
evaporation at a temperature not greater than room temperature. The residue was diluted with a 2:1
mixture of ethyl acetate and hexanes (150 mL), and the resulting solution was extracted with water (20
mL). The aqueous layer was back-extracted with ethyl acetate (30 mL). The combined organic layers
were dried over Na.sub.2SO.sub.4, concentrated by rotary evaporation, and further dried under high
vacuum. The product was carried to the subsequent step without further purification. Yield 5.03 g,
99.2%. Ion found by LCMS: [M+H].sup.+=547.2.

(330) Step c.

(331) ##STR00537##

(332) The step-b product (713 mg, 1.3 mmol) in anhydrous DCM (6 mL) was cooled in an ice-water
bath and 4-dimethylaminopyridine (159.8 mg, 1.3 mmol) and DIPEA (520 mg, 4 mmol) were added.
The mixture was then dropwise added with a solution of 4-nitrophenyl chloroformate (356.6 mg, 2.2
mmol) in anhydrous DCM (2 mL). The ice-water bath was then removed, and the reaction mixture was
stirred for 3 hours and monitored by LCMS (additional 4-nitrophenyl chloroformate may be added if
needed). After the reaction was complete, it was quenched with water (10 mL), and the organic layer
was extracted and concentrated by rotary evaporation. The residue was purified by C18 reversed
phase column chromatography (100 g, 20 to 70% acetonitrile and water). Acetonitrile in the collected
fractions was removed by rotary evaporation at room temperature. The aqueous layer was then
extracted with a 1:1 mixture of ethyl acetate and hexane (120 mL). The aqueous layer was back-
extracted with ethyl acetate (30 mL). The combined organic layers were dried over Na.sub.2SO.sub.4,
concentrated by rotary evaporation, and further dried under high vacuum to afford the title compound
as a white solid. Yield 520 mg, 70%. Ion found by LCMS: [M+H].sup.+=573.2.

Example 3. Synthesis of Linker-1

(333) Step a.

(334) ##STR00538##

(335) To a mixture of Z-D-glutamic γ-methyl ester (2.0 g, 6.77 mmol) and glycine methyl ester HCl
(1.282 g, 10.2 mmol) in anhydrous DMF (7 mL) was added DIPEA (2.02 g, 15.57 mmol) followed by
HATU (2.66 g, 7.0 mmol) in portions over 25 minutes. After HATU was dissolved, an additional amount
of DIPEA (1.15 g, 8.8 mmol) was added. The reaction mixture was stirred for 1.5 hours, then extracted
with 5% aqueous HCl (100 mL) and EtOAc (100 mL×2). The organic layer was concentrated by rotary
evaporation. The residue was re-extracted with water (100 mL) and EtOAc/hexanes (2:1, 150 mL).
The organic layer was dried over Na.sub.2SO.sub.4, concentrated by rotary evaporation and further
dried under high vacuum to a white solid. The crude product was carried on to the subsequent step
without further purification. Yield 2.3 g, 93%. Ion found by LCMS: [M+H].sup.+=367.

(336) Step b.

(337) ##STR00539##

(338) The step-a product (2.3 g, 6.29 mmol) was dissolved in a 1:1 mixture of MeOH and THE (10
mL).

(339) After the solution was cooled in an ice-water bath, a LiOH monohydrate (630 mg, 15 mmol)
solution in water (9 mL) was added in portions over 1.5 hours. After stirring for 2 additional hours, the
reaction mixture was neutralized with 4N HCl in dioxane (3.7 mL). The organic solvent was partially
removed by rotary evaporation at room temperature. The remaining material was directly purified by
RPLC (150 g, 0 to 39% acetonitrile and water). Yield 2.03 g, 88.7% over two steps. Ion found by
LCMS: [M+H].sup.+=339.2.

(340) Step c.
(341) ##STR00540##

(342) To a mixture of the step-b product (1.41 g, 4.17 mmol) and N-Boc-1,6-diaminohexane (1.99 g,
9.2 mmol) in anhydrous DMF (6 mL) and DIPEA (1.3 g, 10 mmol), a solution of HATU (3.5 g, 9.2
mmol) in DMF (10 mL) was added by way of syringe pump at a rate of 11 mL/hr. Upon complete
addition of the HATU, the reaction was stirred for 30 more minutes and directly purified by RPLC (150
g, 10 to 50% acetonitrile and water, using 0.1% TFA as modifier). Yield 1.3 g, 42.4%. Ion found by
LCMS: [M+H].sup.+=735, [M-Boc+H]+=635.4.

(343) Step d.

(344) ##STR00541##

(345) The step-c product (1.3 g, 1.77 mmol) was dissolved in MeOH (20 mL), and Pd/C was added to
the solution. The mixture was stirred under hydrogen for 4 hours. Pd/C was filtered off, and the filtrate
was concentrated by rotary evaporation and further dried under high vacuum. Yield 1.03 g, 96.9%. Ion
found by LCMS: [M+H].sup.+=601.

(346) Step e.

(347) ##STR00542##

(348) A flamed-dried reaction flask was flushed with nitrogen and charged with 4-azidobutyric acid (77
mg, 0.6 mmol), N-hydroxy succinimide (92 mg, 0.8 mmol) and anhydrous DMF (0.5 mL). The mixture
was stirred to dissolve the solids, and then DCC (125.5 mg, 0.608 mmol) was added. After stirring for
one hour, the step-d product (300 mg, 0.5 mmol) was added to the reaction mixture. The reaction was
stirred for 6 hours and directly purified using RPLC (150 g, 10 to 70% acetonitrile and water, using
01.% TFA as modifier). The collected fractions were lyophilized to a white solid (LCMS:
[M+H].sup.+=712, [M-Boc+H].sup.+=612). The material was re-dissolved in DCM (˜2 mL) and TFA (˜1
mL) and stirred for 15 minutes. It was then concentrated by rotary evaporation, and the residue was
purified by RPLC (50 g, 0 to 40% acetonitrile and water). Yield 255 mg, 68. 9%. Ions found by LCMS:
[M+H].sup.+=512, [(M+2H)/2].sup.+=256.

Example 4. Synthesis of Int-1

(349) ##STR00543##

(350) To a solution of the Zanamivir intermediate (Example 2) (532.5 mg, 0.93 mmol) in anhydrous
THE (2 mL) was added DMAP (490 mg, 4 mmol), followed by bis(pentafluorophenyl)carbonate (385
mg, 0.911 mmol). After stirring overnight, a solution of Linker-1 (Example 3) (245.6 mg, 0.332 mmol) in
anhydrous DMF (1 mL) and DIPEA (91 mg, 0.3 mmol) was added to the reaction mixture. The reaction
was continued for 2 hours, then purified by RPLC (100 g, 5 to 67% acetonitrile and water). Yield 135
mg, 23.8%. Ion found by LCMS: [(M+2H)/2].sup.+=845.9.

(351) Step b.

(352) ##STR00544##

(353) The step-a product (135 mg, 0.079 mmol) was dissolved in TFA (0.5 mL), and the solution was
stirred at room temperature for 20 minutes. It was then directly purified by RPLC (50 g, 5 to 32%
acetonitrile and water). Yield 88 mg, 85.1%. Ion found by LCMS: [(M+2H)/2].sup.+=654.8.

(354) Step c.

(355) ##STR00545##

(356) A solution of the step-b product (88 mg, 0.0673 mmol) in MeOH (1.5 mL) was cooled in an ice-
water bath and LiOH (5 mg, 0.2 mmol) in water (0.5 mL) was added. After the mixture was stirred for 5
hours, it was acidified with 4N HCl solution in dioxane (0.1 mL) and purified by HPLC (5 to 20%
acetonitrile and water, using 0.1% TFA as modifier). Yield 76.4 mg, 78%. Ion found by LCM:
[(M+2H)/2].sup.+=614.8.

Example 5. Synthesis of Linker-2

(357) ##STR00546##

(358) Step a.

(359) ##STR00547##

(360) To a solution of propargyl-PEG4-acid (364.4 mg, 1.4 mmol) in anhydrous DMF (2 mL) was
added HATU (558.9 mg, 1.47 mmol). After stirring to dissolve all the coupling reagent, DIPEA (390
mg, 3 mmol) was added and stirred for 10 minutes. A solution of the Linker-1 step-d product (Example
3, step d) (701.1 mg, 1.167 mmol) in anhydrous DMF (1 mL) was added. The resulting mixture was
stirred for 30 minutes and directly purified by RPLC (100 g, 5 to 60% acetonitrile and water). Yield 830
mg, 84.4%. Ion found by LCMS: [M+H].sup.+=843.

(361) Step b.

(362) ##STR00548##

(363) The step-a product was dissolved in THE (5 mL) and treated with 4N HCl solution in dioxane
(2.5 mL). After stirring at room temperature overnight, the reaction mixture was concentrated by rotary
evaporation. The residue was re-dissolved in acetonitrile/water (1:1, 16 mL), and the solution was
lyophilized. The crude product was carried on to the subsequent step without further purification. Yield
761.8 mg, 100%. Ion found by LCMS: [M+H].sup.+=643.8.

Example 6. Synthesis of Int-2

(364) ##STR00549##

(365) A flame-dried reaction flask was flushed with nitrogen and charged with Zanamivir intermediate
(Example 2) (533.6 mg, 0.812 mmol), DMAP (99.8 mg, 0.81 mmol), and anhydrous DCM (1 mL). After
stirring to dissolve the starting material, the solution was cooled in an ice-water bath and 4-nitrophenyl
chloroformate (242 mg, 1 0.2 mmol) was added. The resulting mixture was stirred for 5 hours, then
added into a solution of the Linker-2 (Example 5) (228.4 mg, 0.319 mmol) in anhydrous DMF (1 mL)
and DIPEA (130 mg, 1 mmol). The reaction was stirred overnight and purified by RPLC (150 g, 20 to
65% acetonitrile and water, using 0.1% TEA as modifier). The collected fractions were lyophilized.
Yield 178.3 mg, 30.4%. Ion found by LCMS: [(M+2H)/2].sup.+=920.5, [(M+3H)/3].sup.+=614.2.

(366) Step b.

(367) ##STR00550##

(368) The step-a product (178.3 mg, 0.0969 mmol) was dissolved in TFA (0.5 mL). The solution was
stirred for 20 minutes, then directly purified by HPLC (0 to 25% acetonitrile and water, using 0.1% TFA
as modifier). Yield 91.8 mg, 56.8%. Ions found by LCMS: [(M+2H)/2].sup.+=720.4,
[(M+3H)/3].sup.+=480.6.

(369) Step c.

(370) ##STR00551##

(371) The step-b product (91.8 mg, 0.055 mmol) was dissolved in MeOH (1 mL), and the solution was
cooled in an ice-water bath. LiOH monohydrate (21 mg, 0.5 mmol) in water (1 mL) was added in
portions over 1 hour. After stirring for 2 more hours, the reaction mixture was acidified with a 4N HCl
solution in dioxane (0.3 mL) and purified by HPLC (0 to 20% acetonitrile and water, using 0.1% TFA as
modifier). Yield 36.2 mg, 59.4%.%. Ions found by LCMS: [(M+2H)/2].sup.+=680.3,
[(M+3H)/3].sup.+=454.0.

Example 7. Synthesis of h-IgG1 Fc-PEG.SUB.4.-azide

(372) PEG4-azidoNHS ester (98%, 180 μmol, 9.5 equivalents, 71.4 mg in 0.5 mL of DMF and diluted
to 3.60 mL with pH 7.4 PBS 1× buffer solution) was added to a solution of h-IgG1 Fc (SEQ ID NO: 4)
(1103 mg in 70.0 mL of pH 7.4 PBS, MW-58,000 Da, 19.0 μmol) and the mixture was shaken gently
for 12 hours at ambient temperature. The solution was concentrated using a centrifugal concentrator
(30,000 MWCO) to a volume of ˜1.5 mL. The crude mixture was diluted 1:10 in PBS pH 7.4, and
concentrated again. This wash procedure was repeated for total of three times. The small molecule
reagent was removed with this wash procedure. The concentrated Fc-PEG4-azide was diluted to 70.0
mL with pH 7.4 PBS 1× buffer and ready for Click conjugation. The purified material was quantified
using a Nanodrop™ UV visible spectrophotometer (using a calculated extinction coefficient based on
the amino acid sequence of h-IgG1). Yield is quantitative after purification. DAR=4.3 determined by
MALDI. The DAR value can be adjusted by altering the equivalents of PEG4-azido NHS ester in near
linear relation. For example, when 7.0 equivalents of PEG4-azide NHS ester is used the DAR value
will be at 3.0.

(373) The nucleic acid construct encoding the Fc for any conjugate described herein may include a
nucleic acid sequence encoding for an Fc including the amino acid Lys447 (e.g., a C-terminal lysine
residue). Upon expression, the C-terminal lysine of the Fc is proteolytically cleaved, resulting in an Fc
having the sequence lacking Lys447 (e.g., lacking a C-terminal lysine residue). The presence or
absence of a C-terminal lysine does not alter the properties of the Fc or the corresponding conjugate.

Example 8. Synthesis of Recombinant Mouse Serum Albumin (MSA)-PEG4-Azide

(374) PEG4-azidoNHS ester (98%, 81.7 μmol, 4.5 equivalents, 32.4 mg in 0.3 mL of DMF and diluted
to 1.63 mL with pH 7.4 PBS 1× buffer solution) was added to a solution of recombinant mouse serum
albumin (SEQ ID NO: 80) (1200 mg in 75.0 mL of pH 7.4 PBS, MW-66,000 Da, 18.2 μmol) and the
mixture was shaken gently for 12 hours at ambient temperature. The solution was concentrated using
a centrifugal concentrator (30,000 MWCO) to a volume of ˜1.5 mL. The crude mixture was diluted 1:10
in PBS pH 7.4, and concentrated again. This wash procedure was repeated for total of three times.
The small molecule reagent was removed with this wash procedure. The concentrated MSA-PEG4-
azide was diluted to 75.0 mL with pH 7.4 PBS 1× buffer and ready for Click conjugation. The purified
material was quantified using a Nanodrop™ UV visible spectrophotometer (using a calculated
extinction coefficient based on the amino acid sequence of h-IgG1). Yield is quantitative after
purification. DAR=3.5 determined by MALDI. The DAR value can be adjusted by altering the
equivalents of PEG4-azido NHS ester similar to h-IgG1 Fc (Example 7).

Example 9. Synthesis of Conjugate 1

(375) A PBS solution of h-IgG1 Fc-PEG4-azide (Example 7) (50 mg, 2.815 mL, 0.8591 μmol) was
added into a 15 mL centrifuge tube containing Int-2 (Example 6) (35.2 mg, 0.02217 mmol). After the
mixture was gently shaken to dissolve all Int-2, 344 μl of a solution of L-ascorbic acid sodium (59.4
mg, 0.3 mmol), copper (II) sulfate (10 mg, 0.05 mmol), and THPTA (23 mg, 0.05 mmol) in PBS 7.4
buffer (1 mL) was added. The resulting mixture was gently shaken overnight. It was purified by affinity
chromatography over a protein A column, followed by size exclusion chromatography as described in
Example 8. Maldi TOF analysis of the purified final product gave an average mass of 63797 Da
(DAR=3.4). Yield 27.39 mg, 55% yield. FIG. 9 shows a non-reducing SDS-PAGE of Conjugate 1.

Example 10. Purification of Conjugates Crude mixture was diluted 1:10 in PBS pH 7.4, and purified
using MabSelect Sure Resin (GE

(376) Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. (HiLoad 26/600
Superdex200 pg, GE Healthcare, Chicago, Ill., USA). Fractions containing purified conjugate were
pooled and concentrated to approximately 20 mg/mL using a centrifugal concentrator (30,000
MWCO). Purified material was quantified using a Nanodrop™ UV visible spectrophotometer using a
calculated extinction coefficient based on the amino acid sequence of hIgG1 Fc(myc). Purified
molecules were analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1 μg of each molecule into
the gel, and staining using Instant Blue (Expedeon, San Diego, Calif., USA). Each gel included a
molecular weight ladder with the indicated molecular weight standards. Yields were calculated and
purity determined by Agilent Analytical HPLC. Product peak and MW were found by Maldi MS and a
final DAR calculated.

Example 11. Synthesis of Int-3

(377) ##STR00552## ##STR00553##

(378) To a solution of propargyl-PEG4-acid (260 mg, 1 mmol) and HATU (380.2 mg, 1 mmol) in
anhydrous DMF (1 mL) was added DIPEA (130 mg). After stirring 5 minutes, NH-bis(PEG3-Boc) (500
mg, 0.881 mmol) was added and stirring was continued at room temperature overnight. It was then
directly purified by RPLC 9100 g, 5 to 50% acetonitrile and water, using 0.1% TFA as modifier). Yield
683 mg, 95.8%. Ions found by LCMS: [M+H].sup.+=810.4, [M-Boc+H].sup.+=710.4, [(M-
2Boc+2H)/2].sup.+=305.8.

(379) Step b.

(380) ##STR00554##

(381) The step-a product was dissolved in TFA (1 mL). The solution was stirred for 2 hours and then
directly purified through RPLC (100 g, 0 to 30% acetonitrile and water). Yield 589 mg, 98.7%. Ion
found by LCMS: [(M+2H)/2].sup.+=305.8.

(382) Step c.

(383) ##STR00555##

(384) A flame-dried reaction flask was flushed with nitrogen and charged with Zanamivir intermediate
(Example 2) (572 mg, 1 mmol) and anhydrous DCM (1 mL). After stirring to dissolve the starting
material, the solution was cooled in an ice-water bath and 4-nitrophenyl chloroformate (302.4 mg, 1.5
mmol) was added followed by DMAP (22.4 mg, 0.2 mmol). The resulting mixture was stirred for 5
hours, then quenched water (0.2 mL) was added. After stirring for 10 minutes, the step-b product
(256.7 mg, 0.355 mmol) in anhydrous DMF (1 mL) and DIPEA (163.8 mg, 1.26 mmol) was added.
Stirring was continued for 2 hours and then the reaction was directly purified by RPLC (150 g, 20 to
65% acetonitrile and water, using 0.1% TFA as modifier). The collected fractions were lyophilized.
Yield 422.8 mg of the desired product, which was contaminated with some impurities, <69% yield. The
material was carried on to the subsequent step without further purification. Ion found by LCMS:
[(M+2H)/2].sup.+=903.9, [(M+3H)/3].sup.+=603.2.

(385) Step d.

(386) ##STR00556##

(387) The step-c product (422.8 mg, <0.245 mmol) was dissolved in TFA (1 mL). The solution was
stirred for 20 minutes, then directly purified by HPLC (5 to 25% acetonitrile and water, using 0.1% TFA
as modifier). Yield 169.7 mg, 29.2% over two steps. Ions found by LCMS: [(M+2H)/2].sup.+=704.0,
[(M+3H)/3].sup.+=469.6.

(388) Step e.

(389) ##STR00557##
(390) The step-d product (169.7 mg, 0.0923 mmol) was dissolved in MeOH (1.5 mL), and the solution
was cooled in an ice-water bath. LiOH monohydrate (21 mg, 0.5 mmol) in water (1 mL) was added in
portions over 1 hour. After stirring overnight, the reaction mixture was acidified with Dowex 50W×8
hydrogen form and purified through RPLC (0 to 30% acetonitrile and water, using 0.1% TFA as
modifier). Yield 107.9 mg, 66.9%. Ions found by LCMS: [(M+2H)/2].sup.+=663.8,
[(M+3H)/3].sup.+=442.9.

Example 12. Synthesis of Conjugate 2

(391) The title conjugate was prepared analogously to Conjugate 1 (Example 9) using Int-3 (Example
11). Maldi TOF analysis of the purified final product gave an average mass of 63561 Da (DAR=3.3).
Yield 43.4 mg, 43% yield. FIG. 10 shows a non-reducing SDS-PAGE of Conjugate 2.

Example 13. Synthesis of Int-4

(392) ##STR00558##

(393) Step a.

(394) ##STR00559##

(395) A flame-dried reaction flask was flushed with nitrogen and charged with Zanamivir Intermediate
(Example 2) (343.2 mg, 0.6 mmol) and anhydrous DCM (1.5 mL). The solution was cooled in an ice-
water bath and added with DIPEA (234 mg, 1.8 mmol) followed by 4-nitrophenyl chloroformate (121
mg, 0.6 mmol) and DMAP (67.4 mg, 0.6 mmol). The resulting mixture was stirred for 15 minutes, then
added with an additional amount of 4-nitrophenyl chloroformate (121 mg, 0.6 mmol). After stirring for 2
hours, water (0.2 mL) was added to quench unreacted chloroformate. After stirred for 10 minutes, the
reaction mixture was added with a solution of propargyl-PEG4-amine (185 mg, 0.8 mmol) in
anhydrous DMF (0.5 mL). The reaction was continued for 1 hour and then directly purified through
RPLC (100 g, 5 to 60% acetonitrile and water, using 0.1% TFA as modifier). The collected fractions
were lyophilized. Yield 355 mg, 71.3%. Ion found by LCMS: [M+H].sup.+=830.2.

(396) Step b.

(397) ##STR00560##

(398) The step-a product (355 mg, 0.428 mmol) was dissolved in TFA (1 mL). The solution was stirred
overnight, then directly purified by RPLC (5 to 25% acetonitrile and water, using 0.1% TFA as
modifier). Yield 260.2 mg, 70.9% over two steps. Ion found by LCMS: [M+H].sup.+=630.2.

(399) Step c.

(400) ##STR00561##

(401) The step-b product (260.2 mg, 0.303 mmol) was dissolved in MeOH (1.5 mL). After the solution
was cooled in an ice-water bath, a solution of LiOH monohydrate (42 mg, 1 mmol) in water (1 mL) was
added in portions over 1 hour. The reaction was stirred overnight, then acidified with Dowex 50W×8
hydrogen form and purified by RPLC (0 to 50% acetonitrile and water, using 0.1% TFA as modifier).
Yield 78.1 mg, 99%. Ions found by LCMS: [M+H].sup.+=590.2.

Example 14. Synthesis of Conjugate 3

(402) The title conjugate was prepared analogously to Conjugate 1 (Example 9) using Int-4 (Example
13). Maldi TOF analysis of the purified final product gave an average mass of 61182 Da (DAR=3.4).
Yield 50.89 mg, 51% yield. FIG. 11 shows a non-reducing SDS-PAGE of Conjugate 3.

Example 15. Synthesis of Int-5

(403) ##STR00562## ##STR00563##

(404) A flame-dried reaction flask was flushed with nitrogen and charged with (1S, 2S, 3R, 4R)-methyl
3-((S)-1-acetamido-2-ethylbutyl)-4-(tert-butoxycarbonylamino)-2-hydroxycyclopentanecarboxylate
(320.4 mg, 0.8 mmol) and anhydrous DCM (2 mL). The solution was cooled in an ice-water bath and
DIPEA (312 mg, 2.4 mmol) was added followed by 4-nitrophenyl chloroformate (161.3 mg, 0.8 mmol)
and DMAP (98 mg, 0.8 mmol). The resulting mixture was stirred for 15 minutes, then an additional
amount of 4-nitrophenyl chloroformate (161.3 mg, 0.8 mmol) was added. The reaction was stirred for 2
hours, then water (0.2 mL) was added to quench unreacted chloroformate. After stirring for 10
minutes, a solution of propargyl-PEG4-amine (259 mg, 1.12 mmol) in anhydrous DMF (0.5 mL) was
added. The reaction was stirred for 1 hour and then purified directly by RPLC (100 g, 5 to 60%
acetonitrile and water, using 0.1% TFA as modifier). The collected fractions were lyophilized. Yield
391.2 mg, 74.4%. Ion found by LCMS: [M+H].sup.+=658.3, [M-Boc+H].sup.+=558.3.

(405) Step b.

(406) ##STR00564##

(407) The step-a product (391.2 mg, 0.595 mmol) was dissolved in TFA (0.8 mL), and the solution was
stirred at room temperature for 20 minutes. It was then directly purified by RPLC (100 g, 5 to 60%
acetonitrile and water). Yield 323.2 mg, 81%. Ion found by LCMS: [M+H].sup.+=558.3.

(408) Step c.

(409) ##STR00565##

(410) To a solution of the step-b product (323.2 mg, 0.482 mmol) in THE (2 mL) was added N,N′-Bis-
Boc-1-guanylpyrazole (224.4 mg, 0.723 mmol) and PIPEA (260 mg, 2 mmol). The reaction mixture
was stirred for 1 day and then extracted with water (3 mL) and EtOAc/hexanes (1:1, 8 mL). The
organic layer was dried over Na.sub.2SO.sub.4 and concentrated by rotary evaporation. The residue
was re-dissolved in TFA (˜1 mL), and the solution was stirred overnight. It was then directly purified by
RPLC (100 g, 0 to 30% acetonitrile and water, using 0.1% TFA as modifier). The collected fractions
were lyophilized. Yield 254.2 mg, 83.2%. Ions found by LCMS: [M+H].sup.+=600.3, [M-
Boc+H].sup.+=300.6.

(411) Step d.

(412) ##STR00566##

(413) The step-c product (254.2 mg, 0.356 mmol) was dissolved in THE (1.5 mL), and the solution was
cooled in an ice-water bath. CaCl.sub.2 dihydrate (419 mg, 2.85 mmol) was added, then 1.8 mL of
KOH (112 mg, 2 mmol) in water (2 mL) was added in portions over 1 hour. After stirred for 3 more
hours, the reaction mixture was acidified by Dowex 50W×8 hydrogen form and purified by RPLC (0 to
30% acetonitrile and water, using 0.1% TFA as modifier). Yield 102 mg, 49.8%. Ions found by LCMS:
[M+H].sup.+=586.4, [(M+2H)/2].sup.+=293.8.

Example 16. Synthesis of Conjugate 4

(414) The title conjugate was prepared analogously to Conjugate 1 (Example 9) using Int-5 (Example
15). Maldi TOF analysis of the purified final product gave an average mass of 63002 Da (DAR=3.4).
Yield 49.315 mg, 49% yield. FIG. 12 shows a non-reducing SDS-PAGE of Conjugate 4.

Example 17. Synthesis of Int-6

(415) ##STR00567##

(416) Step a.
(417) ##STR00568##

(418) A flame-dried reaction flask was flushed with nitrogen and charged with (1S, 2S, 3R, 4R)-methyl
3-((S)-1-acetamido-2-ethylbutyl)-4-(tert-butoxycarbonylamino)-2-hydroxy cyclopentanecarboxylate
(280.4 mg, 0.7 mmol) and anhydrous DCM (1 mL). After stirring to dissolve the starting material,
DMAP (85.7 mg, 0.7 mmol) was added to the solution, followed by bis(pentafluorophenyl)carbonate
(295.6 mg, 0.75 mmol). The resulting mixture was stirred for 1 hour, then added into a solution of
Linker-2 (Example 5) (157.5 mg, 0.22 mmol) in anhydrous DMF (1 mL) and DIPEA (130 mg, 1 mmol).
The reaction was stirred overnight and purified by RPLC (50 g, 5 to 90% acetonitrile and water). The
collected fractions were lyophilized. Yield 134.1 mg, 40.7%. Ion found by LCMS:
[(M+2H)/2].sup.+=748.6.

(419) Step b.

(420) ##STR00569##

(421) The step-a product (134.1 mg, 0.0896 mmol) was dissolved in TFA (0.5 mL). The solution was
stirred for 20 minutes, then directly purified by RPLC (50 g, 5 to 60% acetonitrile and water). Yield
108.4 mg, 93.4%. Ions found by LCMS: [(M+2H)/2].sup.+=648.3, [(M+3H)/3].sup.+=432.8.

(422) Step c.

(423) ##STR00570##

(424) To a solution of the step-b product (108.4 mg, 0.0837 mmol) in anhydrous THE (1 mL) was
added N,N′-bis-boc-1-guanylpyrazole (81.3 mg, 0.285 mmol) and DIEPA (65 mg, 0.5 mmol). The
reaction was stirred at room temperature for 2.5 days, then directly purified by RPLC (100 g, 40 to
75% acetonitrile and water). Yield 73.6 mg, 49.4%. Ion found by LCMS: [(M+3H)/3].sup.+=594.2.

(425) Step d.

(426) ##STR00571##

(427) The step-c product (73.6 mg, 0.041 mmol) was dissolved in TFA (0.5 mL). The solution was
stirred for 20 minutes, then directly purified by RPLC (50 g, 5 to 60% acetonitrile and water). Yield 62.1
mg, 94.1%. Ions found by LCMS: [(M+2H)/2].sup.+=690.4, [(M+3H)/3].sup.+=460.8.

(428) Step e.

(429) ##STR00572##

(430) The step-d product (62.1 mg, 0.0386 mmol) in THE (3 mL) was cooled in an ice-water bath and
a 45% w/w solution of KOH (0.2 mL) was added in portions over 1 hour. The reaction was stirred for 2
more hours, then acidified with 4N HCl solution in dioxane (0.8 mL) and extracted with hexanes (10
mL) and water (1.5 mL). The aqueous layer was purified by HPLC (0 to 20% acetonitrile and water,
using 0.1% TFA as modifier). Yield 36.2 mg, 59.4%. Ions found by LCMS: [(M+2H)/2].sup.+=676.5,
[(M+3H)/3].sup.+=451.4.

Example 18. Synthesis of Conjugate 5

(431) The title conjugate was prepared analogously to Conjugate 1 (Example 9) using Int-6 (Example
17). Maldi TOF analysis of the purified final product gave an average mass of 63561 Da (DAR=3.3).
Yield 43.4 mg, 43% yield. FIG. 13 shows a non-reducing SDS-PAGE of Conjugate 5.

Example 19. Synthesis of Int-7

(432) ##STR00573## ##STR00574##

(433) Step a.

(434) ##STR00575##

(435) To a solution of propargyl-PEG4-acid (609 mg, 2.34 mmol) and NH-bis(PEG1-azide) (500 mg,
2.055 mmol) in anhydrous DMF (2 mL) was added HATU (889.7 mg, 2.34 mmol) in portions over 5
minutes. After stirring to dissolve all the coupling reagent, DIPEA (390 mg, 3 mmol) was added and
stirring continued for 1 hour. It was then purified directly by RPLC (100 g, 5 to 40% acetonitrile and
water). Yield 918 mg, 92%. Ion found by LCMS: [M+H].sup.+=486.2.

(436) Step b.

(437) ##STR00576##

(438) The step-a product (918 mg, 1.89 mmol) was dissolved in THF, and the solution was cooled to
13° C. Triphenylphosphine (1.141 g, 4.35 mmol) was added in portions over 10 minutes. The resulting
mixture was stirred at ˜ 13° C. to room temperature for 2 hours. A solution of LiOH monohydrate (42
mg, 1 mmol) in water (2 mL) and MeOH (1 mL) was added. After stirring was continued for 20 hours,
the reaction was purified by RPLC (100 g, 0 to 30% acetonitrile and water, using 0.1% TFA as
modifier). Yield 825 mg, 65.9%. Ion found by LCMS: [M+H].sup.+=434.4.

(439) Step c.

(440) ##STR00577##

(441) A flame-dried reaction flask was flushed with nitrogen and charged with Zanamivir intermediate
(Example 2) (865 mg, 1.51 mmol) and anhydrous DCM (5 mL). After stirring to dissolve the starting
material, the solution was cooled in an ice-water bath and 4-nitrophenyl chloroformate (365.3 mg, 1.81
mmol) was added followed by DMAP (152.2 mg, 0.755 mmol). The ice-water bath was removed, and
the mixture was stirred for 3 hours. An additional amount of 4-nitrophenyl chloroformate (304.4 mg,
1.51 mmol) was added, and stirring was continued for 1 hour. The reaction was then quenched with
water (1 mL). After vigorously stirred for 1 hour, the reaction mixture was extracted with water (20
mL×2) and DCM (20 mL). The organic layer was stirred overnight, dried over Na.sub.2SO.sub.4, and
concentrated by rotary evaporation. The material was carried on to the subsequent step without further
purification. Ion found by LCMS: [M+H].sup.+=738.2.

(442) Step d.

(443) ##STR00578##

(444) A solution of a mixture of the step-b product (429.7 mg, 0.65 mmol) and DIPEA (260 mg, 2
mmol) in anhydrous THE (1 mL) was added dropwise to the step-c product. The resulting mixture was
stirred for 2 hours, then directly purified by RPLC (100 g, 10 to 65% acetonitrile and water).
Acetonitrile in the collected fractions was removed by rotary evaporation at room temperature. The
heterogeneous aqueous layer was extracted with EtOAc (200 mL), then back-extracted with EtOAc
(50 mL). The combined organic layers were dried over Na.sub.2SO.sub.4 and concentrated by rotary
evaporation to dryness. Yield 442 mg, 41.7%. Ions found by LCMS: [(M+2H)/2].sup.+=815.8, [(M-
Boc+2H)/2].sup.+=765.8, [(M-2Boc+2H)/2].sup.+=716.

(445) Step e.

(446) ##STR00579##

(447) The step-d product (441 mg, 0.271 mmol) was dissolved in TFA (1 mL). The solution was stirred
for 20 minutes, then directly purified by HPLC (5 to 20% acetonitrile and water, using 0.1% TFA as
modifier). Yield 308 mg, 77.9%. Ions found by LCMS: [(M+2H)/2].sup.+=615.8, [(M+3H)/3].sup.+=411.

(448) Step f.
(449) ##STR00580##

(450) The step-e product (308 mg, 0.211 mmol) was dissolved in MeOH (1 mL) and water (0.5 mL),
and the solution was cooled in an ice-water bath. A solution of LiOH monohydrate (42 mg, 1 mmol) in
water (1 mL) was added in portions over 1 hour. The reaction was stirred overnight, acidified by 4 N
HCl solution in dioxane (0.25 mL), and purified by RPLC (0 to 20% acetonitrile and water, using 0.1%
TFA as modifier). Yield 198 mg, 64%. Ions found by LCMS: [(M+2H)/2].sup.+=575.8,
[(M+3H)/3].sup.+=384.2.

Example 20. Synthesis of Conjugate 6

(451) The title conjugate is prepared analogously to Conjugate 1 (Example 9) using Int-7 (Example 19)
(SEQ ID NO: 18). Maldi TOF analysis of the purified final product gave an average mass of 62854. Da
(DAR=3.1). Yield 175.4 mg, 50% yield. FIG. 14 shows a non-reducing SDS-PAGE of Conjugate 6. The
resulting conjugate is depicted in FIG. 43.

Example 21. Synthesis of Int-1

(452) ##STR00581##

(453) Step a.

(454) ##STR00582##

(455) Methyl 5-acetoamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-

non-2-enonate (1.8 g, 4.0 mmol) was dissolved into 40 mL methanol and then treated with 400 mg of
5% Pd/C and 1.1 g Boc anhydride (5.0 mmol), then the reaction mixture was stirred at room
temperature under a hydrogen atmosphere for 1 hour. The palladium-charcoal was removed by
filtration. The filtrate was concentrated and used in next step without purification.

(456) Step b.

(457) ##STR00583##

(458) The product from the previous step was dissolved into 20 mL dry methanol and then treated with
2 mL sodium methoxide in methanol (0.5 M) dropwise with cooling in an ice-water bath. After 2 hours,
the progress of reaction was determined by LCMS. The reaction was quenched with 1 N HCl to pH 5-
6. The resulting solution was concentrated and purified by reversed phase liquid chromatography
(RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 5% to 100% acetonitrile and
water, using 0.1% TFA as the modifier. Ion(s) found by LCMS: (M+H).sup.+=405

(459) Step c.

(460) ##STR00584##

(461) Methyl 5-acetamido-2,6-anhydro-4-[(tert-butoxycarbonyl)amino]-3,4,5-trideoxy-D-erythro-non-2-

enonate (0.4 g, 1 mmol) was dissolved in 10 mL of acetone, 4 mL of 2,2-dimethoxypropane and 20 mg
of p-toluenesulfonic acid hydrate (0.1 mmol). The resulting solution was stirred at room temperature
overnight, then quenched with 1 mL saturated NaHCO.sub.3. The mixture was concentrated and
purified by reversed phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid
chromatograph eluted with 5% to 100% acetonitrile and water without modifier. Ion(s) found by LCMS:
(M+H).sup.+=445.

(462) Step d.

(463) ##STR00585##
(464) Sodium hydride (40 mg, 60% in oil, 1.0 mmol) was added into methyl 5-acetamido-2,6-anhydro-
4-[(tert-butoxycarbonyl)amino]-3,4,5-trideoxy-8,9-O-(1-methylethylidene)-D-erythro-non-2-enonate
(0.25 g, 0.50 mmol) in 5 mL dry THE with cooling from an ice-water bath. The resulting solution was
stirred for 0.5 hour, then benzyl bromoacetate (0.23 g, 1.0 mmol) was added. The resulting solution
was stirred for 2 hours and quenched with 5 mL 10% ammonium chloride in water. Then the solution
was diluted with 50 mL ethyl acetate. The organic layer was separated and dried with sodium sulfate.
The dried organic solution was concentrated and purified by reversed phase liquid chromatography
(RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 5% to 100% acetonitrile and
water without modifier. Ion(s) found by LCMS: (M+H).sup.+=593.

Example 22. Synthesis of Int-9

(465) ##STR00586## ##STR00587## ##STR00588##

(466) Step a.

(467) ##STR00589##

(468) Methyl 5-acetoamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-

non-2-enonate (10 g, 22 mmol) was dissolved into 100 ml methanol and then heated to 60° C. with an
oil bath, the SnCl.sub.2 (5.7 g, 20 mmol) was added to the solution in 3 portions (caution, gas
evolves). The reaction mixture was stirred for 10 min, at which time the reaction was complete by
HPLC. The reaction solution was slowly added to a solution of 50 ml Sat NaHCO.sub.3 and 50 g celite
with vigorous stirring. The resulting slurry was filtered. The filtrate was treated with Boc.sub.2O (6.6 g,
30 mmol, 1.5 equiv). After 2 hour at room temperature, the solution was concentrated to remove most
of the methanol, dissolved in 200 ml DCM, and extracted twice with 100 ml DCM. The combined
extracts were dried with sodium sulfate, filtered and used for next step without further purification.
Crude yield 12 g, 100%. Ion(s) found by LCMS: M+H=531.

(469) Step b.

(470) ##STR00590##

(471) The material from the previous step was dissolved into 60 ml dry methanol, then treated with 10
m1 sodium methoxide in methanol (0.5 M) while cooling with an ice-water bath. Progress of reaction
was monitored by LCMS which was complete after 2 h. The reaction was quenched with 1 N HCl to a
pH of 5-6. The resulting solution was concentrated and purified by reverse phase liquid
chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 0% to 30%
acetonitrile and water, using 0.1% TFA as the modifier. Yield of the products 7.2 g, 80%. Ion(s) found
by LCMS: M+H=405.

(472) Step c.

(473) ##STR00591##

(474) A solution of the product from the previous step (3.5 g, 8.5 mmol), CDI (2.8 g, 2 equiv),
trimethylamine (4.2 ml, 30 mmol) and DMAP (240 mg, 2 mmol) were heated in acetonitrile (50 ml)
overnight, then concentrated and purified by reverse phase liquid chromatography (RPLC) using an
Isco COMBIFLASH® liquid chromatograph eluted with 0% to 30% acetonitrile and water without
modifier. Yield of desired product 2.3 g, 60%. Ion(s) found by LCMS: M+H=431.

(475) Step d.

(476) ##STR00592##

(477) Sodium hydride (400 mg, 60% in oil, 10 mmol) was added to the product from the previous step
(1.45 g, 3.3 mmol) in 50 ml dry THE (moisture-sensitive reaction) under the ice-water bath. The
resulted solution was stirred for 0.5 hour, then tertbutyl bromo-acetate (2 g, 10 mmol) was added to
the above solution, the resulted solution was heated up to 60° C. for overnight and quenched with
acetic acid. The resulting solution was concentrated and purified by reverse phase liquid
chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 0% to 50%
acetonitrile and water with TFA as modifier. Yield of 1 g, 57%. Ion(s) found by LCMS: M+H=593.

(478) Step e.

(479) ##STR00593##

(480) The product from the previous step (1.2 g, 2.2 mmol) was stirred with 10 ml TFA at room
temperature for overnight, and the progress of deprotection was monitored by LCMS. The resulted
solution was concentrated and used for next step without purification. The residue was re-dissolved
into 20 ml THF, then N,N′-bis-boc-1-guanylpyrazole (1 g, 3.3 mmol), 4-dimethylaminopyridine (120 mg,
1 mmol) and triethylamine (0.7 ml, 5 mmol) were added to the solution, and the resulting solution was
heated to 60° C. for 2 hours. The resulting solution was concentrated and purified by reverse phase
liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 0% to
50% acetonitrile and water with no modifier. Yield of 700 mg, 84%. Ion(s) found by LCMS: M+H=631.

(481) Step f.

(482) ##STR00594##

(483) To a solution of linker-3 (prepared as described in Example 19) (73 mg, 0.14 mmol) and the
product of the previous step (200 mg, 0.32 mmol, 2.2 equi) in DMF (30 ml) was added EDC (100 mg,
0.5 mmol), HOAt (65 mg, 3 mmol), and DIEA (0.14 ml, 1 mmol) at room temperature. The solution was
stirred overnight. The resulting solution was concentrated and purified by and purified by reverse
phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with
0% to 50% acetonitrile and water with no modifier. Yield of 120 mg, 52%. Ion(s) found by LCMS:
M/2+H=830.

(484) Step g.

(485) ##STR00595##

(486) Lithium hydroxide (24 mg, 1 mmol) in 2 ml H2O was added into the solution of the product from
the previous step (120 mg, 0.07 mmol) in 2 ml THE and 1 ml MeOH, LCMS monitored the progress of
the reaction. After the completion, the solution was added AMBERLITE® IRN-77, ion exchange resin
to adjust to pH1, then the resulting solution was filtered and the filtrate was concentrated and used for
next step without purification. The resulting compound was treated with 2 ml TFA at room temperature,
the solution was stirred for overnight at 40° C., then concentrated and purified by HPLC eluted with 0%
to 20% acetonitrile and water, using TFA as the modifier. Yield 60 mg, 74% yield. Ion(s) found by
LCMS: [M/2]+1=589.8.

Example 23. Neuraminidase Inhibition Assay

(487) A neuraminidase inhibition assay using 2′-(4-Methylumbelliferyl)-alpha-D-N-aceylneuraminic acid

(MUNANA) substrate was performed as described below. Briefly, 50 μL of purified, recombinant
influenza virus neuraminidase (0.1 ng/μL, 50 mM Tris, 5 mM CaCl.sub.2, 200 mM NaCl, pH 7.5) was
mixed with 50 μL of inhibitor and incubated for 30 min at room temperature. At least 5 concentrations
of each inhibitor at an appropriate range were used for each repeat. Following incubation, 50 μL of
400 μM MUNANA in 50 mM Tris, 5 mM CaCl.sub.2, 200 mM NaCl, (pH 7.5) was added to the solution
to start the reaction using a 12-tip pipette (Eppendorf). A positive and a negative control were included
in each 12-well lane. After starting the reaction for each lane on the plate, the reaction mixture was
immediately loaded on a SpectraMax M5 (Molecular Devices) where fluorescence was quantified over
the course of 25 min at an excitation wavelength of 365 nm and an emission wavelength of 445 nm.
Single time points were chosen where the positive control produced a fluorescence signal of
approximately 1,000. All assays were done in triplicates and IC50 values for each inhibitor were
calculated with sigmoidal fitting of the log[inhibitor] vs. inhibition percentage using GraphPad Prism.
FIG. 15 shows the plot of the kinetic data as RFU/min over a linear range for Conjugate 1 and Int-2
showing the greater efficacy of Conjugate 1 as a neuraminidase inhibitor. FIG. 16 and FIG. 17 show
the assay results for Conjugates 1-6 against rH1N1 Neuraminidase and rH3N2 Neuraminidase,
respectively (Table 2).

(488) TABLE-US-00005 TABLE 2 IC50 values for conjugates against H1N1 and H3N2 Neuraminidase
Conjugate Number H1N1 IC50 (nM) H3N2 IC50 (nM) Conjugate 1 3.9 32.5 Conjugate 2 3.7 17.6
Conjugate 3 3.6 38 Conjugate 4 1.9 17.2 Conjugate 5 1.1 14.3 Conjugate 6 4.1 29.8

Example 24: Cytotoxicity Assay

(489) Conjugates 1, 2, 3, and 6 were tested for cytotoxicity. Ten two-fold serial dilutions of each
conjugate starting at 10 μM were prepared in triplicate for inoculation with MDCK cells in 96-well
culture plates. The cell viability was determined four days post treatment using CellTiter-GLO kit. 50%
of cytotoxicity concentration (CC.sub.50) was calculated using XLfit dose response model (Table 3).
For all compounds tested, cytotoxicity was not observed up to the 10 μM, with the exception of
conjugate 3. In the case of Conjugate 3, The EC.sub.50 in the cytopathic effect (CPE) assay (see
Example 23) is 725-fold below the CC.sub.50 in this assay.

(490) TABLE-US-00006 TABLE 3 Cytotoxicity test Conjugate Number CC50 (μM) Conjugate 1 >10
Conjugate 2 >10 Conjugate 3 7.98 Conjugate 6 >10 PBS >10 Zanamivir >10

Example 25. Cytopathic Effect Assay

(491) CPE-Based Microneutralization Assay #1

(492) To measure the ability of neuraminidase conjugates to protect mammalian cells from infection
and destruction by influenza virus, cytopathic effect (CPE)-based microneutralization assays were
conducted. Briefly, twenty two-fold serial dilutions of each conjugate starting at 0.25 μM were prepared
in duplicate for one-hour inoculation with MDCK cells seeded in 96-well plates. INFV CA/09 virus was
added to cells at a multiplicity of infection (MOI) 0.001 for one-hour incubation. On day four post
incubation, cells were stained with crystal violet and optical density was read for calculation of 50
percent effective concentration (EC50) of each TA using XLfit dose response model. Zanamivir was
used as a comparator and positive control. human Fc was included as a negative control. Conjugates
1, 2, 3, and 6 all demonstrated superior performance to the zanamivir control, demonstrating EC50s
42- to 227-fold lower than zanamivir (Table 4).

(493) TABLE-US-00007 TABLE 4 CPE-based microneutralization assay #1 Conjugate Number EC50

(nM) 1 2.0574 2 4.1487 3 11.007 6 5.2167 PBS N/C Human Fc N/C Zanamivir.sup.† 468.7 N/C: Not
calculatable .sup.†Starting concentration 10000 nM

CPE-Based Microneutralization Assay #2

(494) An additional CPE-based microneutralization assay was run to further evaluate the in vitro
activity of Conjugate 6 and Conjugate 7. Briefly, test articles were prepared at a starting concentration
of 160 nM, and a total of ten, 2-fold dilutions were made. The test article dilutions were then pre-mixed
with Influenza A virus (INFV CA/09) at a multiplicity of infection of 0.001 relative to the monolayer.
After one hour the test article+viral mix was added to Madin-Darby Canine Kidney (MDCK) cells grown
to 70-80% confluence under standard conditions in a 96-well plate. The zanamivir control was treated
the same, except the starting concentration was 9600 nM since it was expected to be less potent than
the Conjugates being tested. After four days of incubation the monolayer was stained with crystal
violet and optical density (reflective of monolayer health) was determined to calculate the 50%
effective concentration (EC50) using the XLfit dose response model (idbs; https://www.idbs.com). As
shown in Table 5, zanamivir at a concentration of 496 nM reduced the virus mediated cytopathic
effects by half. In contrast, conjugate 6 was approximately 100× more active with an EC50 of only 4.64
nM. Conjugate 7, further improved activity by approximately 15-fold, reducing the EC50 to the sub-nM
level.
(495) TABLE-US-00008 TABLE 5 CPE-based microneutralization assay #2 Conjugate Number EC50
(nM) 6 4.64 7 0.31 Zanamivir 496

Example 26. Cell Viability Assay

(496) A549 cells were seeded in 96-well plates one day prior to compound treatment. Cells were
treated with either Conjugate 3 (FIG. 18A), Conjugate 4 (FIG. 18B), or Conjugate 6 (FIG. 18C) at
concentrations 1 μM-10 nM for 24 hours. Cell viability was then measured using Cell Titer Glow
(Promega). Results represent average and SD of three biological replicates. No effects were observed
on cell viability for any of the conjugates at any concentrations at any of the concentrations used in the
viral growth assay (Example 27).

Example 27. Viral Growth Assay

(497) To measure the ability of neuraminidase conjugates to inhibit the growth of pathogenic influenza
viral strains of interest in human epithelial cells, viral plaque reduction assays were conducted. Briefly,
A549 cells were seeded in 24-well plates one day prior to compound treatment. Cells were treated
with compounds at concentrations 1 μM-10 nM for 2 hours and infected with indicated viral strains at
MOI 0.01 for one-hour incubation. Virus was removed, cells were washed and compounds were re-
applied at concentrations 1 μM-10 nM. Supernatants were collected at indicated time points and
titrated using plaque assay method in MDCK cells. Results represent average and SD of three
biological replicates. Oseltamivir was used as a comparator and a positive control. Conjugate 3 (FIGS.
19A-19E), Conjugate 4 (FIGS. 20A-E), or Conjugate 6 (FIGS. 21A-21E and FIGS. 22A-22E) all
demonstrated superior performance to the oseltamivir control, showing similar or (usually) superior
plaque reduction to oseltamivir at 100-fold lower concentrations. The effects of the compounds (to
evaluate test articles for potential cytotoxicity) on the A549 cells was evaluated for each test article
using a cell viability assay (Example 26). Briefly, A549 cells were seeded in 96-well plates one day
prior to compound treatment. Cells were treated with compounds at concentrations 1 μM-10 nM for 24
hours. Cell viability was then measured using Cell Titer Glow (Promega). Results represent average
and SD of three biological replicates.

Example 28. Mouse Serum Half-Life

(498) Pharmacokinetic (PK) studies used female CD-1 mice (Charles River Laboratories) between 20
and 22 grams. Mice were injected IV by way of the tail vein, with 50 mg/kg of test article (10 m1/kg
dose volume). Animals were housed under standard IACUC approved housing conditions. At
appropriate times animals were non-terminally bled (retro-orbital, cheek, or by tail vein) with blood
collected in EDTA tubes to prevent coagulation. Collected blood was centrifuged (2,000×g, for 10
minutes) and plasma withdrawn for analysis of test article concentrations over time.

(499) The plasma concentrations for Conjugate 6 or hIgG1 Fc at each time point were measured by
sandwich ELISA as follows: Conjugate 6 molecules were captured either on Neuraminidase coated
plates or anti-hIgG1 antibody coated plates and then detected using an HRP-conjugated anti-human
IgG-Fc antibody. hIgG1 was captured using anti-hIgG1 Fc antibody. Protein concentration was
calculated in GraphPad Prism using 4PL non-linear regression of Conjugate 6 (or hIgG1 Fc) standard
curves. A more detailed method description is provided below.

(500) Qiagen Ni-NTA HisSorb plates (Cat No. 35061, Qiagen) were coated with either Neuraminidase
from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Bio) or anti-IgG1 Fc antibody in 1×KPL
coating buffer (5150-0041, SeraCare). In the cases where the anti-IgG1 Fc antibody was used to
capture test article, capture and detection anti-IgG1 Fc antibodies were selected that bind different
epitopes. Plates were incubated at room temperature for 1 hour. Serial dilutions of the plasma samples
were plated and incubated at room temperature for 2 hrs (sample diluent: 0.5% nonfat dry milk+3%
Goat serum in PBS 0.05% Tween20; naïve mouse plasma final concentration of 1:900). Conjugate 6
or hIgG1 Fc standard curves ranging from 500-0.230 ng/mL, in duplicate were run on each plate.
(501) Following the 2 hr incubation, plates were washed 5× in 300 uL PBS with 0.05% Tween20.
Conjugate 6 bound to neuraminidase on the plates (or anti-hIgG1 Fc antibody) was then probed with
an HRP conjugated anti-human IgG Fc F(ab′)2 (Jackson 709-036-098) diluted 1:2000 in sample
diluent for 1 hour at room temp. Plates were then washed 8× in 300 uL PBS with 0.05% Tween20 and
developed with TMB substrate for 7 minutes. The reaction was stopped with 1 N H2SO4. Absorbance
was read at 450 nm. A similar protocol was used for hIgG1 Fc, where only anti-hIgG1 Fc antibody was
used for capture. The quantities of Conjugate 6 measured at different timepoints using either
neuraminidase or anti-hIgG1 Fc antibody capture were similar within experimental error, suggesting
that the intact conjugate is stable in vivo.

(502) Total Conjugate 6 (or hIgG1 Fc) in test samples was interpolated using in GraphPad Prism
Version 6 following nonlinear regression analysis (Sigmoidal, 4PL analysis) of the Conjugate 6 (or
hIgG1 Fc) standard curves. PK parameters were calculated using WinNonlin software. The curves
comparing Conjugate 6 and hIgG1 Fc are shown in FIG. 23 and a summary of key PK parameters is
provided in Table 6. Unexpectedly, plasma exposures and terminal half-life are significantly better for
Conjugate 6 than for wild-type hIgG1 Fc. The AUCs over 8-days are 3× higher for Conjugate 6 than for
hIgG1 Fc, and the terminal half-life for Conjugate 6 is 214 hours, versus 52 hours for human IgG1 Fc.

(503) TABLE-US-00009 TABLE 6 Mouse PK of Conjugate 6 compared to hIgG1 Fc Half-life (hrs)

AUClast (hr*mg/mL) Conjugate 6 214 33100 hIgG1 51.8 10600

Example 29. Efficacy of Conjugate 6 in a Lethal Mouse Influenza Model: Study #1

(504) Conjugate 6 was evaluated against a lethal INFV A H1N1 influenza infection in female BALB/c
mice. The experiment comprised 7 groups of 5 mice. At day 0, all mice were challenged with 1×LD90
H1N1 A/Texas/36/91. Groups 1-6 received treatment IV, 4 hours before challenge (Table 7). Human
IgG1 (Fc alone) was included as an additional negative control. Group 7 received Oseltamivir
phosphate by way of oral delivery, starting 8 hours post infection twice daily for 5 days. All mice were
monitored for weight loss (FIG. 24, Table 8) and survival (FIG. 25, Table 9) for 15 days after challenge.

(505) Mice treated with Conjugate 6 showed 100% survival with single doses in all the concentrations
tested, compared with 20% and 0% survival in the vehicle control and hIgG1 control groups,
respectively. The results were statistically significant when compared to the vehicle group (p=0.0135)
despite the small group size (n=5). When compared to the Oseltamivir phosphate group, conjugate 6
demonstrated similar efficacy at a 500× lower cumulative dose (in mg/kg). Mice at all Conjugate 6
doses maintained their weight through the entire course of the experiment, superior to the Oseltamivir
control group.

(506) TABLE-US-00010 TABLE 7 Study design Group Challenge Dose Treatment n = 5 Day 0
Compound (mg/kg) Route/Schedule 1 Influenza A virus, Vehicle (PBS) N/A IV, q.d. 4 hours 2 H1N1
strain Fc alone 50 pre-challenge 3 A/Texas/36/91 by Conjugate 6 50 4 way of IN route. Conjugate 6 10
5 Conjugate 6 2 6 Conjugate 6 0.4 7 Oseltamivir 20 PO, b.i.d. 8 hours (Tamiflu ™) after challenge for 5
days

(507) TABLE-US-00011 TABLE 8 Daily Weight Average Daily average weight (The mice number is
added only for Group 1 & 2) Days Oseltamivir post Vehicle Fc alone Conjugate 6 phosphate infection
(PBS) 50 mg/kg 50 mg/kg 10 mg/kg 2 mg/kg 0.4 mg/kg 20 mg/kg 0 17.9 (5) 17.9 (5) 18.7 19.3 18.5
18.8 18.8 1 18.0 (5) 18.0 (5) 18.8 19.36 18.6 18.8 18.9 2 18.5 (5) 18.8 (5) 19.2 19.6 19.4 18.8 19.2
3 17.7 (5) 17.7 (5) 18.9 19.5 19.1 18.9 19.1 4 17.2 (5) 17.0 (5) 19.5 19.9 19.3 18.9 18.9 5 16.3 (5)
15.8 (5) 19.2 19.8 19.5 18.7 18.8 6 15.6 (4) 15.0 (5) 19.1 19.9 19.5 18.7 18.4 7 15.5 (2) 14.0 (2)
18.8 19.6 19.2 18.9 17.3 8 17.2 (1) 19.1 20.0 19.7 19.2 17.1 9 20.1 (1) 18.7 19.3 19.1 18.8 17.8 10
20.0 (1) 19.1 20.1 19.5 19.3 18.9 11 20.4 (1) 19.0 19.8 19.2 19.2 18.7 12 20.6 (1) 19.6 20.3 19.8 19.3
19.2 13 20.3 (1) 19.2 20.4 19.7 19.7 19.3 14 20.7 (1) 19.5 20.6 19.9 19.8 19.6

(508) TABLE-US-00012 TABLE 9 Mouse survival Mean % Significance Compound Dosage Survival
Survival to vehicle (p) Vehicle (PBS) N/A 7 20 N/A Fc alone 50 mg/kg 7 0 0.8335 Conjugate 6 50
mg/kg 15 100 0.0135 Conjugate 6 10 mg/kg 15 100 0.0135 Conjugate 6 2 mg/kg 15 100 0.0135
Conjugate 6 0.4 mg/kg 15 100 0.0135 Oseltamivir 20 mg/kg 15 100 0.0135 phosphate B.I.D

Example 30. Efficacy of Conjugate 6 in a Lethal Mouse Influenza Model: Study #2

(509) Conjugate 6 was evaluated against a lethal INFV A H3N2 influenza infection in female BALB/c
mice. The experiment comprised 11 groups of 5 mice. At day 0, all mice were challenged with 1×LD90
H3N2 A/Hong Kong/1/68. Groups 1-10 received treatment IV, 4 hours before challenge (Table 10).
Human IgG1 (Fc alone) was included as an additional negative control. Group 11 received Oseltamivir
phosphate by way of oral delivery, starting 8 hours post infection twice daily for 5 days. All mice were
monitored for weight loss (FIG. 26) and survival (FIG. 27, Table 11) for 15 days after challenge.

(510) Mice treated with Conjugate 6 showed 100% survival with single doses in down to 0.4 mg/kg,
and 80% survival with a single dose of 0.2 mg/kg compared with 0% survival in the vehicle control and
hIgG1 control groups, respectively. 80% of the mice survived in the oseltamivir control group. The
results were statistically significant when compared to the vehicle group (p=0.0128) despite the small
group size (n=5). When compared to the Oseltamivir phosphate group, conjugate 6 demonstrated
similar efficacy at a 1000× lower cumulative dose (in mg/kg). Mice at all Conjugate 6 doses maintained
down to 0.4 mg/kg their weight within 5%, superior to the Oseltamivir control group.

(511) TABLE-US-00013 TABLE 10 Study design Treatment Group Challenge Dose Route/ Readout/ n
= 5 Day 0 Test Article (mg/kg) Schedule Endpoint 1 H3N2 Vehicle N/A IV, q.d. 4 Daily weight A/Hong
(PBS) hours pre- and 2 Kong/1/68 Fc alone 50 challenge health score 3 by way of Conjugate 6 50
monitoring 4 IN route 2 for 15 5 0.4 days after 6 0.2 challenge 7 0.1 % Survival 8 0.05 9 0.025 10
0.0125 11 Oseltamivir 20 PO, b.i.d. 8 (Tamiflu ™) hours after challenge for 5 days

(512) TABLE-US-00014 TABLE 11 Mouse survival Mean Significance Survival % to vehicle Compound
Dosage (days) Survival (p-value) Vehicle (PBS) N/A 9 0 N/A Fc control 50 mg/kg 8 0 0.2498
Conjugate 6 50 mg/kg 15 100 0.002 2 mg/kg 15 100 0.002 0.4 mg/kg 15 100 0.002 0.2 mg/kg 15 80
0.0128 0.1 mg/kg 9 20 0.8264 0.05 mg/kg 9 20 0.5769 0.025 mg/kg 8 20 >0.9999 0.0125 mg/kg
11 0 0.4703 Oseltamivir 20 mg/kg 15 80 0.0052 phosphate (B.I.D for 5 days)

Example 31. Synthesis of Int-10

(513) ##STR00596## ##STR00597##

(514) Methyl 5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-

non-2-enonate (SM-1, 10 g, 22 mmol) was dissolved into 100 ml methanol and then heated to 60° C.
with an oil bath, the SnCl.sub.2 (5.7 g, 20 mmol) was added to the solution in 3 portions (caution, gas
evolves). The reaction mixture was stirred for 10 min, at which time the reaction was complete by
HPLC. The reaction solution was slowly added to a solution of 50 ml Sat NaHCO.sub.3 and 50 g celite
with vigorous stirring. The resulting slurry was filtered. The filtrate was treated with Boc.sub.2O (6.6 g,
30 mmol, 1.5 equiv). After 2 hour at room temperature, the solution was concentrated to remove most
of the methanol, dissolved in 200 ml DCM, and extracted twice with 100 ml DCM. The combined
extracts were dried with sodium sulfate, filtered and used for next step without further purification.
Crude yield 12 g, 100%. Ion(s) found by LCMS: M+H=531.

(515) Step b.

(516) ##STR00598##

(517) The material from the previous step was dissolved into 60 ml dry methanol, then treated with 10
m1 sodium methoxide in methanol (0.5 M) while cooling with an ice-water bath. Progress of reaction
was monitored by LCMS which was complete after 2 h. The reaction was quenched with 1 N HCl to a
pH of 5-6. The resulting solution was concentrated and purified by reverse phase liquid
chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 0% to 30%
acetonitrile and water, using 0.1% TFA as the modifier. Yield of the products 7.2 g, 80%. Ion(s) found
by LCMS: M+H=405.
(518) Step c.

(519) ##STR00599##

(520) A solution of intermediate from the previous step (3.5 g, 8.5 mmol), CDI (2.8 g, 2 equiv),
trimethylamine (4.2 ml, 30 mmol) and DMAP (240 mg, 2 mmol) were heated at 60° C. in DMF (50 ml)
overnight, then concentrated and purified by reverse phase liquid chromatography (RPLC) using an
Isco COMBIFLASH® liquid chromatograph eluted with 0% to 30% acetonitrile and resulting without
modifier. Yield of desired product 2.3 g, 60%. Ion(s) found by LCMS: M+H=431.

(521) Step d.

(522) ##STR00600##

(523) Sodium hydride (400 mg, 60% in oil, 10 mmol) was added to Int-4 (1.45 g, 3.3 mmol) in 50 ml
dry THE (moisture-sensitive reaction) under the ice-water bath. The resulting solution was stirred for
0.5 hour, then tert-butyl bromo acetate (2 g, 10 mmol) was added to the above solution. The resulting
solution was heated to 60° C. overnight and quenched with acetic acid, concentrated and purified by
reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph
eluted with 0% to 50% acetonitrile and water with TFA as modifier. Yield of 1 g, 57%. Ion(s) found by
LCMS: M+H=593. (The reaction is pretty clean by HPLC, but low isolated yield)

(524) Step f.

(525) ##STR00601##

(526) Intermediate from the previous step (1.2 g, 2.2 mmol) was stirred with 10 ml TFA at room
temperature overnight. The progress of deprotection was monitored by LCMS. The resulting solution
was concentrated and used in the next step without purification.

(527) The residue from the previous reaction was dissolved into 20 ml THF, then N,N′-bis-boc-1-
guanylpyrazole (1 g, 3.3 mmol), 4-dimethylaminopyridine (120 mg, 1 mmol) and triethyl amine(0.7 ml,
5 mmol) were added to the solution, and the resulting solution was heated to 60° C. for 2 hours. The
resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC)
using an Isco COMBIFLASH® liquid chromatograph eluted with 0% to 50% acetonitrile and water with
no modifier. Yield of 700 mg, 84%. Ion(s) found by LCMS: M+H=631.

(528) Step g.

(529) ##STR00602##

(530) To a solution of linker-3 (prepared as described in Example 19) (73 mg, 0.14 mmol) and
intermediate from the previous step. (200 mg, 0.32 mmol, 2.2 equi) in DMF (30 ml) was added EDC
(100 mg, 0.5 mmol), HOAt (65 mg, 3 mmol), and DIEA (0.14 ml, 1 mmol) at room temperature. The
solution was stirred overnight. The resulting solution was concentrated and purified by and purified by
reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph
eluted with 0% to 50% acetonitrile and water with no modifier. Yield of 120 mg, 52%. Ion(s) found by
LCMS: M/2+H=830.

(531) Step h.

(532) ##STR00603##

(533) Lithium hydroxide (24 mg, 1 mmol) in 2 ml water was added to a solution of intermediate from
the previous step (120 mg, 0.07 mmol) in 2 ml THE and 1 ml MeOH. After the reaction is complete by
LCMS, the solution was quenched with AMBERLITE® IRN-77, ion exchange resin to adjust to pH=1,
then the solution was filtered and the filtrate was concentrated and in the next step without further
purification. Intermediate from the previous step was treated in 2 ml TFA at room temperature
overnight at 40° C. The crude reaction was concentrated and purified by HPLC with 0% to 20%
acetonitrile and water, using TFA as the modifier. Yield 60 mg, 74% yield. Ion(s) found by LCMS:
[M/2]+1=589.8.

Example 32. Synthesis of Conjugate 7

(534) A solution of Fc-PEG4-azide (each Fc domain monomer having the sequence of SEQ ID NO:
38) in PBS×1 buffer solution (100 mg, 1.28 μmol, 6.1 mL, MW=57260 Da, DAR=3.6) were added to
Int-10 (prepared as described in Example 31) (TFA salt, 44 mg, 0.031 mmol) and freshly prepared pH
7.4 PBS solutions of CuSO.sub.4 (0.7 mL of 50.0 mM, 20 eq), tris(3-hydroxypropyltriazolylmethyl)-
amine (THPTA, 0.7 mL of 50.0 mM, 20 eq), and sodium ascorbate (1.05 mL of 50.0 mM, 30 eq). The
resulting homogeneous solution was agitated by rocker table for 12 h. The crude solutions were
diluted with pH 7.4 PBS to a final concentration of 1 mg/mL, and ultra-filtered (10,000 MWCO) to a
volume of 1 mL, two times. The crude mixtures were then diluted 1:10 in PBS pH 7.4, and purified
using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion
chromatography. Purified material was quantified using a NANODROP™ UV visible
spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of the
Fc used in the conjugation, and concentrated to approximately 10 mg/mL using a centrifugal
concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE
gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel
included a molecular weight ladder with the indicated molecular weight standards. Yields are typically
40-60%. MALDI MS analysis showed a range of masses (60000-90000) with an average of mass of
63633. Average DAR=4.5.

Example 33. Efficacy of Conjugate 6 in a Lethal Mouse Influenza Model: Study #3

(535) Conjugate 6 was evaluated against a lethal INFV A H1N1 influenza infection in female BALB/c
mice. The experiment comprised 7 groups of 5 mice. At day 0, all mice were challenged with 1×LD90
H1N1 A/Texas/36/91. Groups 1-6 received treatment IV, 28 days before challenge (Table 12). Vehicle
(PBS) was included as an additional negative control. Group 7 received Oseltamivir phosphate by way
of oral delivery, starting 8 hours post infection twice daily for 5 days. All mice were monitored for
survival (FIGS. 31A-31F) for 15 days after challenge.

(536) Mice treated with Conjugate 6 showed 100% survival with single doses in all the concentrations
down to 2.5 mg/kg, and 80% survival at 1.25 mg/kg, compared with 0% survival in the vehicle control
group. All mice in the Oseltamivir phosphate control group survived. When compared to the
Oseltamivir phosphate group, conjugate 6 demonstrated similar efficacy to Oseltamivir at a 20× lower
cumulative dose (in mg/kg), despite the fact that all Conjugate 6 groups were dosed once, 28 days
prior to infection.

(537) TABLE-US-00015 TABLE 12 Study design Group Challenge Dose Treatment n = 5 Day 0
Compound (mg/kg) Route/Schedule 1 Influenza A virus, Vehicle (PBS) N/A IV, q.d. 28 days 2 H1N1
strain Conjugate 6 50 pre-challenge 3 A/Texas/36/91 by Conjugate 6 10 4 way of IN route. Conjugate
6 5 5 Conjugate 6 2.5 6 Conjugate 6 1.25 7 Oseltamivir 20 PO, b.i.d. 8 hours (Tamiflu ™) after
challenge for 5 days

Example 34. Efficacy of Conjugate 6 in a Lethal Mouse Influenza Model: Study #4

(538) Conjugate 6 was evaluated against a lethal INFV A H1N1 influenza infection in female BALB/c
mice. The experiment comprised 13 groups of 5 mice. At day 0, all mice were challenged with 1×LD90
H1N1 A/Texas/36/91. All Conjugate 6 groups (8-13) received single 10 mg/kg IV doses at different
times, pre- and post-infection, as outlined in Table 13. Vehicle (PBS) and Fc only were included as
negative controls. Groups 3-7 received Oseltamivir phosphate (20 mg/kg) by way of oral delivery,
starting at different time points post-infection twice daily for 5 days, as outlined in Table 13. All mice
were monitored for survival (FIGS. 32A-32F) for 15 days after challenge.
(539) Mice treated with Conjugate 6 showed 100% survival with single 10 mg/kg doses when treated
out to 24 hrs post-infection, and 60 and 80% survival at 48 and 72 hrs post-infection, respectively. In
the Oseltamivir phosphate group, survival dropped sharply to 0% and 20%, respectively, in the groups
where treatment was initiated at 48 and 72 hrs post-infection. These results suggest that Conjugate 6
potentially possesses a superior treatment window versus Oseltamivir for treating influenza A
infections.

(540) TABLE-US-00016 TABLE 13 Study design Dose Group Influenza Dose timing (N = 5) A strain
Test Article Route, Schedule (mg/kg) (hours) 1 A/Texas/36/ Vehicle IV, single — T − 4 91 (H1N1)
(PBS) 2 by way Fc only IV, single 10 T − 4 3 of IN Oseltamivir PO, bid × 5 days 20 T + 8 4
Oseltamivir PO, bid × 5 days 20 T + 24 5 Oseltamivir PO, bid × 5 days 20 T + 48 6 Oseltamivir PO, bid
× 5 days 20 T + 72 7 Oseltamivir PO, bid × 5 days 20 T + 96 8 Conjugate 6 IV, single 10 T − 4 9
Conjugate 6 IV, single 10 T + 8 10 Conjugate 6 IV, single 10 T + 24 11 Conjugate 6 IV, single 10 T +
48 12 Conjugate 6 IV, single 10 T + 72 13 Conjugate 6 IV, single 10 T + 96

Example 35. Conjugate 6 Toxicity Study

(541) The safety of Conjugate 6 was evaluated in a 14 day rat dose-range finder toxicity study. Rats
were administered either 5 mpk, 20 mpk, or 50 mpk of Conjugate 6 by intravenous administration on
days 0 and 7 of the study. Compared with vehicle controls, no significant effects on body weight gain
(FIG. 33), organ weights, food consumption were observed at any dose tested. Plasma exposures
(measured by AUC) increased proportionally with dose. These preclinical safety results are consistent
with a high therapeutic index. A summary of observations is provided in Table 14.

(542) TABLE-US-00017 TABLE 14 Summary of 14-day rat dose-range finder toxicity study Findings at
highest dose (50 mpk), Parameter compared to vehicle Clinical observations No findings Hematology
No change from vehicle Clinical Chemistry No change from vehicle Coagulation No change from
vehicle Urinalysis No change from vehicle Histopathology No observations

Example 36. Efficacy of Conjugate 6 Against in a Lethal Mouse Influenza Model Dose by Three
Different Routes: Study #5

(543) Conjugate 6 was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c mice
(Charles River Laboratories, 6-8 weeks). The challenge virus (A/Texas/36/91) is a mouse-adapted
isolate capable of causing lethal infections in mice. The experiment comprised 15 groups of 5 mice. At
day 0, all mice were challenged with virus at 1× the LD90 by intranasal inoculation in a volume of 50
μl, after being lightly anesthetized with isoflurane. Groups 1-14 received a single treatment, 4 hours
before challenge (Table 15). In order to determine the potency of Conjugate 6 by different dose routes
matching concentrations of Conjugate (10, 2, 0.4, and 0.1 mg/kg) were dosed either IV, IM, or SC. In
addition to the vehicle (PBS) only group, Human IgG1 (Fc alone) was included as an additional
negative control (group 2). Group 15 received Oseltamivir phosphate by way of oral delivery, starting 8
hours post infection twice daily for 5 days. All mice were monitored for survival (Table 15) for 14 days.

(544) Mice treated with Conjugate 6 showed 100% survival at day 14 against challenge by influenza
(H1N1) with single doses of 10, 2, or 0.4 mg/kg, regardless of dose route. Only at the lowest test
article concentration were single mouse differences seen between IV, IM, and SC dosing (Table 16;
60, 80, and 100% survival, respectively). These results strongly suggest that protective lung
concentrations of Conjugate 6 is achievable by multiple dose routes.

(545) TABLE-US-00018 TABLE 15 Study design Group Challenge Dose Dose Treatment n = 5 Day 0
Compound (mg/kg) route Route/Schedule 1 Influenza A Vehicle (PBS) N/A Single dose, 4 2 virus
(H1N1) Fc alone 50 IV hours before 3 A/Texas/36/91 Conjugate 6 10 IV viral challenge 4 by way of IN
Conjugate 6 2 5 Conjugate 6 0.4 6 Conjugate 6 0.1 7 Conjugate 6 10 IM 8 Conjugate 6 2 9 Conjugate
6 0.4 10 Conjugate 6 0.1 11 Conjugate 6 10 SC 12 Conjugate 6 2 13 Conjugate 6 0.4 14 Conjugate 6
0.1 15 Oseltamivir 20 PO b.i.d. 8 hours (Tamiflu ™) after challenge for 5 days
(546) TABLE-US-00019 TABLE 16 Mouse survival Dose % Survival Test article mg/kg route (Day 14)
Vehicle (PBS) na IV 0 hIgG1 (Fc only) 10 IV 0 Conjugate 6 10 IV 100 2 100 0.4 100 0.1 60 Conjugate
6 10 IM 100 2 100 0.4 100 0.1 80 Conjugate 6 10 SC 100 2 100 0.4 100 0.1 100 oseltamivir 20 PO
100

Example 37. Efficacy of Conjugate 6 Against an Oseltamivir-Resistant Isolate in a Lethal Mouse

Influenza Model: Study #6

(547) Conjugate 6 was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c mice
(Charles River Laboratories, 6-8 weeks). The challenge virus (A/Perth/261/2009) is a mouse-adapted
isolate that carries the H275Y mutation resulting in resistance to the neuraminidase inhibitor
oseltamivir. The experiment comprised 10 groups of 5 mice. At day 0, all mice were challenged with
A/Perth/261/2009 (H1N1) at 1× the LD90 by intranasal inoculation in a volume of 50 μl, to mice lightly
anesthetized with isoflurane. Groups 1-9 received a single treatment by IV, 4 hours before challenge
(Table 17). In addition to the vehicle (PBS) only group, Human IgG1 (Fc alone) was included as an
additional negative control. Group 10 received Oseltamivir phosphate by way of oral delivery, starting
8 hours post infection twice daily for 5 days. All mice were monitored for survival (FIGS. 34A-34D,
Table 18) and weight loss (FIG. 35, Table 19) for 15 days after challenge.

(548) Mice treated with Conjugate 6 showed 100% survival against challenge by influenza
(A/Perth/261/2009) with single doses at 50, 10, and 2 mg/kg. Furthermore, despite the small group
size (n=5) these results were statistically significant relative to the vehicle control (Table 18).

(549) At Conjugate 6 concentrations of 0.4, 0.2, or 0.1 mg/kg survival was 60%, while no mice
survived to the end of the study if dosed with vehicle (PBS) or hIgG1 (Fc only) only. The oseltamivir
group only had a single animal surviving (20% efficacy) despite treatment with a dose shown to be
protective against oseltamivir sensitive isolates previously (20 mg/kg, bid×5 days). These results
confirm that the challenge virus is resistant to oseltamivir, and sensitive to Conjugate 6.

(550) The potency of Conjugate 6 against mutants containing the H275Y mutation was further
supported by body weight data (FIG. 35, Table 19). Groups receiving a single dose of Conjugate 6 at
concentrations of 2 mg/kg or more demonstrated 5%, or less, transient weight loss.

(551) TABLE-US-00020 TABLE 17 Study design Group Challenge Dose Treatment n = 5 Day 0
Compound (mg/kg) Route/Schedule 1 Influenza A virus Vehicle (PBS) N/A IV, q.d. 4 hours 2 (H1N1)
Fc alone 50 pre-challenge 3 A/Perth/261/2009 Conjugate 6 50 4 by way of IN route. Conjugate 6 10 5
Conjugate 6 2 6 Conjugate 6 0.4 7 Conjugate 6 0.2 8 Conjugate 6 0.1 9 Conjugate 6 0.05 10
Oseltamivir 20 PO, b.i.d. 8 hours (Tamiflu ™) after challenge for 5 days

(552) TABLE-US-00021 TABLE 18 Mouse survival Median % Significance Compound Dosage Survival
Survival to vehicle (p) Vehicle (PBS) N/A 7 0 N/A Fc alone 50 mg/kg 6 0 ns Conjugate 6 50 mg/kg
Undef 100 0.0027 Conjugate 6 10 mg/kg Undef 100 0.0027 Conjugate 6 2 mg/kg Undef 100 0.0027
Conjugate 6 0.4 mg/kg Undef 60 0.1167 Conjugate 6 0.2 mg/kg Undef 60 0.0185 Conjugate 6 0.1
mg/kg Undef 60 0.1047 Conjugate 6 0.05 mg/kg 7 0 0.9241 Oseltamivir 20 mg/kg 7 20 0.3470
phosphate B.I.D

(553) TABLE-US-00022 TABLE 19 Daily Weight Average Daily average weight (#of mice shown in
parenthesis if less than 5) Days Fc Oseltamivir post Vehicle alone Conjugate 6 phosphate infection
(PBS) 50 mg/kg 50 mg/kg 10 mg/kg 2 mg/kg 0.4 mg/kg 0.2 mg/kg 0.1 mg/kg 0.05 mg/kg 20 mg/kg 0
100 (5) 100 (5) 100 100 100 100 100 100 100 100 1 103 (5) 104 (5) 101 101 102 102 103 105 102
105 2 102 (5) 105 (5) 103 101 103 100 102 102 101 102 3 93 (5) 97 (5) 101 101 98 92 98 95
94 95 4 88 (5) 90 (5) 95 98 99 91 98 92 90 93 5 85 (5) 85 (5) 94 98 98 91 98
92 89 92 6 83 (4) 88 (3) 100 100 99 88 95 85 84 (4) 86 7 79 (3) 90 (2) 103 99 95
90 (3) 94 83 (4) 77 (1) 86 (3) 8 75 (2) 81 (1) 102 99 97 93 (3) 94 (4) 87 (3) 78 (1) 85
(2) 9 106 104 104 101 (3) 101 (4) 95 (3) 86 (2) 10 105 102 103 100 (3) 99 (4) 97 (3) 95 (1) 11
106 102 104 102 (3) 104 (4) 102 (3) 97 (1) 12 108 104 105 102 (3) 105 (3) 107 (3) 104 (1) 13 107
103 105 102 (3) 105 (3) 107 (3) 104 (1) 14 107 103 105 102 (3) 106 (3) 108 (3) 104 (1) 15 100 100
100 100 (3) 100 (3) 100 (3) 100 (1)

Example 38. 7-Day Rat PK Study Following IV Administration of Test Article

(554) Rat pharmacokinetic (PK) studies were performed by Seventh Wave Laboratories (Maryland
Heights, Mo.) using male Sprague Dawley Rats 46-49 days of age. Rats were injected IV by way of
the tail vein with 75 mg/kg of test article (5 m1/kg dose volume). Animals were housed under standard
IACUC approved housing conditions. At appropriate times animals were non-terminally bled (retro-
orbital, cheek, or by tail vein) with blood collected in K2EDTA tubes to prevent coagulation. Collected
blood was centrifuged (2,000×g, for 10 minutes) and plasma withdrawn for analysis of test article
concentrations over time.

(555) The plasma concentrations for Conjugate 6 or hIgG1 Fc at each time point were measured by
sandwich ELISA as follows: Conjugate 6 molecules were captured either on neuraminidase coated
plates or anti-hIgG1 antibody coated plates and then detected using an HRP-conjugated anti-human
IgG-Fc antibody. hIgG1 was captured using anti-hIgG1 Fc antibody. Protein concentration was
calculated in GraphPad Prism using 4PL non-linear regression of Conjugate 6 (or hIgG1 Fc) standard
curves. A more detailed method description is provided below.

(556) Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) were coated with either
neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Biological) or anti-IgG1 Fc
antibody in 1×KPL coating buffer (5150-0041, SeraCare). In the cases where the anti-IgG1 Fc
antibody was used to capture test article, capture and detection anti-IgG1 Fc antibodies were selected
that bind different epitopes. Plates were incubated at room temperature for 1 hr on an orbital plate
shaker (500 rpm). Serial dilutions of the plasma samples were plated and incubated at room
temperature for 2 hrs (sample diluent: 1% BSA in PBS 0.05% Tween 20+naïve rat plasma final
concentration of 1:900). Conjugate 6 or hIgG1 Fc standard curves ranging from 500-0.230 ng/mL, in
duplicate were run on each plate.

(557) Following the 2 hr incubation, plates were washed 5× in 300 μL PBS with 0.05% Tween 20.
Conjugate 6 bound to neuraminidase on the plates (or anti-hIgG1 Fc antibody) was then probed with
an HRP conjugated anti-human IgG Fc F(ab′)2 (709-036-098, Jackson) diluted 1:1,000 in sample
diluent for 1 hr at room temp. Plates were then washed 8× in 300 μL PBS with 0.05% Tween 20 and
developed with TMB substrate for 7-8 minutes. The reaction was stopped with 1 N H.sub.2SO.sub.4.
Absorbance was read at 450 nm. A similar protocol was used for hIgG1 Fc, where only anti-hIgG1 Fc
antibody was used for capture. The quantities of Conjugate 6 measured at different time points using
either neuraminidase or anti-hIgG1 Fc antibody capture were similar within experimental error,
suggesting that the intact conjugate is stable in vivo.

(558) Total Conjugate 6 (or hIgG1 Fc) in test samples was interpolated using in GraphPad Prism
Version 6 following nonlinear regression analysis (Sigmoidal, 4PL analysis) of the Conjugate 6 (or
hIgG1 Fc) standard curves. The curves comparing Conjugate 6 and hIgG1 Fc are shown in FIG. 36.
The quantities of Conjugate 6 measured at different time points using either neuraminidase or anti-
hIgG1 Fc antibody capture were similar within experimental error, suggesting that the intact conjugate
is stable in vivo.

Example 39. 14-Day Rat PK Study Following IV Administration of Test Article

(559) Rat PK studies were performed by Seventh Wave Laboratories (Maryland Heights, Mo.) using
male Sprague Dawley Rats 46-49 days of age. Rats were injected IV by way of the tail vein with 5, 20,
or 50 mg/kg of test article (5 m1/kg dose volume) at days 1 and 8. Animals were housed under
standard IACUC approved housing conditions. At appropriate times animals were non-terminally bled
(retro-orbital, cheek, or by tail vein) with blood collected in K2EDTA tubes to prevent coagulation.
Collected blood was centrifuged (2,000×g, for 10 minutes) and plasma withdrawn for analysis of test
article concentrations over time.
(560) The plasma concentrations for Conjugate 6 at each time point were measured by sandwich
ELISA as follows: Conjugate 6 molecules were captured either on neuraminidase coated plates and
then detected using an HRP-conjugated anti-human IgG-Fc antibody. hIgG1 was captured using anti-
hIgG1 Fc antibody. Protein concentration was calculated in GraphPad Prism using 4PL non-linear
regression of Conjugate 6 standard curves. A more detailed method description is provided below.

(561) Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) were coated with either
neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Biological) in 1×KPL coating
buffer (5150-0041, SeraCare). Plates were incubated at room temperature for 1 hr on an orbital plate
shaker (500 rpm). Serial dilutions of the plasma samples were plated and incubated at room
temperature for 2 hrs (sample diluent: 1% BSA in PBS 0.05% Tween 20+naïve rat plasma final
concentration of 1:900). Conjugate 6 standard curves ranging from 500-0.230 ng/mL, in duplicate were
run on each plate.

(562) Following the 2 hr incubation, plates were washed 5× in 300 μL PBS with 0.05% Tween 20.
Conjugate 6 bound to neuraminidase on the plates was then probed with an HRP conjugated anti-
human IgG Fc F(ab′)2 (709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1 hr at room temp.
Plates were then washed 8× in 300 μL PBS with 0.05% Tween 20 and developed with TMB substrate
for 7-8 minutes. The reaction was stopped with 1 N H.sub.2SO.sub.4. Absorbance was read at 450
nm. Total Conjugate 6 (or hIgG1 Fc) in test samples was interpolated using in GraphPad Prism
Version 6 following nonlinear regression analysis (Sigmoidal, 4PL analysis) of the Conjugate 6
standard curves. The curves comparing Conjugate 6 shown in FIG. 37 demonstrate linear dose
proportionality.

Example 40. 28-Day Rat PK Study Comparing IV and SC Administration of Test Article

(563) Rat PK studies were performed by Seventh Wave Laboratories (Maryland Heights, Mo.) using
male Sprague Dawley Rats 46-49 days of age. Rats were injected IV or SC with 5 mg/kg of test article
(5 ml/kg dose volume). Animals were housed under standard IACUC approved housing conditions. At
appropriate times animals were non-terminally bled (retro-orbital, cheek, or by tail vein) with blood
collected in K2EDTA tubes to prevent coagulation. Collected blood was centrifuged (2,000×g, for 10
minutes) and plasma withdrawn for analysis of test article concentrations overtime.

(564) The plasma concentrations for Conjugate 6 or hIgG1 Fc at each time point were measured by
sandwich ELISA as follows: Conjugate 6 molecules were captured on neuraminidase coated plates
and then detected using an HRP-conjugated anti-human IgG-Fc antibody. Protein concentration was
calculated in GraphPad Prism using 4PL non-linear regression of Conjugate 6 standard curves. A
more detailed method description is provided below.

(565) Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) were coated with either
neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Biological) in 1×KPL coating
buffer (5150-0041, SeraCare). Plates were incubated at room temperature for 1 hr on an orbital plate
shaker (500 rpm). Serial dilutions of the plasma samples were plated and incubated at room
temperature for 2 hrs (sample diluent: 1% BSA in PBS 0.05% Tween 20+naïve rat plasma final
concentration of 1:900). Conjugate 6 standard curves ranging from 500-0.230 ng/mL, in duplicate were
run on each plate.

(566) Following the 2 hr incubation, plates were washed 5× in 300 μL PBS with 0.05% Tween 20.
Conjugate 6 bound to neuraminidase on the plates was then probed with an HRP conjugated anti-
human IgG Fc F(ab′)2 (709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1 hr at room temp.
Plates were then washed 8× in 300 μL PBS with 0.05% Tween 20 and developed with TMB substrate
for 7-8 minutes. The reaction was stopped with 1 N H.sub.2SO.sub.4. Absorbance was read at 450
nm.

(567) Total Conjugate 6 (or hIgG1 Fc) in test samples was interpolated using in GraphPad Prism
Version 6 following nonlinear regression analysis (Sigmoidal, 4PL analysis) of the Conjugate 6
standard curves. The curves comparing Conjugate 6 in FIG. 38 show that SC and IV plasma levels
converged at 24 h and were similar within experimental error out to 336 h.

Example 41. 28-Day Non-Human Primate PK Study Following IV Administration of Test Article

(568) Non-human primate (NHP) PK studies were performed by BTS Research (San Diego, Calif.)
using male and female Cynomolgus monkeys 4.5-8 years old with body weights ranging from 2.5-6.5
kg. NHPs were injected IV by way of the saphenous or cephalic vein with 5 or 20 mg/kg of test article
(5 m1/kg dose volume). Animals were housed under standard IACUC approved housing conditions. At
appropriate times animals were non-terminally bled (by way of femoral or cephalic veins) with blood
collected in K2EDTA tubes to prevent coagulation. Collected blood was centrifuged (2,000×g, for 10
minutes) and plasma withdrawn for analysis of test article concentrations overtime.

(569) The plasma concentrations for Conjugate 6 at each time point were measured by sandwich
ELISA as follows: Conjugate 6 molecules were captured on neuraminidase coated plates and then
detected using an HRP-conjugated anti-human IgG-Fc antibody. Protein concentration was calculated
in GraphPad Prism using 4PL non-linear regression of Conjugate 6 standard curves. A more detailed
method description is provided below.

(570) Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) were coated with
neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Biological) in 1×KPL coating
buffer (5150-0041, SeraCare). Plates were incubated at room temperature for 1 hr on an orbital plate
shaker (500 rpm). Serial dilutions of the plasma samples were plated and incubated at room
temperature for 2 hrs (sample diluent: 1% BSA in PBS 0.05% Tween 20+naïve cynomolgus monkey
plasma final concentration of 1:2,500). Conjugate 6 standard curves ranging from 500-0.230 ng/mL, in
duplicate were run on each plate.

(571) Following the 2 hr incubation, plates were washed 5× in 300 μL PBS with 0.05% Tween 20.
Conjugate 6 bound to neuraminidase on the plates was then probed with an HRP conjugated anti-
human IgG Fc F(ab′)2 (709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1 hr at room temp.
Plates were then washed 8× in 300 μL PBS with 0.05% Tween 20 and developed with TMB substrate
for 7-8 minutes. The reaction was stopped with 1 N H.sub.2SO.sub.4. Absorbance was read at 450
nm.

(572) Total Conjugate 6 in test samples was interpolated using in GraphPad Prism Version 6 following
nonlinear regression analysis (Sigmoidal, 4PL analysis) of the Conjugate 6 (or hIgG1 Fc) standard
curves. PK parameters were calculated using WinNonlin software. The curves comparing Conjugate 6
are shown in FIG. 39 and a summary of key PK parameters is provided in Table 20. The dose
response is linear between 5 and 20 mg/kg IV, resulting in a half-life of approximately 9 days across
both doses (comparable to mouse/rat).

(573) TABLE-US-00023 TABLE 20 Cynomolgus monkey PK of Conjugate 6 Half-life AUClast

Conjugate 6 (hrs) (hr * mg/mL) 5 mg/kg IV 230 6240 20 mg/kg IV 197 25400

Example 42. Mouse Lung Distribution PK Study Following IV Administration of Test Article

(574) Mouse PK studies were performed using male CD-1 mice 6 weeks of age. Mice were injected IV
by way of the tail vein with 10 mg/kg of test article (5 m1/kg dose volume). Animals were housed under
standard IACUC approved housing conditions. At the indicated time points, the animals were
euthanized to harvest blood (by way of cardiac puncture) in K2EDTA tubes and lungs. Blood was
centrifuged (2,000×g, for 10 minutes) to obtain plasma. The lungs were weighed and homogenized in
1.5 ml tubes with a sterile disposable pestle (Z359947, Sigma) in 100 μL of homogenization buffer
comprised of 11.64 mL tissue protein extraction reagent (78510, Thermo Scientific), 0.24 ml of
protease inhibitor cocktail (78410, Thermo Scientific), and 0.12 ml of EDTA. After 1-2 min
homogenization, the volume was adjusted to 1 mL with homogenization buffer and the samples
incubated on ice for 20 min with periodic mixing by gentle vortexing. The homogenates were
centrifuged at 8,000×g for 10 min and the supernatant retained for analysis of test article
concentrations over time.

(575) The plasma and lung concentrations for Conjugate 6 at each time point were measured by
sandwich ELISA as follows: Conjugate 6 molecules were captured on neuraminidase coated plates
and detected using an HRP-conjugated anti-human IgG-Fc antibody. Protein concentration was
calculated in GraphPad Prism using 4PL non-linear regression of Conjugate 6 standard curves. A
more detailed method description is provided below.

(576) Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) were coated with
neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Biological) in 1×KPL coating
buffer (5150-0041, SeraCare). Plates were incubated at room temperature for 1 hr on an orbital plate
shaker (500 rpm). Serial dilutions of the plasma samples were plated and incubated at room
temperature for 2 hrs (sample diluent: 1% BSA in PBS 0.05% Tween 20+naïve mouse plasma final
concentration of 1:100). Conjugate 6 standard curves ranging from 500-0.230 ng/mL, in duplicate were
run on each plate.

(577) Following the 2 hr incubation, plates were washed 5× in 300 μL PBS with 0.05% Tween 20.
Conjugate 6 bound to neuraminidase on the plates was then probed with an HRP conjugated anti-
human IgG Fc F(ab′)2 (709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1 hr at room temp.
Plates were then washed 8× in 300 μL PBS with 0.05% Tween 20 and developed with TMB substrate
for 7-8 minutes. The reaction was stopped with 1 N H.sub.2SO.sub.4. Absorbance was read at 450
nm.

(578) Total Conjugate 6 in test samples was interpolated using in GraphPad Prism Version 6 following
nonlinear regression analysis (Sigmoidal, 4PL analysis) of the Conjugate 6 standard curves. The
curves comparing Conjugate 6 plasma and lung concentrations are shown in FIG. 40. Unexpectedly,
lung C.sub.max was reached at t=1 h (19.5 μg/g lung tissue, ˜310 nM). There was approximately 10%
lung exposure of Conjugate 6, relative to plasma

Example 43. 5-Day Mouse PK Study Comparing IV, SC and IM Administration of Test Article

(579) Mouse PK studies were performed using male CD-1 mice 6 weeks of age. Mice were injected IV
by way of the tail vein with 5 mg/kg of test article (5 m1/kg dose volume). Animals were housed under
standard IACUC approved housing conditions. At appropriate times animals were non-terminally bled
(retro-orbital, cheek, or by tail vein) with blood collected in K.sub.2EDTA tubes to prevent coagulation.
Collected blood was centrifuged (2,000×g, for 10 minutes) and plasma withdrawn for analysis of test
article concentrations over time.

(580) The plasma concentrations for Conjugate 6 at each time point were measured by sandwich
ELISA as follows: Conjugate 6 molecules were captured on neuraminidase coated plates and then
detected using an HRP-conjugated anti-human IgG-Fc antibody. Protein concentration was calculated
in GraphPad Prism using 4PL non-linear regression of Conjugate 6 (or hIgG1 Fc) standard curves. A
more detailed method description is provided below.

(581) Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) were coated with
neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Biological) in 1×KPL coating
buffer (5150-0041, SeraCare). Plates were incubated at room temperature for 1 hr on an orbital plate
shaker (500 rpm). Serial dilutions of the plasma samples were plated and incubated at room
temperature for 2 hrs (sample diluent: 1% BSA in PBS 0.05% Tween 20+naïve mouse plasma final
concentration of 1:100). Conjugate 6 standard curves ranging from 500-0.230 ng/mL, in duplicate were
run on each plate.

(582) Following the 2 hr incubation, plates were washed 5× in 300 μL PBS with 0.05% Tween 20.
Conjugate 6 bound to neuraminidase on the plates was then probed with an HRP conjugated anti-
human IgG Fc F(ab′)2 (709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1 hr at room temp.
Plates were then washed 8× in 300 μL PBS with 0.05% Tween 20 and developed with TMB substrate
for 7-8 minutes. The reaction was stopped with 1 N H.sub.2SO.sub.4. Absorbance was read at 450
nm.

(583) Total Conjugate 6 in test samples was interpolated using in GraphPad Prism Version 6 following
nonlinear regression analysis (Sigmoidal, 4PL analysis) of the Conjugate 6 standard curves. The
curves comparing Conjugate 6 are shown in FIG. 41. The exposure levels for IV, IM and SC were
similar with AUCs of 2082, 1944 and 2500, respectively.

Example 44. Mouse Efficacy and Multi-Species PK Supports Infrequent Prophylactic Dosing in Human
Subjects

(584) Allometric scaling of mouse efficacy dosing based on mouse, rat, and primate PK studies was
used to predict dosing and PK parameters in human subjects. Allometric scaling was based on the
Area Under the Curve (AUC) at a 2.5 mg/kg efficacious dose in a 28-day mouse prevention study (see
Example 33).

(585) For cynomolgus monkey-only allometric scaling, human clearance (CL) was calculated based on
cynomolgus monkey PK data (Example 41) only using a simple allometric equation:
CL(human)=CL(monkey).Math.[BW(human)/BW(monkey)].sup.w, where BW=bodyweight and w is the
scaling exponent fixed at 0.85. The results of cynomolgus monkey-only scaling are provided in Table
21.

(586) For Mouse-rat-cynomolgus allometric scaling, human clearance (CL) from animal species was
plotted against the animal body weight (BW) on a log-log scale according to the following allometric
equation: CL=a.Math.BW.sup.x, where a is the coefficient and x is the exponent of the allometric
equation. The coefficient a and exponent x were calculated from the intercept and slope of the linear
regression line, respectively. The results of mouse-rat-cyno scaling are provided in Table 22.

(587) Human clearance values calculated by respective algorithms above were then used to calculate
the corresponding human dose needed to achieve the Efficacy AUC target of 3700 ug-hr/mL (from
mouse 2.5 mg/kg dose, Example 33) using the following equation: Dose=CL.Math.AUC.

(588) TABLE-US-00024 TABLE 21 Cynomolgus monkey-only allometric scaling (β = 0.85) Efficacy

Area Under the Curve (AUC) 3700 μg-hr/mL Human Clearance (CL) 0.43 mL/min Human Dose 95.46
mg, 1.4 mg/kg

(589) TABLE-US-00025 TABLE 22 Mouse-rat-cyno allometric scaling (β = 0.97) Efficacy Area Under
the Curve (AUC) 3700 μg-hr/mL Human Clearance (CL) 0.59 mL/min Human Dose 130.98 mg, 1.9
mg/kg

Example 45. Synthesis of Int-11

(590) ##STR00604## ##STR00605##

(591) A solution of the tert-butyl (4-bromobutyl)carbamate (11.2 g, 60 mmol) and tert-butyl (4-
aminobutyl)carbamate (10 g, 40 mmol) dissolved in DMF (150 mL) was treated with potassium
carbonate (16.4 g, 120 mmol), then stirred at 80° C. for 6 hrs. The reaction mixture was partitioned
between DCM (500 ml) and brine (100 ml). The organic layer was separated and washed with brine
then dried with sodium sulfate. The solution was filtered, concentrated, and purified by reverse phase
liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 10% to
100% acetonitrile and water with 0.1% TFA as modifier. Yield of the product 11.0 g, 77%. Ion(s) found
by LCMS: M+H=360.4

(592) Step b.

(593) ##STR00606##
(594) Product from the previous step (0.4 g, 1.11 mmol) and Fmoc-OSu (0.45 mg, 1.3 mmol) were
dissolved in DCM (10 mL), then treated with N-methylmorpholine (0.22 ml, 2 mmol), and stirred for 1
hr at room temperature. The reaction was concentrated and purified by and purified by reverse phase
liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 10% to
100% acetonitrile and water with no 0.1% TFA as modifier. Yield of the products 450 mg, 69%. Ion(s)
found by LCMS: M/2+H=582.4.

(595) Step c.

(596) ##STR00607##

(597) Product from the previous step (0.4 g, 0.7 mmol) was treated with TFA (5 mL) at room
temperature for 0.5 hour, then concentrated to dryness and used for next step without further
purification. Yield was quantitative for this step. Ion(s) found by LCMS: M/2+H=382.3.

(598) Step d.

(599) ##STR00608##

(600) Fmoc diamine (24 mg, 0.063 mmol) from the previous step was added to a solution of carboxylic
acid (80 mg, 0.126 mmol, described in the synthesis Int-10, step f) in DMF (5 mL), then treated with
HATU (50 mg, 0.13 mmol), followed N-methylmorpholine (0.06 ml, 0.50 mmol). After stirring for 1 h the
resulting solution was concentrated and purified by reverse phase liquid chromatography (RPLC)
using an Isco COMBIFLASH® liquid chromatograph eluted with 10% to 100% acetonitrile and water
without TFA as a modifier. Yield of the products 80 mg, 79%. Ion(s) found by LCMS: M/2+H=803.9.

(601) Step e.

(602) ##STR00609##

(603) Product from the previous step (80 mg, 0.050 mmol) was treated with 1% DBU (1,8-
diazabicyclo[5.4.0]undec-7-ene) in DMF (2 mL) and stirred at room temperature. When the reaction
was complete by LCMS (15 min), it was concentrated, then treated with TFA (2 mL) and stirred for 30
min at room temperature. The reaction solution was concentrated and purified by reverse phase liquid
chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 10% to
100% acetonitrile and water, using 0.1% TFA as the modifier. Yield of product was 52 mg as TFA salt.
Ion(s) found by LCMS: M/2+H=492.7.

(604) Step f.

(605) ##STR00610##

(606) Product from the previous step was dissolved into water (2 mL), then treated with a lithium
hydroxide (12 mg, 0.50 mmol, in 1 mL water) solution. The resulting reaction was monitored by LCMS.
After stirring for 10 min the reaction was quenched with 0.1 ml acetic acid. The resulting solution was
concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco
COMBIFLASH® liquid chromatograph eluted with 0% to 30% acetonitrile and water, using 0.1% TFA
as the modifier. Yield of the product: 30 mg as TFA salt, 66%. Ion(s) found by LCMS: M/2+H=452.7.
M/3+H=302.1.

Example 46. Synthesis of Int-12

(607) ##STR00611## ##STR00612##

(608) To a solution of tert-butyl (4-bromobutyl)carbamate (4.8 g, 19 mmol) and propargyl-PEG4 amine

(2 g, 8.6 mmol) in DMF (50 mL) was added potassium carbonate (3.6 g, 26 mmol). The solution was
stirred at 80° C. for 6 hrs, then partitioned between DCM (200 ml) and brine (50 ml). The organic layer
was separated, washed with brine, dried with sodium sulfate, filtered, and concentrated, then purified
by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph
eluted with 10% to 100% acetonitrile and water with 0.1% TFA as modifier. Yield of product 3.5 g,
70%. Ion(s) found by LCMS: M/2+H=574.4.

(609) Step b.

(610) ##STR00613##

(611) Product from the previous step (3.5 g, 8.6 mmol) was treated with TFA (20 ml) at room
temperature for 0.5 hour, then concentrated to dryness, dissolved in water, frozen, lyophilized, and
used in the next step without further purification. The yield was quantitative for this step. Ion(s) found
by LCMS: M/2+H=374.3.

(612) Step c.

(613) ##STR00614##

(614) Diamine TFA salt from the previous step (37 mg, 0.1 mmol) was added to a solution of the
carboxylic acid (130 mg, 0.2 mmol, described in the synthesis Int-10, step f) in 10 ml DMF, then
treated with HATU (80 mg, 0.2 mmol), and N-methylmorpholine (0.25 ml, 2 mmol). The resulting
solution was stirred for 1 hr, then concentrated and purified by reverse phase liquid chromatography
(RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 10% to 100% acetonitrile and
water with no 0.1% TFA as modifier. Yield of the product 120 mg, 75%. Ion(s) found by LCMS:
M/2+H=799.9.

(615) Step d.

(616) ##STR00615##

(617) Product from the previous step (120 mg, 0.075 mmol) was treated with 2 ml trifluoroacetic acid
and stirred for 30 min at room temperature. The resulting solution was concentrated, dissolved into
water (2 mL), then treated with a solution of lithium hydroxide (12 mg, 0.5 mmol) dissolved in water (1
mL). The resulting solution was stirred 10 min, then made slightly acidic with 0.1 ml acetic acid,
concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco
COMBIFLASH® liquid chromatograph eluted with 0% to 30% acetonitrile and water, using 0.1% TFA
as the modifier. Yield of the products 48 mg as TFA salt, 57%. Ion(s) found by LCMS: M/2+H=559.757.
M/3+H=373.5.

Example 47. Synthesis of Conjugate 8

(618) A solution of h-IgG1 Fc-PEG4-azide in PBS×1 buffer solution (100 mg, 1.71 μmol, 7.011 mL,
MW=58200 Da, DAR=3.7) were added alkyne derivatized small molecule (Int-12 TFA salt, 45 mg,
0.031 mmol) and freshly prepared pH 7.4 PBS solutions of CuSO4 (0.7 mL of 50.0 mM, 20 eq), tris(3-
hydroxypropyltriazolylmethyl)-amine (THPTA, 0.7 mL of 50.0 mM, 20 eq), and sodium ascorbate (1.05
mL of 50.0 mM, 30 eq). The resulting homogeneous solution was agitated by rocker table for 12 h.
The crude solutions were diluted with pH 7.4 PBS to a final concentration of 1 mg/mL, and ultra filtered
(10,000 MWCO) to a volume of 1 mL, two times. The crude mixtures were then diluted 1:10 in PBS pH
7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size
exclusion chromatography. Purified material was quantified using a NANODROP™ UV visible
spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of the
Fc used in the conjugation, and concentrated to approximately 10 mg/mL using a centrifugal
concentrator (10,000 MWCO). Purified molecules were analyzed using 4-12% Bis Tris SDS PAGE
gels by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel
included a molecular weight ladder with the indicated molecular weight standards (FIG. 42). Yields are
typically 40-60%. MALDI MS analysis showed a range of masses (60000-90000) with an average of
mass of 62358. Average DAR=3.
Example 48. Comparison of In Vitro and In Vivo Potency of Selected Inhibitors with their Fc-
Conjugates in CPE Assays and in a Lethal Mouse Influenza Model

(619) To demonstrate that conjugation of neuraminidase inhibitors described herein to Fc enhances

their activities in viral replication assays and in in vivo efficacy models, we compared the activities of
selected unconjugated inhibitors to their Fc conjugates in Cytopathic Effect (CPE) assays, and in a
lethal mouse influenza infection model. For the CPE microneutralization assay, ten two-fold serial
dilutions of each test article (TA), starting at 160 nM or 400 nM (9600 nM for zanamivir and oseltamivir
controls), were prepared in duplicate for one-hour inoculation with INFV A (A/CA/09 H1N1) at a
multiplicity of infection (MOI) 0.001 and INFV B at a MOI of 0.01 for one-hour incubation. The TA-virus
mix was then added to MDCK cells seeded in 96-well plates, and incubated for one hour. On day 3
post infection for INFV A and 5 for INFV B (B/Brisbane), Cells were fixed and stained with crystal violet
and optical density was read for calculation of 50 percent effective concentration (EC.sub.50) of each
TA using XLfit dose response model. The intrinsic cytotoxicity of each TA was also evaluated using ten
two-fold serial dilutions of each TA starting at 160 nM or 400 nM were prepared in duplicate for
inoculation with MDCK cells in 96-well culture plates. The cell viability was determined 3 and 5 days
post treatment using CellTiter-Glo kit. 50% of cytotoxicity concentration (CC.sub.50) was calculated
using XLfit dose response model. No cytotoxicity was observed for any of the TAs to the highest
concentrations tested. A summary of the CPE-based microneutralization assay is shown in Table 23.

(620) TABLE-US-00026 TABLE 23 CPE-based microneutralization assay EC.sub.50 vs INFV A

EC.sub.50 vs INFV B (nM) (nM) Oseltamivir 390.0 1065.0 Zanamivir 264.8 382.6 Int -7 665.7 106.6
(Targeting Moiety corresponding to Conjugate 6) Int-12 15.4 0.6 (Targeting Moiety corresponding to
Conjugate 8) Conjugate 6 4.0 52.1 Conjugate 8 0.6 0.3

(621) The data summarized in Table 23 demonstrate that the Fc-conjugated forms of the
neuraminidase dimers possess superior activity in CPE microneutralization assay to their
unconjugated counterpart. Enhancements of 166-fold and 2-fold, versus INFV A and INFV B,
respectively, were observed when comparing Conjugate 6 to Int-7. Enhancements of 26-fold and 2-
fold, versus INFV A and INFV B, respectively, were observed when comparing Conjugate 8 to Int-12.

(622) Conjugate 6 was compared to a the most potent neuraminidase inhibitor dimer from the study
summarized in Table 23 (Int-11, dimer-only corresponding to Int-12 without the trimeric linker allowing
for conjugation to an Fc) in a lethal IAV H1N1 influenza infection model using female BALB/c mice
(Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/08/1934, aka PR8) is a
mouse-adapted isolate with a LD.sub.90 of approximately 30 plaque-forming units (pfu) per mouse.

(623) The experiment comprised 8 groups of 5 mice. At day 0, all mice were challenged with PR8 at
10× the LD.sub.90 by intranasal inoculation in a volume of 50 μl, to mice anesthetized with a mixture
of ketamine/xylazine (150 and 10 mg/kg respectively). Groups received a single treatment of vehicle,
hIgG1 Fc only, conjugate 6, or Int-11 by IV, 2 hours after challenge (Table 24; groups 1-6). Group 7
received Int-11 (15 mg/kg) twice daily (bid) for 5 days, also by IV. Group 8 received oseltamivir (15
mg/kg) orally (PO), bid, for 5 days. All mice were monitored for survival (Table 25) and weight loss
(data not shown) for 10 days after viral challenge.

(624) As expected, mice treated with vehicle or hIgG1 Fc only succumbed to the infection by day 7
(Table 25). Mice treated with 10 doses (150 mg/mouse in total) of oseltamivir demonstrated a
statistically significant delay in death but only a single animal survived until day 10. All mice receiving a
single dose of conjugate 6 at 0.3 or 3 mg/kg survived to the end of the study and showed a net weight
gain over the course of the experiment. Importantly, all mice receiving the Int-11 at 0.3 or 3 mg/kg died
over the course of the study, with only a minimal (2 days or less) delay in death relative to the vehicle
and hIgG1 Fc only controls. As the hIgG1 Fc has no inherent antiviral activity (group 2) this suggests
the greatly improved activity of conjugate 6 is the result of the improved avidity resulting from the
multivalent display on an Fc, as suggested by the results summarized in Table 23, as well as improved
pharmacokinetics and contribution from Fc mediated immune engagement. The difference in activity
between the inhibitor dimer alone and conjugate 6 was statistically significant at both dose
concentrations (compare groups 3 and 6, and groups 4 and 5; Table 25). On a mass basis, a 500×
greater cumulative dose of Int-11 was required to observe equivalent efficacy to conjugate 6.

(625) TABLE-US-00027 TABLE 24 Study design Treatment Group Challenge Dose Route/ n = 5 Day 0
Compound (mg/kg) Schedule 1 Influenza Vehicle N/A IV, Singe A virus (PBS) dose starting 2 (H1N1)
hIgG1 Fc 3 2 hours A/Puerto only post infection 3 Rico/08/1934 Conjugate 6 3 4 by way Conjugate 6
0.3 5 of IN route. Int-11 0.3 6 Int-11 3 7 Int-11 15 IV, bid for 5 days starting 2 hours post infection 8
Oseltamivir 20 PO, bid for (TAMIFLU ™) 5 days starting 2 hours post infection

(626) TABLE-US-00028 TABLE 25 Mouse survival Significance Dose to Int-11 Test (mg/ Dosing %
Significance 0.3 mg/kg* Group article kg) schedule Survival to vehicle or 3 mg/kg.sup.∧ 1 Vehicle IV,
Single 0 (PBS) 2 hIgG1 3 IV, Single 0 NS Fc only 3 Conjugate 3 IV, Single 100 0.0027 0.0035 6
4 Conjugate 0.3 IV, Single 100 0.0027 0.0027* 6 5 Int-11 0.3 IV, Single 0 0.0027 6 Int-11 3 IV,
Single 0 0.0027 7 Int-11 15 IV, bid × 100 0.0027 5 days 8 Oselta- 15 PO, bid × 20 0.0027 mivir 5
days

Example 49. Synthesis of Int-13 ((5R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-E/Z-

[3-(propargyl-PEG4)-propenyl]-pyrrolidine-(2R)-carboxylic acid)

(627) ##STR00616##

(628) Step a. (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxy-(3S)-Z-

propenylpyrrolidine-1-carboxylic acid tert-butyl ester.

(629) To a stirring mixture of (5R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-Z-

propenylpyrrolidine-(2R)-carboxylic acid, HCl salt (prepared accordingly to reference JACS, 2002, 124,
4716-4721; 1.0 mmol) in acetonitrile (10 mL) it is added trimethylammonium hydroxide (1.5 mmol).
After stirring for 3 h at room temperature the di-tert-butyldicarbonate (4 eqmol) is added. Upon reaction
completion, all the volatiles are evaporated per vacuum techniques. The residue is diluted with water
(10 ml). Ethyl acetate (10 ml) is added, and 1 M sulfuric acid aqueous solution is added until the water
layer reaches pH ˜3. The water layer is washed with two additional aliquots of ethyl acetate (10 mL).
The combined organics are dried over sodium sulfate, filtered and concentrated. The residue is
purified by chromatographic techniques to afford the desired product.

(630) Step b. (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-Z-

propenylpyrrolidine-1-carboxylic acid tert-butyl ester.

(631) To a 0° C. stirring mixture of (2R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-

carboxy-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester (1.0 mmol) in dichloromethane
(5.0 mL) and methanol (1.0 mL) it is slowly added (trimethylsilyl)diazomethane (1.1 mmol). The
mixture is stirred until completion, while temperature is gently allowed to reach ambient. All the
volatiles are evaporated per vacuum techniques. If necessary, the residue is purified by
chromatographic techniques to afford the desired product.

(632) Step c. (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-

formylpyrrolidine-1-carboxylic acid tert-butyl ester.

(633) A room temperature mixture of (2R)-((1R)-acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-

carboxymethyl-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester (1 mmol) in
dichloromethane (15 mL) is cooled to −78° C. Ozone is bubbled through the solution until a faint blue
color of dissolved ozone persists. Nitrogen is bubbled through the solution until blue color disappears,
then dimethyl sulfide (4.0 mmol) is added, the flask transferred into a freezer (−20° C.) and let sit for 1
hour. The solution is concentrated and the residue is purified by chromatographic techniques to afford
the desired product.

(634) Step d. (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxymethyl-(3S)-E/Z-

[3-(propargyl-PEG4)-propenyl]pyrrolidine-1-carboxylic acid tert-butyl ester.
(635) To a 0° C. stirring mixture of propargyl-PEG4-phosphonium bromide (1.0 mmol) in DMF (5.0 mL)
it is added sodium hydride (1.1 mmol), and after 10 minutes temperature is raised to ambient. Stirring
is continued for 1 h, then (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-
carboxymethyl-(3S)-formylpyrrolidine-1-carboxylic acid tert-butyl ester (1.0 mmol) is added in DMF
(1.0 mL). Upon completion, the reaction is quenched by saturated ammonium chloride solution. The
aqueous solution is extracted several times with ethyl acetate, and the combined organic phases are
washed with brine, dried, and evaporated. The residue is purified by chromatographic techniques to
afford the desired product.

(636) Step e. (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-carboxy-(3S) E/Z-[3-

(propargyl-PEG4)-propenyl]pyrrolidine-1-carboxylic acid tert-butyl ester.

(637) To a 0° C. stirring mixture of (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-

carboxymethyl-(3S)-E/Z-[3-(propargyl-PEG4)-propenyl]pyrrolidine-1-carboxylic acid tert-butyl ester
(1.0 mmol) in tetrahydrofuran (12.0 mL) and water (3.0 mL) it is added lithium hydroxide (1.1 mmol).
Stirring is continued and the temperature is raised to ambient after 15 minutes. Upon completion, the
solution is brought to acidic pH by the means of the excess addition of AMBERLITE® IRN-77 resin.
The mixture is filtered and the filtrate is concentrated per vacuum techniques, yielding to the title
compound. If necessary, the residue is purified by chromatographic techniques to afford the desired
product.

(638) Step f. (5R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-E/Z-[3-(propargyl-PEG4)-

propenyl]-pyrrolidine-(2R)-carboxylic acid.

(639) To a 0° C. stirring mixture of (2R)-((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(5R)-

carboxy-(3S) E/Z-[3-(propargyl-PEG4)-propenyl]pyrrolidine-1-carboxylic acid tert-butyl ester (1.0
mmol) and 2-methyl-2-butene (0.5 mL) in dichloromethane (8.0 mL), it is added trifluoroacetic acid (4.0
mL).

(640) After 10 minutes, the temperature is raised to ambient. Upon completion, all the volatiles are
evaporated per vacuum techniques. The residue is purified by chromatographic techniques to afford
the desired product.

Example 50. Synthesis of Conjugate 9

(641) A solution of hIgG1 Fc-PEG4-azide in pH 7.4 PBS×1 buffer solution (100 mg, 1.71 μmol, 7.011
mL, MW=58,200 Da, DAR=3.7) is added to a pH 7.4 PBS×1 buffer solution (2.45 mL) of Int-13 ((5R)-
((1R)-Acetylamino-(2S)-methoxy-(2S)-methylpentyl)-(4S)-E/Z-[3-(propargyl-PEG4)-propenyl]-
pyrrolidine-(2R)-carboxylic acid) (0.031 mmol), cupric sulfate (0.62 mmol), tris(3-
hydroxypropyltriazolylmethyl)-amine (0.62 mmol), and sodium ascorbate (0.93 mmol). The resulting
homogeneous solution is gently shaken with a rocker table for 12 h. The crude solution is diluted with
pH 7.4 PBS to a final concentration of 1 mg/mL, and ultra-filtered (10,000 MWCO) to a volume of 1
mL, for two times. The crude mixture is then diluted 1:10 in PBS pH 7.4, and purified using MabSelect
Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified
material is quantified using a NANODROP™ UV visible spectrophotometer (using a calculated
extinction coefficient based on the amino acid sequence of the Fc used in the conjugation, and
concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified
molecules are analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 ug of each molecule into
the gel, and staining using instant Blue staining. Each gel includes a molecular weight ladder with the
indicated molecular weight standards. MALDI MS analysis is used to determine the average DAR.

Example 51. Synthesis of propargyl-PEG4-phosphonium bromide

(642) ##STR00617##

(643) A mixture of propargyl-PEG4-bromide (1.0 mmol) and triphenylphosphine (1.2 mmol) in toluene
(10 mL) are refluxed. Upon completion, the mixture is cooled to ambient. The solids are filtered and
used in the next step without any additional purification.
Example 52. Synthesis of Int-14 ((5R)-[(1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-
methylpentyl]-(4S)-Z-propenylpyrrolidine-(2R)-carboxylic acid, HCl salt)

(644) ##STR00618##

(645) Step a. N-{(2.S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-

(2S)-methylpentyl}-(propargyl-PEG4)-carboxyamide.

(646) To 0° C. a stirring mixture of {(2S)-Methoxy-(1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-

propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-carbamic acid tert-butyl ester (it is prepared accordingly
to reference JACS, 2002, 124, 4716-4721; 1.0 mmol) in dry dichloromethane (5 mL) is added
trifluoroacetic acid (10 mmol), and the temperature is raised to ambient. Upon completion, the solvent
is removed under reduced pressure. The resulting residue is dissolved in dichloromethane (20 mL)
and extracted with a saturated aqueous solution of sodium bicarbonate. The organic layer is
separated, dried (sodium sulfate), filtered and evaporated. The crude amine is dissolved in DMF (5
mL) and treated at 0° C. under stirring with propargyl-PEG4-acid (1.1 mmol), diisopropylethylamine
(3.0 mmol) and HATU (1.1 mmol). Upon reaction completion, all the volatiles are evaporated per
vacuum techniques. The residue is taken up in ethyl acetate (15 ml), and washed with saturated
aqueous solution of sodium bicarbonate (10 mL), then 1 M sulfuric acid aqueous solution (10 mL). The
combined organics are dried over sodium sulfate, filtered and concentrated. The residue is purified by
chromatographic techniques to afford the desired product.

(647) Step b. (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-oxo-

(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester.

(648) To a stirring solution of N-{(2.S)-methoxy-(1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-

propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-(propargyl-PEG4)-carboxyamide (1.0 mmol) in a mixture
of acetonitrile and water (10:1, 5 mL) is added ceric ammonium nitrate (2.0 mmol) in small portions at
45° C. during 1 h, and stirring is continued until completion. The reaction is quenched with a saturated
aqueous solution of sodium bicarbonate (5 mL). The aqueous layer is extracted with EtOAc (3×10
mL), the combined organic layers are dried (sodium sulfate) and evaporated to give a crude which is
used for the next step without further purification. The material is dissolved in acetonitrile (5 mL), di-
tert-butylcarbonate is added (1.5 mmol) followed by triethylamine (2.0 mmol) and DMAP (catalytic).
Upon completion, the reaction is quenched with a saturated solution of ammonium chloride (5 mL).
The aqueous layer is extracted with EtOAc (3×10 mL), and the combined organic layers are dried
(sodium sulfate). All the volatiles are removed per vacuum techniques. If necessary, the residue is
purified by chromatographic techniques to afford the desired product.

(649) Step c. (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R/S)-

methoxy-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester.

(650) To a −78° C. stirring solution of (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-

methylpentyl)-(5R)-oxo-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester (1.0 mmol) in THE
(8 mL) is added SUPER-HYDRIDE® (1 M in THF, 2.2 mmol). After 30 min the reaction mixture is
quenched with a saturated aqueous solution of sodium bicarbonate (4 mL) and 30% hydrogen
peroxyde (5 drops). The mixture is warmed up to rt and stirred for another 30 min and the aqueous
layer is extracted with EtOAc (3×10 mL). The combined organic layers are dried (sodium sulfate) and
the solvent is evaporated to give the hemiaminal, which is used without further purification. To a
solution of the above product in methanol (16 mL) is added p-toluenesulfonic acid hydrate (0.1 mmol)
at rt. The reaction mixture is stirred overnight and is quenched with a saturated aqueous solution of
sodium bicarbonate (10 mL). Methanol is removed under reduced pressure, water (10 mL) is added to
the resulting residue and extracted with EtOAc (3×10 mL). The organics are separated and dried with
brine and sodium sulfate, filtered and concentrated. If necessary, the residue is purified by
chromatographic techniques to afford the desired product.

(651) Step d. (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-

cyano-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester.
(652) To a −78° C. stirring solution of (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-
methylpentyl)-(5R/S)-methoxy-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester (1.0 mmol)
in dichloromethane (20 mL) it is added trimethylsilyl cyanide (2.0 mmol) followed by boron trifluoride
diethyl etherate (1.2 mmol). The reaction mixture is stirred from −78° C. to −50° C. over a period of 3
h. A saturated aqueous solution of sodium bicarbonate (40 mL) is added and the aqueous layer is
extracted with EtOAc (3×15 mL). The combined organic layers are dried (sodium sulfate), filtered and
concentrated under reduced pressure. The resulting residue consisting of a mixture of epimeric cyano
derivatives is purified by chromatographic techniques to afford the desired product.

(653) Step e. (5R)-[(1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl]-(4S)-Z-

propenylpyrrolidine-(2R)-carboxylic acid, HCl salt.

(654) To a solution of (2R)-((1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl)-

(5R)-cyano-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester (1.0 mmol) in AcOH (10 mL),
12N HCl (10 mL) is added at rt. The solution is stirred at rt until completion, the solvent is evaporated
under reduced pressure. If necessary, the residue is purified by chromatographic techniques to afford
the desired product.

Example 53. Synthesis of Conjugate 10

(655) A solution of hIgG1 Fc-PEG4-azide in pH 7.4 PBS×1 buffer solution (100 mg, 1.71 μmol, 7.011
mL, MW=58,200 Da, DAR=3.7) is added to a pH 7.4 PBS×1 buffer solution (2.45 mL) of Int-14 ((5R)-
[(1R)-(propargyl-PEG4-carboxyamide)-(2S)-methoxy-(2S)-methylpentyl]-(4S)-Z-propenylpyrrolidine-
(2R)-carboxylic acid, HCl salt) (0.031 mmol), cupric sulfate (0.62 mmol), tris(3-
hydroxypropyltriazolylmethyl)-amine (0.62 mmol), and sodium ascorbate (0.93 mmol). The resulting
homogeneous solution is gently shaken with a rocker table for 12 h. The crude solution is diluted with
pH 7.4 PBS to a final concentration of 1 mg/mL, and ultra-filtered (10,000 MWCO) to a volume of 1
mL, for two times. The crude mixture is then diluted 1:10 in PBS pH 7.4, and purified using MabSelect
Sure Resin (GE Healthcare, Chicago, Ill., USA), followed by size exclusion chromatography. Purified
material is quantified using a NANODROP™ UV visible spectrophotometer (using a calculated
extinction coefficient based on the amino acid sequence of the Fc used in the conjugation, and
concentrated to approximately 10 mg/mL using a centrifugal concentrator (10,000 MWCO). Purified
molecules are analyzed using 4-12% Bis Tris SDS PAGE gels by loading 1-2 μg of each molecule into
the gel, and staining using instant Blue staining. Each gel includes a molecular weight ladder with the
indicated molecular weight standards. MALDI MS analysis is used to determine the average DAR.

Example 54. Synthesis of Int-15, HCl salt

(656) ##STR00619## ##STR00620##

(657) Step a. N-{(2.S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-propenylpyrrolidin-(2R)-yl]-

(2S)-methylpentyl}-(3-butynyl)-carboxyamide.

(658) To 0° C. a stirring mixture of {(2S)-Methoxy-(1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-

propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-carbamic acid tert-butyl ester (it is prepared accordingly
to reference JACS, 2002, 124, 4716-4721; 1.0 mmol) in dry dichloromethane (5 mL) is added
trifluoroacetic acid (10 mmol), and the temperature is raised to ambient. Upon completion, the solvent
is removed under reduced pressure. The resulting residue is dissolved in dichloromethane (20 mL)
and extracted with a saturated aqueous solution of sodium bicarbonate. The organic layer is
separated, dried (sodium sulfate), filtered and evaporated. The crude amine is dissolved in DMF (5
mL) and treated at 0° C. under stirring with 4-pentynoic acid (1.1 mmol), diisopropylethylamine (3.0
mmol) and HATU (1.1 mmol). Upon reaction completion, all the volatiles are evaporated per vacuum
techniques. The residue is taken up in ethyl acetate (15 ml), and washed with saturated aqueous
solution of sodium bicarbonate (10 mL), then 1 M sulfuric acid aqueous solution (10 mL). The
combined organics are dried over sodium sulfate, filtered and concentrated. The residue is purified by
chromatographic techniques to afford the desired product.
(659) Step b. (2R)-((1R)-(4-pentynoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-oxo-(3S)-Z-
propenylpyrrolidine-1-carboxylic acid tert-butyl ester.

(660) To a stirring solution of N-{(2.S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-

propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-(3-butynyl)-carboxyamide (1.0 mmol) in a mixture of
acetonitrile and water (10:1, 5 mL) is added ceric ammonium nitrate (2.0 mmol) in small portions at 45°
C. during 1 h, and stirring is continued until completion. The reaction is quenched with a saturated
aqueous solution of sodium bicarbonate (5 mL). The aqueous layer is extracted with EtOAc (3×10
mL), the combined organic layers are dried (sodium sulfate) and evaporated to give a crude which is
used for the next step without further purification. The material is dissolved in acetonitrile (5 mL), di-
tert-butylcarbonate is added (1.5 mmol) followed by triethylamine (2.0 mmol) and DMAP (catalytic).
Upon completion, the reaction is quenched with a saturated solution of ammonium chloride (5 mL).
The aqueous layer is extracted with EtOAc (3×10 mL), and the combined organic layers are dried
(sodium sulfate). All the volatiles are removed per vacuum techniques. If necessary, the residue is
purified by chromatographic techniques to afford the desired product.

(661) Step c. (2R)-((1R)-(4-pentynoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R/S)-methoxy-(3S)-Z-

propenylpyrrolidine-1-carboxylic acid tert-butyl ester.

(662) To a −78° C. stirring solution of (2R)-((1R)-(2R)-((1R)-(4-pentynoyl)-(2S)-methoxy-(2S)-

(663) Step d. (2R)-((1R)-(4-pentynoyl)-(2S)-methoxy-(2S)-methylpentyl)-(5R)-cyano-(3S)-Z-

propenylpyrrolidine-1-carboxylic acid tert-butyl ester.

(664) To a −78° C. stirring solution of (2R)-((1R)-(4-pentynoyl)-(2S)-methoxy-(2S)-methylpentyl)-

(5R/S)-methoxy-(3S)-Z-propenylpyrrolidine-1-carboxylic acid tert-butyl ester (1.0 mmol) in
dichloromethane (20 mL) it is added trimethylsilyl cyanide (2.0 mmol) followed by boron trifluoride
diethyl etherate (1.2 mmol). The reaction mixture is stirred from −78° C. to −50° C. over a period of 3
h. A saturated aqueous solution of sodium bicarbonate (40 mL) is added and the aqueous layer is
extracted with EtOAc (3×15 mL). The combined organic layers are dried (sodium sulfate), filtered and
concentrated under reduced pressure. The resulting residue consisting of a mixture of epimeric cyano
derivatives is purified by chromatographic techniques to afford the desired product.

(665) Step e.

(666) To a stirring mixture of N-{(2.S)-methoxy-((1R)-[1-(4-methoxybenzyl)-5-oxo-(3S)-Z-

propenylpyrrolidin-(2R)-yl]-(2S)-methylpentyl}-(3-butynyl)-carboxyamide (1.0 mmol), bis-[N′-(2-
azidoethyl)]-iminodiacetic amide (0.5 mmol), 1H-1,2,3-Triazole-4-methanamine, 1-(phenylmethyl)-N,N-
bis[1-(phenylmethyl)-1 H-1,2,3-triazol-4-yl]methyl]-(0.1 mmol) and sodium ascorbate (1.0 mmol) in
ethanol (5 mL) and water (5 mL), it is added copper sulfate (0.1 mmol). Upon completion, the reaction
is treated with SiliaMetS TAAcONa (0.3 mmol) for 30 minutes. The reaction is filtered and all the
volatiles are evaporated per vacuum techniques. The residue is purified by chromatographic
techniques to afford the desired product.

(667) Step f.
(668) To 0° C. a stirring solution of the intermediate from step e. (1.0 mmol), propargyl-PEG4-acid
(1.05 mmol), and DIPEA (3.0 mmol) in DMF (10 mL) it is added HATU (2.0 mmol). All the volatiles are
evaporated per vacuum techniques and the residue is purified by chromatographic techniques to
afford the desired product.

(669) Step g. Int-15, HCl salt

(670) To a solution of the intermediate from step f. (1.0 mmol) in AcOH (10 mL), 12N HCl (10 mL) is
added at rt. The solution is stirred at rt until completion, the solvent is evaporated under reduced
pressure. If necessary, the residue is purified by chromatographic techniques to afford the desired
product.

Example 55. Synthesis of Conjugate 11

(671) A solution of hIgG1 Fc-PEG4-azide in pH 7.4 PBS×1 buffer solution (100 mg, 1.71 μmol, 7.011
mL, MW=58,200 Da, DAR=3.7) is added to a pH 7.4 PBS×1 buffer solution (2.45 mL) of Int-15 HCl salt
(0.031 mmol), cupric sulfate (0.62 mmol), tris(3-hydroxypropyltriazolylmethyl)-amine (0.62 mmol), and
sodium ascorbate (0.93 mmol). The resulting homogeneous solution is gently shaken with a rocker
table for 12 h. The crude solution is diluted with pH 7.4 PBS to a final concentration of 1 mg/mL, and
ultra-filtered (10,000 MWCO) to a volume of 1 mL, for two times. The crude mixture is then diluted
1:10 in PBS pH 7.4, and purified using MabSelect Sure Resin (GE Healthcare, Chicago, Ill., USA),
followed by size exclusion chromatography. Purified material is quantified using a NANODROP™ UV
visible spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence
of the Fc used in the conjugation, and concentrated to approximately 10 mg/mL using a centrifugal
concentrator (10,000 MWCO). Purified molecules are analyzed using 4-12% Bis Tris SDS PAGE gels
by loading 1-2 μg of each molecule into the gel, and staining using instant Blue staining. Each gel
includes a molecular weight ladder with the indicated molecular weight standards. MALDI MS analysis
is used to determine the average DAR.

Example 56. Synthesis of bis-[N′-(2-azidoethyl)]-iminodiacetic amide

(672) ##STR00621##

(673) Step a. Bis-[N′-(2-azidoethyl)]-N-Boc-iminodiacetic amide.

(674) To 0° C. a stirring solution of N-Boc-iminodiacetic acid (1.0 mmol), 2-azidoethan-1-amine

hydrochloride (2.0 mmol), and DIPEA (6.0 mmol) in DMF (10 mL) it is added HATU (2.0 mmol). All the
volatiles are evaporated per vacuum techniques and the residue is purified by chromatographic
techniques to afford the desired product.

(675) Step b. Bis-[N′-(2-azidoethyl)]-iminodiacetic amide.

(676) To a 0° C. stirring mixture of Bis-[N′-(2-azidoethyl)]-N-Boc-iminodiacetic amide (1.0 mmol) and 2-

methyl-2-butene (0.5 mL) in dichloromethane (8.0 mL), it is added trifluoroacetic acid (4.0 mL). After
10 minutes, the temperature is raised to ambient. Upon completion, all the volatiles are evaporated per
vacuum techniques. The residue is purified by chromatographic techniques to afford the desired
product.

Example 57. Synthesis of Int-16

(677) A monomer of sulfozanamivir conjugated to a PEG4-alkyne linker and which may be further
conjugated to an Fc domain or an albumin protein is produced according to the following synthetic
scheme. Sulfozanamivir starting material is produced according to Hadházi et al. A sulfozanamivir
analogue has potent anti-influenza virus activity. ChemMedChem Comm. 13:785-789 (2018).

(678) ##STR00622##

Example 58. Synthesis of Int-17

(679) A dimer of sulfozanamivir conjugated to a PEG4-alkyne linker and which may be further
conjugated to an Fc domain or an albumin protein is produced according to the following synthetic
scheme. Sulfozanamivir starting material is produced according to Hadházi et al. A sulfozanamivir
analogue has potent anti-influenza virus activity. ChemMedChem Comm. 13:785-789 (2018).

(680) ##STR00623##

Example 59. Synthesis of Conjugate 12

(681) A solution of azido functionalized aglycosylated Fc (70 mg, 4.7 mL, 1.3709 μmol) was added to
a 15 mL centrifuge tube containing alkyne derivatized small molecule (29.8 mg, 0.0216 mmol, Int-7).
After gently shaking to dissolve all solids, the mixture was added with 206 μl of a mixture solution of L-
ascorbic acid sodium (59.4 mg, 0.3 mmol), copper (II) sulfate (15.9 mg, 0.1 mmol), and THPTA (43.5
mg, 0.1 mmol) in PBS 7.4 buffer (1 ml). The resulting mixture was gently shaken overnight. It was
purified by affinity chromatography over a protein A column, followed by size exclusion
chromatography as described in Example 8. Maldi TOF analysis of the purified final product gave an
average mass of 56,177 Da (DAR=3.6). Yield 10.1 mg, 14% yield. A non-reducing SDS-PAGE of
Conjugate 12 is provided in FIG. 54.

Example 60. Synthesis of Int-18

(682) ##STR00624## ##STR00625##

(683) Step a. Synthesis of tert-butyl (17-{4-[(tert-butoxycarbonyl)amino]butyl}-16-oxo-4,7,10,13-

tetraoxa-17-azahenicos-1-yn-21-yl)carbamate

(684) ##STR00626##

(685) To the solution of di-tert-butyl [azanediyldi(butane-4,1-diyl)]biscarbamate (1.5 g, 4.17 mmol from

Example 45, step a) and propargyl-PEG4-acid (1.08 g, 4.17 mmol) in DCM (40 mL) was added EDC
(1.0 g, 5 mmol), HOBt (650 mg, 5 mmol), and DIEA (1.4 ml, 10 mmol) at room temperature, then
stirred overnight at room temperature. The resulting solution was concentrated and purified by and
purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid
chromatograph eluted with 10% to 100% acetonitrile and water with no modifier. Yield of 1.9 g, 76%.
Ion(s) found by LCMS: M+H=602.4.

(686) Step b. Synthesis of N,N-bis(4-aminobutyl)-4,7,10,13-tetraoxahexadec-15-yn-1-amide NH.sub.2

(687) ##STR00627##

(688) Tert-butyl (17-{4-[(tert-butoxycarbonyl)amino]butyl}-16-oxo-4,7,10,13-tetraoxa-17-azahenicos-1-

yn-21-yl)carbamate (1.90, 3.1 mmol) was treated with 20 ml TFA at room temperature for 0.5 hour,
then concentrated to dryness and used in the next step without further purification. Yield is quantitative
for this step. Ion(s) found by LCMS: M/2+H=402.3.

(689) Step c. Synthesis of fully protected Int-18

(690) ##STR00628##

(691) N,N-bis(4-aminobutyl)-4,7,10,13-tetraoxahexadec-15-yn-1-amide (0.150 g, 0.32 mmol) was

added to a solution of the ether linked of zanamivir acid (0.400 g, 0.63 mmol, described in Example
31, step f) in DCM (10 mL), then treated with EDC (0.200 g, 1.0 mmol), HOBt (0.135 g, 1.00 mmol),
and DIEA (0.14 ml, 1.00 mmol) at room temperature for overnight. The resulting solution was
concentrated and purified by and purified by reverse phase liquid chromatography (RPLC) using an
Isco COMBIFLASH® liquid chromatograph eluted with 10% to 100% acetonitrile and water with no
0.1% TFA as modifier. Yield of the products 310 mg, 60.3%. Ion(s) found by LCMS: M/2+H=813.9.

(692) Step d. Synthesis of Int-18

(693) ##STR00629##

(694) Product from the previous step (300 mg, 0.18 mmol) was treated with trifluoroacetic acid (2 mL)
acid and stirred for 30 min at room temperature. The resulting solution was concentrated, re-dissolved
in water (2 mL), then treated with a solution of lithium hydroxide (24 mg, 1 mmol) dissolved in water (1
mL). The reaction was stirred 10 min then quenched with 0.1 ml acetic acid, concentrated and purified
by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph
eluted with 0% to 50% acetonitrile and water, using 0.1% TFA as the modifier. Yield of the products
140 mg, 52%. Ion(s) found by LCMS: M/2+H=573.8, M/3+H=382.9.

Example 61. Synthesis of PEG4-azido Fc for Conjugate 13a

(695) Preparation of 0.05M PEG4-azidoNHS Ester Solution in DMF/PBS

(696) PEG4-azido NHS ester (80.5 mg) was dissolved in DMF (0.50 mL) at 0° C. and diluted to 4.063
mL by adding 3.50 mL of PBS 1× buffer at 0° C. This solution was used for preparing other PEG4-
azido Fc with variety of drug-antibody ratio (DAR) values by adjusting the equivalents of this PEG4-
azido NHS ester PBS×1 solution.

(697) Preparation of PEG4-Azido Fc

(698) 0.05M PEG4-azidoNHS ester PBS×1 buffer solution (0.0984 mL, 4.92 μmol, 2.5 equivalents)
was added to a solution of h-IgG1 Fc (105 mg in 5.031 mL of pH 7.4 PBS, MW-53,360 Da, 1.968
μmol) and the mixture was shaken gently for 12 hours at ambient temperature. The solution was
concentrated using a centrifugal concentrator (30,000 MWCO) to a volume of—1.5 mL. The crude
mixture was diluted 1:10 in PBS pH 7.4, and concentrated again. This wash procedure was repeated
for total of three times. The unreacted azido reagent was removed with this procedure. The
concentrated Fc-PEG4-azide was diluted to 5.03 mL with pH 7.4 PBS 1× buffer and ready for click
conjugation. The purified material was quantified using a NANODROP™ UV visible spectrophotometer
(using a calculated extinction coefficient based on the amino acid sequence of h-IgG1). The yield was
quantitative after buffer exchange/purification.

(699) The nucleic acid construct encoding the Fc for any conjugate described herein may include a
nucleic acid sequence encoding the amino acid sequence of an Fc including Lys447 (e.g., a C-
terminal lysine residue) and/or an N-terminal murine IgG signal sequence. Upon expression, the C-
terminal lysine and, when present, the N-terminal murine IgG signal sequence of the Fc are
proteolytically cleaved, resulting in an Fc having the amino acid sequence lacking Lys447 (e.g.,
lacking a C-terminal lysine residue) and, when present in the expression construct, the N-terminal
murine IgG signal sequence. The presence or absence of a C-terminal lysine does not alter the
properties of the Fc or the corresponding conjugate.

Example 62. Synthesis of Conjugate 13a

(700) Preparation of click reagent solution: 0.0050M CuSO.sub.4 in PBS buffer solution: 20.0 mg
CuSO.sub.4 was dissolved in 25.0 mL PBS×1, than took 22.0 mL above CuSO.sub.4 solution and
added 189.4 mg BTTAA and 1090 mg Na Ascorbate to give a clear solution (0.0050M CuSO.sub.4,
0.020M BTTAA and 0.25M Sodium Ascorbate).

(701) A solution of azido functionalized Fc (100 mg, 4.79 mL, 1.87 μmol, SEQ ID NO: 35) was added
to a 15 mL centrifuge tube containing alkyne derivatized small molecule, Int-18 (11.2 mg, 0.00750
mmol, prepared in Example 60). After gently shaking to dissolve all solids, the solution was treated
with 3.00 mL of above click reagent solution. The resulting colorless homogeneous solution was gently
shaken overnight. It was purified by affinity chromatography over a protein A column, followed by size
exclusion chromatography (see general conjugate purification protocol in Example 10). Maldi TOF
analysis of the purified final product gave an average mass of 55,913 Da (DAR=1.7). Yield 54.0 mg,
54% yield.
(702) The nucleic acid construct encoding the Fc for conjugate 13a included a nucleic acid encoding
the amino acid sequence of SEQ ID NO: 35, which includes a C-terminal lysine residue and N-terminal
murine IgG signal sequence. Upon expression, the C-terminal lysine and the N-terminal murine IgG
signal sequence of the Fc of conjugate 13a are proteolytically cleaved, resulting in an Fc having the
sequence lacking Lys447 (e.g., lacking a C-terminal lysine residue) and the N-terminal murine IgG
signal sequence. The presence or absence of a C-terminal lysine does not alter the properties of the
Fc or the corresponding conjugate.

(703) Example 63. Syntheses of Conjugate 13b, Conjugate 13c, Conjugate 13d, Conjugate 13e,
Conjugate 13f, and Conjugate 13g

(704) The PEG4-azido Fcs for Conjugate 13b, Conjugate 13c, Conjugate 13d, Conjugate 13e,
Conjugate 13f, and Conjugate 13g were prepared analogously to the PEG4-azido Fc of Conjugate 13a
(Example 61) adjusting the number of equivalents of PEG4-azido NHS ester as described in the table
below. Conjugate 13b, Conjugate 13c, Conjugate 13d, Conjugate 13e, Conjugate 13f, and Conjugate
13g were prepared analogously to Conjugate 13a in Example 62 where the number of equivalents of
targeting moiety (Int-18) was adjusted based on the desired DAR value (Table 26), and the volume of
click reagent solution used was the same volume as employed in the procedure for Example 62. The
DAR values, molecular weights and yields are listed in the table below. Product conjugates were
purified by affinity chromatography over a protein A column, followed by size exclusion
chromatography as described in Example 8. A non-reducing SDS-PAGE of Conjugates 13a-13g is
provided in FIG. 55.

(705) TABLE-US-00029 TABLE 26 Yields of Conjugates 13a-13g Equivalents of PEG4- MALDI

Targeting Sample azido NHS DAR mass Da moiety Equivalents Protein Yield Name ester (Average)
(Average) (TM) of TM Type (%) Conjugate 13a 2.5 1.7 55913 Int-18 4 hIgG1 Fc 54% Conjugate 13b
4.5 2.7 57278 Int-18 6 hIgG1 Fc 50% Conjugate 13c 6.5 3.8 58908 Int-18 8 hIgG1 Fc 60% Conjugate
13d 8.5 4.7 60149 Int-18 10 hIgG1 Fc 54% Conjugate 13e 11 5.8 61726 Int-18 12 hIgG1 Fc 55%
Conjugate 13f 18 8.2 65146 Int-18 16 hIgG1 Fc 53% Conjugate 13g 25 10.3 68153 Int-18 20 hIgG1 Fc
45%

Example 64. In vitro stability of Conjugate 6

(706) To demonstrate the in vitro stability of Conjugate 6 using both mouse and human fresh K2EDTA
treated plasma and liver microsomes. The in vitro mouse and human plasma stability were determined
by comparing the Drug to Antibody Ratio (DAR) envelope after a 24 hr incubation in plasma at 37° C.
by MALDI-TOF mass spectrometric detection. Liver microsomal stability using mouse and human
microsomes was also performed after incubation for 24 hr at 37° C. with MALDI-TOF mass
spectrometric detection. This was to identify potential metabolically labile sites on the Fc protein,
linker, or target moiety.

(707) 64.1 Plasma Stability Sample Preparation

(708) First, 60 μL of Conjugate 6 at 3 mg/ml was mixed with 120 μL plasma. Each plasma type was
aliquoted into 2 tubes. One aliquot was immediately frozen from each plasma type. The remaining
aliquot was placed in a water bath (37° C.) for 24 hours. MAGNE® Protein A beads (Promega) were
equilibrated by gently vortexing the beads into suspension. In duplicate for both plasma types, 50 μL
of bead slutty was added to a 1.5 mL microcentrifuge tube and placed on the magnetic stand for 10
seconds. After 10 seconds, the storage buffer was removed and discarded. 500 μL of bind/wash buffer
(0.1% BSA in 1×PBS pH 7.4) was added to the 1.5 mL microcentrifuge tube containing the beads. The
beads were mixed (vortexing) and placed on a magnetic stand for 10 seconds. After 10 seconds, the
bind/wash buffer was removed and discarded. 50 μL of buffer (1×PBS, pH 7.4) was added to the
microcentrifuge tube containing the beads. 50 μL of the plasma mixture was added to the beads and
gently vortexed to mix. Using a tube shaker, the sample was mixed at room temperature for 60
minutes, ensuring the beads remained in suspension. After mixing, the tube was placed on a magnetic
stand for seconds and the supernatant was removed. 500 μL of buffer (1×PBS, pH 7.4) was added
and gently vortexed to mix. After mixing, the tube was placed on a magnetic stand for 10 seconds,
followed by removal and discarding of wash buffer. The wash step was repeated for a total of 2
washes. Following 2 washes with 500 μL of buffer (1×PBS, pH 7.4), 3 washes with 500 μL, 200 μL,
and 100 μL, respectively, of water were performed. The appropriate volume of water was added to the
tube and gently vortexed to mix well and then placed on a magnetic stand for 10 seconds prior to
removal and discarding of the water. After the third wash with water, 30 μL of elution buffer (90:10:0.4
Water:Acetonitrile:TFA) was added to the beads. Using a tube shaker, the elution buffer and sample
were mixed for 30 minutes at room temperature. After mixing, the tube was placed on a magnetic
stand for 10 seconds, the elution buffer, containing the sample, was removed and kept. 2 μL of sample
were mixed with 2 μL of MALDI matrix (20 mg/mL Sinapic Acid in 70:30:0.1 water:acetonitrile:TFA)
and spotted onto a MALDI target plate using a dual layer technique. The sample was then analyzed by
MALDI-TOF mass spectrometry.

(709) 64.2 Liver Microsomal Sample Preparation

(710) A 10× buffer was made with 500 mM Tris-HCl at pH 7.5 and 50 mM magnesium chloride
hexahydrate. ACV-006 was diluted to 50 μM in 1×PBS, pH 7.4. Liver microsomes were thawed and
vortexed. An aliquot of each species of liver microsomes (human and mouse) were heat killed at 70°
C. for 15 minutes for use as a control. Reaction mixtures were prepared for both species according to
Table 27. Tubes were incubated in a water bath (37° C.) for 24 hours. Samples were extracted for
analysis using MAGNE® Protein A beads (Promega) following the protocol from 59.1.

(711) TABLE-US-00030 TABLE 27 0.5 mg/mL final microsomes concentration with 5 μM Conjugate 6
Heat Killed Live Total 400 μL 400 μL Water 286 μL 286 μL 10x Buffer 40 μL 40 μL Microsomes 10
μL 10 μL (20 mg/mL) Compound 40 μL 40 μL (50 μM) NADPH 20 μL 20 μL Regenerating
Solution A NADPH 4 μL 4 μL Regenerating Solution B

64.3 Sample and Data Analysis

(712) Samples were acquired using Bruker Compass Flex Control version 3.4 to obtain full scan
MALDI-TOF mass spectra (Table 28). BSA was used as an internal calibrant for the acquisition mass
range. Data was further analyzed with Bruker Compass Flex Analysis version 3.4 software. In addition,
the DAR pattern of the control is compared to the DAR pattern of the test sample.

(713) TABLE-US-00031 TABLE 28 Mass Spectrometer (MS) Parameters Mass Spectrometer Bruker
Microflex LT Detection Mass Range 17-141 kDa Sample Rate and Digitizer Settings 0.5 Detector Gain
2.05x Baseline Offset Adjustment 0% Analog Offset 0.5 mV Laser Frequency 60 Hz Spectrometer
Ion Source 1 (IS1) 20.03 kV Ion Source 2 (IS2) 18.12 kV (90.5% IS1) Lens 7.17 kV (35.8% IS1)
Pulsed Ion Extraction 1010 ns Sample Carrier Random Walk Partial Sample Laser Shots 100/section
Total Laser Shots 500 Shots at Raster 20 Raster Shot Diameter Limit 2000 μm Setup Laser Global
Attenuator Offset 0% Laser Attenuator Offset 20% Laser Attenuator Range 30% Digitizer Sensitivity
(Full Scale) 100 mV Digitizer Analog Offset Linear 0.5 mV Digitizer Digital Offset Linear 0 cnt
Detector Gain Voltage—Linear Base 2500 V Detector Gain Voltage—Linear Boost 0 V Calibration
Bovine Serum Albumin [M + 2H], [M + H], [2M + H]

64.4 Results

(714) The test compound, conjugate 6 (FIG. 43), was tested for in vitro stability in both mouse and
human fresh K.sub.2EDTA treated plasma and liver microsomes.

(715) Conjugate 6 was spiked into fresh K2EDTA mouse and human plasma at a final concentration of
1 mg/mL. The plasma was split into 2 aliquots, one being frozen immediately, and the other incubated
in a water bath at 37° C. for 24 hours. At the end of the incubation, samples were extracted from the
plasma matrix by MAGNE® Protein A magnetic beads. Following plasma incubations, samples were
analyzed by MALDI-TOF mass spectrometry for changes in DAR. Conjugate 6 in either mouse (FIG.
44) or human (FIG. 45) plasma incubations were not found to generate any changes in DAR.

(716) Conjugate 6 liver microsomal stability was tested at a final concentration of 5 μM into a 50 mM,
pH 7.5 Tris-HCl buffer solution that contained either active or heat killed liver microsomes at a final
concentration of 0.5 mg/mL and MgCl.sub.2 at a final concentration of 5 mM. All samples were
incubated at a constant temperature of 37° C., and nicotinamide adenine dinucleotide phosphate
(NADPH) regenerating solution was utilized to provide continuous cofactor availability during the
incubation. Incubations were carried out for 24 hours. At the end of the incubation, samples were
extracted from the microsomal matrix by Protein A magnetic beads. Following liver microsomal
incubations, samples were analyzed by MALDI-TOF mass spectrometry for changes in DAR.
Conjugate 6 in either mouse (FIG. 46) or human (FIG. 47) liver microsomal incubations were not found
to generate any changes in DAR.

(717) The in vitro plasma stability after incubation at 37° C. for 24 hr, suggests a lack of degradation of
the Conjugate 6 Fc, linker, or targeting moiety in either mouse or human. Similarly a lack of
degradation was observed after incubation in both mouse and human liver microsomes, suggesting
the absence of metabolites. The results of these in vitro stability studies support that this is a stable
compound with degradants that could have biological liabilities.

Example 65. Efficacy of Conjugate 13 at Different Drug-to-Antibody Ratios (DARs) Against Influenza a
(H1N1) in a Lethal Mouse Model

(718) Conjugate 13 was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/1934) is a mouse-
adapted isolate capable of causing lethal infections in mice. The experiment comprised 13 groups of 5
mice. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal inoculation in a
volume of 30 μl, after being anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg
respectively). Groups 1-13 received a single IV treatment, 2 hours post viral challenge of test article or
vehicle (PBS). The study evaluated 4 different OAR constructs of Conjugate 13 corresponding to
Conjugate 13a, Conjugate 13c, Conjugate 13d, and Conjugate 13g (DARs of 1.7, 3.8, 5.8, and 10.3,
respectively). The synthesis of Conjugate 13a, Conjugate 13c, Conjugate 13d, and Conjugate 13g is
described in Example 61-Example 63. Each construct was evaluated at 0.03, 0.1, and 0.3 mg/kg. The
general study design for each conjugate is summarized in Table 29.

(719) TABLE-US-00032 TABLE 29 General study design of DAR scan Dose Dose volume Group
Conjugate DAR Route/Schedule (mg/kg) (ml/kg) 1 Conjugate 13a 1.7 IV, T + 2 hours 0.3 5 2
Conjugate 13a 1.7 IV, T + 2 hours 0.1 5 3 Conjugate 13a 1.7 IV, T + 2 hours 0.03 5 4 Conjugate 13c
3.8 IV, T + 2 hours 0.3 5 5 Conjugate 13c 3.8 IV, T + 2 hours 0.1 5 6 Conjugate 13c 3.8 IV, T + 2 hours
0.03 5 7 Conjugate 13d 5.8 IV, T + 2 hours 0.3 5 8 Conjugate 13d 5.8 IV, T + 2 hours 0.1 5 9
Conjugate 13d 5.8 IV, T + 2 hours 0.03 5 10 Conjugate 13g 10.3 IV, T + 2 hours 0.3 5 11 Conjugate
13g 10.3 IV, T + 2 hours 0.1 5 12 Conjugate 13g 10.3 IV, T + 2 hours 0.03 5 13 Vehicle (PBS) Na IV, T
+ 2 hours na 5

(720) All constructs were fully protective at 0.3 mg/kg, in contrast, no construct was active at 0.03
mg/kg (0% survival for all groups) indicating the low dose was below the threshold efficacious dose.
However, groups receiving 0.1 mg/kg of conjugates could be discriminated (Table 30). At this dose
level conjugates with DARs of 1.7, 3.8, and 5.8 were significantly more protective than vehicle only
treated mice (p=0.0027). The high OAR construct (10.3) however was not significantly more protective
than vehicle only treated mice (p=0.091). The underlying mechanism by which the high OAR construct
loses activity is currently unknown but could be caused by several factors, including interference with
antibody recycling, resulting in shorter half-life.

(721) TABLE-US-00033 TABLE 30 DAR Range Study (0.1 mg/kg dose groups) Conjugate DAR %
Survival Significance* Conjugate 13a 1.7 60 p = 0.0027 Conjugate 13c 3.8 40 p = 0.0027 Conjugate
13d 5.8 80 p = 0.0027 Conjugate 13g 10.3 0 p = 0.091** * = Significance relative to vehicle (PBS) only
treated mice by the Log-rank (Mantel-Cox) test. ** = Not significant

Example 66. In vivo Efficacy of Conjugate 6 and Conjugate 12

(722) Conjugate 6 and Conjugate 12, analog with Fc mutation at N297A, was evaluated against a
lethal IAV H1N1 influenza infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks).
The challenge virus (A/Puerto Rico/8/34) is a mouse-adapted isolate. The experiment comprised 5
mice per group. Mice were anesthetized with ketamine/Xylazine (150/10 mg/kg) and were challenged
with influenza virus at 3-5× the LD.sub.95 by intranasal inoculation in a volume of 30 μL. A single 1
dose of treatment was administered by IV, 2 h post-infection. PBS was given as negative control. All
mice were monitored for % body weight loss (FIG. 48) and for survival (FIG. 49) for 15 days after
challenge.

Example 67. In Vitro Fcγ Receptor IIA Binding of Conjugate 6 and Conjugate 12

(723) Binding of Conjugate 6 and Conjugate 12, analog with Fc mutation at N297A, was evaluated
against FcγRIIIA by ELISA. The plate was coated with 1 μg/mL recombinant human FcγRIIIA
overnight. The next day, the plate was blocked with 1% BSA solution for 1 h. Conjugates were added
to plate in dose-response ranging from 0.01-1000 nM and incubated for 2 h. Binding was detected by
incubation with peroxidase-conjugated anti-human Fc for 1 h and subsequent incubation with TMB
substrate reagent for 10-15 min.

(724) Binding was determined by reading absorbance at 450 nm (FIG. 50 and FIG. 51).

Example 68. In Vivo Conjugate 6 Plasma Sample Analysis

(725) Conjugate 6 in plasma samples were quantified by a neuraminidase capture detection ELISA.
Briefly, molecules were captured on neuraminidase coated plates and then detected using a HRP-
conjugated anti-human IgG-Fc antibody. Protein concentration was calculated in GraphPad Prism
using 4PL non-linear regression of Conjugate 6 standard curves. A more detailed method description
is provided below.

(726) Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) were coated with 0.1 U/well
neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Biological) in 1×KPL coating
buffer (5150-0041, SeraCare). Plates were incubated at room temperature for 1 hr on an orbital plate
shaker (500 rpm). Serial dilutions of the plasma samples were plated and incubated at room
temperature for 2 hours (sample diluent: 0.5% BSA in PBS 0.025% Tween 20+naïve cynomolgus
monkey plasma final concentration of 1:2,500). Conjugate 6 standard curves ranging from 0.230 to
500 ng/mL, in duplicate were run on each plate. Following the 2 hr incubation, plates were washed 5×
in 300 μL PBS with 0.05% Tween 20. Conjugate bound to neuraminidase on the plates was then
probed with an HRP conjugated anti-human IgG Fc F(ab′)2 (709-036-098, Jackson) diluted 1:1,000 in
sample diluent for 1 hr at room temp. Plates were then washed 8× in 300 μL PBS with 0.05% Tween
20 and developed with TMB substrate for 7-8 minutes. The reaction was stopped with 1 N
H.sub.2SO.sub.4. Absorbance was read at 450 nm. Conjugate 6 in plasma samples was interpolated
using GraphPad Prism Version 6 following nonlinear regression analysis (Sigmoidal, 4PL analysis) of
the standard curves.

(727) The resulting mean plasma concentrations were then used to calculate pharmacokinetic
parameters by non-compartmental analysis using Phoenix WinNonlin 7.0.

(728) Toxicokinetics (TK), Groups 2 (IV, 5 mg/kg) and 3 (IV, 20 mg/kg)

(729) Concentrations were comparable between male and female animals within the same dose group
on day 1 (Table 31) and day 8 (Table 32) after administration. Mean plasma exposures appeared to
increase approximately dose-proportionally across both days. After the 2nd dose administration, a
slight accumulation of about 30% was noted across the different dose groups. A plot of days 1 and 8
mean plasma concentrations across the different dose groups is shown in FIG. 52.

(730) Table 31: Toxicokinetics Day 1

(731) TABLE-US-00034 TABLE 31 Toxicokinetics Day 1 Dose Conc (μg/mL) at Time (hr) Tmax Cmax
AUC0-t (mg/kg) Route Sex 0.083 1 2 4 8 24 72 120 168 (hr) (μg/mL) (μg•hr/mL) 5 IV F 112 78.8 85.8
71.0 75.1 52.5 38.6 18.5 22.3 0.083 112 6190 IV M 101 84.6 107 75.3 72.5 38.1 29.8 35.3 15.8 2 107
5970 Mean 107 81.7 96.5 73.2 73.8 45.3 34.2 26.9 19.0 1.04 110 6080 20 IV F 449 510 353 331 303
134 105 63.6 46.6 1 510 18800 IV M 448 422 485 373 329 160 96.0 76.2 53.4 2 485 20500 Mean 449
466 419 352 316 147 100 69.9 50.0 1.50 497 19600

(732) TABLE-US-00035 TABLE 32 Toxicokinetics Day 8 Dose Conc (μg/mL) at Time (hr) Tmax Cmax
AUCO-t (mg/kg) Route Sex 0.083 1 2 4 8 24 72 120 168 (hr) (μg/mL) (μg•hr/mL) 5 IV F 111 111 99.3
82.3 100 52.2 46.7 35.0 33.9 1 111 7970 IV M 113 114 113 78.4 74.4 54.7 35.2 47.1 28.2 1 114 7700
Mean 112 112 106 80.4 87.3 53.5 41.0 41.1 31.0 1 112 7830 20 IV F 398 359 319 315 298 190 126
99.3 85.5 0.083 398 23900 IV M 391 443 354 435 263 219 143 142 83.8 1 443 27800 Mean 394 401
337 375 280 205 134 120 84.7 0.542 420 25800

Pharmacokinetics (PK), Groups 4 (IV, 10 mg/kg) and 5 (SC, 10 mg/kg)

(733) Following IV administration, plasma concentrations from the male and female animals were
comparable. Very low clearance, resulting in a long terminal half-life was observed following IV
administration (Table 33A and Table 33B).

(734) TABLE-US-00036 TABLE 33A Plasma concentrations for male and female animals Dose Conc
(μg/mL) at Time (hr) (mg/kg) Route Sex 0.083 1 2 4 8 24 72 120 168 336 504 672 5 IV F 166 174 220
153 144 58.6 40.6 21.2 20.7 14.7 n/a 2.7 5 IV M 191 135 258 161 154 71 43.7 47.6 32 22.9 n/a 5.3

(735) TABLE-US-00037 TABLE 33B PK parameters for male and female animals Tmax C0 Cmax
AUClast AUCINF_obs Half-life CI_obs Vss_obs Vz_obs (hr) (μg/mL) (μg/mL) (hr*μg/mL) (hr*μg/mL)
(hr) (mL/min/kg) (mL/kg) (mL/kg) 1 166 220 13700 14400 170 0.00579 68.9 85.1 1 204 258 19400
20800 183 0.004 56.6 63.2

(736) Following SC administration, the time to reach maximum concentrations was reached 72 hours
after dosing but concentrations were measurable through 672 hours post-dose (Table 34A and Table
34B). Bioavailability after SC dosing was high at approximately 139%. A comparison of the plasma
concentration over time between 10 mg/kg IV and SC administration is shown in FIG. 53.

(737) TABLE-US-00038 TABLE 34A Plasma concentrations for male and female animals Dose Conc
(μg/mL) at Time (hr) Route (mg/kg) Sex 0.083 0.5 1 4 8 24 72 120 168 336 504 672 SC 10 F n/a 1.9
5.3 23.1 39.5 54 58.6 66.9 54 28.4 18.9 6.4 SC 10 M n/a 0.3 2.1 19.9 28.1 46.8 64.9 54.7 57.2 26.8
25.5 8.6

(738) TABLE-US-00039 TABLE 34B PK parameters for male and female animals T.sub.max
C.sub.max AUC.sub.0-t F (hr) (μg/mL) (μg × hr/mL) (%) 120 66.9 22600 137% 72 64.9 23300 141%

Example 69. Efficacy of Conjugate 6 Against Influenza A/Puerto Rico/8/1934 (H1N1) in a Lethal
Mouse Model

(739) Conjugate 6 was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c mice
(Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/1934) is a mouse-
adapted isolate capable of causing lethal infections in mice. The experiment comprised 7 groups of 5
mice. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal inoculation in a
volume of 30 μl, after being anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg
respectively). Mortality and body weights were recorded daily and any animal with a 20% loss of body
weight was scored as a death. Test groups received a single IV treatment, 2 hours post viral challenge
of conjugate 6, hIgG1 Fc control, or vehicle (PBS). Animals receiving oseltamivir were dosed orally,
twice daily, for 5 days, starting 2 hours after viral challenge. The study design is summarized in Table
35.

(740) TABLE-US-00040 TABLE 35 Study design for Influenza A/PR/8/34 (h1N4) study Dose Dose
volume # of Group Test article Route/Schedule (mq/kq) (ml/kq) mice 1 Vehicle IV, T + 2 hrs. na 5 5 2
hIgG1 Fc IV, T + 2 hrs. 10 5 5 3 Oseltamivir PO, bid × 5, 20 5 5 T + 2 hrs. 4 Oseltamivir PO, bid × 5, 5
5 5 T + 2 hrs. 5 Conjugate 6 IV, T + 2 hrs. 10 5 5 6 Conjugate 6 IV, T + 2 hrs. 2 5 5 7 Conjugate 6 IV, T
+ 2 hrs. 0.4 5 5
(741) As expected, mice receiving vehicle or the hIgG Fc only succumbed to the infection by day 6.
Similarly, mice treated with oseltamivir at the low dose (5 mg/kg; bid for 5 days) reached mortality by
day 8 (Table 36). However, mice receiving 20 mg/kg of oseltamivir with the same dosing schedule
were fully protected (p=0.0027). In contrast to that seen with oseltamivir, mice treated with conjugate 6
were fully protected at all dose levels (i0, 2, and 0.4 mg/kg) from a single IV dose (p=0.0027).

(742) TABLE-US-00041 TABLE 36 Survival for Influenza A/PR/8/34 (H1N1) study (Day 14) Test article
Dose % Survival Significance* hIgG1 Fc 10 0 p = 0.85** Oseltamivir 20 100 p = 0.0027 Oseltamivir 5 0
p = 0.091** Conjugate 6 10 100 p = 0.0027 Conjugate 6 2 100 p = 0.0027 Conjugate 6 0.4 100 p =
0.0027 * = Significance relative to vehicle (PBS) only treated mice by the Log-rank (Mantel-Cox) test.
** = Not significant

(743) The potency of conjugate 6 was further supported by daily body weight measurements. As
expected, mice treated with vehicle or hIgG1 Fc demonstrated a steady drop in body weight until it
exceeded 20%, at which time they were scored as a death (Table 37). The group treated with
oseltamivir at 5 mg/kg also displayed a consistent loss of weight until reaching mortality at day 8. Mice
treated with 15 oseltamivir at the high dose (20 mg/kg) showed a steady, but reduced loss of body
weight, which reached i4% at day 8, before recovering.

(744) In contrast to control and oseltamivir treated mice, those groups receiving conjugate 6
maintained healthy body weights throughout the study even at the lowest dose concentration (0.4
mg/kg) (Table 37). The largest transient loss of weight among conjugate 6 treated mice was only 2% at
day 14 in the 2 mg/kg dose group. By both survival and body weight measurements conjugate 6
demonstrated robust protection from Influenza A/Puerto Rico/8/1934 with a single IV dose as low as
0.4 mg/kg.

(745) TABLE-US-00042 TABLE 37 Mouse body weight data (% BW relative to day 0). Average of 5
mice; *data not included once the first animal reaches mortality within a group hIgG1 Fc Oseltamivir
Conjugate 6 Day post Vehicle (mg/kg) (mg/kg) (mg/kg) challenge (PBS) 10 20 5 10 2 0.4 0 100 100
100 100 100 100 100 1 99 99 102 99 103 102 101 2 102 102 103 96 104 102 103 3 96 96 102 95 102
102 100 4 89 88 100 90 103 100 100 5 82 81 95 89 102 99 99 6 77 77 98 90 102 100 101 7 90 84 103
101 102 8 86 78 105 101 102 9 91 101 102 102 10 95 102 101 103 11 97 102 101 102 12 97 101 99
102 13 97 103 100 102 14 96 104 98 101

Example 70. Synthesis of Conjugate 14

(746) The title conjugate was prepared analogously to Conjugate 13a (Example 62) using PEG-azido-
Fc (SEQ ID NO:35) and Int-7 (Example 19). Maldi TOE analysis of the purified final product gave an
average mass of 56,502. Da (DAR=2.1). Yield 43.4 mg, 43.4%.

Example 71. Synthesis of Conjugate 15

(747) This conjugate was prepared analogously to Example 62 (Conjugate 13a) by PEG4-azido-Fc
(SEQ ID NO: 35, Example 61) and Int-12 (Example 46). Maldi TOE analysis of the purified final
product gave an average mass of 56,528. Da (OAR=2.2). Yield 40.0 mg, 40.0%.

Example 72. Synthesis of Conjugate 16

(748) This conjugate was prepared analogously to Example 62 (Conjugate 13a) by PEG4-azido-Fc
(SEQ ID NO: 35, Example 61) and Int-10 (Example 31). Maldi TOF analysis of the purified final
product gave an average mass of 56,507. Da (DAR=2.1). Yield 41.7 mg, 42%.

Example 73. Synthesis of Int-19

(749) ##STR00630## ##STR00631##

(750) To a solution of tert-butyl [2-(2-bromoethoxy)ethyl]carbamate (1.8 g, 6.6 mmol) and propargyl-

PEG4 amine (0.7 g, 3.0 mmol) in 30 ml DMF was added potassium carbonate (1.2 g, 9 mmol). The
reaction was stirred at 80° C. for 6 hrs, and then partitioned between DCM (200 mL) and brine (50
mL). The organic layer was separated and washed with brine and dried with anhydrous sodium
sulfate. Upon filtration, the resulting filtrate was concentrated and purified by reverse phase liquid
chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 10% to
100% acetonitrile and water with 0.1% TFA as modifier. Yield of the products 1.0 g, 65%. Ion(s) found
by LCMS: M/2+H=606.4.

(751) Step b.

(752) ##STR00632##

(753) Product from the previous step (1.0 g, 1.6 mmol) was treated with TFA (10 mL) at room
temperature for 0.5 hour, then concentrated to dryness and used in next step without further
purification. Yield was quantitative for this step. Ion(s) found by LCMS: M/2+H=406.3.

(754) Step c.

(755) ##STR00633##

(756) Product from the previous step (120 mg, 0.17 mmol) was treated with a solution of ether
zanamivir acid (230 mg, 0.38 mmol, Example 31) in 10 ml DMF. To this solution was added EDC (100
mg, 0.5 mmol), HOBt (65 mg, 0.5 mmol), and DIEA (0.14 ml, 1 mmol). The resulting solution was
stirred at room temperature overnight, then purified by and purified by reverse phase liquid
chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 10% to
100% acetonitrile and water with no TFA as modifier. Yield of the products 180 mg, 60.2%. Ion(s)
found by LCMS: M/2+H=816.9.

(757) Step d.

(758) ##STR00634##

(759) Product from the previous step (180 mg, 0.11 mmol) was treated with trifluoroacetic acid (2 mL)
for 30 min at room temperature. The resulting solution was concentrated and was dissolved into water
(2 mL), then treated with a solution of lithium hydroxide (24 mg, 1 mmol) dissolved in H.sub.2O (1 mL).
The resulting reaction was stirred 10 min then quenched with 0.1 ml acetic acid. The solution was
concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco
COMBIFLASH® liquid chromatograph eluted with 0% to 50% acetonitrile and water, using 0.1% TFA
as the modifier. Yield of product 140 mg, 52%. Ion(s) found by LCMS: M/2+H=575.8, M/3+H=384.2.

Example 74. Synthesis of Conjugate 17

(760) This conjugate was prepared analogously to Example 62 (Conjugate 13a) by PEG4-azido-Fc
(SEQ ID NO: 35, Example 61) and Int-19 (Example 73). Maldi TOF analysis of the purified final
product gave an average mass of 56,672. Da (DAR=2.2). Yield 36.7 mg, 36.7%.

Example 75. Synthesis of Int-20

(761) ##STR00635## ##STR00636##

(762) To a solution of diethyl iminodiacetate (3.1 g, 16.06 mmol) in anhydrous DMF (36 mL) was
added benzyl bromide (2.38 mL, 19.64 mmol) and potassium carbonate (6.44 g, 46.46 mmol). The
resulting mixture was heated at 70° C. for 16 hours. After cooling to room temperature, the reaction
mixture was diluted water and extracted with tertiarybutylmethylether (3×100 mL). The organic layers
were washed with brine, dried over Na.sub.2SO.sub.4 and filtered then concentrated. The residue was
purified by normal phase silica gel chromatography (Isco, 0 to 10% ethyl acetate and hexane). Yield
3.13 g, 69.8%. Ion found by LCMS: [M+H].sup.+=280.2.

(763) Step b.
(764) ##STR00637##

(765) A solution of di-ethyl ester from step-a (3.2 g, 11.2 mmol) in anhydrous THE (5 mL) was added
slowly to a round bottom flask containing LiALH.sub.4 (425 mg, 14.2 mmol) in THE (2 mL) under
nitrogen gas at 0° C. The syringe was rinsed with THE (2×5 mL). The resulting mixture was slowly
warmed up to room temperature overnight. Methanol (2 mL) was added slowly to quench the reaction
following by addition of NaOH aqueous (1 mL). The resulting mixture was stirred for 1 hour then
filtered under vacuum. The filtrate was concentrated and used in the next step without purification.
Yield 2.4 g, 109%. Ion found by LCMS: [M+H].sup.+=196.2.

(766) Step c.

(767) ##STR00638##

(768) Product from the previous step (1.02 g, 5.24 mmol) in anhydrous THE (4 mL) was added slowly
into a flask containing NaH (60% purity, 2.09 g, 52.4 mmol) and THE (5 mL) at 0° C. under nitrogen
gas. The resulting mixture was stirred for 1 hour followed by dropwise addition of 3-(Boc-
amino)propylbromide (3.8 g, 15.7 mmol) in THE (20 mL). The reaction mixture was slowly warmed to
room temperature and stirred for 3 days. The reaction was cooled down to 0° C. then quenched with
water (6 mL) and stirred for 1 hour. It was extracted with ethyl acetate (2×100 mL). The combined
organic layers were washed with 1 N HCl aqueous and brine. It was dried over Na.sub.2SO.sub.4,
filtered and concentrated. The residue was purified by normal phase chromatography (Isco, 0 to 5%
methanol and dichloromethane). Yield 984 mg, 37%. Ion found by LCMS [M+H].sup.+=510.0.

(769) Step d.

(770) ##STR00639##

(771) Palladium hydroxide (543 mg, 0.77 mmol) was added into a flask containing the step-c product
(985.3 mg, 1.93 mmol) in anhydrous methanol (19.5 mL), under an H2 atmosphere. The resulting
mixture was stirred at room temperature for 16 hours. It was then filtered through a pad of celite and
washed with methanol, and concentrated. The residue was carried on to the next step without
purification. Yield 828.6 mg, 102%. Ion found by LCMS [M+H].sup.+=420.0.

(772) Step e.

(773) ##STR00640##

(774) The step-d product (829 mg, 1.97 mmol) in H.sub.2O:THF (1:1, 16 mL) was cooled down to 0°
C. To this solution was added Na.sub.2CO.sub.3 (314 mg, 2.96 mmol) following by addition of Fmoc
N-hydroxysuccinimide ester (826 mg, 2.37 mmol). The resulting mixture was warmed up to room
temperature and stirred until complete by LCMS, then extracted with ethyl acetate. The organic layer
was washed with brine and dried over Na.sub.2SO.sub.4, filtered and concentrated. The residue was
purified by normal phase chromatography (Isco, 0 to 60% ethyl acetate and hexane). Yield 784 mg,
62%. Ion found by LCMS [M+H-Boc].sup.+=542.0.

(775) Step f.

(776) ##STR00641##

(777) The step-e product (1.01 g, 1.57 mmol) was stirred in TFA (5 mL) and CH.sub.2Cl.sub.2 (9 ml)
at room temperature for 1 hour then concentrated under reduced pressure. The residue was purified
by RPLP (Isco, 5 to 100% methanol and water without modifier). Yield 903 mg, 86%. Ion found by
LCMS [M+H].sup.+=442.2.

(778) Step g.

(779) ##STR00642##
(780) To a mixture of ether zanamivir acid (340 mg, 0.49 mmol), step-f product (111 mg, 0.25 mmol,
Example 31) and HATU (206 mg, 0.53 mmol) in anhydrous DMF (3 mL) was added DIEA (162 mg,
1.23 mmol). The resulting mixture was stirred at room temperature for 1 hour, then directly purified by
RPLC (Isco, 30 to 100% methanol and water without modifier). Yield 249 mg, 61%. Ion found by
LCMS [(M+2H)/2].sup.+=833.8.

(781) Step h.

(782) ##STR00643##

(783) To a solution of the step-g product (249 mg, 0.15 mmol) in anhydrous DMF (0.5 mL) was added
SilaMetS Thiol (1.2 g, 1.47 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (12 mg, 0.07 mmol). The
resulting mixture was stirred for 1.5 hours, then filtered directly into a vial containing HATU (69 mg,
0.18 mmol), propargyl PEG-4 acid (43 mg, 0.16 mmol) and DIEA (43 mg, 0.33 mmol). The reaction
mixture was stirred for 1 hour, then directly purified by RPLC (Isco, 30 to 100% methanol and water
without modifier). Yield 298 mg, 118%. Ion found by LCMS [(M+2H-Boc)/2].sup.+=843.9.

(784) Step i.

(785) ##STR00644##

(786) The step-h product (298 mg, 0.18 mmol) was dissolved in TFA (3 mL) and CH.sub.2Cl2 (3 mL),
and the solution was stirred at room temperature for 16 hours. It was then concentrated under reduced
pressure and purified by HPLC (ACCQ Isco, 0 to 25% acetonitrile and water, using 0.1% TFA as a
modifier). Yield 112 mg, 44%. Ions found by LCMS [(M+2H)/2].sup.+=643.8 and
[(M+3H)/3].sup.+=429.6.

(787) Step j.

(788) ##STR00645##

(789) To a solution of the step-i product (112 mg, 0.072 mmol) in MeOH (4 mL) and water (2 mL) was
added LiOH (10.6 mg, 0.43 mmol). The resulting solution was stirred at room temperature for 1 hour,
then acidified with TFA and concentrated under reduced pressure. The residue was purified by HPLC
(ACCQ Isco, 0 to 25% acetonitrile and water, using 0.1% TFA as modifier). Yield 37 mg, 36%. Ions
found by LCMS [(M+2H)/2].sup.+=603.8, [(M+3H)/3].sup.+=402.9.

Example 76. Synthesis of Conjugate 18

(790) A solution of azido functionalized aglycosylated Fc (100 mg, 5.4 mL, 1.87 μmol, SEQ ID NO: 35)
was added to a 15 mL centrifuge tube containing alkyne derivatized small molecule (16.1 mg, 0.011
mmol, Int-20). After gently shaking to dissolve all solids, the mixture was added to 3 mL premixed
solution of L-ascorbic acid sodium (149 mg, 0.75 mmol, 0.25 M), copper (II) sulfate (2.4 mg, 0.015
mmol, 0.005 M) and BTTAA (25.8 mg, 0.6 mmol, 0.02 M) in PBS 7.4 buffer. The resulting solution was
gently shaken overnight. It was purified by affinity chromatography over a protein A column, followed
by size exclusion chromatography (see general conjugate purification protocol in Example 10). Maldi
TOF analysis of the purified final product gave an average mass of 56826 Da (DAR 2.2). Yield 36.64
mg, 37%.

(791) The nucleic acid construct encoding the Fc for conjugate 18 included a nucleic acid encoding the
amino acid sequence of SEQ ID NO: 35, which includes a C-terminal lysine residue and N-terminal
murine IgG signal sequence. Upon expression, the C-terminal lysine and the N-terminal murine IgG
signal sequence of the Fc of conjugate 18 are proteolytically cleaved, resulting in an Fc having the
sequence lacking Lys447 (e.g., lacking a C-terminal lysine residue) and the N-terminal murine IgG
signal sequence. The presence or absence of a C-terminal lysine does not alter the properties of the
Fc or the corresponding conjugate.

Example 77. Synthesis of Int-21

(792) ##STR00646## ##STR00647##

(793) To a solution of 2-(2-Boc-aminoethoxy) ethanol (6.15 g, 30 mmol) in anhydrous DCM (60 ml)
was added DIPEA (7.8 g, 60 mmol) and DMAP (366.6 mg, 3 mmol). P-toluenesulfonyl chloride (6.86
g, 36 mmol) was then added in portions over 30 minutes. After the resulting mixture was stirred for 3
days, it was concentrated by rotary evaporation and purified by RPLC (20% to 70% acetonitrile/water
with no modifier). Yield 3.71 g, 34.4%. Ion found by LCMS: [M-Boc+H].sup.+=260.

(794) Step b.

(795) ##STR00648##

(796) To a solution of the step-a product (2.1 g, 5.83 mmol) in anhydrous THE (10 ml) was added
sodium carbonate (1.24 g, 11.7 mmol) and mono-N-Boc-1,4-diaminobutane (1.32 g, 7 mmol). The
resulting mixture was heated at 60° C. for 1 day. The salt was then filtered off, and the filtrate was
concentrated by rotary evaporation. The residue was purified through RPLC (100 g, 5 to 50%
acetonitrile and water, using 0.1% TFA as modifier). Yield 1.94 g, 88.6%. Ion found by LCMS:
[M+H].sup.+=376.0.

(797) Step c.

(798) ##STR00649##

(799) To a solution of propargyl PEG-4 acid (781 mg, 3 mmol) and HATU (1.14 g, 3 mmol) in
anhydrous DMF (3 ml) was added DIPEA (390 mg, 3 mmol), followed by the addition of the solution
step-b product (940 mg, 2.5 mmol) and DIPEA (390 mg, 3 mmol) in anhydrous DMF (3 ml). The
reaction mixture was stirred for 30 minutes, then directly purified through RPLC (100 g, 5 to 80%
acetonitrile and water, using 0.1% TFA as modifier). Yield 960.2 mg, 65.3%. Ion found by LCMS:
[M+H].sup.+=618.3, [M-Boc+H].sup.+=518.3.

(800) Step d.

(801) ##STR00650##

(802) The step-c product (960.2 mg, 1.63 mmol) was dissolved in anhydrous THE (6 ml). 4N HCl
solution in dioxane (4 ml) was added, and the reaction mixture was stirred overnight. It was then
concentrated by rotary evaporation. The residue was extracted with water (3 ml×3) and ethyl acetate
(10 m1). The combined aqueous layers were lyophilized. Yield 760 mg, 95.1%. Ion found by LCMS:
[M+H].sup.+=418.0.

(803) Step e.

(804) ##STR00651##

(805) To a mixture of ether zanamivir acid (315 mg, 0.5 mmol) and HATU (190 mg, 0.5 mmol) in
anhydrous DMF (1 ml) was added in portions a solution of the step-d diamine product (148 mg, 0.3
mmol) and DIPEA (165 mg, 1.5 mmol) in anhydrous DMF (1 ml) over 20 minutes. After the addition,
the reaction was stirred for 30 more minutes and directly purified by RPLC (50 g, 30 to 90%
acetonitrile and water, using 0.1% TFA as modifier). Yield 233 mg, 56.7%. Ion found by LCMS:
[(M+2H)/2].sup.+=821.3.

(806) Step f.

(807) ##STR00652##

(808) The step-e product (233 mg, 0.142 mmol) was dissolved in TFA (1.5 ml), and the solution was
heated at 30° C. for 30 minutes. It was then concentrated and directly purified by RPLC (0% to 30%
acetonitrile and water, using 0.1% TFA as modifier). Yield 120 mg, 57.4%. Ions found by LCMS:
[(M+2H)/2].sup.+=621.4, [(M+3H)/3].sup.+=414.7.

(809) Step g.

(810) ##STR00653##

(811) To a solution of the step-f product (120 mg, 0.0816 mmol) in MeOH (2 ml) was added LiOH
monohydrate (63 mg, 1.5 mmol) solution in water (2 ml). The resulting mixture was stirred for 1.5
hours and then concentrated by rotary evaporation. The residue was acidified by 4N HCl solution in
dioxane (0.5 ml) and was purified by HPLC (0 to 15% acetonitrile and water, using 0.1% TFA as
modifier). Yield 98.2 mg, 86.6%. Ions found by LCMS: [(M+2H)/2].sup.+=581.8,
[(M+3H)/3].sup.+=388.2.

Example 78. Synthesis of Conjugate 19

(812) This conjugate was prepared analogously to Example 62 (Conjugate 13a) by PEG4-azido-Fc
(SEQ ID NO: 35, Example 61) and Int-21 (Example 78). Maldi TOF analysis of the purified final
product gave an average mass of 56,548. Da (DAR=2.1). Yield 39.7 mg, 39.7%.

Example 79. Synthesis of Int-22

(813) ##STR00654##

(814) Step a.

(815) ##STR00655##

(816) To a mixture of ether zanamivir acid (1.8 g, 2.8 mmol Example 31) and propargyl-PEG4-amine
(0.82 g, 3.5 mmol. 1.2 eq.) in dichloromethane (50 mL), was added EDC (1.0 g, 5 mmol), HOBt (0.65
g, 5 mmol), and DIEA (1.4 ml, 10 mmol). The resulting solution was stirred at room temperature
overnight, concentrated, and purified by reverse phase liquid chromatography (RPLC) using an Isco
COMBIFLASH® liquid chromatograph eluted with 10% to 100% acetonitrile and water with no 0.1%
TFA as modifier. Yield of products 1.35 g, 50.2%. Ion(s) found by LCMS: M+H=830.4.

(817) Step b.

(818) ##STR00656##

(819) Product from the previous step (1.35 g, 1.6 mmol) was treated with trifluoacetic acid (20 mL) for
30 min at room temperature. The resulting solution was concentrated, dissolved in water (10 mL) and
MeOH (10 mL), then treated with a lithium hydroxide (120 mg, 5 mmol) solution in water (10 mL). The
reaction solution was stirred 10 min then quenched with 0.5 ml acetic acid. The reaction was
concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco
COMBIFLASH® liquid chromatograph eluted with 0% to 50% acetonitrile and water, using 0.1% TFA
as the modifier. Yield of the products 510 mg, 52.8%. Ion(s) found by LCMS: M+H=604.3.

Example 80. Synthesis of Conjugate 20

(820) This conjugate was prepared analogously to Example 62 (Conjugate 13a) by PEG4-azido-Fc
(SEQ ID NO: 35, Example 61) and Int-22 (Example 79). Maldi TOF analysis of the purified final
product gave an average mass of 55,508. Da (DAR=2.3). Yield 37.0 mg, 37.0%.

Example 81. Synthesis of Int-23

(821) ##STR00657## ##STR00658##

(822) To a solution of N-Boc-1.4-diaminobutane (941.5 mg, 5 mmol) in anhydrous THE (10 ml) was
added sodium carbonate (1.06 g, 10 mmol) and 3-(Boc-amino)propylbromide (1.43 g, 6 mmol). The
resulting mixture was heated at 50° C. for 1 day. The salt was then filtered, and concentrated by rotary
evaporation. The residue was purified by RPLC (100 g, 5 to 50% acetonitrile and water, using 0.1%
TFA as modifier). Yield 1.35 g, 58.8%. Ion found by LCMS: [M+H].sup.+=346.0.

(823) Step b.

(824) ##STR00659##

(825) To a solution of propargyl PEG-4 acid (781 mg, 3 mmol) and HATU (1.14 g, 3 mmol) in
anhydrous DMF (3 ml) was added DIPEA (390 mg, 3 mmol), followed by the addition of the solution
step-a product (863.8 mg, 2.5 mmol) and DIPEA (390 mg, 3 mmol) in anhydrous DMF (3 ml). The
reaction mixture was stirred for 30 minutes, then directly purified by RPLC (100 g, 5 to 80% acetonitrile
and water, using 0.1% TFA as modifier). Yield 1.19 g, 81%. Ion found by LCMS: [M+H].sup.+=588.3.

(826) Step c.

(827) ##STR00660##

(828) The step-b product (1.19 g, 2.02 mmol) was dissolved in anhydrous THE (6 ml). 4N HCl solution
in dioxane (4.5 ml) was added, and the reaction mixture was stirred for 1 day. It was then concentrated
by rotary evaporation. The residue was extracted with water (3 ml×3) and ethyl acetate (15 ml). The
combined aqueous layers were lyophilized. Yield 940 mg, quantitative yield. Ion found by LCMS:
[M+H].sup.+=388.3.

(829) Step d.

(830) ##STR00661##

(831) To a mixture of ether zanamivir acid (315 mg, 0.5 mmol, Example 31) and HATU (209.1 mg, 0.55
mmol) in anhydrous DMF (1 ml) was added DIPEA (65 mg, 0.5 mmol). After 5 minutes, a solution of
the step-c product (170 mg, 0.439 mmol) and DIPEA (130 mg, 1 mmol) in anhydrous DMF (1 ml) was
added in portions over 20 minutes. The reaction was stirred for an additional 30 minutes, then directly
purified by RPLC (50 g, 30 to 90% acetonitrile and water, using 0.1% TFA as modifier). Yield 208 mg,
51.6%. Ion found by LCMS: [(M+2H)/2].sup.+=806.7.

(832) Step e.

(833) ##STR00662##

(834) The step-d product (208 mg, 0.129 mmol) was dissolved in TFA (1.5 ml), and the solution was
heated at 30° C. for 30 minutes. It was then directly purified by RPLC (100 g, 0 to 30% acetonitrile and
water, using 0.1% TFA as modifier). Yield 134 mg, 72%. Ions found by LCMS:
[(M+2H)/2].sup.+=606.8, [(M+3H)/3].sup.+=405.0.

(835) Step f.

(836) ##STR00663##

(837) To a solution of the step-e product (134 mg, 0.093 mmol) in MeOH (2 ml) was added a solution
of LiOH monohydrate (63 mg, 1.5 mmol) in water (2 ml). The resulting mixture was stirred for 1.5
hours and then concentrated by rotary evaporation. The residue was acidified by 4N HCl solution in
dioxane (0.5 m1) and was purified by HPLC (0 to 15% acetonitrile and water, using 0.1% TFA as
modifier). Yield 78.4 mg, 62%. Ions found by LCMS: [(M+2H)/2].sup.+=566.4, [(M+3H)/3].sup.+=378.4.

Example 82. Synthesis of Conjugate 21

(838) This conjugate was prepared analogously to Example 62 (Conjugate 13a) by PEG4-azido-Fc
(SEQ ID NO: 35, Example 61) and Int-23 (Example 81). Maldi TOF analysis of the purified final
product gave an average mass of 56,503. Da (DAR=2.2). Yield 34.4 mg, 34%.

Example 83. Activity of Conjugates 13 to 21 Against High Path H7N9 Influenza A, and Two Influenza B
Isolates in a Cytopathic Effects (CPE) Assay

(839) A total of 9 conjugates (Table 38) were run at concentrations of 100, 10, 1, and 0.1 nM and
compared to ribavirin in a CPE assay. The CPE assay followed standard methodology, but briefly,
utilized 80-100% confluent monolayers of MDCK cells in a 96-well plate. To these, test articles were
added in triplicate and allowed to incubate at 37° C. (+5% CO.sub.2) until CPE effects were visually
apparent. Once CPE was noted, cell layers were stained with 0.011% neutral red for approximately 2
hours. Afterwards, a 50:50 mix of Sorensen citrate buffer/ethanol was added and allowed to incubate
for 30 m, then the A.sub.540 was read on a spectrophotometer and EC.sub.50/CC.sub.50 values
calculated by regression analysis. All conjugates had significant activity against the Influenza
B/Florida/4/2006 isolate, with an EC.sub.50 values ranging from 3.05 to 33.5 nm (Table 39). On
average, conjugates demonstrated a 275-fold potency advantage over ribavirin (EC.sub.50 of 3,250
nM). Activity of conjugates against Influenza B/Brisbane/60/2008 was very similar to that seen against
B/Florida with the exception of conjugate 14, which had an EC.sub.50 of greater than 100 nM

(840) Importantly, all conjugates were highly active against the high path Influenza A/Anhui/1/2013
(H7N9) isolate as well. The average EC.sub.50 for all conjugates was 21.2 nM against this isolate
(ranging from 12 to 28.5 nM), compared to 14,000 nM for ribavirin. Lastly, no direct cytotoxic effects of
the conjugates on MOCK monolayers were detected at the concentrations tested.

(841) TABLE-US-00043 TABLE 38 Conjugates and properties Conjugate Int Fc domain DAR Linker
Notes 13 18 SEQ ID NO: 35 2.2 15 atom ether, dimer 14 7 SEQ ID NO: 35 2.1 15 atom carbamate,
dimer 15 12 SEQ ID NO: 35 2.2 15 atom ether, dimer 16 9 SEQ ID NO: 35 2.1 17 atom ether, dimer 17
19 SEQ ID NO: 35 2.2 17 atom ether, dimer 18 20 SEQ ID NO: 35 2.2 19 atom ether, dimer 19 21
SEQ ID NO: 35 2.1 16 atom ether, dimer 20 22 SEQ ID NO: 35 2.3 na ether, monomer 21 23 SEQ ID
NO: 35 2.2 14 atom ether, dimer

(842) TABLE-US-00044 TABLE 39 Activity of conjugates in a CPE assay against Influenza subtypes
EC50 (nM)* A/Anhui/1/2013 (H7N9) B/Brisbane/60/2008 B/Florida/4/2006 Conjugate (zoonotic avian)
(Victoria lineage) (Yamagata lineage) Ribavirin 14000 3300 3250 13 24 21 33.5 14 12 >100 18.5 15
21.5 5.95 4.6 16 28.5 14.5 11 17 28** 11 13 18 31 4.35 3.05 19 14.5 6.8 4.55 20 20.5 12.5 14.5 21
17.5 4.05 3.05 *Average of 2 values (VIS + NR) **EC50 of 28 by VIS, >100 by NR

Example 84. Syntheses of Conjugate 22

(843) Step a. Synthesis of PEG4-Azido IVIG

(844) Preparation of 0.05M PEG4-azidoNHS ester solution in DMF/PBS: 6.05 mg of PEG4-azido NHS
ester was dissolved in 0.050 mL of DMF at 0° C. and diluted to 0.305 mL by adding 0.250 mL of PBS
1× buffer at 0° C. This solution was used for preparing other PEG4-azido IVIG with variety of DAR
values by adjusting the equivalents of this PEG4-azido NHS ester PBS solution.

(845) Preparation of PEG4-azido IVIG: 0.05M PEG4-azidoNHS ester PBS buffer solution (0.301 mL,
15.0 μmol, 5.5 equivalents) was added to a solution of IVIG (Intravenous Immune Globulin, Baxter))
(407 mg in 9.25 mL of pH 7.4 PBS, MW-148863 Da, 1.968 μmol) and the mixture was shaken gently
for 12 hours at ambient temperature. The solution was concentrated using a centrifugal concentrator
(100,000 MWCO) to a volume of ˜1.5 mL. The crude mixture was diluted 1:10 in PBS pH 7.4, and
concentrated again. This wash procedure was repeated for total of three times. The small molecule
reagent was removed with this wash procedure. The concentrated IVIG-PEG4-azide was diluted to
9.25 mL with pH 7.4 PBS 1× buffer and ready for Click conjugation. The purified material was
quantified using a NANODROP™ UV visible spectrophotometer (using a calculated extinction
coefficient based on the amino acid sequence of IVIG). Yield is quantitative after purification.
(846) Step b. Synthesis of Conjugates

(847) Prepared the Click reagent solution: 0.0050M CuSO.sub.4 in PBS buffer solution: 10.0 mg
CuSO.sub.4 was dissolved in 12.53 mL PBS×1, than took 6.00 mL 0.0050M CuSO.sub.4 solution and
added 57.7 mg BTTAA (CAS number 1334179-85-9) and 297.6 mg Na Ascorbate to give the Click
reagent solution (0.0050M CuSO.sub.4, 0.020M BTTAA and 0.25M Sodium Ascorbate).

(848) A solution of azido functionalized IVIG (140 mg, 3.17 mL, 0.936 μmol, IVIG-Linker-1-Azide) was
added to a 15 mL centrifuge tube containing alkyne derivatized small molecule (8.4 mg, 0.00618
mmol, 6.6 eq, described in Example 60). After gently shaking to dissolve all solids, 1.50 mL of above
click reagent solution of (L-ascorbic acid sodium, 0.25 M, 74.2 mg, 0.374 mmol, copper (II) sulfate
0.0050M, 1.2 mg, 0.0075 mmol, and BTTAA 0.020M, 12.9 mg, 0.0300 mmol). The resulting mixture
was gently shaken overnight. It was purified by affinity chromatography over a Protein A column,
followed by size exclusion chromatography (see conjugate purification protocol in Example 10). Maldi
TOF analysis of the purified final product gave an average mass of 151873 Da (DAR=2.7). Yield 51.0
mg, 36% yield.

Example 85. Syntheses of Conjugate 23

(849) Conjugate 23 was prepared analogously to Conjugate 22, substituting the appropriate alkyne
functionalized small molecule (Int-23 described in Example 81) in the click conjugation step.

Example 86. Syntheses of Conjugate 24

(850) Conjugate 24 was prepared analogously to Conjugate 22, substituting the appropriate alkyne
functionalized small molecule (described in Example 19) in the click conjugation step.

Example 87. Efficacy of Conjugates 13, 14, and 21 against Influenza B in a lethal mouse model

(851) Conjugates were evaluated against a lethal Influenza B influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (B/Malaysia/2506/04) is a mouse-
adapted isolate capable of causing lethal infections in mice. The experiment comprised 11 groups of 5
mice. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal inoculation in a
volume of 30 μl (approx. 1 E4 per mouse), after being anesthetized with a mixture of
ketamine/xylazine (150 and 10 mg/kg respectively).

(852) All groups received a single IV treatment, 2 hours post viral challenge of test article, vehicle
(PBS), or Fc only control (hIgG1 Fc). The study evaluated Int-18, Int-7, and Int-23 conjugated to
identical Fc monomers (conjugates 13, 14, and 21, respectively), tested at 3.0, 1.0, and 0.3 mg/kg.
Mice were monitored for 2 weeks and animals exceeding 20% body weight loss, or were found
moribund, were scored as a mortality.

(853) All mice treated with vehicle or the Fc only control, reached mortality by day 7. In contrast, mice
receiving conjugates 13, 14, and 21 were fully protected after receiving a single IV dose at 0.3 mg/kg
(Table 40). As expected, groups receiving the conjugates at 1.0 or 3.0 mg/kg were fully protected as
well.

(854) The potency of all conjugates against Influenza B was further supported by the daily body
measurements (Table 41), which show a less than 5% transient drop across the entire study for any
conjugate treated group. The activity of conjugates 13, 14, and 21 is comparable by dose to the
activity of conjugate 6 against Influenza A H1N1 and H3N2 subtypes. Since conjugates, 6 and 14 have
identical targeting moieties (corresponding to Int-7), a single conjugate may be active against the
dominant seasonal influenza types (Influenza A (H1N1), Influenza A (H3N2), and Influenza B).

(855) TABLE-US-00045 TABLE 40 Mortality data at study end (day 14). Dosage Significance to
Compound (mg/kg) % Survival vehicle (p-value) Vehicle (PBS) na 0 na Fc alone 3.0 0 p = 1 Conjugate
13 0.3 100 (0.0027) 1.0 100 (0.0027) 3.0 100 (0.0027) Conjugate 14 0.3 100 p = 0.0027 1.0 100 p =
0.0027 3.0 100 p = 0.0027 Conjugate 21 0.3 100 (0.0027) 1.0 100 (0.0027) 3.0 100 (0.0027) Average
of 5 mice; p-values calculated by Log-rank (Mantel-Cox) test

(856) TABLE-US-00046 TABLE 41 Mouse body weight data (% BW relative to day 0). Day post
Vehicle Fc alone Conjugate 14 (mg/kg) challenge (PBS) 3.0 0.3 1.0 3.0 0 100.0 100.0 100.0 100.0
100.0 1 98.1 98.1 99.3 99.1 96.5 2 99.6 96.7 98.6 98.6 98.3 3 96.4 94.9 96.8 97.0 98.7 4 89.8 87.8
98.4 97.5 97.8 5 83.6 80.8 97.4 97.8 98.0 6 79.0 76.1 96.9 98.3 99.7 7 * * 99.2 100.5 100.6 8 * * 100.3
101.9 101.1 9 * * 102.5 100.4 100.6 10 * * 100.6 102.0 101.2 11 * * 100.9 100.9 100.9 12 * * 101.8
101.7 101.8 13 * * 101.4 101.7 101.4 14 * * 102.2 102.6 102.2 Average of 5 mice; *data not included
once the first animal reaches mortality within a group

Example 88. Synthesis of PEG4-azido Fc for Conjugate 25, Conjugate 26, Conjugate 27, and
Conjugate 28

(857) Preparation of 0.05M PEG4-azidoNHS ester solution in DMF/PBS×1:27.40 mg of PEG4-azido

NHS ester was dissolved in 0.155 mL of DMF at 0° C. and diluted to by adding 1.200 mL of PBS 1×
buffer at 0° C. This solution was used for preparing other PEG4-azido Fc with variety of OAR values
by adjusting the equivalents of this PEG4-azido NHS ester PBS×1 solution.

(858) Preparation of PEG4-azido Fc (SEQ ID NO: 48): 0.05M PEG4-azidoNHS ester PBS×1 buffer
solution (0.0984 mL, 4.92 μmol, 2.5 equivalents) was added to a solution of h-IgG1 Fc (SEQ ID NO:
48) (234 mg in 13.605 mL of pH 7.4 PBS, MW-57,976 Da, 4.036 μmol) and the mixture was shaken
gently for 2 hours at ambient temperature. The solution was concentrated using a centrifugal
concentrator (30,000 MWCO) to a volume of ˜2 mL. The crude mixture was diluted 1:7 in PBS pH 7.4,
and concentrated again. This wash procedure was repeated for total of three times. The small
molecule reagent was removed with this wash procedure. The concentrated Fc (SEQ ID NO: 48)-
PEG4-azide was diluted to 13.60 mL with pH 7.4 PBS 1× buffer and ready for Click conjugation. The
purified material was quantified using a NANODROP™ UV visible spectrophotometer (using a
calculated extinction coefficient based on the amino acid sequence of h-IgG1).

(859) Preparation of PEG4-azido Fc (SEQ ID NO: 50) was analogous to above PEG4-azido Fc (SEQ
ID NO: 48).

Example 89. Synthesis of Conjugate 25

(860) Preparation of the Click reagent solution: 0.0050M CuSO.sub.4 in PBS×1 buffer solution: 10.0
mg CuSO.sub.4 was dissolved in 12.53 mL PBS×1, than took 10.00 mL this CuSO.sub.4 solution and
added 86.1 mg BTTAA and 495.3 mg Na Ascorbate to give the Click reagent solution (0.0050M
CuSO.sub.4, 0.020M BTTAA and 0.25M Sodium Ascorbate). This Click reagent solution will be used
for Conjugate 25 and Conjugate 26.

(861) A solution of azido functionalized Fc (78.0 mg, 4.535 mL, 1.35 μmol, SEQ ID NO: 48-PEG4-
Azide) was added to a 15 mL centrifuge tube containing alkyne derivatized small molecule (13.2 mg,
8.88 μmol, Int-23). After gently shaking to dissolve all solids, the mixture was added with 2.153 mL of
above Click reagent solution of (L-ascorbic acid sodium, 0.25 M, 106.6 mg, 0.538 mmol, copper (II)
sulfate 0.0050M, 1.72 mg, 0.0107 mmol, and BTTAA 0.020M, 18.5 mg, 0.0431 mmol). The resulting
mixture was gently shaken for 6 hours at ambient temperature. It was purified by affinity
chromatography over a protein A column, followed by size exclusion chromatography (see conjugate
purification protocol in Example 10). Maldi TOF analysis of the purified final product gave an average
mass of 60973 Da (DAR=2.1). Yield 50.3 mg, 64% yield.

(862) The nucleic acid construct encoding the Fc for conjugate 25 included a nucleic acid encoding the
amino acid sequence of SEQ ID NO: 48, which includes a C-terminal lysine residue and N-terminal
murine IgG signal sequence. Upon expression, the C-terminal lysine and the N-terminal murine IgG
signal sequence of the Fc of conjugate 25 are proteolytically cleaved, resulting in an Fc having the
sequence lacking Lys447 (e.g., lacking a C-terminal lysine residue) and the N-terminal murine IgG
signal sequence. The presence or absence of a C-terminal lysine does not alter the properties of the
Fc or the corresponding conjugate.

Example 90. Synthesis of Conjugate 26

(863) Preparation of Conjugate 26 was analogous to Conjugate 25 by using the same batch of PEG4-
azido Fc(SEQ ID NO: 48) and an alkyne-derivatized small molecule (Int-7). Maldi TOF analysis of the
purified final product gave an average mass of 61068 Da (DAR=2.2). Yield 49.5 mg, 63% yield.

Example 91. Synthesis of Conjugate 27

(864) Preparation of the Click reagent solution: 0.0050M CuSO.sub.4 in PBS×1 buffer solution: 10.0
mg CuSO.sub.4 was dissolved in 12.53 mL PBS×1, than took 12.00 mL this CuSO.sub.4 solution and
added 103.3 mg BTTAA and 594.3 mg Na Ascorbate to give the Click reagent solution (0.0050M
CuSO.sub.4, 0.020M BTTAA and 0.25M Sodium Ascorbate). This Click reagent solution will be used
for Conjugate 28.

(865) A solution of azido functionalized Fc (80.0 mg, 4.535 mL, 1.38 μmol, SEQ ID NO: 50-PEG4-
Azide) was added to a 15 mL centrifuge tube containing alkyne derivatized small molecule (13.5 mg,
9.10 μmol, Int-23). After gently shaking to dissolve all solids, the mixture was added with 2.21 mL of
above Click reagent solution of (L-ascorbic acid sodium, 0.25 M, 109.3. mg, 0.552 mmol, copper (II)
sulfate 0.0050M, 1.76 mg, 0.0110 mmol, and BTTAA 0.020M, 19.0 mg, 0.0441 mmol). The resulting
mixture was gently shaken for 6 hours at ambient temperature. It was purified by affinity
chromatography over a protein A column, followed by size exclusion chromatography (see conjugate
purification protocol in Example 10). Maldi TOF analysis of the purified final product gave an average
mass of 61447 Da (DAR=2.5). Yield 37.1 mg, 46% yield.

(866) The nucleic acid construct encoding the Fc for conjugate 27 included a nucleic acid encoding the
amino acid sequence of SEQ ID NO: 50, which includes a C-terminal lysine residue and N-terminal
murine IgG signal sequence. Upon expression, the C-terminal lysine and the N-terminal murine IgG
signal sequence of the Fc of conjugate 27 are proteolytically cleaved, resulting in an Fc having the
sequence lacking Lys447 (e.g., lacking a C-terminal lysine residue) and the N-terminal murine IgG
signal sequence. The presence or absence of a C-terminal lysine does not alter the properties of the
Fc or the corresponding conjugate.

Example 92. Synthesis of Conjugate 28

(867) Preparation of Conjugate 28 was analogous to Conjugate 27 by using the same batch of PEG4-
azido Fc(SEQ ID NO: 50) and an alkyne-derivatized small molecule (Int-7). Maldi TOF analysis of the
purified final product gave an average mass of 61388 Da (DAR=2.4). Yield 44.6 mg, 56% yield.

Example 93. Activity of Conjugate 6 and Conjugate 21 Against High Path Influenza A (H5N1, H7N9) in
a Cytopathic Effects (CPE) Assay

(868) An in vitro assay to determine the potency of conjugates of the invention was conducted against
BSL-3 (high path) influenza A, and generally followed standard procedures. Briefly, different
concentrations of conjugates were mixed with virus (approximately 250 TC.sub.ID50) and allowed to
incubate at 35° C. for one hour. After incubation, the mixture was added to an 80-90% confluent
monolayer of MDCK cells. After a 90 minute incubation cells were washed and conjugates re-applied.
The monolayer was subsequently overlayed with carboxymethylcellulose to minimize viral spreading
and allowed to incubate for two days. After two days of culture cells were washed with PBS and fixed
with 10% formalin. After fixation the MDCK monolayer was permeabilized with Triton X-100 and
immunostained with a mouse mAb against influenza nucleoprotein. Monolayers were read, and the
stained area per well was calculated to determine EC.sub.50/100 values.

(869) The results of the study are summarized in Table 42 and demonstrate the potency of Conjugate
6 and Conjugate 21 against highly pathogenic strains with pandemic potential. Importantly, both
conjugates generated EC.sub.100 values at, or below, 15 nM against four H5N1 and one H7N9
isolate. In contrast, oseltamivir had an EC.sub.100 of approximately 15 nM only against one isolate
(A/Vietnam/i194/2004) and values ranging from 125 to >1000 nM against the other high path strains.
These results suggest that the potential of Conjugate 6 and Conjugate 21 to treat pandemics caused
by highly-virulent influenza to be superior to that of oseltamivir.

(870) TABLE-US-00047 TABLE 42 In vitro activity of Conjugate 6 and Conjugate 12 against high path
influenza isolates EC100 (nM) (CPE in MDCK cells) Conjugate Conjugate High Path Influenza Vehicle
6 21 Oseltamivir H5N1 A/Indonesia/5/2005 N/A ~15 <15 ~200-500 (clade 2.1) A/Vietnam/1194/2004
N/A ~15 <15 ~15 (clade 1) A/turkey/Turkey/1/2005 N/A <15 <15 ~125-250 (clade 2.2) A/Hong
Kong/156/1997 N/A ~15 <15 >1000 (clade 0) H7N9 A/Anhui/01/2013 N/A <15 <15 ~500-1000 H1N1
(positive control) A/Netherlands/602/2009 N/A <15 <15 ~1000

Example 94. Activity of Conjugate 6 and Conjugate 21 Against Influenza A (HI NI) at Different
Multiplicities of Infection (MOI) in a Cytopathic Effects (CPE) Assay

(871) MOCK cells were seeded at 4×10.sup.4 cells/well in MEM media in 96 well plate (TC-treated)
and incubated at 37° C., 5% C02 for 18-24 h. Test articles (Zanamivir, Oseltamivir, Baloxavir,
Conjugate 6, and Conjugate 21) at dose-range between 1.93-10000 nM were incubated with influenza
AMWSN/1933 at an multiplicity of infection (MOI) of virus:cell between 0.001-1 for 1 h at room
temperature (RT). After 1 h, pre-incubated virus and test article were added to 90-100% confluent
monolayer of MOCK cells and incubated for 1 h at RT.

(872) After 1 h, MEM media supplemented with L-glutamine and penicillin/streptomycin was added to
wells. Infected cells were incubated at 37° C., 5% CO.sub.2 for 72 h. CPE was determined after fixing
and staining cells with crystal violet. EC50 was calculated with non-linear regression analysis using
GraphPad Prism 6 software. The results of the CPE assay provided in Table 43 indicate that
Conjugate 6 and Conjugate 21 outperform standard of care agents in vitro, particularly at high MOIs.

(873) TABLE-US-00048 TABLE 43 In vitro activity of Conjugate 6 and Conjugate 12 against influenza
A (H1N1) at differentmultiplicities of infection EC.sub.50 (nM) H1N1 A/WSN/1933 (CPE in MDCK
cells) MOI 0.001 MOI 0.01 MOI 0.1 MOI 1 Zanamivir 137 938 >10000 >10000 Oseltamivir 525 1896
>10000 >10000 Baloxavir 5 4 79 >100 Conjugate 6 9 15 >100 >100 Conjugate 12 0.6 2 7 10

Example 95. Efficacy of Conjugate 6 Against Influenza A (H1N1) in a Lethal Severe Combined
Immunodeficiency Mouse Model

(874) Conjugates were evaluated against a lethal Influenza A infection in male BALB/c Severe
Combined Immunodeficiency (SCID) mice (Stock #001803; Jackson Laboratories, 6-8 weeks old). The
challenge virus (A/Puerto Rico/08/1934) is a mouse-adapted isolate capable of causing lethal
infections in mice. The experiment comprised 5 groups of 5 mice each. At day 0, all mice were
challenged with virus at 3× the LD95 by intranasal inoculation in a volume of 30 μl (approx. 1 E3 per
mouse), after being anesthetized with a mixture of ketamine and xylazine (150 and 10 mg/kg,
respectively). All groups received a single IV treatment of conjugate 6, 2 hours post viral challenge of
test article, vehicle (PBS), or Fc only control (hIgG1 Fc). The study evaluated 3 different dose
concentrations of conjugate 6 (0.3, 1.0, or 3.0 mg/kg). Mice were monitored for 5 weeks and animals
exceeding 20% body weight loss, or who were found moribund, were scored as a mortality. Body
weights were also recorded to monitor the general health of the animals.

(875) All mice treated with vehicle, or the Fc only control, reached mortality by week 2. In contrast,
mice receiving conjugate 6 were fully protected after receiving single IV doses of 1 or 3 mg/kg for the
duration of the study (FIG. 56, Table 44). When dose of conjugate 6 was lowered to 0.3 mg/kg survival
dropped to 20% by study end. The 0.3 mg/kg dose was fully protected for 3 weeks. The potency of
conjugate 6 in this model of severe immunodeficiency was further supported by body weight data
(FIG. 57, Table 45). Groups receiving conjugate 6 at the 1 or 3 mg/kg dose concentrations
demonstrated no more than a transient body weight loss of less than 3% over the entire course of the
study. Furthermore, at study end both dose groups showed a net gain in weight (7.5 and 2.2%,
respectively). The group dosed with the lowest concentration of conjugate 6 (0.3 mg/kg) had less than
a 4% transient loss of body weight over the first 3 weeks of the study before showing signs of infection
in week 4, which ultimately resulted in death for four of five animals.

(876) Collectively these data demonstrate the potency of conjugate 6 by protecting lethally challenged
mice with single IV doses of conjugate as low as 1 mg/kg. Additionally, this protection was long lasting,
extending over the 5 week duration of the study. This was accomplished in an extreme model of
immunodeficiency in mice completely lacking T & B immune cells, which are essential in clearing
influenza infections. This data supports the use of conjugate 6 to treat both immune competent and
deficient patient populations.

(877) TABLE-US-00049 TABLE 44 Mortality of study dose groups per week % Survival (Day post viral
challenge) Test article 7 14 21 28 35 Vehicle 80 0 0 0 0 Fc only 80 0 0 0 0 Conjugate 6 (3 mg/kg) 100
100 100 100 100 Conjugate 6 (1 mg/kg) 100 100 100 100 100 Conjugate 6 (0.3 mg/kg) 100 100 100
80 20

(878) TABLE-US-00050 TABLE 45 Average group body weight for study animals over 35 days or until
first death within a group Conjugate 6 dose Day Vehicle Fc only 3 mg/kg 1 mg/kg 0.3 mg/kg 0 100 100
100 100 100 1 99.5 99.8 99.6 99.4 98.6 2 98.8 99.2 99.9 99.1 98.9 3 97.7 96.8 101 100.6 97.9 4 94.4
95.4 99.4 98.4 98.5 5 93.7 90.7 100.6 97.9 99.3 6 88.7 83.9 101.2 98.7 98.1 7 81.1 77.6 101.8 98 96.9
8 101.3 99.7 98.9 9 102.6 100.7 98.8 10 101.6 99.8 99.4 11 101.6 99.7 100.3 12 101.8 100.2 100.7 13
102.3 101 100.7 14 103.9 100.5 99.9 15 104.3 101.8 102.3 16 104.3 102 101.3 17 104.3 100.6 102 18
105.4 103.9 100.5 21 105.7 105.9 96.8 22 105.5 103.4 93.9 23 105.6 106.9 91 24 108.4 102.9 25
106.4 102.7 26 108.4 103.9 27 108.6 103.5 28 109.1 104.7 29 108.96 104.5 30 109 105.8 31 108.2
104.9 35 107.5 102.2

Example 96. Conjugate 6 Dose-Dependent Viral Clearance in Lungs

(879) Efficacy studies were conducted in 6-8 weeks female BALB/c mice (Charles River) challenged
intranasally with 3×10.sup.2 PFU/mouse (3× the LD( ) of mouse-adapted influenza A/Puerto
Rico/8/934 (Hi N1). Conjugate 6 or human IgG1 Fc control was administered as a single intravenous
(IV) dose 2 h post-challenge at 0.1-3 mg/kg. Oseltamivir was dosed orally, twice daily for 4 days
starting 2 h post-infection at 5 or 15 mg/kg. Body weights (BW) were recorded for 4 days. At 4 days
post-infection, mice were sacrificed by C02 and both lung lobes were harvested. Lungs were
homogenized with 1 mm silica beads in 1 mL PBS using a MagNA Lyser (Roche). Homogenization
was carried out at 6,000 rpm for 60 s and chilled on ice for 5 min in-between runs. After lung
homogenization tubes were centrifuged for 10 min at 600×g and supernatant was transferred into new
tube.

(880) To determine the viral burden in lungs (measured as Plaque Forming Units (PFUs)),
supernatants of lung homogenate were diluted in infection buffer ranging from 10-i to 10-6. 100 μL of
virus dilutions were added to confluent monolayer of MOCK cells in 24 well plates and incubated for 1
h at room temperature with rocking every 15 min. After removing the virus, liquid overlay media
containing Avicel was added to MOCK cells. Cells were incubation at 37° C., 5% C02 for 40 h. After
incubation, the media was removed and cells were stained with crystal violet to enumerate plaques.
PFUs were calculated relative to weight of the lung (PFU/g lung).

(881) The results of this study demonstrate that low doses of conjugate 6 rapidly lower the viral burden
orders of magnitude better than Oseltamivir (TAMIFLUO) (FIG. 58, Table 46). This observation has
clinical significance since severe influenza infections are caused by the virus moving from an initial
upper respiratory tract infection to the lungs.

(882) TABLE-US-00051 TABLE 46 Viral burden on day 4 post-infection Log reduction Log reduction
Test article [mg/kg] (PFU/mL) (PFU/g) PBS [0] 0.00 0.00 hIgG1 Fc [3] −0.04 0.26 Oseltamivir [5] 0.24
0.43 Oseltamivir [15] 0.52 0.75 Conjugate 6 [0.1] 0.60 0.79 Conjugate 6 [0.3] 1.33 1.55 Conjugate 6 [1]
2.25 2.34 Conjugate 6 [3] 3.20 3.40

Example 97. Conjugate 6 Dose-Dependent Reduction in Inflammatory Cytokines in Lungs

(883) Efficacy studies were conducted in 6-8 weeks female BALB/c mice (Charles River) challenged
intranasally with 3×10.sup.2 PFU/mouse (3× the LD.sub.95) of mouse-adapted influenza A/Puerto
Rico/8/1934 (H1N1). Conjugate 6 or human IgG1 Fc control was administered as a single intravenous
(IV) dose 2 h post-challenge at 0.1-3 mg/kg. Oseltamivir was dosed orally, twice daily for 4 days
starting 2 h post-infection at 5 or 15 mg/kg. Body weights (BW) were recorded for 4 days. At 4 days
post-infection, mice were sacrificed by C02 and both lung lobes were harvested. Lungs were
homogenized with 1 mm silica beads in 1 mL PBS using a MagNA Lyser (Roche). Homogenization
was carried out at 6,000 rpm for 60 s and chilled on ice for 5 mi in-between runs. After lung
homogenization tubes were centrifuged for 10 m at 600×g and supernatant was transferred into new
tube.

(884) For cytokine analysis, supernatants of lung homogenate were serially diluted 2-fold in 96 well
plate. Cytokine levels for INF-γ, TNF-α, IL-6, MIP-1α and MCP-1 were determined by ELISA according
to manufacturer's instructions (R&D Systems).

(885) Morbidity and mortality from severe influenza is ultimately caused by virally induced influx of pro-
inflammatory cytokines in the lungs. One potential concern of using an Fc-conjugate to treat influenza
is whether the Fc fragment would exacerbate cytokine induced inflammation. The results of the H1N1
lethal infection model show just the opposite: a conjugate 6 dose-dependent decrease in pro-
inflammatory cytokines (e.g., TNFα and IL-6) in infected lung tissues (FIG. 59, Table 47).

(886) TABLE-US-00052 TABLE 47 Cytokine response on day 4 post-infection Fold-shift compared to

uninfected control Test article [mg/kg] INFy TNFa IL-6 MCP-1 MIP-1a PBS [0] 0.4 2.2 4.6 16.3 16.4
hIgG1 Fc [3] 0.5 2.2 6.2 20.2 16.8 Oseltamivir [5] 0.2 1.7 4.0 11.7 7.5 Oseltamivir [15] 0.2 1.8 3.5 10.5
6.1 Conjugate 6 [0.1] 0.3 1.5 3.0 10.8 7.9 Conjugate 6 [0.3] 0.2 1.2 1.9 6.7 7.4 Conjugate 6 [1] 0.2 1.1
1.9 6.1 3.6 Conjugate 6 [3] 0.8 1.1 1.4 2.8 2.6 Uninfected 1.0 1.0 1.0 1.0 1.0

Example 98. In Vivo Conjugate 6 Plasma Sample Analysis. Comparison of PK in CD-1 and BALB/c
Severe Combined Immune Deficient Mice

(887) Conjugate 6 in plasma samples were quantified by a neuraminidase capture detection ELISA.
Briefly, molecules were captured on neuraminidase coated plates and then detected using a HRP-
conjugated anti-human IgG-Fc antibody. Protein concentration was calculated in GraphPad Prism
using 4PL non-linear regression of Conjugate 6 standard curves. A more detailed method description
is provided below.

(888) Nunc Maxisorp 96-well plates (Cat No. 12-565-136, ThermoFisher) were coated with 0.1 U/well
neuraminidase from A/California/04/2009 (H1N1) (11058-VNAHC, Sino Biological) in 1×KPL coating
buffer (5150-0041, SeraCare). Plates were incubated at room temperature for 1 hr on an orbital plate
shaker (500 rpm). Serial dilutions of the plasma samples were plated and incubated at room
temperature for 2 hours (sample diluent: 0.5% BSA in PBS 0.025% Tween 20+naïve mouse plasma
final concentration of 1:2,500). Conjugate 6 standard curves ranging from 0.230 to 500 ng/mL, in
duplicate were run on each plate. Following the 2 hr incubation, plates were washed 5× in 300 μL PBS
with 0.05% Tween 20. Conjugate bound to neuraminidase on the plates was then probed with an HRP
conjugated anti-human IgG Fc F(ab′)2 (709-036-098, Jackson) diluted 1:1,000 in sample diluent for 1
hr at room temp. Plates were then washed 8× in 300 μL PBS with 0.05% Tween 20 and developed
with TMB substrate for 7-8 minutes. The reaction was stopped with 1 N H.sub.2SO.sub.4. Absorbance
was read at 450 nm. Conjugate 6 in plasma samples was interpolated using GraphPad Prism Version
6 following nonlinear regression analysis (Sigmoidal, 4PL analysis) of the standard curves.

(889) PK Profiles, CD-1 vs BALB/c SCID Mouse

(890) Conjugate 6 administered intravenously to SCID and CD-1 (immune competent) mice at 5 mg/kg
demonstrated similar PK profiles (FIG. 60). Concentrations were comparable at the sampled time
points. The two-phase PK profiles comprise 24-hour distribution phases followed by a shallow
elimination phase. Conjugate 6 plasma levels remained high (˜10 μg/ml) relative to C.sub.max levels
over the one-week course of the study.
Example 99. Synthesis of the Propargyl Diamine Central Linker

(891) ##STR00664##

(892) Step a.

(893) ##STR00665##

(894) A solution of 2-(2-Boc-Aminoethoxy)ethanol (16.0 g, 78.0 mmol) and CBr.sub.4 (31.0 g, 93.5
mmol) in DCM (100 mL) at 0° C. was treated with PPh.sub.3 (24.5 g, 93.5 mmol) slowly over 15
minutes (exothermic). During the course of the addition the internal temperature was kept below 30°
C. After addition of PPh.sub.3 the reaction was stirred overnight at room temperature. The crude
reaction was concentrated to an oil then purified by normal phase chromatography, eluting with 10%
ethyl acetate/hexanes to 80% ethyl acetate/hexanes. Fractions containing oil droplets on the inside of
the collection tubes were combined and concentrated to a colorless oil. Yield 18.1 g, 86%.

(895) Step b.

(896) ##STR00666##

(897) A solution of the step-a product (10 g, 37.3 mmol), benzylamine (1.60 g, 14.9 mmol), and
K.sub.2CO.sub.3 (6.19 g, 44.8 mmol) in DMF (20 mL) were heated in an oil bath at 75° C. for 8 h. The
mixture was filtered, concentrated and purified by RPLC (5% ACN/water to 100% ACN). Yield 6.8 g,
95%.

(898) Step c.

(899) ##STR00667##

(900) To a solution of the step-b product (5.35 g, 8.98 mmol) in CHCl.sub.3/EtOH (1:20, 100 mL) was
added 20% Pd(OH).sub.2/C (1.26 g, 1/80 mmol). The reaction was stirred overnight under hydrogen
balloon at ambient temperature. The reaction mixture was filtered through a Celite pad. The solvents
were removed and carried to the subsequent step without purification.

(901) Step d.

(902) ##STR00668##

(903) The step-c product was re-dissolved in 20 mL of DMF/dichloromethane (1:5). To this free amine
solution propargyl PEG4 acid (2.36 g, 8.98 mmol), EDCl (2.57 g, 13.5 mmol), HOAt (1.83 g, 13.5
mmol) and Hunig's base (3.13 mL, 18.0 mmol) were added. The reaction mixture was stirred for four
hours, then concentrated and purified by RPLC (10% ACN/water to 60% ACN/water. Yield 4.00 g, 70%
over two steps. Ions found by LCMS: [M-Boc+H].sup.+=534.2, [M+H].sup.+=634.2.

(904) Step e.

(905) ##STR00669##

(906) The step-d product (4.00 g, 6.31 mmol) was treated with 4N HCl in dioxane (30 mL) for 2 hours.
Extra HCl and dioxane were removed by rotary evaporation, and the remaining was further dried
under high vacuum to give Int-10 as 2HCL salt. Yield 3.15 g, 99%. Ion found by LCMS:
[M+H].sup.+=434.2.

Example 100. Synthesis of Int-7a (C7-C7 isomer)

(907) ##STR00670## ##STR00671##

(908) Methyl 5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-

non-2-enonate (30 g, 65.7 mmol) was dissolved into methanol (100 mL) and combined with Lindlar
catalyst (15 g). The resulting mixture was flushed with hydrogen and stirred for 5 hours, flushing
hydrogen through the headspace with hydrogen every 30 minutes. After complete reaction as
determined by HPLC, the catalyst is filtered through celite. The filtrate was used in the next step.

(909) Crude amine from the previous step (18.9 g, 43.8 mmol) was treated with N,N′-bis-boc-1-
guanylpyrazole (14.3 g, 46.0 mmol), and DIEA (9.9 ml, 57.0 mmol) in methanol (100 mL). The
resulting solution was stirred at room temperature until all starting material was consumed as
determined by LCMS (˜30 min). The solution was concentrated to a foam and stored under high
vacuum overnight then used without further purification in the next step. Crude tri-acetate from the
previous step (43.8 mmol) was dissolved in 100 ml dry methanol, then treated with sodium methoxide
in methanol (1.9 mL, 25% solution in methanol, 8.76 mmol) at room temperature. Progress of reaction
was monitored by LCMS which was complete after 10 minutes. The reaction was quenched with 1 N
HCl to a pH of ˜7. The resulting solution was concentrated and purified by reverse phase liquid
chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 10% to
100% acetonitrile and water. No TFA modifier was used for this purification. Yield of product 15.6 g,
65%.

(910) Step b.

(911) ##STR00672##

(912) A mixture of the step-a product (5.47 g, 10 mmol) and DMAP (1.222 g, 10 mmol) was dissolved
in anhydrous THE (30 ml). After cooling in an ice-water bath, the solution was slowly treated with 1,1′-
carbonyldiimidazole (2.6 g, 16 mmol), then stirred for 30 minutes at 0° C., followed by heating at 60°
C. for 2 hours. It was then cooled to room temperature and extracted with water (50 ml) and
EtOAc/hexane (1:1, 100 ml). The organic layer was washed with water (50 ml×3), dried over
Na.sub.2SO.sub.4 and concentrated by rotary evaporation. The white foam product was further dried
under high vacuum and carried to the subsequent step without further purification. Ion found by LCMS:
[M+H].sup.+=573.2.

(913) Step c.

(914) ##STR00673##

(915) A reaction flask containing the step-b product was vacuum flushed with nitrogen and dissolved in
anhydrous DCM (50 ml), then cooled in an ice-water bath. To the cooled solution was added DMAP
(4.89 g, 40 mmol), followed by 4-nitrophenylchloroformate (6.05 g, 30 mmol) was added in portions
over 20 minutes. The solution was stirred at 0° C. then warmed to room temperature for 1 hour. LCMS
shows starting material 1 hr so additional DMAP (1.22 g, 10 mmol) and 4-nitrophenylchloroformate (1
g, 5 mmol) were added. The reaction was continued for 4 hours, then purified with two silica gel
columns (220 g, pre-wet by 20% EtOAc and hexane) and eluted with 20% to 80% EtOAc and hexane.
Yield 4.42 g, 59.9% for two steps. Ion found by LCMS: [M+H].sup.+=738.2.

(916) Step d.

(917) ##STR00674##

(918) To a solution of the step-c product (3.2 g. 4.34 mmol) in anhydrous DCM (3 ml) was added in-
portions over 30 minutes a mixture of propargyl diamine central linker (1.26 g, 2.5 mmol, described in
Example 99) and DIPEA (1.68 g, 13 mmol) in anhydrous DMF (5 ml). The reaction was stirred at room
temperature for 2 hours. It was then concentrated and purified by RPLC (30% to 90% acetonitrile and
water, no TFA modifier). Ions found by LCMS: [(M+2H)/2].sup.+=815.8, [(M-Boc+2H)/2].sup.+=765.8,
[(M-2Boc+2H)/2].sup.+=716. Yield 3.33 g, 94.2%.

(919) Step e.

(920) ##STR00675##
(921) The step-d product (3.33 g, 2.04 mmol) was dissolved in DCM (5 ml) and TFA (5 ml) then stirred
at 35° C. for ˜6 hours. The reaction was monitored by LCMS. When complete the solution was
concentrated and purified by RPLC (5 to 30% acetonitrile and water, no TFA). Ions found by LCMS:
[(M+2H)/2].sup.+=615.8, [(M+3H)/3].sup.+=411. Yield 2.29 g, 91.3%.

(922) Step f.

(923) ##STR00676##

(924) The step-e product (61.5 mg, 0.05 mmol) was dissolved in MeOH/water (1:1, 0.6 ml). After the
solution was cooled to −6° C. (salt/ice bath), 1.0 M LiOH (0.3 ml, 0.3 mmol) was added drop-wise and
the reaction was stirred for 30 minutes. It was then quenched to pH ˜7.0 with 4N HCl in dioxane
solution (75 μl) and directly purified by prep HPLC (Isco ACCQ prep, Luna 5 μm C18(2) 100 Å LC
column 100 mm×30 mm; Gradient: 0% acetonitrile/water for 2 min, then 0% to 15% acetonitrile/water
over 12 min, then isocratic at 15% acetonitrile for 10 min, using 0.1% TFA). Yield 45 mg, 65.3%. Ions
found by LCMS: [(M+2H)/2].sup.+=575.8, [(M+3H)/3].sup.+=384.2. Analytical retention time: 6.013
min. Conditions: Phenomenex Gemini HPLC column, 3 μm WX-C18 110 Å, 100 mm×3 mm, eluted
over 25 minutes with 5-95% acetonitrile and water gradient, using 0.1% TFA.

Example 101. Synthesis of Int-7b (C7-C9 isomer)

(925) ##STR00677##

(926) The C7-C9 heterodimer (Int-7b) was prepared analogously to Int-7a (C7-C7 isomer, Example
100), with the exception that the reaction is conducted at 0° C. and is monitored by HPLC (retention
time: 6.112 min. Conditions: see Example 100, synthesis of Int-7a and stopped when the C7-C9
isomer predominates (˜3 h). This isomer is isolated using the same conditions that were used to
isolate Int-7a. Ions found by LCMS: [(M+2H)/2].sup.+=575.8, [(M+3H)/3].sup.+=384.2.

Example 102. Synthesis of Int-7c (C9-C9 isomer)

(927) ##STR00678##

(928) Int-7c (C9-C9 isomer) was prepared analogously to Int-7a (C7-C7 isomer, Example 100), with
the exception that the reaction conducted at 0° C. and is monitored by HPLC (retention time: 6.232
min. Conditions: see Example 100, synthesis of Int-7a and stopped when the C9-C9 isomer
predominates (˜6 h). This isomer is isolated using the same conditions that were used to isolate Int-7a.
Ions found by LCMS: [(M+2H)/2].sup.+=575.8, [(M+3H)/3].sup.+=384.2.

Example 103. Synthesis of Int-7 (acetonide route)

(929) ##STR00679## ##STR00680##

(930) Step a.

(931) ##STR00681##

(932) Methyl 5-acetamido-7,8,9-O-triacetyl-2,6-anhydro-4-azido-3,4,5-trideoxy-D-glycero-D-galacto-

non-2-enonate (10.0 g, 22 mmol) was dissolved in 60 ml dry methanol, then treated with 20 ml sodium
methoxide in methanol (0.5 M in methanol, 10 mmol) while cooling in an ice-water bath. Progress of
the reaction was monitored by LCMS, which was complete after 2 hours. The pH of the reaction
solution was then adjusted to a value of 5 to 6 by using Amberlite IRN-77 ion exchange resin. The
mixture was filtered to remove the resin and evaporated to dryness under vacuum. The resulting oil
was used for next step without further purification. Ion(s) found by LCMS: M+H=331.1.

(933) Step b.

(934) ##STR00682##
(935) To a solution of the products from the previous step in 70 ml of acetone were added 30 ml of 2,2-
dimethoxypropane and p-toluenesulfonic acid 1 hydrate (400 mg, 2.0 mmol), the resulting solution was
stirred at room temperature overnight. At the end of this time, sodium bicarbonate (170 mg, 2.0 mmol)
was added, and the mixture was concentrated to dryness. The resulting residue was used in next step
without purification. Ion(s) found by LCMS: M+H=371.2.

(936) Step c.

(937) ##STR00683##

(938) To a solution of material from the previous step in 60 ml of methanol was added 5.0 g of a
Lindlar catalyst. The resulting mixture was flushed with hydrogen every 30 minutes and stirred for 5
hours. After complete reaction as determined by HPLC, the catalyst was filtered off through celite. The
filtrate was concentrated and used in the next step without purification.

(939) Crude product from the previous step in 60 ml THE was treated with N,N′-bis-boc-1-
guanylpyrazole (9.3 g, 30.0 mmol), and DIEA (9.9 ml, 57.0 mmol). The resulting solution was stirred at
room temperature until all starting material was consumed as determined by LCMS (4 h). The solution
was concentrated and purified by flash chromatography eluted with 20% to 80% ethyl
acetate/dichloromethane. Yield 8.8 g, 59.0% for four steps. Ion(s) found by LCMS: M+H 587.3.

(940) Step d.

(941) ##STR00684##

(942) A reaction flask containing product from the previous step (6.5 g, 11 mmol) was vacuum flushed
with nitrogen and dissolved in anhydrous dichloromethane (100 ml). After the solution was cooled in
an ice-water bath, DMAP (4.89 g, 40 mmol) was added and stirred to dissolve, then 4-
nitrophenylchloroformate (5.58 g, 28 mmol) was added in portions, while stirring at 0° C. to room
temperature for 1 hour. LCMS shows starting material 1 hr so additional DMAP (1.22 g, 10 mmol) and
4-nitrophenylchloroformate (1.0 g, 5 mmol) were added. The reaction was continued for 4 more hours,
then concentrated and purified by flash chromatography eluting with 20% to 80% ethyl
acetate/dichloromethane. Yield 5.1 g, 59.9%. Ion found by LCMS: [M+H].sup.+=752.2.

(943) Step e.

(944) ##STR00685##

(945) To a solution of the nitrophenyl carbonate from the previous step (1.8 g. 2.3 mmol) in anhydrous
dichloromethane (20 ml) was added a mixture of central linker (0.51 g, 1.0 mmol, added in portions
over 30 minutes) and DIPEA (1.4 ml, 10 mmol) in anhydrous DMF (20 ml). The reaction was stirred at
room temperature overnight, then concentrated and purified by flash chromatography eluting with 0%
to 10% methanol/dichloromethane. Yield 1.35 g, 80%. Ions found by LCMS: [(M+2H)/2].sup.+=830.4,
[(M-Boc+2H)/2].sup.+=780.4, [(M-2Boc+2H)/2].sup.+=730.4.

(946) Step f.

(947) ##STR00686##

(948) Product from the previous step (200 mg, 0.2 mmol) was dissolved into 2 ml MeOH and 2 ml
THF, then treated with a solution of lithium hydroxide (24 mg, 1 mmol) dissolved in 2 ml water. The
reaction was stirred for 10 min at room temperature at which time HPLC showed the reaction was
complete. The pH of the reaction solution was adjusted to the value of 5 to 6 by using Amberlite IRN-
77 ion exchange resin the filtered to remove the resin. The crude product was evaporated to dryness
under a vacuum and used in the next step with purification. Ion(s) found by LCMS:
[(M+2H)/2].sup.+=815.4, [(M-Boc+2H)/2].sup.+=765.4, [(M-2Boc+2H)/2].sup.+=715.4.

(949) Step g.
(950) ##STR00687##

(951) The product from step g (400 mg, 0.25 mmol) was dissolved into 5 ml dichloromethane and 5
m1 TFA, and the resulting reaction solution was stirred at room temperature. The progress of the
reaction was monitored by LCMS. After the completion of the reaction (6 h), the solution was stripped
to dryness and then dissolved in 4 ml water and 4 ml acetonitrile. The resulting solution was stirred for
another 2 hour at room temperature at which LCMS show complete deprotection of the acetonide
protecting groups. This mixture was concentrated and purified by reverse phase liquid
chromatography (RPLC) using an Isco COMBIFLASH® liquid chromatograph eluted with 5% to 40%
acetonitrile/water with 0.1% TFA as the modifier. Yield 270 mg, 68.0%. Ion(s) found by LCMS:
[(M+2H)/2].sup.+=575.8, [(M+3H)/3].sup.+=384.2.

Example 104. NMR Results for Int-7a, Int-7b, and Int-7c

(952) ##STR00688##

(953) .sup.1H NMR (500 MHz, Methanol-d.sub.4) δ 5.91-5.89 (m, 2H), 5.00-4.96 (m, 2H), 4.58-4.53
(m, 2H), 4.42-4.37 (m, 2H), 4.20-4.17 (m, 6H), 4.02-3.97 (m, 2H), 3.78-3.49 (m, 28H), 3.29-3.18 (m,
4H), 2.86 (t, J=2.6 Hz, 1H), 2.85-2.72 (m, 2H), 1.96 (s, 3H), 1.95 (s, 3H).

(954) .sup.13C NMR (125 MHz, MeOD) δ 171.73, 170.80, 170.29, 161.95, 156.06, 155.08, 154.99,
144.07, 143.93, 116.41, 114.10, 106.03, 105.86, 77.71, 74.46, 73.22, 68.62, 68.54, 68.50, 68.38,
68.12, 68.00, 67.94, 67.67, 67.64, 67.53, 67.20, 66.96, 65.44, 61.53, 56.17, 49.74, 49.64, 47.37,
45.17, 39.16, 39.06, 31.73, 19.96, 19.92.

(955) ##STR00689##

(956) .sup.1H NMR (500 MHz, Methanol-d.sub.4) δ 5.91-5.87 (m, 2H), 5.04-4.95 (m, 1H), 4.58-4.48
(m, 2H), 4.45-4.37 (m, 3H), 4.20-4.10 (m, 5H), 4.08-3.98 (m, 2H), 3.79-3.44 (m, 29H), 3.29-3.18 (m,
4H), 2.88-2.70 (m, 3H), 2.02 (s, 3H), 1.96-1.94 (m, 3H).

(957) .sup.1H NMR (500 MHz, DMSO-d.sub.6) δ: 8.28 (d, J=8.4 Hz, 2H, —NH), 7.98 (d, J=11.5 Hz,
2H, —NH), 7.78 (d, J=8.7 Hz, 2H, —NH), 7.60 (t, J=8.5 Hz, 2H, —NH), 7.20 (bs, 2H, —NH), 7.06 (bs,
2H, —NH), 5.69 (bs, 1H), 5.67 (d, J=2.4 Hz, 1H), 4.83 (dd, J=9.2, 2.2 Hz, 1H), 4.45 (dt, J=9, 2.5 Hz,
1H), 4.37 (d, J=2.2 Hz, 1H), 4.33-4.24 (m, 2H), 4.13 (d, J=2.5 Hz, 2H), 4.05-4.32 (m, 41H), 3.23 (m,
1H), 3.15-3.09 (m, 2H), 3.06-3.02 (m, 2H), 2.59 (t, J=6.7 Hz, 2H), 1.91 (s, 3H), 1.78 (s, 3H).

(958) .sup.13C NMR (125 MHz, MeOD) δ: 173.19, 172.98, 172.25, 171.75, 164.26, 163.82, 157.88,
157.55, 156.51, 146.09, 107.21, 106.93, 106.59, 79.22, 76.19, 75.92, 74.72, 74.68, 70.12, 70.08,
70.03, 69.99, 69.92, 69.88, 69.66, 69.48, 69.12, 69.01, 68.94, 68.73, 68.69, 68.50, 68.19, 67.08,
66.93, 66.79, 63.04, 57.68, 51.24, 51.15, 50.13, 48.87, 48.72, 46.72, 46.58, 46.23, 40.64, 40.41,
33.21, 21.49, 21.45, 21.40.

(959) ##STR00690##

(960) .sup.1H NMR (500 MHz, Methanol-d.sub.4) δ: 5.88 (d, J=2.6 Hz, 2H), 4.50 (dt, J=8.5, 2.7 Hz,
2H), 4.43 (ddd, J=9.7, 3.9, 1.5 Hz, 2H), 4.39 (dd, J=11.5, 2.4 Hz, 2H), 4.25-4.16 (m, 2H), 4.19 (d,
J=2.4 Hz, 2H), 4.13 (dt, J=11.5, 5.9 Hz, 2H), 4.05 (m, 2H), 3.77 (t, J=6.2 Hz, 2H), 3.72-3.55 (m, 22H),
3.51 (m, 4H), 3.35-3.23 (m, 4H), 2.86 (t, J=2.4 Hz, 1H), 2.74 (t, J=6.2 Hz, 2H), 2.02 (s, 6H).

(961) .sup.13C NMR (125 MHz, MeOD) δ:173.03, 163.76, 157.89, 157.54, 145.60, 107.21, 79.28,
76.29, 74.70, 70.11, 70.07, 70.03, 69.92, 69.70, 69.47, 68.96, 68.89, 68.72, 68.54, 68.19, 67.05,
66.75, 57.69, 50.09, 48.69, 48.08, 46.10, 40.44, 40.38, 33.23, 21.42.

Example 105. Synthesis of Int-60

(962) ##STR00691## ##STR00692##

(963) Step a.

(964) To a 0° C. stirring solution of previously prepared Ether-zanamivir acid (1.00 g, 1.586 mmol,
Example 31), 2-azidoethylamine hydrochloride (213 mg, 1.744 mmol) and DIPEA (1.105 mL, 6.343
mmol) in DMF (8.0 mL), it was added HATU (615 mg, 1.618 mmol). The temperature was raised to
ambient and stirring was continued until completion. All the volatiles were removed per vacuum
techniques. The residue was taken up in ethyl acetate, washed with a 1 M aqueous solution of sulfuric
acid (1×50 mL), then a saturated aqueous solution of sodium bicarbonate (3×20 mL), and brine (1×50
mL). The resulting organic layers was dried with magnesium sulfate, filtered, and all the volatiles were
removed per vacuum techniques. In this way, 816 mg of the desired intermediate azide was obtained
in high purity and used in the next step without any further purification. (Ion found by LCMS:
[M+H].sup.+=699.2). To a stirring solution of the described crude material (816 mg, 1.168 mmol),
dipropargylamine (54 mg, 0.584 mmol), tris((1-benzyl-4-triazolyl)methyl)amine (31 mg, 0.058 mmol),
and sodium ascorbate (58 mg, 0.292 mmol) in ethanol (10 mL) and water (5 mL), it was added cupric
sulfate (10 mg, 0.061 mmol). Upon completion, copper scavenger SiliaMetS TAAcONa (300 mg,
loading 0.45 mmol/g) was added and stirring was continued for 1 h. The mixture was filtered with the
aid of dichloromethane. The filtrate was washed with a saturated solution of sodium bicarbonate. The
aqueous layer was additionally washed with dichloromethane (3 times). The combined organics were
dried with magnesium sulfate, filtered and concentrated. The residue was purified by silica column
using an Isco COMBIFLASH® liquid chromatography eluted with 0% to 100% hexanes and ethyl
acetate, followed by 0% to 30% dichloromethane and. Yield 817 mg, 94% yield. Ions found by LCMS:
[(M+2H)/2].sup.+=745.8, [(M+3H)/3].sup.+=497.5.

(965) Step b.

(966) To a 0° C. stirring solution of step a product (817 mg, 0.548 mmol), propargyl-PEG4-acid (185
mg, 0.712 mmol) and DIPEA (286 μL, 1.644 mmol) in DMF (7.0 mL), it was added HATU (212 mg,
0.544 mmol). The temperature was raised to ambient and stirring was continued until completion. All
the volatiles were removed per vacuum techniques. The residue was purified by HPLC (0 to 90%
methanol and water). Yield 520 mg, 56%. Ions found by LCMS: [(M+2H)/2].sup.+=866.8,
[(M+3H)/3].sup.+=578.4.

(967) Step c.

(968) A stirring solution of step b compound (520 mg, 0.300 mmol) in 2-methyl-2-butene (0.25 mL),
dichloromethane (4.0 mL) and TFA (2.0 mL) was stirred until gas evolution ceased. All the volatiles
were removed per vacuum techniques. The residue was purified by HPLC (0 to 30% methanol and
water, using 0.1% TFA as modifier). Yield 221 mg, 47%. Ions found by LCMS:
[(M+2H)/2].sup.+=666.8, [(M+3H)/3].sup.+=444.8.

(969) Step d.

(970) To a 0° C. stirring solution of step c product (221 mg, 0.142 mmol) in water (3.0 mL) it was
added lithium hydroxide (20 mg, 0.850 mmol). The reaction was quenched with acetic acid (120 μL),
and all the volatiles were removed per vacuum techniques. The residue was purified by HPLC (0 to
20% methanol and water, using 0.1% TFA as modifier). Yield 130 mg, 62%. Ions found by LCMS:
[(M+2H)/2].sup.+=626.8, [(M+3H)/3].sup.+=418.2.

Example 106. Synthesis of Conjugate 29

(971) A solution of azido functionalized aglycosylated Fc (SEQ ID NO: 35) in pH 7.4 PBS×1 buffer
solution (100 mg, 10 mL, 1.874 μmol) is added to a centrifuge tube containing a pH 7.4 PBS×1 buffer
solution (10.50 mL) of alkyne derivatized small molecule (17 mg, 0.0112 mmol; Example 105, Int-60),
cupric sulfate (4 mg, 0.0225 mmol), tris(3-hydroxypropyltriazolylmethyl)-amine (39 mg, 0.0900 mmol),
and sodium ascorbate (7.4 mg, 0.375 mmol). The resulting mixture was gently shaken overnight. It
was purified by affinity chromatography over a protein A column, followed by size exclusion
chromatography (see Example 10). Maldi TOF analysis of the purified final product gave an average
mass of 56938 Da (DAR=2.3). Yield 49.9 mg, 49% yield.

(972) The nucleic acid construct encoding the Fc for conjugate 29 included a nucleic acid encoding the
amino acid sequence of SEQ ID NO: 35, which includes a C-terminal lysine residue and N-terminal
murine IgG signal sequence. Upon expression, the C-terminal lysine and the N-terminal murine IgG
signal sequence of the Fc of conjugate 29 are proteolytically cleaved, resulting in an Fc having the
sequence lacking Lys447 (e.g., lacking a C-terminal lysine residue) and the N-terminal murine IgG
signal sequence. The presence or absence of a C-terminal lysine does not alter the properties of the
Fc or the corresponding conjugate.

Example 107. Synthesis of Int-65

(973) ##STR00693## ##STR00694##

(974) Step a.

(975) To a 0° C. stirring solution of previously prepared ether-zanamivir acid (2.00 g, 3.172 mmol,
Example 31) and 4-methylmorpholine (0.628 mL, 5.709 mmol) in tetrahydrofuran (30 mL) it was added
isobutyl chloroformate (0.617 mL, 4.7574 mmol). After 10 minutes, the temperature was increased to
ambient and stirring continued for 20 minutes. The temperature was decreased back to 0° C., and
sodium borohydride (360 mg, 9.515 mmol) was added in one portion, followed by dropwise addition of
(10 mL) over 5 minutes. Upon completion, the reaction was quenched with acetic acid (2.860 mL, 50
mmol), and after 5 minutes the temperature was raised to ambient, while stirring was continued until
gas evolution ceased. All the volatiles were evaporated and the residue was suspended in
dichloromethane and filtered. The filtrate was concentrated and the residue was purified by silica
column using an Isco COMBIFLASH® liquid chromatography eluted with 20% to 100% hexanes and
ethyl acetate, using 3% methanol as a modifier. Yield 1.302 g, 66% yield. Ions found by LCMS:
[(M+H)].sup.+=617.2.

(976) Step b.

(977) To a 0° C. stirring solution of step a product (1.25 g, 2.027 mmol) and DIPEA (1.095 mL, 6.284
mmol) in dichloromethane (15 mL), it was added methanesulfonyl chloride (0.314 mmol, 4.054 mmol).

(978) Upon completion, the reaction was treated with water (15 mL). The layers were separated, and
the dichloromethane layer was dried with brine, then magnesium sulfate, and filtered. The solution was
concentrated and the residue was dissolved in DMF (10 mL), and sodium azide (264 mg, 4.054 mmol)
was added, while temperature was raised to 50° C. Completion was observed after 18 h, and all the
volatiles were evaporated per vacuum techniques. The residue was purified by silica column using an
Isco COMBIFLASH® liquid chromatography eluted with 20% to 100% hexanes and ethyl acetate,
using 3% methanol as a modifier. Yield 767 mg, 59% yield. Ions found by LCMS: [(M+H)].sup.+=642.2.

(979) Step c.

(980) To a stirring solution of step b product (496 mg, 0.773 mmol), dipropargylamine (36 mg, 0.386
mmol), tris((1-benzyl-4-triazolyl)methyl)amine (41 mg, 0.077 mmol), and sodium ascorbate (115 mg,
0.580 mmol) in ethanol (16 mL) and water (8 mL), it was added cupric sulfate (13 mg, 0.081 mmol).
Upon completion, copper scavenger SiliaMetS TAAcONa (600 mg, loading 0.45 mmol/g) was added
and stirring was continued for 1 h. The mixture was filtered with the aid of dichloromethane. The filtrate
was washed with a saturated solution of sodium bicarbonate. The aqueous layer was additionally
washed with dichloromethane (3 times). The combined organics were dried with magnesium sulfate,
filtered and all the volatiles were evaporated per vacuum techniques. To a 0° C. stirring solution of the
residue, propargyl-PEG4-acid (151 mg, 0.580 mmol)) and DIPEA (337 μL, 1.933 mmol) in DMF (10.0
mL), it was added HATU (220 mg, 0.580 mmol). The temperature was raised to ambient and stirring
was continued until completion. All the volatiles were removed per vacuum techniques. The residue
was purified by HPLC (0 to 90% methanol and water). Yield 457 mg, 73%. Ions found by LCMS:
[(M+2H)/2].sup.+=809.8, [(M+2H-Boc)/2].sup.+=759.8.
(981) Step d.

(982) A stirring solution of step c compound (451 mg, 0.279 mmol) in 2-methyl-2-butene (0.25 mL),
dichloromethane (4.0 mL) and TFA (2.0 mL) was stirred until gas evolution ceased. All the volatiles
were removed per vacuum techniques. Ions found by LCMS: [(M+2H)/2].sup.+=609.8,
[(M+3H)/3].sup.+=407.0. To a 0° C. stirring solution of the residue in tetrahydrofuran (6 mL) and water
(6 mL), it was added lithium hydroxide (240 mg, 10.03 mmol). Upon completion, the reaction was
quenched with acetic acid (0.638 mL, 11.14 mmol), and all the volatiles were removed per vacuum
techniques. The residue was purified by HPLC (0 to 90% methanol and water, using 0.1% TFA as
modifier). Yield 209 mg, 67%. Ions found by LCMS: [(M+2H)/2].sup.+=569.8, [(M+2H-
Boc)/2].sup.+=380.3.

Example 108. Synthesis of Conjugate 30

(983) A solution of azido functionalized aglycosylated Fc (Example 7, SEQ ID NO: 35) in pH 7.4
PBS×1 buffer solution (50 mg, 5 mL, 1.874 μmol) is added to a centrifuge tube containing a pH 7.4
PBS×1 buffer solution (9.50 mL) of alkyne derivatized small molecule (8.7 mg, 0.0064 mmol, Int-65),
cupric sulfate (2 mg, 0.013 mmol), tris(3-hydroxypropyltriazolylmethyl)-amine (22 mg, 0.0508 mmol),
and sodium ascorbate (25 mg, 0.127 mmol). The resulting mixture was gently shaken overnight. It was
purified by affinity chromatography over a protein A column, followed by size exclusion
chromatography (see Example 10). Maldi TOF analysis of the purified final product gave an average
mass of 61548 Da (DAR=2.4). Yield 32.33 mg, 67% yield.

(984) The nucleic acid construct encoding the Fc for conjugate 30 included a nucleic acid encoding the
amino acid sequence of SEQ ID NO: 35, which includes a C-terminal lysine residue and N-terminal
murine IgG signal sequence. Upon expression, the C-terminal lysine and the N-terminal murine IgG
signal sequence of the Fc of conjugate 30 are proteolytically cleaved, resulting in an Fc having the
sequence lacking Lys447 (e.g., lacking a C-terminal lysine residue) and the N-terminal murine IgG
signal sequence. The presence or absence of a C-terminal lysine does not alter the properties of the
Fc or the corresponding conjugate.

Example 109: Synthesis of p-Nitrophenyl Carbonate Zanamivir Intermediate

(985) ##STR00695## ##STR00696##

(986) Step a.

(987) ##STR00697##

(988) Triacetoxy-azido Zanamivir intermediate (10.0 g, 22 mmol) was dissolved in 60 ml dry methanol,
then treated with 20 ml sodium methoxide in methanol (0.5 M in methanol, 10 mmol) while cooling in
an ice-water bath. Progress of the reaction was monitored by LCMS, which was complete after 2
hours. The pH of the reaction solution was then adjusted to a value of 5 to 6 by using Amberlite IRN-
77 ion exchange resin. The mixture was filtered to remove the resin and evaporated to dryness under
vacuum. The resulting oil was used for next step without further purification. Ion(s) found by LCMS:
M+H=331.1.

(989) Step b.

(990) ##STR00698##

(991) To a solution of the products from the previous step in 70 ml of acetone were added 30 ml of 2,2-
dimethoxypropane and p-toluenesulfonic acid 1 hydrate (400 mg, 2.0 mmol), the resulting solution was
stirred at room temperature overnight. At the end of this time, sodium bicarbonate (170 mg, 2.0 mmol)
was added, and the mixture was concentrated to dryness. The resulting residue was used in next step
without purification. Ion(s) found by LCMS: M+H=371.2.

(992) Steps c & d.

(993) ##STR00699##

(994) To a solution of material from the previous step in 60 ml of methanol was added 5.0 g of a
Lindlar catalyst. The resulting mixture was flushed with hydrogen every 30 minutes and stirred for 5
hours. After complete reaction as determined by HPLC, the catalyst was filtered off through celite. The
filtrate was concentrated and used in the next step without purification.

(995) Crude product from the previous step in 60 ml THE was treated with N,N′-bis-boc-1-
guanylpyrazole (9.3 g, 30.0 mmol), and DIEA (9.9 ml, 57.0 mmol). The resulting solution was stirred at
room temperature until all starting material was consumed as determined by LCMS (4 h). The solution
was concentrated and purified by flash chromatography eluted with 20% to 80% ethyl
acetate/dichloromethane. Yield 8.8 g, 59.0% for four steps. Ion(s) found by LCMS: M+H 587.3.

(996) Step e.

(997) ##STR00700##

(998) A reaction flask containing product from the previous step (6.5 g, 11 mmol) was vacuum flushed
with nitrogen and dissolved in anhydrous dichloromethane (100 ml). After the solution was cooled in
an ice-water bath, DMAP (4.89 g, 40 mmol) was added and stirred to dissolve, then 4-
nitrophenylchloroformate (5.58 g, 28 mmol) was added in portions, while stirring at 0° C. to room
temperature for 1 hour. LCMS shows starting material 1 hr so additional DMAP (1.22 g, 10 mmol) and
4-nitrophenylchloroformate (1.0 g, 5 mmol) were added. The reaction was continued for 4 more hours,
then concentrated and purified by flash chromatography eluting with 20% to 80% ethyl
acetate/dichloromethane. Yield 5.1 g, 59.9%. Ion found by LCMS: [M+H].sup.+=752.2.

Example 110. Synthesis of Int-71

(999) ##STR00701## ##STR00702##

(1000) To a solution of 2-(2-Boc-aminoethoxy) ethanol (6.15 g, 30 mmol) in anhydrous DCM (60 ml)
was added DIPEA (7.8 g, 60 mmol) and DMAP (366.6 mg, 3 mmol). P-toluenesulfonyl chloride (6.86
g, 36 mmol) was then added in portions over 30 minutes. After the resulting mixture was stirred for 3
days, it was concentrated by rotary evaporation and purified by RPLC (20% to 70% acetonitrile/water).
Yield 3.71 g, 34.4%. Ion found by LCMS: [M-Boc+H].sup.+=260.

(1001) Step b.

(1002) ##STR00703##

(1003) To a solution of the step-a product (2.1 g, 5.83 mmol) in anhydrous THE (10 ml) was added
sodium carbonate (1.24 g, 11.7 mmol) and N-Boc-1,4-diaminobutane (1.32 g, 7 mmol). The resulting
mixture was heated at 60° C. for 1 day. The salt was then filtered off, and the filtrate was concentrated
by rotary evaporation. The residue was purified by RPLC (100 g, 5 to 50% acetonitrile and water).
Yield 1.94 g, 88.6%. Ion found by LCMS: [M+H].sup.+=376.0.

(1004) Step c.

(1005) ##STR00704##

(1006) To a solution of propargyl PEG-4 acid (781 mg, 3 mmol) and HATU (1.14 g, 3 mmol) in
anhydrous DMF (3 ml) was added DIPEA (390 mg, 3 mmol), followed by the addition of the solution
step-b product (940 mg, 2.5 mmol) and DIPEA (390 mg, 3 mmol) in anhydrous DMF (3 ml). The
reaction mixture was stirred for 30 minutes, then directly purified by RPLC (5% to 80% acetonitrile and
water, using 0.1% TFA as modifier). Yield 960.2 mg, 65.3%. Ion found by LCMS: [M+H].sup.+=618.3,
[M-Boc+H].sup.+=518.3.

(1007) Step d.
(1008) ##STR00705##

(1009) The step-c product (960.2 mg, 1.63 mmol) was dissolved in anhydrous THE (6 ml). 4N HCl
solution in dioxane (4 ml) was added, and the reaction mixture was stirred overnight. It was then
concentrated by rotary evaporation. The residue was extracted with water (3×3 ml) and ethyl acetate
(10 ml). The combined aqueous layers were lyophilized. Yield 760 mg, 95.1%. Ion found by LCMS:
[M+H].sup.+=418.0.

(1010) Step e.

(1011) ##STR00706##

(1012) To a mixture of the step-d product (556.2 mg, 1.13 mmol) and DIPEA (741 mg, 5.7 mmol) in
anhydrous DMF (3 ml) was added in portions p-nitrophenyl carbonate of zanamivir (1.67 g, 2.26 mmol,
described in Example 109) over 20 minutes. The reaction was stirred for 1 hour, then directly purified
by RPLC (30% to 90% acetonitrile/water, using 0.1% TFA as modifier). Yield 1.35 g, 74%. Ion found by
LCMS: [(M+2H)/2].sup.+=807.9.

(1013) Step f.

(1014) ##STR00707##

(1015) The step-e product (1.35 g, 0.836 mmol) was dissolved in TFA (5 ml). The reaction was heated
at 30° C. for 1 hour, it was directly purified by RPLC (0% to 35% acetonitrile/water, using 0.1% TFA as
modifier). Yield 1.00 g, 82.9%. Ions found by LCMS: [(M+2H)/2].sup.+=607.8.

(1016) Step g.

(1017) ##STR00708##

(1018) The step-f product (1.00 g, 0.693 mmol) was dissolved in MeOH (12 ml), then cooled in an ice-
water bath. It was then treated with a solution of LiOH monohydrate (286 mg, 6.6 mmol) in water (9
ml). The resulting mixture was stirred overnight and then acidified by 4N HCl solution in dioxane (2
ml). After organic solvents were removed by rotary evaporation, the residue was purified by
preparative HPLC (Isco ACCQ prep, Luna 5 μm C18(2) 100 Å LC column 100 mm×30 mm; Gradient:
0% acetonitrile/water for 2 min, then 0% to 15% acetonitrile/water over 12 min, then isocratic at 15%
acetonitrile for 10 min, using 0.1% TFA). Yield 275.9 mg, 29.2%. Ions found by LCMS:
[(M+2H)/2].sup.+=567.8, [(M+3H)/3].sup.+=378.9.

Example 111. Synthesis of Conjugate 31

(1019) A 15-ml sterile centrifuge tube was charged with sodium ascorbate (68.1 mg, 0.344 mmol),
THPTA (14.9 mg, 0.0344 mmol), alkyne derivatized small molecule (17.5 mg, 0.00953 mmol,
described in Example 110) and buffer PBS 7.4 (1 ml). After stirred by vortex to dissolve everything,
Peg 4 azido Fc (50 mg, 0.0008588 mmol, described in Example 7 SEQ ID No. 18) was added followed
by a solution of CuSO.sub.4 (2.05 mg, 0.0129 mmol) in PBS (0.5 ml). The mixture was gently rotated
for 20 hours then purified by affinity chromatography over a protein-A column, followed size exclusion
chromatography. Maldi TOF analysis of the purified final product gave an average mass of 63586 Da
(DAR=3.8). Yield 32.8 mg, 66% yield.

Example 112. Synthesis of Int-72

(1020) ##STR00709## ##STR00710##

(1021) To a solution of N-Boc-1,4-diaminobutane (1.56 g, 8.28 mmol) in anhydrous DMF (7 ml) was
added sodium carbonate (742 mg, 7 mmol) and 5-(Boc-amino)-1-pentylbromide (1.73 g, 6.5 mmol).
The resulting mixture was heated at 50° C. for 24 hours. The salt was then filtered off, and the filtrate
was concentrated by rotary evaporation. The residue was purified by RPLC (5% to 50%
acetonitrile/water, using 0.1% TFA as modifier). Yield 2 g, 63.2%. Ion found by LCMS:
[M+H].sup.+=374.4.

(1022) Step b.

(1023) ##STR00711##

(1024) To a solution of propargyl PEG-4 acid (1.3 g, 5 mmol) and HATU (2.1 g, 5.5 mmol) in
anhydrous DMF (6 ml) was added DIPEA (1.3 g, 10 mmol). After 5 minutes, the reaction mixture was
added to the step-a product (2 g, 4.1 mmol) and stirred for 1 hour. It was then directly purified by
RPLC (5% to 80% acetonitrile/water, using 0.1% TFA as modifier). Yield 2.34 g, 95%. Ion found by
LCMS: [M+H].sup.+=616.4, [M-Boc+H]=516.4.

(1025) Step c.

(1026) ##STR00712##

(1027) The step-b product (2.34 g, 3.8 mmol) was dissolved in anhydrous THE (12 ml). 4N HCl
solution in dioxane (10 ml) was added, and the reaction mixture was stirred overnight. It was then
concentrated by rotary evaporation. The residue was re-dissolved in acetonitrile/water (1:1, ˜16 ml),
and the solution was lyophilized. The crude product was carried to the subsequent step without further
purification. Yield 1.91 g, quantitative yield. Ion found by LCMS: [M+H].sup.+=416.4.

(1028) Step d.

(1029) ##STR00713##

(1030) To a solution of p-nitrophenyl carbonate of zanamivir (2.25 g, 3.05 mmol, described in Example
109) in anhydrous DCM (3 ml) was added in-portions a mixture of the step-c product (610 mg, 1.249
mmol) and DIPEA (1.05 g, 8.1 mmol) in anhydrous DMF (4 ml) over 20 minutes. After stirring for 1
hour, the reaction mixture was concentrated and purified by RPLC (30% to 80% acetonitrile/water,
using 0.1% TFA as modifier). Yield 1.73 g, 85.9%. Ion found by LCMS: [(M+2H)/2].sup.+=806.8, [(M-
Boc+2H)/2].sup.+=756.8.

(1031) Step e.

(1032) ##STR00714##

(1033) The step-d product (1.73 g, 1.073 mmol) was dissolved in TFA (5 ml). After the solution was
heated at 30° C. for 3 hours, it was directly purified by RPLC (0% to 35% acetonitrile/water, using
0.1% TFA as modifier). Yield 1.176 g, 76.1%. Ions found by LCMS: [(M+2H)/2].sup.+=606.8,
[(M+3H)/3].sup.+=405.

(1034) Step f.

(1035) ##STR00715##

(1036) The step-e product (1.176 g, 0.817 mmol) was dissolved in MeOH (12 ml), and the solution
was cooled in an ice-water bath. It was treated dropwise with a solution of LiOH monohydrate (344.4
mg, 8.2 mmol) in water (9 ml). The resulting mixture was stirred overnight and then acidified by 4N HCl
solution in dioxane (2 ml). After organic solvents were removed by rotary evaporation, the residue was
purified by preparative HPLC (Isco ACCQ prep, Luna 5 μm C18(2) 100 Å LC column 100 mm×30 mm;
Gradient: 0% acetonitrile/water for 2 min, then 0% to 15% acetonitrile/water over 12 min, then isocratic
at 15% acetonitrile for 10 min, using 0.1% TFA). Yield 108 mg, 9.7%. Ions found by LCMS:
[(M+2H)/2].sup.+=566.8, [(M+3H)/3].sup.+=378.2.

Example 113. Synthesis of Conjugate 32

(1037) A 15-ml sterile centrifuge tube was charged with sodium ascorbate (68.1 mg, 0.344 mmol),
THPTA (14.9 mg, 0.0344 mmol), product from Example 112, Int-72 (17.5 mg, 0.00953 mmol) and PBS
7.4 (1 ml). After stirring by vortex to dissolve everything, azido Fc (50 mg, 0.0008588 mmol, described
in Example 7 with SEQ ID NO: 4) was added followed by a solution of CuSO.sub.4 (2.05 mg, 0.0129
mmol) in PBS (0.5 ml). The mixture was rotated for 20 hours. It was purified by affinity
chromatography over a protein A column, followed size exclusion chromatography. Maldi TOF analysis
of the purified final product gave an average mass of 63588. Da (DAR=3.8). Yield 30.9 mg, 62% yield.

(1038) The nucleic acid construct encoding the Fc for conjugate 32 included a nucleic acid encoding
the amino acid sequence of SEQ ID NO: 4, which includes a C-terminal lysine residue. Upon
expression, the C-terminal lysine of the Fc of conjugate 32 is proteolytically cleaved, resulting in an Fc
having the sequence lacking Lys447 (e.g., lacking a C-terminal lysine residue). The presence or
absence of a C-terminal lysine does not alter the properties of the Fc or the corresponding conjugate.

Example 114. Synthesis of Int-73

(1039) ##STR00716## ##STR00717##

(1040) To a solution of the p-nitrophenyl carbonate of zanamivir (698.4 mg, 0.95 mmol, described in
Example 109) in anhydrous DCM (2 ml) was added in-portions over 10 minutes a mixture of propargyl
diamine central linker (209 mg, 0.426 mmol, described in Example 110) and DIPEA (330.8 mg, 2.56
mmol) in anhydrous DMF (2 ml). The reaction was stirred at room temperature for 1 hour. It was then
concentrated and purified by RPLC (30% to 85% acetonitrile/water, no TFA modifier). Yield 531 mg,
69.2%. Ions found by LCMS: [(M+2H)/2].sup.+=807.8, [(M-Boc+2H)/2].sup.+=757.8.

(1041) Step b.

(1042) ##STR00718##

(1043) The step-b product (531 mg, 0.329 mmol) was dissolved in DCM (1.5 ml) and TFA (1.5 ml),
then stirred at 35° C. for 3 hours. It was concentrated and purified by RPLC (5% to 30%
acetonitrile/water, no TFA modifier). Yield 387 mg, 97.1%. Ions found by LCMS:
[(M+2H)/2].sup.+=607.6, [(M+3H)/3].sup.+=405.4.

(1044) Step c.

(1045) ##STR00719##

(1046) The step-b product (121.4 mg, 0.1 mmol) was dissolved in 1.0 M NaCl solution (3 ml) and
acetonitrile (1 ml). After the solution was cooled to −8° C. (salt/ice bath), 1.0 M NaOH (0.4 ml, 0.4
mmol) was added dropwise and the reaction was stirred at −14° C. to −8° C. for 6 hours. It was then
neutralized with 4N HCl in dioxane solution (100 μl) and directly purified by preparative HPLC (Isco
ACCQ prep, Luna μm C18(2) 100 Å LC column 100 mm×30 mm; Gradient: 0% acetonitrile/water for 2
min, then 0% to 17.8% acetonitrile/water over 12 min, then isocratic at 17.8% acetonitrile for 10 min,
using 0.1% TFA). Yield 85.5 mg, 62.8%. Ions found by LCMS: [(M+2H)/2].sup.+=567.8,
[(M+3H)/3].sup.+=378.9.

Example 115. Synthesis of Int-74

(1047) ##STR00720## ##STR00721##

(1048) To a solution of N-Boc-2-(2-amino-ethoxy)ethylamine (1.2 g, 5 mmol) in anhydrous DMF (5 ml)

was added sodium carbonate (691 mg, 5 mmol) and N-Boc-6-bromo-hexylamine (1.4 g, 5 mmol). The
resulting mixture was heated at 70° C. for 24 hours. The salt was then filtered off, and the filtrate was
concentrated by rotary evaporation. The residue was purified by RPLC (5% to 50% acetonitrile/water,
using 0.1% TFA as modifier). Yield 444 mg, 22%. Ion found by LCMS: [M+H].sup.+=404.3.

(1049) Step b.
(1050) ##STR00722##

(1051) To a solution of propargyl PEG-4 acid (342.6 mg, 1.32 mmol) and HATU (601.5 mg, 1.58 mmol)
in anhydrous DMF (2 ml) was added DIPEA (258 mg, 2 mmol). After 5 minutes, the reaction mixture
was added into the step-a product (444.3 mg, 0.858 mmol) and stirred for 1 hour. It was then directly
purified by RPLC (5% to 80% acetonitrile/water, using 0.1% TFA as modifier). Yield 297.1 mg, 53.6%.
Ion found by LCMS: [M+H].sup.+=646.2, [M-Boc+H].sup.+=546.2.

(1052) Step c.

(1053) ##STR00723##

(1054) The step-b product (297.1 mg, 0.46 mmol) was dissolved in anhydrous THE (2 ml). 4N HCl
solution in dioxane (4 ml) was added, and the reaction mixture was stirred overnight. It was then
concentrated by rotary evaporation and purified by preparative HPLC (5% to 50% acetonitrile/water,
using 0.1% TFA as modifier). Yield 239.6 mg, 77.3%. Ion found by LCMS: [M+H].sup.+=446.2.

(1055) Step d.

(1056) ##STR00724##

(1057) To a solution of the p-nitrophenyl carbonate of zanamivir (523.8 mg, 0.71 mmol, described in
Example 109) in anhydrous DCM (2 ml) was added in portions over 10 minutes a mixture of the step-c
product (239.6 mg, 0.356 mmol) and DIPEA (245.5 mg, 1.9 mmol) in anhydrous DMF (2 ml). The
reaction was stirred at room temperature for 1 hour. It was then concentrated and purified by RPLC
(30% to 85% acetonitrile/water, no TFA modifier). Yield 553 mg, 96.5%. Ions found by LCMS:
[(M+2H)/2].sup.+=821.8, [(M-Boc+2H)/2].sup.+=771.8.

(1058) Step e.

(1059) ##STR00725##

(1060) The step-d product (553 mg, 0.343 mmol) was dissolved in DCM (1.5 ml) and TFA (1.5 ml),
then stirred at 35° C. for 3 hours. It was concentrated and purified by RPLC (5% to 30%
acetonitrile/water, no TFA modifier). Yield 424.6 mg, 99.6%. Ions found by LCMS:
[(M+2H)/2].sup.+=621.6, [(M+3H)/3].sup.+=415.0.

(1061) Step f.

(1062) ##STR00726##

(1063) The step-e product (424.6 mg, 0342 mmol) was dissolved in 1.0 M NaCl solution (4 ml) and
acetonitrile (4 ml). After the solution was cooled to −11° C. (salt/ice bath), 1.0 M NaOH (1.53 ml, 1.53
mmol) was added dropwise and the reaction was stirred at −14° C. to −8° C. for 6 hours. It was then
quenched with 4N HCl in dioxane solution (375 μl) and directly purified by preparative HPLC (Isco
ACCQ prep, Luna 5 μm C18(2) 100 Å L.sup.C column 100 mm×30 mm; Gradient: 0%
acetonitrile/water for 2 min, then 0% to 20.9% acetonitrile/water over 13.6 min, then isocratic at 20.9%
acetonitrile for 10 min, using 0.1% TFA). Yield 439 mg, 92.3%. Ions found by LCMS:
[(M+2H)/2].sup.+=581.8, [(M+3H)/3].sup.+=388.2.

Example 116. Synthesis of Int-75

(1064) ##STR00727##

(1065) A mixture of tert-butyl (4-oxobutyl) carbamate (850 mg, 4.54 mg) and tert-butyl N-{2-[2-(2-
aminoethoxy) ethoxy] ethyl} carbamate (1.99 g, 8 mmol) was dissolved in DCM (30 ml) and dried over
Na.sub.2SO.sub.4. After filtering and concentrating, the residue was redissolved in anhydrous DCM
(20 ml). Acetic acid (641 mg, 11 mmol) was added, followed by sodium triacetoxyborohydride (3.4 g,
16 mmol) in portions over 1 hour. The reaction mixture was stirred overnight, then quenched with
AcOH (3 ml) and MeOH (10 ml). The mixture was filtered, and the filtrate was concentrated by rotary
evaporation and purified by RPLC (5% to 45% acetonitrile and water). Yield 618 mg, 32.5%. Ion found
by LCMS: [M+H].sup.+=420.4.

(1066) Step b.

(1067) ##STR00728##

(1068) This compound was prepared analogously to the step-b product of Example 115. Ions found by
LCMS: [M+H].sup.+=662.4, [M-Boc+H].sup.+=562.4.

(1069) Step c.

(1070) ##STR00729##

(1071) This compound was prepared analogously to the step-c product of Example 115. Ion found by
LCMS: [M+H].sup.+=462.4.

(1072) Step d.

(1073) ##STR00730##

(1074) This compound was prepared analogously to the step-d product of Example 115. Ions found by
LCMS: [(M+2H)/2].sup.+=829.8, [(M-Boc+2H)/2].sup.+=779.8.

(1075) Step e.

(1076) ##STR00731##

(1077) This compound was prepared analogously to the step-e product of Example 115. Ions found by
LCMS: [(M+2H)/2].sup.+=629.8, [(M+3H)/3].sup.+=420.2.

(1078) Step f.

(1079) ##STR00732##

(1080) This compound was prepared analogously to the step-f product of Example 115. Ions found by
LCMS: [(M+2H)/2].sup.+=598.8, [(M+3H)/3].sup.+=393.6.

Example 117. Synthesis of Int-76

(1081) ##STR00733##

(1082) To a solution of the N-Boc-peg-1 tosylate (1.54 g, 4.28 mmol, described in Example 110) in
anhydrous THE (8 ml) was added tert-butyl N-2{2-[2-(2-aminoethoxy) ethoxy] ethyl} carbamate (1.59
g, 6.42 mmol) and sodium carbonate (453.7 mg, 4.28 mmol). The resulting mixture was heated at 50°
C. for 24 hours. The solid was filtered and washed with acetonitrile. The filtrate was concentrated and
purified by RPLC (100 g, 5 to 90% acetonitrile and water). Yield 1.15 g, 61.7%. Ion found by LCMS:
[M+H].sup.+=436.4.

(1083) Step b.

(1084) ##STR00734##

(1085) This compound was prepared analogously to the product from step-b of Example 115. Ions
found by LCMS: [M+H].sup.+=678.2, [M-Boc+H].sup.+=578.2.

(1086) Step c.
(1087) ##STR00735##

(1088) This compound was prepared analogously to the product of step-c of Example 115. Ion found
by LCMS: [M+H].sup.+=478.2.

(1089) Step d.

(1090) ##STR00736##

(1091) This compound was prepared analogously to the product from step-d of Example 115. Ions
found by LCMS: [(M+2H)/2].sup.+=837.6, [(M-Boc+2H)/2].sup.+=767.8.

(1092) Step e.

(1093) ##STR00737##

(1094) This compound was prepared analogously to the product from step-e of Example 115. Ions
found by LCMS: [(M+2H)/2].sup.+=637.5, [(M+3H)/3].sup.+=425.6.

(1095) Step f.

(1096) ##STR00738##

(1097) This compound was prepared analogously to the product from step-f of Example 115. Ions
found by LCMS: [(M+2H)/2].sup.+=597.8, [(M+3H)/3].sup.+=399.0.

Example 118 Synthesis of Int-67

(1098) ##STR00739##

(1099) Previously prepared ether zanamivir acid starting material (0.90 g, 1.43 mmol, described in
Example 22) and N-methyl morpholine (0.23 mL, 2.14 mmol) were dissolved in THE (35 mL) and
cooled to 0° C. (ice water bath) under an atmosphere of nitrogen. Isobutyl chloroformate (0.24 mL,
1.85 mmol, in 2 mL DCM) was added dropwise, by way of syringe over a 5 minute period. The mixture
was stirred at 0° C. for 30 minutes then 15 min at ambient temperature, and then cooled to 0° C.,
where sodium borohydride (540 mg, 14.3 mmol, dissolved in 5 mL of methanol) was added, dropwise
over 5 minutes. The reaction was stirred for 15 minutes at which point all starting material had been
consumed (by LC/MS). A few drops (˜1 mL) of glacial acetic acid was added to acidify the mixture
(pH˜5). The mixture was diluted with ethyl acetate and water and extracted into ethyl acetate (3×). The
organic layer was washed with brine, and the organic extracts were dried over sodium sulfate, and
concentrated on a rotary evaporator. The crude material was purified by silica gel chromatography,
dried onto celite first, and then eluted with 0%-10% methanol in DCM over 30 min. Yield 0.66 g, 75%.
Ion found by LCMS: [M+H].sup.+=617.2.

(1100) Step b.

(1101) ##STR00740##

(1102) To a stirring mixture of product from the previous step (0.66 g, 1.10 mmol), in 20 mL DCM, was
added triethylamine (0.30 mL, 1.3 mmol). The mixture was cooled to 0° C. (ice-water bath) under an
atmosphere of nitrogen then treated with mesyl chloride (0.15 g, 1.3 mmol), dropwise over 5 minutes
by way of syringe. The ice bath was removed and the reaction was stirred for 45 minutes. The reaction
was quenched with saturated aqueous sodium bicarbonate, then extracted into DCM (3×). The
combined organic extracts were washed with brine, dried over sodium sulfate and concentrated on the
rotary evaporator. Yield 0.74 g, 99%. Ion found by LCMS: [M+H].sup.+=695.2. The intermediate was
taken to the next step without purification.

(1103) Step c.
(1104) ##STR00741##

(1105) Mesylate from the previous step (0.74 g, 1.1 mmol) was stirred in DMF (5 mL) at 80° C. with 3
eq of sodium azide (0.21 g, 3.3 mmol) for 5 hours. The mixture was diluted with water, extracted into
DCM (3×). The combined organic extracts were washed with brine, dried over sodium sulfate and
concentrated. Yield 0.67 g, 95%. Ion found by LCMS: [M+H].sup.+=642.4. The azide was taken to the
next step without purification.

(1106) Step d.

(1107) ##STR00742##

(1108) Azide from the previous step (0.67 g, 1.04 mmol) was stirred in methanol (20 mL) in the
presence of Lindlar catalyst (300 mg) under 1 atmosphere of hydrogen gas for 12 hours. The mixture
was filtered through celite and concentrated to afford the title compound as a clear oil. The amine was
taken to the next step without purification. Yield 0.37 g, 54%, 3 steps. Ion found by LCMS:
[M+H].sup.+=616.2.

(1109) Step e.

(1110) ##STR00743##

(1111) EDC (150 mg, 0.78 mmol) was added to a stirring solution of amine from the previous step (370
mg, 0.60 mmol), propargyl-peg4-carboxylic acid (188 mg, 0.72 mmol) and triethylamine (0.100 mL,
0.72 mmoL), dissolved in DMF (4 mL). The reaction was stirred for 2 hours at ambient temperature
then purified directly RPLC (10%-95% acetonitrile/water, no modifier, 30 minute gradient). The pure
fractions were pooled and lyophilized and taken directly to the next step. Yield 490 mg, 80%. Ion found
by LC/MS: [M+H].sup.+=858.2.

(1112) Step f.

(1113) ##STR00744##

(1114) Product from the previous step (490 mg, 0.57 mmol) was stirred in TFA (4 mL) for 45 minutes
then concentrated and azeotroped with methanol (3×) on the rotary evaporator. Ion found by LC/MS:
[M+H].sup.+=658.2. The residue was stirred in 1/1 methanol/water containing LiOH (43 mg, 1.8 mmol)
for 30 minutes. The reaction was neutralized with a few drops of glacial acetic acid and the volume
was reduced by half on the rotary evaporator. The crude product was purified by semi-preparative
HPLC (0%-75% acetonitrile/water, 0.1% TFA, 30 minute gradient). The pure fractions were pooled and
lyophilized to afford the title compound. Yield 105 mg, 29%, 3 steps. Ion found by LC/MS:
[M+H].sup.+=618.2.

Example 119. Synthesis of Int-68

(1115) ##STR00745##

(1116) p-Nitrophenyl chloroformate (0.23 g, 1.14 mmol) was added to a stirring mixture of the primary
alcohol (0.47 g, 0.76 mmol described in Example 118) and triethylamine (0.21 mL, 1.52 mmol)
dissolved in DCM (15 mL, anhydrous). The reaction was stirred for 1 hour then additional
trimethylamine (0.21 mL) and p-nitrophenyl chloroformate (230 mg) were added, and stirring was
continued another hour, at which time the mixture was diluted with water, and extracted into DCM (3×).
The combined organic extracts were washed with brine and dried over sodium sulfate. The solvent
was removed by rotary evaporation. The crude residue was dissolved in DCM (3 mL), loaded onto
celite and purified by silica gel chromatography (0% to 70% ethyl acetate/hexanes, 30 minute
gradient). The pure fractions were pooled and concentrated to afford the title compound as a white
solid. Yield 525 mg, 88%. Ion found by LCMS: [M+H].sup.+=782.2.

(1117) Step b.
(1118) ##STR00746##

(1119) Triethylamine (0.14 mL, 0.97 mmol) was added to a stirring solution of propargyl-peg4-amine
(181 mg, 0.78 mmol) in 1 mL of acetonitrile. The triethylamine/propargyl-peg4-amine mixture was
added to the product from the previous step (510 mg, 0.65 mmol, in 2 mL acetonitrile) and stirred for 1
hour at which point all starting materials had been consumed. The solvent was removed on a rotary
evaporator. The crude residue was purified RPLC (20-95% acetonitrile/water, 30 min gradient, no
modifier). Yield 475 mg, 83%. Ion found by LC/MS: [M+H].sup.+=874.2.

(1120) Step c.

(1121) ##STR00747##

(1122) The product from the previous step (475 mg, 0.54 mmol), was stirred in TFA (5 mL) for 2 hours
and then concentrated and azeotroped with methanol (3×) on a rotary evaporator. Ion found by
LC/MS: [M+H].sup.+=674.2. The residue was stirred in 1:1 mixture of methanol/water containing LiOH
(49 mg, 1.6 mmol) for 30 minutes. The reaction was neutralized with glacial acetic acid and then
concentrated by rotary evaporation. The product was purified by semi-preparative HPLC (0%-75%
acetonitrile/water, 0.1% TFA, 30 minute gradient). The pure fractions were pooled and lyophilized.
Yield 204 mg, 59%. Ion found by LCMS: [M+H].sup.+=634.2.

Example 120 Synthesis of Int-77

(1123) ##STR00748##

(1124) HATU (0.44 g, 1.16 mmol, in 1.5 mL of DMF) was added dropwise to a stirring solution of
propargyl-peg4-amine (0.24 g, 1.05 mmol), bis-Boc-D-ornithine (0.35 g, 1.05 mmol), and triethylamine
(0.59 mL, 4.21 mmol) in DMF (2 mL). The reaction was stirred for 1 hour at ambient temperature, then
purified directly by RPLC (5%-90% acetonitrile/water, 0.1% TFA, 35 minute gradient). The pure
fractions were pooled and lyophilized to afford the product as a viscous clear oil. Ion found by LC/MS:
[M+H].sup.+=546.2.

(1125) The Boc-protected intermediate was stirred in 4N HCL in dioxane (10 mL) for 45 minutes at
ambient temperature. The solvent was removed by rotary evaporation, then the resulting residue was
dissolved in DI water (20 mL), frozen, and lyophilized to afford the product as a clear oil. Yield 310 mg,
70%, 2 steps. Ion found by LCMS: [M+H].sup.+=346.2.

(1126) Step b.

(1127) ##STR00749##

(1128) The product from the previous step (96 mg, 0.28 mmol) and triethylamine (0.15 mL, 1.11 mmol)
in acetonitrile (2 mL) were added to a stirring solution of p-nitrophenyl carbonate of zanamivir (435 mg,
0.56 mmol, described in Example 118, in 6 mL of acetonitrile), and stirred for 12 hours at ambient
temperature, then 2 hours at 80° C. The solvent was removed and the crude residue was purified by
RPLC (10-95% acetonitrile/water, no modifier, 35 minute gradient). The pure fractions were pooled
and concentrated on a rotary evaporator. Ion found by LC/MS: [(M+2H)/2].sup.+=815.8.

(1129) This intermediate was stirred in TFA (5 mL) for 45 minutes then concentrated and dried under
high vacuum. Ion found by LC/MS[(M+2H)/2].sup.+=615.8.

(1130) This TFA salt was stirred in (1/1) MeOH/DI water (5 mL) containing LiOH (27 mg, 1.11 mmol)
for 30 minutes at 0° C. The mixture was acidified (˜pH-5) with glacial acetic acid. The methanol was
removed by rotary evaporation and the resulting residue was purified by semi-preparative HPLC
(5%-70% acetonitrile/water, 0.1% TFA, 35 minute gradient). The pure fractions were pooled and
lyophilized. Yield 35 mg, 11%, 3 steps. Ion found by LC/MS: [(M+2H)/2].sup.+=575.8.

Example 121. Synthesis of Int-78

(1131) ##STR00750## ##STR00751##

(1132) Propargyl-Peg4-acid (640 mg, 2.46 mmol), diol-HCl salt (350 mg, 2.46 mmol), EDC (471 mg,
2.46 mmol), HOBt (377 mg, 2.46 mmol), and triethylamine (249 mg, 2.46 mmol) were stirred in DMF (3
mL) at ambient temperature for 4 hours. The mixture purified directly by RPLC (0%-80%
acetonitrile/water, no modifier, 35 minute gradient). The pure fractions were pooled and concentrated
to afford the product as a clear oil. Yield 580 mg, 68%. Ion found by LCMS: [M+H].sup.+=348.4.

(1133) Step b.

(1134) ##STR00752##

(1135) p-Nitro-phenyl chloroformate (1.23 g, 1.25 mmol) was added to a stirring solution of product
from the previous step (530 mg, 6.10 mmol) and triethylamine (925 mg, 9.15 mmol) in DCM (25 mL),
then cooled to 0° C. under a nitrogen atmosphere. The mixture was stirred for 15 minutes at 0° C. and
then at room temperature for 2 hours. The reaction was diluted with DI water and extracted into DCM
(3×20 mL). The combined organic extracts were washed with brine and dried over sodium sulfate. The
crude residue was purified by silica gel chromatography (10%-100% ethyl acetate/hexanes, 25 minute
gradient) to afford the product as a white solid. Yield, 250 mg, 24%. Ion found by LCMS:
[M+H].sup.+=678.2.

(1136) Step c.

(1137) ##STR00753##

(1138) Amine functionalized zanamivir (505 mg, 0.82 mmol, described in Example 118) and
triethylamine (0.15 mL, 1.11 mmol) in acetonitrile (2 mL) were added to a stirring solution of the
product of the previous step (220 mg, 0.37 mmol) in acetonitrile (10 mL), and stirred for 4 hours at
ambient temperature. The reaction was concentrated on the rotary evaporator. The resulting residue
was purified by RPLC (15%-95% acetonitrile/water, no modifier, 35 minute gradient). The pure
fractions were pooled and concentrated on a rotary evaporator. Ion found by LC/MS:
[(M+2H)/2].sup.+=815.2.

(1139) This intermediate was stirred in TFA (5 mL) for 45 minutes then concentrated and dried under
high vac. Ion found by LC/MS: [(M+2H)/2].sup.+=615.2.

(1140) The resulting TFA salt was stirred in (1/1) MeOH/DI water (5 mL) containing LiOH (71 mg, 2.98
mmol) for 30 minutes at 0° C. The mixture was acidified (˜pH-5) with glacial acetic acid. The methanol
was removed by rotary evaporator and purified by semi-preparative HPLC (5%-70% acetonitrile/water,
0.1% TFA, 35 minute gradient). The pure fractions were pooled and lyophilized. Yield 125 mg, 29% for
3 steps. Ion found by LC/MS: [(M+2H)/2].sup.+=575.8.

Example 122. Synthesis of Int-4a

(1141) ##STR00754##

(1142) Propargyl-Peg 4-amine (165 mg, 0.71 mmol) was added to a stirring solution of p-nitrophenyl
carbonate of zanamivir (350 mg, 0.47 mmol, described in Example 109) in acetonitrile (20 mL). The
reaction was stirred for 1 hour at ambient temperature, then solvent was removed by rotary
evaporator. The residue was purified by RPLC (10%-95% acetonitrile/water, no modifier, 30 minute
gradient). The pure fractions were pooled and lyophilized to afford the boc protected intermediate as a
white solid. Ion found by LCMS: [M+H].sup.+=830.2.

(1143) This intermediate was stirred in TFA (5 mL) at ambient temperature for 30 minutes. The solvent
was removed by rotary evaporator and the residue was purified by RPLC (10%-95% acetonitrile/water,
0.1% TFA, 30 minute gradient). The pure fractions were pooled and lyophilized to afford the product as
a white solid. Yield 155 mg, 52%, 2 steps. Ion found by LCMS: [M+H].sup.+=630.2.
(1144) Step b.

(1145) ##STR00755##

(1146) The product from the previous step (140 mg, 0.22 mmol) was stirred in a 1:3 mixture of
methanol:water (8 mL) containing LiOH (21 mg, 0.89 mmol) at 0° C. for 20 minutes. The mixture was
acidified with a few drops of glacial acetic acid and concentrated on a rotary evaporator. The crude
material was purified by semi-preparative HPLC (5%-95% acetonitrile/water, 0.1% TFA, 30 minute
gradient). The pure fractions were pooled and lyophilized to afford the product as a white solid. Yield
60 mg, 45%. Ion found by LCMS: [M+H].sup.+=590.2.

(1147) .sup.1H NMR (500 MHz, Methanol-d.sub.4) δ 5.86 (d, J=2.6 Hz, 1H), 4.99 (dd, J=9.0, 2.7 Hz,
1H), 4.55 (dd, J=9.7, 2.7 Hz, 1H), 4.38 (dd, J=8.6, 2.6 Hz, 1H), 4.24-4.14 (m, 1H), 4.19 (d, J=2.6 Hz,
2H), 4.02-3.98 (m, 1H), 3.74-3.59 (m, 13H), 3.54 (t, J=5.6 Hz, 2H), 3.52-3.48 (m, 1H), 3.29-3.22 (m,
2H), 2.84 (t, J=2.4 Hz, 1H), 1.96 (s, 3H).

(1148) .sup.13C NMR (126 MHz, MeOD) δ 172.33, 163.68, 157.54, 156.58, 106.80, 79.23, 75.91,
74.56, 70.18, 70.13, 69.96, 69.87, 69.59, 69.51, 69.14, 68.74, 62.98, 57.67, 51.11, 40.54, 21.37.

Example 123. Synthesis of Int-4b

(1149) ##STR00756##

(1150) Peg functionalized intermediate (100 mg, 0.13 mmol, described in Example 122) was stirred in
a 1:3 mixture of methanol:water (5 mL) containing LiOH (12 mg, 0.52 mmol) at ambient temperature
for 2 hours. The mixture was acidified with glacial acetic acid and concentrated by rotary evaporator.
The crude material was purified by RPLC (5-95% acetonitrile in DI water, 0.1% TFA, 30 minute
gradient). The pure fractions were pooled and lyophilized to afford the product as a white solid. Yield
21 mg, 31%. Ion found by LCMS: [M+H].sup.+=590.2.

(1151) .sup.1H NMR (Methanol-d.sub.4) δ: 5.86 (d, J=2.6 Hz, 1H), 4.48 (dd, J=8.6, 2.7 Hz, 1H), 4.42
(dd, J=9.6, 1.4 Hz, 1H), 4.38 (d, J=12 Hz, 1H), 4.22-4.19 (m, 1H), 4.19 (d, J=2.4 Hz, 2H), 4.16-4.12
(dd, J=11.5, 6 Hz, 1H), 4.09-4.02 (m, 1H), 3.75-3.58 (m, 13H), 3.55 (t, J=5.5 Hz, 2H), 3.33-3.29 (m,
2H), 2.84 (t, J=2.4 Hz, 1H), 2.02 (s, 3H).

(1152) .sup.13C NMR (126 MHz, MeOD) δ 172.94, 163.82, 157.90, 157.53, 106.80, 79.23, 76.18,
74.55, 70.19, 70.13, 69.96, 69.90, 69.61, 68.96, 68.74, 68.17, 66.69, 57.67, 50.11, 40.43, 21.33.

Example 124. General Procedure for Synthesis of Azido Fc

(1153) Preparation of PEG4-azido NHS ester solution (0.050 M) in DMF/PBS: 16.75 mg of PEG4-
azido NHS ester was dissolved in 0.100 mL of DMF at 0° C. and diluted to 0.837 mL by adding PBS
1× buffer at 0° C. This solution was used for preparing other PEG4-azido Fc with a variety of DAR
values by adjusting the equivalents of this PEG4-azido NHS ester PBS solution.

(1154) The nucleic acid construct encoding the Fc for any conjugate described herein may include a
nucleic acid encoding the amino acid sequence of an Fc including a C-terminal lysine residue and/or
N-terminal murine IgG signal sequence (e.g., any one of SEQ ID NOs: 48-53). Upon expression, the
C-terminal lysine and the N-terminal murine IgG signal sequence of the Fc of the conjugate are
proteolytically cleaved, resulting in an Fc having the sequence lacking Lys447 (e.g., lacking a C-
terminal lysine residue) and the N-terminal murine IgG signal sequence. The presence or absence of a
C-terminal lysine does not alter the properties of the Fc or the corresponding conjugate.

(1155) Pretreatment of h-IgG1 Fc, SEQ ID NO: 48 (107.2 mg in 8.800 mL of pH 7.4 PBS, MW-57891
Da, 1.852 μmol): The Fc solution was transferred into four centrifugal concentrators (30,000 MWCO,
15 mL) and diluted to 15 mL with PBS×1 buffer and concentrated to a volume of ˜1.5 mL. The residue
was diluted 1:10 in PBS pH 7.4, and concentrated again. This wash procedure was repeated for total
of four times followed by dilution to 8.80 mL.
(1156) Preparation of PEG4-azido Fc: 0.050M PEG4-azidoNHS ester PBS buffer solution (0.593 mL,
29.6 μmol, 16 equivalents) was added to above solution of h-IgG1 Fc (SEQ ID NO: 48) and the
mixture was shaken rotated for 2 hours at ambient temperature. The solution was concentrated by
using four centrifugal concentrators (30,000 MWCO, 15 mL) to a volume of ˜1.5 mL. The crude mixture
was diluted 1:10 in PBS pH 7.4, and concentrated again. This wash procedure was repeated for total
of three times. The concentrated Fc-PEG4-azide was diluted to 8.80 mL with pH 7.4 PBS buffer and
ready for Click conjugation. The purified material was quantified using a NANODROP™ UV visible
spectrophotometer (using a calculated extinction coefficient based on the amino acid sequence of h-
IgG1). Yield was quantitative after purification.

Example 125. Synthesis of Conjugate 34

(1157) A solution of azido functionalized Fc (40 mg, 2.5 mL, 0.69 μmol, described in the general
preparation of azido Fc, Example 124, SEQ ID No. 18) was added to a 15 mL centrifuge tube
containing alkyne derivatized small molecule (6.0 mg, 8.23 μmol, Int-67, Example 118). After gently
agitating to dissolve all solids, the mixture was added to a solution of L-ascorbic acid sodium salt (54
mg, 0.27 mmol), copper (II) sulfate (0.88 mg, 5.5 μmol), and BTTA (9.4 mg, 22 μmol) in PBS 7.4 buffer
(1.09 mL). The resulting mixture was gently rotated overnight. It was then purified by affinity
chromatography over a protein A column, followed size exclusion chromatography (See general
conjugate purification protocol). Maldi TOF analysis of the purified final product gave an average mass
of 64,678 Da (DAR=7.2). Yield 24 mg, 54% yield.

Example 126. Synthesis of Conjugate 35

(1158) A solution of azido functionalized Fc (40 mg, 2.5 mL, 0.69 μmol, described in the general
preparation of azido Fc, Example 124, SEQ ID No. 18) was added to a 15 mL centrifuge tube
containing alkyne derivatized small molecule (6.1 mg, 8.23 μmol, Int-68 Example 119). After gently
agitating to dissolve all solids, the mixture was added to solution of L-ascorbic acid sodium salt (54
mg, 0.27 mmol), copper (II) sulfate (0.88 mg, 5.5 μmol), and BTTA (9.4 mg, 22 μmol) in PBS 7.4 buffer
(1.09 mL). The resulting solution was gently rotated overnight. It was then purified by affinity
chromatography over a protein A column, followed size exclusion chromatography (See general
conjugate purification protocol). Maldi TOF analysis of the purified final product gave an average mass
of 64,830 Da (DAR=7.3). Yield 23 mg, 52% yield.

Example 127. Synthesis of Conjugate 36

(1159) A solution of azido functionalized Fc (50 mg, 2.5 mL, 0.86 μmol, described in the general
preparation of azido Fc, Example 124, SEQ ID No. 18) was added to a 15 mL centrifuge tube
containing alkyne derivatized small molecule (7.1 mg, 5.2 μmol, Int-77, Example 120). After gently
agitating to dissolve all solids, the mixture was added to a solution of L-ascorbic acid sodium salt (68
mg, 0.34 mmol), copper (II) sulfate (1.1 mg, 6.9 μmol), and BTTA (12 mg, 27 μmol) in PBS 7.4 buffer
(1.37 mL). The resulting mixture was gently rotated overnight. It was then purified by affinity
chromatography over a protein A column, followed size exclusion chromatography (See general
conjugate purification protocol). Maldi TOF analysis of the purified final product gave an average mass
of 63,300 Da (DAR=3.6). Yield 37 mg, 73% yield.

Example 128. Synthesis of Conjugate 37

(1160) A solution of azido functionalized Fc (70 mg, 2.5 mL, 1.2 μmol, described in the general
preparation of azido Fc, Example 124, SEQ ID No. 18) was added to a 15 mL centrifuge tube
containing alkyne derivatized small molecule (13.3 mg, 9.6 μmol, Int 78, Example 121). After gently
agitating to dissolve all solids, the mixture was added to a solution of L-ascorbic acid sodium salt (96
mg, 0.48 mmol), copper (II) sulfate (1.6 mg, 10 μmol), and BTTA (17 mg, 38 μmol) in PBS 7.4 buffer
(1.92 mL). The resulting mixture was gently shaken overnight. It was then purified by affinity
chromatography over a protein A column, followed size exclusion chromatography (See general
conjugate purification protocol). Maldi TOF analysis of the purified final product gave an average mass
of 63,574 Da (DAR=3.6). Yield 42 mg, 61% yield.
Example 129. Synthesis of Conjugate 33

(1161) Preparation of the Click reagent solution: 0.0050M CuSO.sub.4 in PBS buffer solution: 10.0 mg
CuSO.sub.4 was dissolved in 12.53 mL PBS, then took 5.00 mL this CuSO.sub.4 solution and added
43.1 mg BTTAA (CAS #1334179-85-9) and 247.5 mg sodium ascorbate to give the Click reagent
solution (0.0050M CuSO.sub.4, 0.020M BTTAA and 0.25M sodium ascorbate).

(1162) To a solution of azido functionalized Fc (104.9 mg, 8.60 mL, 18.1 μmol; Example 124, SEQ ID
NO: 73 in a 15 mL centrifuge tube was added to alkyne derivatized small molecule (29.7 mg, 19.9
μmol, described in Example 100, 2.5 equivalents for each azido on the Fc). After gently agitating to
dissolve all solids, the mixture was treated with the Click reagent solution(4.34 mL) of (L-ascorbic acid
sodium, 0.25 M, 1086 μmol, copper (II) sulfate 0.0050M, 21.7 μmol, and BTTAA 0.020M, 86.9 μmol).
The resulting mixture was gently rotated for 6 hours at ambient temperature. It was purified by affinity
chromatography over a protein A column, followed size exclusion chromatography (see general
conjugate purification protocol). Maldi TOF analysis of the purified final product gave an average mass
of 64550 Da (DAR=4.6). Yield 90.7 mg with 98% purity. The resulting conjugate is depicted in FIG. 61.

(1163) The nucleic acid construct encoding the Fc for Conjugate 33 included a nucleic acid encoding
the amino acid sequence of SEQ ID NO: 64, which includes a C-terminal lysine residue. Upon
expression, the C-terminal lysine of the Fc of Conjugate 33 is proteolytically cleaved, resulting in an Fc
having the sequence of SEQ ID NO: 73. The presence or absence of a C-terminal lysine does not alter
the properties of the Fc or the corresponding conjugate.

Example 130. Efficacy of Conjugate 33 Against Influenza B/Brisbane/60/2008 in a Lethal Mouse

Model

(1164) Test articles were evaluated against a lethal Influenza B influenza infection in female BALB/c
mice (Jackson Laboratories, 6-8 weeks old). The challenge virus (B/Brisbane/60/2008) is a mouse-
adapted isolate capable of causing lethal infections in mice. The experiment comprised 10 groups of 5
mice each. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal inoculation in a
volume of 50 μl (approx. 1 E5 virus per mouse), after being anesthetized with isoflurane.

(1165) All groups received a single IV treatment of conjugate 33 (Example 129), vehicle (PBS), or Fc
only control (hIgG1 Fc) two hours post viral challenge. An additional group was treated orally with
oseltamivir (20 mg/kg, bid, for 5 days) starting 8 hours after viral challenge.

(1166) The study evaluated 7 different dose concentrations of conjugate 33 (10, 3, 1, 0.3, 0.1, 0.03,
and 0.01 mg/kg). Mice were monitored for 2 weeks and animals exceeding 20% body weight loss, or
were found moribund, were scored as a mortality. Body weights were also recorded to monitor the
general health of the animals.

(1167) All mice treated with vehicle, or the Fc only control, reached mortality by Day 8 as expected. In
contrast, all mice receiving conjugate 33 were fully protected after receiving single IV doses from 10
down to 0.3 mg/kg for the duration of the study (Table 48). In contrast, the oseltamivir treated group
only resulted in 40% survival although the cumulative dose these mice received was 200 mg/kg over
the course of the experiment. The potency of conjugate 33 was further supported by daily body weight
measurements (Table 49; data only shown until the first death within a group). Mice treated with
conjugate 33 at 0.3 mg/kg only demonstrated a transient loss of body weight reaching a maximum of
7%, for a single day. Collectively this study demonstrates the potency of conjugate 33 as measured by
two parameters indicative of Influenza B infection (survival and body weight) against an influenza
strain of the Yamagata lineage.

(1168) TABLE-US-00053 TABLE 48 % survival Day Fc Conjugate 33 post- (10 (10 to 0.3 (0.1 (0.03
(0.01 infection PBS mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) Oseltamivir 0 100 100 100 100 100 100 100
1 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 3 100 100 100 100 100 100 100 4 60
100 100 100 100 100 100 5 40 40 100 100 100 100 100 6 0 0 100 60 60 80 40 7 0 0 100 60 60 40 0 8
0 0 100 60 60 0 0 9 0 0 100 60 60 0 0 10 0 0 100 60 60 0 0 11 0 0 100 60 60 0 0 12 0 0 100 60 60 0 0
13 0 0 100 60 60 0 0 14 0 0 100 60 60 0 0

(1169) TABLE-US-00054 TABLE 49 Body weights (gm) Day Fc Conjugate 33 post- (10 (10 (3 (1 (0.3
(0.1 (0.03 (0.01 infection PBS mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) Oseltamivir
0 100 100 100 100 100 100 100 100 100 100 1 99 101 103 98 98 98 98 99 98 98 2 95 97 100 99 97
101 97 97 97 97 3 87 90 101 98 96 97 93 95 92 89 4 82 84 104 99 99 97 93 93 88 85 5 78 79 102 99
99 97 92 84 79 6 98 96 98 93 79 7 102 98 100 96 8 101 98 98 97 9 104 101 102 101 10 103 100 101
101 11 104 102 101 101 12 104 101 102 102 13 103 102 102 102 14 103 102 102 101

Example 131. Characterization of Regioisomers

(1170) Monomer and dimer intermediates may be produced as a particular regioisomer or a mixture of
regioisomers. Examples 100-102 showed how to prepare Int-7 regioisomers (C7-C7, Example 100;
07-09, Example 101; C9-C9, Example 102; C7-C7 optimized, Example 103). The methods described
therein can be used to separate mixtures of regioisomers of any intermediate described herein. Table
50 provides the characterization of the relative percent (%) amounts of 07 and 09 linked monomers
and 07-07, C7-C9, and C9-C9 linked dimers in the previously described syntheses of the pre-
conjugation intermediate.

(1171) TABLE-US-00055 TABLE 50 Regiosomer Analysis Monomer (M) Int Example or Dimer (D) %
C7 % C7-C9 % C9 Int-2 Example 6 D 95 5 Int-3 Example 11 D 70 30 Int-4 Example 13 M 58 42 Int-4a
Example 122 M 97 Int-7 Example 19 D 3 2 95 Int-7a Example 100 D 100 Int-7b Example 101 D 5 95
Int-7c Example 102 D 100 Int-25 N/A D 5 95 Int-26 N/A D 25 50 25 Int-27 N/A D 29 35 36 Int-28 N/A D
5 28 67 Int-31 N/A D 90 Int-34 N/A D 12 6 82 Int-37 N/A M 95 Int-71 Example 110 D 3 97 Int-72
Example 112 D 100

Example 132. Efficacy of Conjugate 33 Subcutaneously Dosed Against Influenza A/Puerto Rico/8/34
(HI NI) in a Lethal Mouse Model

(1172) Conjugate 33 was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/1934) is a mouse-
adapted isolate capable of causing lethal infections in mice. The experiment comprised 6 groups of 5
mice. At day 0, all mice were challenged with virus at 3× the LD.sub.95 by intranasal inoculation in a
volume of 30 μl, after being anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg
respectively). Mortality and body weights were recorded daily and any animal with a 20% loss of body
weight was scored as a death.

(1173) Test groups received a single subcutaneous (SC) treatment of conjugate 33, hIgG1 Fc control,
or vehicle (PBS) 2 hours post viral challenge. The study design is summarized in Table 51.

(1174) TABLE-US-00056 TABLE 51 Study design for Influenza A/PR/8/34 (H1N1) study Dose Dose
volume # of Group Test article Route/Schedule (mq/kq) (ml/kq) mice 1 Vehicle SC, T + 2 hrs. na 10 5 2
hIgG1 Fc SC, T + 2 hrs. 1 10 5 3 Conjugate 33 SC, T + 2 hrs. 1 10 5 4 Conjugate 33 SC, T + 2 hrs.
0.3 10 5 5 Conjugate 33 SC, T + 2 hrs. 0.1 10 5 6 Conjugate 33 SC, T + 2 hrs. 0.03 10 5

(1175) As expected, mice receiving vehicle or the hIgG1 Fc control succumbed to infection on Days 6-
7 (Table 52). However, mice treated with conjugate 33 were fully protected at 1, 0.3, and 0.1 mg/kg
dose levels. Mortality with conjugate 33 was only seen at the lowest dose concentration of 0.03 mg/kg.

(1176) TABLE-US-00057 TABLE 52 Percent survival by study day (n = 5) Day Post hIgG1 Fc
Conjugate 33 Infection Vehicle (1.0 mg/kg) (1.0 mg/kg) (0.3 mg/kg) (0.1 mg/kg) (0.03 mg/kg) 0 100
100 100 100 100 100 1 100 100 100 100 100 100 2 100 100 100 100 100 100 3 100 100 100 100 100
100 4 100 100 100 100 100 100 5 100 100 100 100 100 100 6 40 60 100 100 100 100 7 0 0 100 100
100 60 8 0 0 100 100 100 0 9 0 0 100 100 100 0 10 0 0 100 100 100 0 11 0 0 100 100 100 0 12 0 0
100 100 100 0 13 0 0 100 100 100 0 14 0 0 100 100 100 0
(1177) The potency of conjugate 33 was further supported by daily body weight measurements. As
expected, mice treated with vehicle or hIgG Fc demonstrated a steady drop in body weight until it
exceeded 20%, at which time they were scored as a mortality (Table 53).

(1178) In contrast to control mice, those groups receiving conjugate 33 at 1, 0.3, and 0.1 mg/kg
maintained healthy body weights throughout the study and never demonstrated more than a transient
body weight drop of less than 8% (0.1 ink/kg dose group, Day 8; Table 53). By both survival and body
weight measurements conjugate 33 demonstrated robust protection from a lethal challenge of
Influenza A/Puerto Rico/8/1934 with a single SC dose as low as 0.1 mg/kg.

(1179) TABLE-US-00058 TABLE 53 Body weight data (gm) Day Post hIgG1 Fc Conjugate 33 Infection
Vehicle (1.0 mg/kg) (1.0 mg/kg) (0.3 mg/kg) (0.1 mg/kg) (0.03 mg/kg) 0 100 100 100 100 100 100 1
98.1 97.6 98 98.6 98.7 98.5 2 100.9 99 100.3 101.3 101.6 99.9 3 94.1 92.6 98.4 100.5 96.5 93.9 4 86
85.7 102.6 99.2 93.5 88.6 5 79.8 79.1 98.2 97.7 94.9 86.5 6 75.3 75.2 102.2 102.3 99.4 84.6 7 103.5
102.9 95.2 78.4 8 101.8 103.2 92.4 9 101.2 102.9 95.4 10 101.3 102.5 98.8 11 102.8 102.6 98.7 12
102.4 100.7 98.6 13 102.2 101.1 98.3 14 100.2 102 99.6

Example 133. Efficacy of Conjugate 33 Against A/PR/8/1934 (H1 N1), Lung PFU Burden and Cytokine
Levels

(1180) Efficacy studies for conjugate 33 were conducted in 6-8 weeks female BALB/c mice (Charles
River) challenged intranasally with 3E2 PFU/mouse (3× the LD.sub.95) of mouse-adapted influenza
A/PR/8/1934 (H1N1). Conjugate 33 or human IgG1 Fc controls was administered as a single
subcutaneously (SC) dose 2 h post-challenge at 0.1-3 mg/kg. Oseltamivir was dosed orally, twice daily
for 4 days starting 2 h post-infection at 5 or 50 mg/kg. Baloxavir was resuspended in 0.5%
methylcellulose and dosed orally, twice daily for 4 days starting 2 h post-infection at 30 mg/kg (Table
54). Body weights (BW) were recorded for 4 days (FIGS. 62A-62B). At 4 days post-infection, mice
were sacrificed by C02 and both lung lobes were harvested. Lungs were homogenized with 1 mm
silica beads in 1 mL PBS using a MagNA Lyser (Roche). Homogenization was carried out at 6,000
rpm for 60 s and chilled on ice for 5 min in-between runs. After lung homogenization tubes were
centrifuged for 10 min at 600×g and supernatant was transferred into new tube.

(1181) TABLE-US-00059 TABLE 54 Study Design Test Article (DAR) Route/Schedule Dose [mg/kg]
hIgG1 Fc IV, T + 2 h 3 Oseltamivir PO, BID × 4 5 Oseltamivir PO, BID × 4 50 Baloxavir SC, BID x 4 30
Conjugate 33 (4.7) SC, T + 2 h 0.1 Conjugate 33 (4.7) SC, T + 2 h 0.3 Conjugate 33 (4.7) SC, T + 2 h
1 Conjugate 33 (4.7) SC, T + 2 h 3 uninfected N/A N/A

(1182) For PFU determination, supernatants of lung homogenate were diluted in infection buffer
ranging from 10.sup.−1 to 10.sup.−6. 100 μL of virus dilutions were added to confluent monolayer of
MDCK cells in 24 well plates and incubated for 1 h at room temperature with rocking every 15 min.
After removing the virus, liquid overlay media containing Avicel was added to MDCK cells. Cells were
incubation at 37° C., 5% CO.sub.2 for 40 h. After incubation, the media was removed and cells were
stained with crystal violet to enumerate plaques and Plaque forming units (PFU) were calculated
relative to weight of the lung (PFU/g lung).

(1183) For cytokine analysis, supernatants of lung homogenate were serially diluted 2-fold in 96 well
plate. Cytokine levels for INF-γ, TNF-α, IL-6, MIP-1α and MCP-1 were determined by ELISA according
to manufacturer's instructions (R&D Systems). After a lethal challenge with influenza in a mouse
model, lung PFU burden (FIGS. 63A-63B) and lung cytokine levels were determined on day 4 post-
infection (Table 55, and Table 56, respectively). Conjugate 33 demonstrated a dose-dependent log
reduction in viral burden resulting in 0.7 at 0.1 mg/kg, 1.88 at 0.3 mg/kg, 3 at 1 mg/kg and 3.8 at 3
mg/kg. Oseltamivir at 5 mg/kg and 50 mg/kg had modest effects on viral burden resulting in 0.86 and 2
log reduction, respectively. Baloxavir reduced the viral burden below the limit of detection, 1e2
PFU/mL, thereby reducing the viral burden by >5.99 logs as compared to PBS control.

(1184) No biological relevant difference was observed between negative controls, PBS and hIgG1 Fc
as expected.
(1185) Conjugate 33 reduces viral burden in dose dependency on day 4 post-infection challenged with
influenza A in a mouse model (Table 55, FIGS. 64A-64B). Similarly, conjugate 33 demonstrated a
dose-dependent fold-reduction in cytokine levels for TNF-α, IL-6, INF-γ, MCP-1, and MIP-1α (FIGS.
65A-65E, respectively) compared to uninfected control on day 4 post-infection challenged with
influenza A in a mouse model (Table 56).

(1186) TABLE-US-00060 TABLE 55 PFU burden Test article [mg/kg] PFU/g Log reduction PBS [0]PBS
3.26E+07 0.0 hIgG1 Fc [3] 1.74E+07 0.364 Oseltamivir [5] 4.78E+06 0.86 Oseltamivir [50] 3.81E+05 2
Baloxavir [30] <1.00E+02* >5.99* conjugate 33 [0.1] 6.85E+06 0.715 conjugate 33 [0.3] 5.31E+05
1.88 conjugate 33 [1] 3.69E+04 2.99 conjugate 33 [3] 5.84E+03 3.77 *Baloxavir reduced the viral
burden below the limit of detection.

(1187) TABLE-US-00061 TABLE 56 Lung cytokine levels Test article [mg/kg] INF-γ TNF-α IL-6 MCP-1
MIP-1a PBS [0] 1.6 1.4 4.2 38.6 18.1 hIgG1 Fc [3] 1.7 1.2 3.3 36.3 15.9 Oseltamivir [5] 1.0 0.8 1.8
24.0 6.3 OseItamivir [50] 0.8 0.8 1.4 16.2 4.0 Baloxavir [30] 1.2 1.0 1.0 1.0 0.8 conjugate 33 [0.1] 1.1
0.7 0.8 19.1 5.6 conjugate 33 [0.3] 1.1 0.9 0.8 9.6 3.9 conjugate 33 [1] 0.9 0.7 1.1 5.7 1.7 conjugate 33
[3] 0.8 0.7 1.1 1.8 1.0 Uninfected 1 1 1 1 1

(1188) The highest concentration tested of conjugate 33 at 3 mg/kg demonstrated no weight loss
throughout the course of infection similar to uninfected control mice (Table 57).

(1189) TABLE-US-00062 TABLE 57 Body weight data (% reduction) Test article [mg/kg] Day 0 Day 1
Day 2 Day 3 Day 4 PBS [0] 0 −3.5 −2.86 −10.96 −17.32 hIgG1 Fc [3] 0 −2.6 −1.3 −9.58 −15.6
Oseltamivir [5] 0 −2.86 −2.42 −8.16 −13.88 Oseltamivir [50] 0 −3.78 −2.12 −3.22 −5.74 Baloxavir [30]
0 −2.38 −2.82 −2.6 −0.18 conjugate 33 [0.1] 0 −3.24 −2.32 −10.12 −11.46 conjugate 33 [0.3] 0 −2.16
1.34 −3.4 −4.36 conjugate 33 [1] 0 −1.5 −0.9 −1.98 −0.9 conjugate 33 [3] 0 −1.7 −1.6 −1.9 0.52
Uninfected 0 1.02 −0.14 −0.16 2.8

Example 134. Efficacy of Conjugate 33 Against Influenza A (H1N1) in a Lethal Severe Combined
Immunodeficiency (SCID) Mouse Model

(1190) Test articles were evaluated against a lethal Influenza A influenza infection in female
BALB/cscid mice (Jackson Laboratories, 6-8 weeks old). The challenge virus (A/Puerto Rico/8/1934) is
a mouse-adapted isolate capable of causing lethal infections in mice. The experiment comprised 11
groups of 5 mice each. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal
inoculation in a volume of 30 μl (approx. 1 E3 virus per mouse), after being anesthetized with a
mixture of ketamine/xylazine (150 and 10 mg/kg respectively).

(1191) Groups received a single SC treatment of vehicle (PBS), hIgG1 Fc control, or conjugate 33 (3,
1, 0.3, 0.1, 0.03 mg/kg) two hours post viral challenge. A separate arm of the study consisted of 3
groups of mice treated with baloxavir marboxil (DC Chemicals, Shanghai, China) orally, twice daily, for
1 day; also starting 2 hours post viral challenge. Mice were monitored for 2 weeks and animals
exceeding 20% body weight loss, or were found moribund, were scored as a mortality.

(1192) At study end (Day 14) mice receiving conjugate 33 were fully protected at all dose
concentrations between 3 and 0.1 mg/kg (Table 58). Conjugate 33 only failed to protect against lethal
viral challenge at the lowest tested concentration of 0.03 mg/kg. As expected, groups receiving vehicle
or hIgG1 Fc were not protected. Mice treated with baloxavir were also protected, but at the
significantly higher cumulative doses of 60 and 20 mg/kg, at a total dose of 6 mg/kg only 40% of mice
survived to Day 14.

(1193) TABLE-US-00063 TABLE 58 % Survival on Day 14. hIgG1 Fc Baloxavir Conjugate 33 Vehicle
(3.0 (30 (10 (3.0 (3.0 (1.0 (0.3 (0.1 (0.03 (PBS) mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) mg/kg)
mg/kg) mg/kg) 0 0 100 100 40 100 100 100 100 0

(1194) The potency of conjugate 33 in this model of severe immunodeficiency was also evident based
on body weights (Table 59). The lowest concentration of conjugate providing full protection based on a
mortality readout was 0.1 mg/kg. At this dose level, the greatest average weight loss for the group was
transient, and resulted in less than a 5% reduction (occurring on Day 2). Furthermore the difference in
body weight for groups at the 1 and 3 mg/kg dose levels showed less than a 2% difference on Day 14
compared to uninfected mice.

(1195) Collectively this data demonstrates the potency of conjugate 33 by protecting lethally
challenged mice with single SC doses of conjugate as low as 0.1 mg/kg. This was accomplished in a
severe model of immunodeficiency with mice completely lacking T & B immune cells, which are
essential in clearing influenza infections. This data supports the use of conjugate 33 to treat immune
deficient patient populations.

(1196) TABLE-US-00064 TABLE 59 % Average body weight by day. (mg/kg). Data only shown until the
first mortality within a group. Day post Vehicle hIgG1 Fc Baloxavir Conjugate 33 infection (PBS) (3
mg/kg) (30 mg/kg) (10 mg/kg) (3 mg/kg) (3 mg/kg) (1 mg/kg) (0.3 mg/kg) (0.1 mg/kg) (0.003 mg/kg)
Uninfected 0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 1 100.0 99.0 98.9
91.5 97.3 99.0 99.6 98.8 97.6 99.7 99.5 2 99.0 100.8 99.1 97.3 100.1 100.1 99.9 100.7 95.8 98.6 99.8
3 94.0 93.0 97.0 96.4 97.4 100.0 98.9 97.6 97.0 96.7 98.8 4 93.0 89.7 99.1 95.9 99.0 101.0 99.8 99.2
97.8 94.4 99.9 5 87.0 84.4 99.1 97.5 96.6 99.0 98.6 96.3 97.4 93.0 10.9 6 82.0 80.1 97.9 97.9 97.0
100.6 99.7 99.9 98.7 91.6 100.2 7 78.0 98.6 97.0 97.2 101.9 99.6 99.6 98.1 87.0 101.0 8 98.6 96.4
95.4 100.9 98. 99.2 98.3 82.2 100.4 9 99.6 95.1 93.1 100.6 98.4 100.0 97.0 100.7 10 96.0 95.9 90.5
98.8 98.8 98.5 95.8 100.2 11 97.3 97.3 99.0 99.3 98.3 96.0 99.7 12 98.7 95.8 99.1 99.4 98.1 96.3
100.4 13 98.6 96.8 98.4 99.0 97.9 97.3 99.7 14 97.9 95.9 99.3 99.3 98.1 97.8 100.5

Example 135. Efficacy of Conjugate 33 Subcutaneously Dosed Against Influenza A/California/07/2009

(H1N1) Pdm in a Lethal Mouse Model

(1197) Conjugate 33 was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/California/07/2009 (H1N1) pdm)
is a pandemic isolate capable of causing lethal infections in mice. The experiment comprised 6 groups
of 5 mice. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal inoculation in a
volume of 30 μl, after being anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg
respectively). Mortality and body weights were recorded daily and any animal with a 20% loss of body
weight was scored as a death.

(1198) Test groups received a single subcutaneous (SC) treatment of conjugate 33, hIgG1 Fc control,
or vehicle (PBS) 2 hours post viral challenge. The study design and dose levels are summarized in
Table 60.

(1199) TABLE-US-00065 TABLE 60 Study design for Influenza A/California/07/2009 (H1N1) pdm study
Dose Dose volume # of Group Test article Route/Schedule (mq/kq) (ml/kq) mice 1 Vehicle SC, T + 2
hrs. na 10 5 2 hIgG1 Fc SC, T + 2 hrs. 1 10 5 3 conjugate 33 SC, T + 2 hrs. 1 10 5 4 conjugate 33 SC,
T + 2 hrs. 0.3 10 5 5 conjugate 33 SC, T + 2 hrs. 0.1 10 5 6 conjugate 33 SC, T + 2 hrs. 0.03 10 5

(1200) As expected, mice receiving vehicle or the hIgG1 Fc control succumbed to infection on Days 6-
7 (Table 61). However, mice treated with conjugate 33 were fully protected at 1 mg/kg, and nearly so
at 0.3 (80% survival). Significant mortality with conjugate 33 was only seen at the lower dose
concentrations of 0.1 and 0.03 mg/kg.

(1201) TABLE-US-00066 TABLE 61 Percent survival by day. (mg/kg) hIgG1 Fc conjugate 33 (1.0 (1.0
(0.3 (0.1 (0.03 Day Vehicle mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) 0 100 100 100 100 100 100 1 100
100 100 100 100 100 2 100 100 100 100 100 100 3 100 100 100 100 100 100 4 100 100 100 100 100
100 5 40 20 100 100 60 80 6 0 20 100 80 20 20 7 0 0 100 80 0 0 8 0 0 100 80 0 0 9 0 0 100 80 0 0 10
0 0 100 80 0 0 11 0 0 100 80 0 0 12 0 0 100 80 0 0 13 0 0 100 80 0 0 14 0 0 100 80 0 0

(1202) The potency of conjugate 33 was further supported by daily body weight measurements. As
expected, mice treated with vehicle or hIgG1 Fc demonstrated a steady drop in body weight until it
exceeded 20%, at which time they were scored as a mortality (Table 62).
(1203) In contrast to control mice, mice receiving conjugate 33 at 1 mg/kg only demonstrated a
transient drop in bodyweight of approximately 10%, peaking on Day 3 (Table 62). By both survival and
body weight measurements conjugate 33 demonstrated robust protection from a lethal challenge of
Influenza A/California/07/2009 (H1N1) pdm with a single 1 mg/kg dose administered SC. Activity
against the clinically relevant pandemic strain used in this study supports the utility of conjugate 33 in
treating serious influenza infections.

(1204) TABLE-US-00067 TABLE 62 % Average body weight by day. (mg/kg). Data only shown until
the first mortality within a group. Day hIgG1 Fc Conjugate 33 post (1.0 (1.0 (0.3 (0.1 (0.03 infection
Vehicle mg/kg) mg/kg) mg/kg) mg/kg) mg/kg) 0 100 100 100 100 100 100 1 99.5 97.9 96.8 96.7 98
98.7 2 97.1 97.2 99.5 99.2 98 98.4 3 86.5 86.0 89.7 89.6 87.6 88.8 4 80.3 80.3 91.6 88.5 81.3 81.9 5
76.2 76.6 92.9 91.0 76.8 77.9 6 94.7 90.5 7 94.2 8 95.4 9 98.0 10 96.8 11 98.8 12 97.7 13 100.1 14
100.5

Example 136. Efficacy of Conjugate 33 Intravenously (IV) Dosed Against Influenza A/Puerto
Rico/8/1934 (H1N1) in a Lethal Mouse Model of Delayed Treatment

(1205) Conjugate 33 was evaluated against a lethal influenza A (H1N1) infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/1934) is a mouse-
adapted isolate capable of causing lethal infections in mice. At day 0, all mice were challenged with
virus at 3× the LD95 by intranasal inoculation in a volume of 30 μl, after being anesthetized with a
mixture of ketamine/xylazine (150 and 10 mg/kg respectively). Mortality and body weights were
recorded daily and any animal with a 20% loss of body weight was scored as a death.

(1206) The study design is detailed in Table 63, and consists of multiple arms. The control arm
comprises vehicle (PBS) and hIgG1 Fc only groups, dosed 24 hours after viral challenge (an
uninfected group was also part of this arm). The second arm consisted of oseltamivir dosed at 4× its
humanized dose, with initiation of treatment delayed for 24, 48, or 72 hours. The final 3 arms consisted
of conjugate 33 administered as single IV doses of 10, 3, or 1 mg/kg; each being dosed on the same
schedule as the oseltamivir arm above.

(1207) As expected, vehicle and hIgG1 Fc were not protective when dosed 24 hours after viral
challenge and resulted in complete mortality by Day 7. In our hands, oseltamivir, even at 4× the
humanized dose (200 mg/kg cumulative dose) was only partially efficacious when dosing was delayed
24 hours (Table 64; 40% survival). However, conjugate 33 was fully protective at all concentrations
(10, 3, & 1 mg/kg) at the same 24 hour dose schedule.

(1208) When dosing was delayed a full 48 hours after viral challenge oseltamivir was no longer
efficacious (0% survival) while conjugate 33 was 80% protective at doses of 10 and 3 mg/kg. When
dosing was delayed until 72 hours, only the 10 mg/kg dose of conjugate 33 demonstrated partial
protection (40%). The efficacy of conjugate 33 was also evident based on daily body weight
measurements (Table 65). This was especially significant in the T+24 hours groups where less than a
3% reduction was observed for any conjugate 33 group, which was transient and occurred on Day 1.
In this study, conjugate 33 is more efficacious than oseltamivir, an approved treatment for influenza.

(1209) TABLE-US-00068 TABLE 63 Study design Dose Influenza A Test Route, First dose Dose
volume N Group strain Article Schedule (hours) (mg/kg) (ml/kg) (balb/c) 1 A/PR/8/34 PBS IV, single T
+ 24 — 5 5 2 (H1N1) hIgG1 Fc IV, single T + 24 10 5 5 3 3E2 Oseltamivir PO, bid × 5 T + 24 20 10 5 4
PFU/mouse days T + 48 5 T + 72 6 Conjugate IV, single T + 24 10 5 5 7 33 T + 48 8 T + 72 9
Conjugate IV, single T + 24 3 5 5 10 33 T + 48 11 T + 72 12 Conjugate IV, single T + 24 1 5 5 13 33 T
+ 48 14 T + 72 15 Uninfected BW control

(1210) TABLE-US-00069 TABLE 64 % Survival by day. Test article/Dose/Time of treatment initiation

Oseltamivir Conjugate 33 Conjugate 33 Conjugate 33 Day Vehicle hIgG1 Fc 20 mpk, 20 mpk, 20 mpk,
10 mpk, 10 mpk, 10 mpk, 3 mpk, 3 mpk, 3 mpk, 1 mpk, 1 mpk, 1 mpk, Unin- post na 10 mpk bid × 5
bid × 5 bid × 5 single single single single single single single single single fected infec- T + T + T + T +
T + T + T + T + T + T + T + T + T + T + na tion 24 hrs. 24 hrs. 24 hrs. 48 hrs. 72 hrs. 24 hrs. 48 hrs. 72
hrs. 24 hrs. 48 hrs. 72 hrs. 24 hrs. 48 hrs. 72 hrs. na 0 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 1 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 2 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100 3 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 4 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 5 100 100 100
100 100 100 100 100 100 100 100 100 80 100 100 6 60 100 100 100 100 100 100 60 100 100 80 100
80 80 100 7 0 0 100 100 20 100 100 40 100 80 0 100 60 40 100 8 0 0 100 100 0 100 80 40 100 80 0
100 40 0 100 9 0 0 60 60 0 100 80 40 100 80 0 100 40 0 100 10 0 0 40 0 0 100 80 40 100 80 0 100 40
0 100 11 0 0 40 0 0 100 80 40 100 80 0 100 40 0 100 12 0 0 40 0 0 100 80 40 100 80 0 100 40 0 100
13 0 0 40 0 0 100 80 40 100 80 0 100 40 0 100 14 0 0 40 0 0 100 80 40 100 80 0 100 40 0 100

(1211) TABLE-US-00070 TABLE 65 Body Weight Data (gm). mpk = mg/kg. Data only shown until the
first death within a group. Test article/Dose/Time of treatment initiation Day Vehi- hIgG1 Oseltamivir
Conjugate 33 Conjugate 33 Conjugate 33 post cle Fc 20 mpk, 20 mpk, 20 mpk, 10 mpk, 10 mpk, 10
mpk, 3 mpk, 3 mpk, 3 mpk, 1 mpk, 1 mpk, 1 mpk, Unin- in- na 10 mpk bid × 5 bid × 5 bid × 5 single
single single single single single single single single fected fec- T + T + T + T + T + T + T + T + T + T +
T + T + T + T + na tion 24 his. 24 hrs. 24 hrs. 48 hrs. 72 hrs. 24 hrs. 48 hrs. 72 hrs. 24 hrs. 48 hrs. 72
his. 24 his. 48 hrs. 72 hrs. na 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 1
99.3 98.8 96.4 97.5 98.2 97.6 97.7 98.5 97.1 98.3 98.7 98.3 99.4 99.4 99.5 2 103.9 101 100.7 101.2
102.6 102.1 101.7 100.1 102 102.5 99.4 104.2 102 101.4 101.7 3 96.9 97.4 95.5 97.5 95.6 102.3 96.2
95.1 100.6 95.9 97.6 102.3 96.9 98.6 105.8 4 88.7 90.1 92.6 89.7 87.7 102.3 88.4 88.8 100 87.8 88.6
100 89.2 89.7 105 5 81.7 83.9 92.2 88.6 81.6 101.5 88.1 82.7 99 85.3 81.7 101.2 83.7 83.2 105.5 6
77.3 79.3 91.8 86 78.6 102.6 91.1 80.4 101.4 87.1 77.5 103.3 78.7 104.9 7 87.8 81 101.9 91.1 102.3
86.1 102.1 103.5 8 83.5 75.8 101.5 91.3 101.1 103.2 102.9 9 81.1 102.2 102.1 104.1 102.9 10 102.5
101.6 105 103.3 11 102.4 102.1 103.9 104.3 12 100.9 101.6 103.4 102.5 13 101 101.8 103.5 102.5 14
102.2 103.6 104 103.1

Example 137. Synthesis of Conjugate 38 (Int-73), Conjugate 39 (Int-74), Conjugate 40 (Int-75), and
Conjugate 41 (Int-76)

(1212) A 15-ml sterile centrifuge tube is charged with sodium ascorbate (68.3 mg, 0.345 mmol),
BTTAA (11.9 mg, 0.0276 mmol), product from Examples 114 (Int-73), 115 (Int-74), 116 (Int-75), or 117
(Int-76) (0.00953 mmol) and PBS 7.4 (1 ml). The reagents were vortexed until homogeneous then
mixed with azido Fc (50 mg, 0.0008624 mmol, described in Example 124, SEQ ID NO: 73) followed by
a solution of CuSO.sub.4 (1.1 mg, 0.0069 mmol) in water (0.5 ml). The mixture is rotated for 12 hours
then purified by affinity chromatography over a protein A column, followed size exclusion
chromatography. Conjugates are characterized by Maldi TOF analysis (DAR typically =4.5). Yields are
typically 50%.

(1213) The nucleic acid construct encoding the Fc for conjugates 38-41 included a nucleic acid
encoding the amino acid sequence of SEQ ID NO: 64, which includes a C-terminal lysine residue.
Upon expression, the C-terminal lysine of the Fc of conjugates 38-41 is proteolytically cleaved,
resulting in an Fc having the sequence of SEQ ID NO: 73. The presence or absence of a C-terminal
lysine does not alter the properties of the Fc or the corresponding conjugate.

Example 138. Synthesis of Int-79

(1214) ##STR00757## ##STR00758##

(1215) Zanamivir-ether-acid (0.90 g, 1.43 mmol, Example 31) and N-methyl morpholine (0.23 mL, 2.14
mmol) were dissolved in THE (35 mL) and cooled to 0° C. (ice water bath) under an atmosphere of
nitrogen. Isobutyl chloroformate (0.24 mL, 1.85 mmol, in 2 mL DCM) was added dropwise, by way of
syringe over a 5 minute period. The mixture was stirred at 0° C. for 30 minutes then 15 min at ambient
temperature, and then cooled to 0° C. Sodium borohydride (540 mg, 14.3 mmol, dissolved in 5 mL of
methanol) was added, dropwise over 5 minutes. The reaction was stirred for 15 minutes at which point
all starting material had been consumed (by LC/MS). A few drops (˜1 mL) of glacial acetic acid was
added to acidify the mixture (pH-5). Dilute with ethyl acetate and water and extract into ethyl acetate
(3×). The organic layer was washed with brine, and the organic extracts were dried over sodium
sulfate, and concentrated on the rotary evaporator. The crude material was purified by silica gel
chromatography (loaded on celite first) (0-10% methanol in DCM, 30 min). Yield 0.66 g, 75%. Ion
found by LCMS: [M+H].sup.+=617.2.

(1216) Step b.

(1217) ##STR00759##

(1218) To a stirring mixture of alcohol (0.66, 1.1 mol, in 20 mL CH.sub.2Cl.sub.2), was added
triethylamine (0.30 mL, 1.3 mmol). The mixture was cooled to 0° C. (ice-water bath) under 1
atmosphere of nitrogen and mesyl chloride (150 mg, 1.3 mmol) was added, dropwise over 5 minutes
by way of syringe. The ice bath was removed and the reaction was stirred for 45 minutes. The mixture
was diluted with saturated, aqueous sodium bicarbonate, extracted into DCM (3×). The combined
organic extracts were washed with brine, dried over sodium sulfate and concentrated on the rotary
evaporator. Ion found by LCMS: [M+H]+=695.2. The intermediate was taken to the next step without
purification.

(1219) The zanamivir mesylate was stirred in DMF at 80° C. with 3 eq of sodium azide for 5 hours.
The mixture was diluted with water, extracted into DCM (3×). The combined organic extracts were
washed with brine, dried over sodium sulfate and concentrated. Ion found by LCMS: [M+H]+=695.2.
The azide was taken to the next step without purification.

(1220) The zanamivir azide (670 mg, 1.04 mmol) was stirred in methanol (20 mL) in the presence of
Lindlar catalyst (300 mg) under 1 atmosphere of hydrogen gas for 12 hours. The mixture was filtered
through celite and concentrated to afford the title compound as a clear oil. The amine was taken to the
next step without purification. Yield 0.37 g, 54%, 3 steps. Ion found by LCMS: [M+H]+=616.2.

(1221) Step c.

(1222) ##STR00760##

(1223) Methyl 3-(benzylamino)propanoate (1 g, 5.2 mmol), methyl 4-bromobutanoate (1.2 g, 6.2

mmol), and diisopropylethylamine (1.34 mL, 7.8 mmol) were stirred in DMF (5 mL) at 65° C. for 4
hours. Most of the solvent was removed by rotary evaporator and the crude material was purified by
silica gel chromatography (Isco, 0 to 9% methanol in DCM, 30 minute gradient) to afford the benzyl
protected intermediate as a clear oil. The benzyl protected intermediate was stirred in methanol (20
mL) under 1 atmosphere of hydrogen gas in the presence of 20% palladium hydroxide on carbon (300
mg) for 12 hours. The mixture was filtered through celite and concentrated to afford the title compound
as a clear oil. Yield 0.72 g, 69%. Ion found by LCMS: [M+H]+=204.2.

(1224) Step d.

(1225) ##STR00761##

(1226) The step-c product (0.70 g, 2.16 mmol), propargyl PEG-4 acid (0.63 mg, 2.38 mmol) HATU
(1.26 g, 3.24 mmol) in DMF (2 mL) were stirred at room temperature following addition of DIEA (1.15
mL, 6.49 mmol). The reaction mixture was stirred for 2 hours then purified by reverse phase liquid
chromatography (Isco, 5 to 50% acetonitrile and water with 0.1% TFA as modifier). Yield 840 mg, 87%.
Ion found by LCMS [M+H]+=446.2.

(1227) Step e.

(1228) ##STR00762##

(1229) A solution of step-d product (840 mg, 1.85 mmol) and LiOH (113.1 mg, 4.72 mmol) in
H.sub.2O:MeOH (1:2, 9 mL) was stirred at room temperature for 2 hours. Then the reaction was
acidified with TFA. The resulted solution was concentrated then purified by reverse phase liquid
chromatography (Isco, 0% to 20% acetonitrile and water). Yield 454.5 mg, 59%. Ion found by LCMS
[M+H]+=418.2.

(1230) Step f.

(1231) ##STR00763##

(1232) To a solution of step-b product (334.7 mg, 0.52 mmol), step-e product (102 mg, 0.24 mmol) and
HATU (273.25 mg, 0.704 mmol) in anhydrous DMF (3 mL) at room temperature was added DIEA (217
mg, 16.4 mmol). The resulting mixture was stirred for 1 hour then was purified by RPLC (20% to 100%
methanol and water without modifier). Yield 206.5 mg, 55%. Ion found by LCMS [(M+2H)/2]+=806.8.

(1233) Step g.

(1234) ##STR00764##

(1235) The step-f product (206.5 mg, 0.128 mmol) and TFA (3 mL) in CH.sub.2Cl.sub.2 (5 mL) was
stirred at room temperature overnight, then concentrated under reduced pressure. The resulting
residue was purified by semi-preparative HPLC (0% to 30% acetonitrile and water with 0.1% TFA as
modifier). Yield 144.5 mg, 69%. Ion found by LCMS [(M+2H)/2]+=606.8.

(1236) Step h.

(1237) ##STR00765##

(1238) To a solution of the step-g product (144.5 mg, 0.119 mmol) in MeOH (9 mL) and water (3 mL)
was added LiOH (18 mg, 0.75 mmol). The resulting solution was stirred at room temperature for 1
hour, then acidified with TFA and concentrated under reduced pressure. The residue was purified by
semi-preparative HPLC (0% to 25% acetonitrile and water, using 0.1% TFA as modifier). Yield 45 mg,
28%. Ions found by LCMS [(M+2H)/2]+=566.8.

Example 139. Synthesis of conjugate 42

(1239) A solution of azido functionalized Fc (50 mg, 5.4 mL, 0.859 μmol, Example 124, SEQ ID NO:
18) was added to a 10 mL centrifuge tube containing alkyne derivatized small molecule (7.87 mg,
0.057 mmol, Example 138). After gently shaking to dissolve all solids, the mixture was added to 3 mL
premixed solution of L-ascorbic acid sodium (0.68 mg, 0.34 mmol, 0.25 M), copper (II) sulfate (1.1 mg,
0.0069 mmol, 0.005 M) and BTTAA (11.8 mg, 0.027 mmol, 0.02 M) in PBS 7.4 buffer. The resulting
solution was gently rotated overnight. It was purified by affinity chromatography over a protein A
column, followed by size exclusion chromatography (See general conjugate purification protocol).
Maldi TOF analysis of the purified final product gave an average mass of 63623 Da (DAR 3.8). Yield
36.01 mg, 72%.

Example 140. Synthesis of Int-80

(1240) ##STR00766##

(1241) To a solution of the p-nitrophenyl carbonate of Zanamivir (0.3 g. 0.4 mmol, Example 103) in
anhydrous dichloromethane (5 ml) was added a propargyl-PEG4-methylamine (0.11 g, 0.44 mmol)
and DIPEA (0.14 ml, 1.0 mmol) in anhydrous DMF (5 ml). The reaction was stirred at room
temperature overnight, then concentrated and purified by flash chromatography eluting with 0% to
10% methanol/dichloromethane. Yield 0.28 g, 81%. Ions found by LCMS: (M+H).sup.+=858.4, (M-
Boc).sup.+H.sup.+=758.4.

(1242) Step b.

(1243) ##STR00767##
(1244) Product from the previous step (280 mg, 0.2 mmol) was dissolved into 2 ml MeOH and 2 ml
THF, then treated with a solution of lithium hydroxide (24 mg, 1 mmol) dissolved in 2 ml water. The
reaction was stirred for 10 min at room temperature at which time HPLC showed the reaction was
complete. The pH of the reaction was adjusted to the value of 5 to 6 by using Amberlite IRN-77 ion
exchange resin, then filtered to remove the resin. The crude filtrate was evaporated to dryness under a
vacuum and used in the next step with purification, and the yield was quantitative. Ion(s) found by
LCMS: (M+H).sup.+=844.4, (M-Boc+H).sup.+=744.4.

(1245) Step c.

(1246) ##STR00768##

(1247) The step-b product was dissolved into 2 ml dichloromethane and 2 ml TFA, and stirred at room
temperature. The progress of the reaction was monitored by LCMS. After the completion of the
reaction (6 h), the solution was stripped to dryness and then dissolved in 2 ml water and 2 ml
acetonitrile. The resulting solution was stirred for another 2 hour at room temperature at which time
LCMS show complete deprotection of the acetonide protecting groups. This mixture was concentrated
and purified by reverse phase liquid chromatography (RPLC) using an Isco COMBIFLASH® liquid
chromatograph eluted with 5% to 40% acetonitrile/water with 0.1% TFA as the modifier. Yield 180 mg,
78.0%. Ion(s) found by LCMS: (M+H).sup.+=604.2.

Example 141. Stability Data for Int-80 Compared to Int-4

(1248) Int-80 (Example 140) and Int-4 (Example 13) were solubilized to 10 mg/mL in deionized water
and then diluted 1:10 into 1×PBS to a final concentration of 1 mg/mL. Samples were incubated at 37°
C. or 60° C. for 1 week. A 25 μL aliquot was diluted into 75 μL of water for HPLC analysis. Using a
Waters Acquity H-Class UPLC with a Phenomenex Biozen PS-C18 column (150×2.1 mm, 1.6 um) a
gradient of 0.1% formic acid in water to 0.1% formic acid in acetonitrile was run as follows: 5% B for 0-
1 min, 5-20% B from 1-20 min. Detection was at 240 nm using a diode array detector. Int-80 had less
than 1% degradation over a week at 60° C., while Int-4 showed 15% degradation over the same time
period (FIG. 66)

Example 142. Synthesis of Conjugate 43

(1249) Preparation of the Click reagent solution: 0.0050M CuSO.sub.4 in PBS×1 buffer solution: 10.0
mg CuSO.sub.4 was dissolved in 12.53 mL PBS×1, than took 10.00 mL this CuSO.sub.4 solution and
added 86.1 mg BTTAA and 495.3 mg Na Ascorbate to give the Click reagent solution (0.0050M
CuSO.sub.4, 0.020M BTTAA and 0.25M Sodium Ascorbate).

(1250) A solution of azido functionalized Fc (78.0 mg, 4.535 mL, 1.35 μmol, Example 124, SEQ ID
NO: 73) was added to a 15 mL centrifuge tube containing alkyne derivatized small molecule (13.2 mg,
8.88 μmol, Int-80, Example 140). After gently shaking to dissolve all solids, the mixture was added with
2.153 mL of above Click reagent solution of (L-ascorbic acid sodium, 0.25 M, 106.6 mg, 0.538 mmol,
copper (II) sulfate 0.0050M, 1.72 mg, 0.0107 mmol, and BTTAA 0.020M, 18.5 mg, 0.0431 mmol). The
resulting mixture was gently rotated for 6 hours at ambient temperature. It was purified by affinity
chromatography over a protein A column, followed size exclusion chromatography (See conjugate
purification protocol). Maldi TOF analysis of the purified final product gave an average mass of 64,012
Da (DAR=7.0). Yield 50.3 mg, 62% yield.

(1251) The nucleic acid construct encoding the Fc for conjugate 43 included a nucleic acid encoding
the amino acid sequence of SEQ ID NO: 64, which includes a C-terminal lysine residue. Upon
expression, the C-terminal lysine of the Fc of conjugate 43 is proteolytically cleaved, resulting in an Fc
having the sequence of SEQ ID NO: 73. The presence or absence of a C-terminal lysine does not alter
the properties of the Fc or the corresponding conjugate.

Example 143. Synthesis of Int-81

(1252) ##STR00769## ##STR00770##

(1253) To a well-stirred solution of N-Boc-N-Me-glycinol (3.5 g, 20 mmol) in DMSO (20 mL) cooled
with an ice-water bath was add allyl bromide (3.6 g, 30.0 mmol), followed by finely ground KOH
powder (3.5 g, 30.0 mmol) over 15 minutes. The resulting solution was stirred for overnight at room
temperature. The resulting mixture was partitioned between 5% aq. HOAc (50 mL) and ethyl acetate
(200 ml). The organic layer was separated, washed with brine, dried with sodium sulfate, filtered, and
concentrated, then purified by flash chromatography eluting with 10% to 80% ethyl acetate/hexane.
Yield of product 4.1 g, 95%. Ion(s) found by LCMS: M+H=216.3.

(1254) Step b.

(1255) ##STR00771##

(1256) Ozone was bubbled through a solution of the compound from step a (8.0 g, 37 mmol) in MeOH
(50 mL) and DCM (50 ml) at −78° C. until the appearance of a light blue color. Unreacted ozone was
removed by bubbling with oxygen for 10 minutes before the addition of NaBH.sub.4 (1.6 g, 40 mmol)
in small portion over 10 minutes. After all NaBH.sub.4 was added the mixture was gradually warmed
to room temperature. The resulting solution was partitioned between ethyl acetate (100 ml) and brine
(50 ml). The organic layer was separated, washed with brine, dried with sodium sulfate, filtered,
concentrated to an oil, and then purified by flash chromatography eluting with 10% to 80% ethyl
acetate/dichloromethane. Yield of product 5.0 g, 62%. Ion(s) found by LCMS: M+H=220.2.

(1257) Step c.

(1258) ##STR00772##

(1259) To a solution of the product (4.4 g, 20 mmol) from the previous step and CBr.sub.4 (10.0 g,
30.0 mmol) in DCM (50 mL) at 0° C. was added PPh.sub.3 (8.0 g, 30 mmol) slowly over 15 minutes
(exothermic). During the course of the addition the internal temperature was kept below 30° C. After
addition of PPh.sub.3 the reaction was stirred overnight at room temperature. The crude reaction was
concentrated to an oil then purified by normal phase chromatography, eluting with 10% ethyl
acetate/hexanes to 80% ethyl acetate/hexanes. Fractions containing oil droplets on the inside of the
collection tubes were combined and concentrated to a colorless oil. 4.0 g, 70.5%. Ion(s) found by
LCMS: M+H=282.1.

(1260) Step d.

(1261) ##STR00773##

(1262) A solution of the step c product (4 g, 14 mmol), benzylamine (0.60 g, 5.7 mmol), and
K.sub.2CO.sub.3 (2.35 g, 17 mmol) in DMF (20 mL) were heated in an oil bath at 75° C. for 8 h. The
mixture was filtered, concentrated, and purified by RPLC (5% ACN/water to 100% ACN). Yield 2.1 g,
72.7%.

(1263) Step e.

(1264) ##STR00774##

(1265) To a solution of the step-d product (1.3 g, 2.0 mmol) dissolved in CHCl.sub.3/EtOH (1:20, 20
mL) was added 20% Pd(OH).sub.2/C (0.50 g). The reaction was stirred overnight under hydrogen
from a balloon at ambient temperature. The reaction mixture was filtered through a Celite pad, then
concentrated by way of rotary evaporator and carried to the subsequent step without further
purification.

(1266) Step f.

(1267) ##STR00775##
(1268) The step-e product was dissolved in 10 mL of DMF, then treated with propargyl PEG4 acid
(0.52 g, 2.0 mmol), EDCl (0.6 g, 3.0 mmol), HOAt (0.45 g, 3 mol) and Hunig's base (0.7 mL, 5.0 mmol)
at room temperature. The reaction mixture was stirred for four hours, then concentrated and purified
by RPLC (10% ACN/water to 60% ACN/water). Yield 0.43 g, 65% for two steps. Ions found by LCMS:
[M-Boc+H].sup.+=562.4, [M+H].sup.+=662.4.

(1269) Step g.

(1270) ##STR00776##

(1271) The step-d product (70 mg, 0.1 mmol) was treated with TFA (2 mL) for 2 hours at room
temperature. TFA was removed by rotary evaporation, and the remaining oil was further dried under
high vacuum for 12 h to give the desired product as bis-TFA salt. Yield was quantative. Ion found by
LCMS: [M+H].sup.+=462.4.

(1272) Step h. 0 O$

(1273) ##STR00777##

(1274) To a solution of the nitrophenyl carbonate described in the Example 109 (0.72 g. 0.95 mmol) in
anhydrous DMF (5 ml) was added a mixture of the step g diamine (0.3 g, 0.43 mmol, added in portions
over 30 minutes) and DIPEA (0.28 ml, 2 mmol) in anhydrous DMF (20 ml). The reaction was stirred at
room temperature overnight, then concentrated and purified by flash chromatography eluting with 0%
to 10% methanol/dichloromethane. Yield 0.63 g, 86%. Ions found by LCMS: [(M+2H)/2].sup.+=844.4,
[(M-Boc+2H)/2].sup.+=794.4, [(M-2Boc+2H)/2].sup.+=744.4.

(1275) Step i.

(1276) ##STR00778##

(1277) Product from the previous step (600 mg, 0.35 mmol) was dissolved into 5 ml MeOH and 5 m1
THF, then treated with a solution of lithium hydroxide (48 mg, 2 mmol) dissolved in 2 ml water. The
reaction was stirred for 10 min at room temperature at which time HPLC showed the reaction was
complete. The pH of the reaction solution was adjusted to a value of 5 to 6 by using Amberlite IRN-77
ion exchange resin, then filtered to remove the resin. The crude product was evaporated to dryness by
rotary evaporation and used in the next step with purification. Ion(s) found by LCMS:
[(M+2H)/2].sup.+=829.9, [(M-Boc+2H)/2].sup.+=779.4, [(M-2Boc+2H)/2].sup.+=729.4.

(1278) Step j.

(1279) ##STR00779##

(1280) The product from step-i was dissolved into 5 ml dichloromethane and 5 ml TFA, and stirred at
room temperature. The progress of the reaction was monitored by LCMS. After the completion of the
reaction (6 h), the solution was stripped to dryness and then dissolved in 4 ml water and 4 ml
methanol. The resulting solution was stirred for another 2 hour at room temperature at which LCMS
show complete deprotection of the acetonide protecting groups. This mixture was concentrated and
purified by reverse phase liquid chromatography (RPLC) using an Isco CombiFlash liquid
chromatograph eluted with 5% to 40% acetonitrile/water with 0.1% TFA as the modifier. Yield 380 mg,
71.0%. Ion(s) found by LCMS: [(M+2H)/2].sup.+=589.8, [(M+3H)/3].sup.+=392.5.

Example 144. Synthesis of Int-82

(1281) ##STR00780##

(1282) The title compound was prepared analogously to Example 143, Int-81, where the N-Boc-N-Me-
glycinol was substituted with N-Boc-N-ethy-glycinol in the step a. Ion(s) found by LCMS:
[M/2]+1=603.8, [M/3]+1=402.9.
Example 145. Synthesis of Int-83

(1283) ##STR00781## ##STR00782##

(1284) Step a.

(1285) ##STR00783##

(1286) The CBZ-protected-amino-peg1-bromide (7.6 g, 25.2 mmol), benzylamine (1.1 g, 10.1 mmol),
and potassium carbonate (2.8 g, 2.8 mmol) were stirred in DMF (10 mL) at 60° C. for 12 hours. The
solution was concentrated on the rotary evaporator and purified by silica gel chromatography (0-10%
methanol in DCM, 30 minute gradient) to afford the product as a clear viscous oil. Yield 3.2 grams,
57%. Ion found by LC/MS [M+H]+=550.2.

(1287) Step b.

(1288) ##STR00784##

(1289) The product of step a (1.9 g, 3.5 mmol) was dissolved in THE (20 mL) and cooled to 0° C. by
way of an ice/water bath under an atmosphere of nitrogen. LAH (6.9 ml, 13.8 mmol, 2M in THF) was
added dropwise by way of syringe over a period of 10 minutes. The mixture was stirred at reflux for 2
hours then cooled to 0° C. by way of an ice/water bath. 1 mL of water was added, dropwise followed
by the dropwise addition of 1 mL of aqueous (15% by weight) NaOH solution. 3 mL of water was
added and the mixture was stirred for 15 minutes at which time 2 g of magnesium sulfate was added.
The mixture was stirred for 10 minutes then filtered through celite, washed with 2 additional 10 ml
portions of THE and the combined filtrates were concentrated by the rotary evaporator. The residue
was taken up in acetonitrile (20 mL), triethylamine (1.4 g, 13.8 mmol) and boc anhydride (3.0 g, 13.8
mmol) were added. The mixture was stirred for 45 minutes, concentrated and purified by reversed
phase HPLC (5-95% acetonitrile/di water, 0.1% TFA modifier, 30 minute gradient). Yield 1.4 g, 79%.
Ion found by LC/MS [M+H]+=510.2.

(1290) Step c.

(1291) ##STR00785##

(1292) The product from step b. (1 g, 1.9 mmol) of this example was stirred in methanol (25 mL) in the
presence of palladium hydroxide (200 mg) under an atmosphere of hydrogen for 2 hours. The mixture
was filtered through celite and concentrated to afford the product as a clear oil which was used with no
further purification. Yield 0.73 g, 89%. Ion found by LC/MS [M+H]+=420.4.

(1293) Step d.

(1294) ##STR00786##

(1295) The product from step c (0.73 g, 1.7 mmol), propargyl-peg4-tosylate (0.91 g, 2.4 mmol), and
diisopropylethylamine (0.76 g, 5.9 mmol) were stirred in DMF (5 mL) at 85° C. for 4 hours. The mixture
was concentrated on the rotary evaporator, then purified by reversed phase HPLC (5-95%
acetonitrile/DI water, 0.1% TFA modifier, 30 minute gradient) to afford the product as a clear viscous
oil. Yield 0.89 g, 82%. Ion fond by LC/MS [M+H]+=634.4.

(1296) Step e.

(1297) ##STR00787##

(1298) The product from step d. (0.89 g, 1.4 mml) was stirred in 4N HCl (in dioxane) for 45 minutes at
ambient temperature. The mixture was concentrated on the rotary evaporator and azeotroped (3×)
with benzene. The resulting residue was taken up in DI water (15 mL) frozen and lyophilized to afford
the product as a clear oil, bis-HCl salt. Yield 0.65 g, 91%. Ion found by LC/MS [M+H}+=434.4.
(1299) Step f.

(1300) ##STR00788##

(1301) The remaining four steps in the synthesis of this compound were analogous to those used in
the synthesis of Example 143, Int-81. Ion(s) found by LCMS: (M+2H)/2=575.8

Example 146a. Alternate Synthesis I of Int-83

(1302) ##STR00789## ##STR00790##

(1303) The product described in Example 145, Int-83, can be prepared alternatively using the above
reaction scheme by a person skilled in the art using methods described in this patent.

Example 146b. Alternate Synthesis II of Int-83

(1304) The product described in Example 145, Int-83, can be prepared alternatively using the reaction
scheme, below, by a person skilled in the art using methods described in this patent.

(1305) ##STR00791## ##STR00792##

(1306) To a well-stirred solution of N-Boc-N-Me-glycinol (3.5 g, 20 mmol) in DMSO (20 mL) cooled
with an ice-water bath was added allyl bromide (3.6 g, 30.0 mmol), followed by finely ground KOH
powder (3.5 g, 30.0 mmol) over 15 minutes. The resulting solution was stirred overnight at room
temperature. The resulting mixture was partitioned between 5% aq. HOAc (50 mL) and ethyl acetate
(200 ml). The organic layer was separated, washed with brine, dried with sodium sulfate, filtered, and
concentrated, then purified by flash chromatography eluting with 10% to 80% ethyl acetate/hexane.
Yield of product 4.1 g, 95%. Ion found by LCMS: M+H=216.3.

(1307) Synthesis of b.

(1308) ##STR00793##

(1309) Ozone was bubbled through a solution of a (8.0 g, 37 mmol) in MeOH (50 mL) and DCM (50
ml) at −78° C. until the appearance of a light blue color. Unreacted ozone was removed by bubbling
with oxygen for 10 minutes before the addition of NaBH.sub.4 (1.6 g, 40 mmol) in small portions over
10 minutes. After all NaBH.sub.4 was added, the mixture was gradually warmed to room temperature.
The resulting solution was partitioned between ethyl acetate (100 ml) and brine (50 ml). The organic
layer was separated, washed with brine, dried with sodium sulfate, filtered, concentrated to an oil, and
then purified by flash chromatography eluting with 10% to 80% ethyl acetate/dichloromethane. Yield of
product 5.0 g, 62%. Ion(s) found by LCMS: M+H=220.2.

(1310) Synthesis of c.

(1311) ##STR00794##

(1312) To a solution of b (4.4 g, 20 mmol) and CBr.sub.4 (10.0 g, 30.0 mmol) in DCM (50 mL) cooled
in an ice bath, was added PPh.sub.3 (8.0 g, 30 mmol) slowly over 15 minutes (exothermic). During the
course of the addition, the internal temperature was kept below 30° C. After addition of PPh.sub.3 the
reaction was stirred overnight at room temperature. The crude reaction was concentrated to an oil,
then purified by normal phase chromatography, eluting with 10% ethyl acetate/hexanes to 80% ethyl
acetate/hexanes. Fractions containing oil droplets on the inside of the collection tubes were checked
by LCMS, then combined and concentrated to a colorless oil. 4.0 g, 70.5%. Ion(s) found by LCMS:
M+H=282.1.

(1313) Synthesis of d.

(1314) ##STR00795##
(1315) A solution of c (4.4 g, 15.5 mmol), propargyl-PEG4-amine (1.5 g, 6.4 mmol), and DIPEA (3.3 g,
25.8 mmol) in DMF (20 mL) were heated in an oil bath at 75° C. for 18 h. The mixture was filtered,
concentrated, and purified by RPLC (5% ACN/water to 100% ACN). Yield 3.85 g, 92%. LC/MS:
[M+H]=634.2.

(1316) Synthesis of e.

(1317) ##STR00796##

(1318) The product d (3.85, 6.1 mmol) was treated with HCl (4 N in dioxane, 15 mL) for 2 hours at
room temperature. The solvent was removed by rotary evaporation, and the remaining oil was
dissolved in di water (20 mL) frozen, and lyophilized to afford the product as a light yellow oil. Yield
was quantitative. Ion found by LCMS: [M+H].sup.+=434.2.

(1319) Synthesis of f.

(1320) ##STR00797##

(1321) To a solution of f (0.68 g, 1.34 mmol) and DIPEA (0.87 g, 6.7 mmol) dissolved in anhydrous
DMF (5 ml) was added f (2.1 g, 2.8 mmol) in portions over 1 hour. The reaction was stirred at room
temperature overnight, then concentrated and purified by flash chromatography, eluting with 0% to
10% methanol/dichloromethane. Yield 1.45 g, 67%. Ion found by LCMS: [(M+2H)/2].sup.+=829.8.

(1322) Synthesis of g.

(1323) ##STR00798##

(1324) The product f (1.45 g, 0.87 mmol) was dissolved into 3 ml MeOH then treated with a solution of
lithium hydroxide (90 mg, 3.8 mmol) dissolved in deionized water (6 mL). The reaction was stirred for
15 minutes at room temperature at which time LCMS showed the reaction was complete. The pH of
the reaction solution was adjusted to a value of 5 to 6 by using Amberlite IRN-77 ion exchange resin,
then filtered to remove the resin. The filtrate was concentrated to dryness by rotary evaporation and
used in the next step without further purification. Ion found by LCMS: [(M+2H)/2].sup.+=815.9.

(1325) The hydrolysis product was dissolved in dichloromethane (5 mL) and TFA (10 mL), and stirred
at room temperature. The progress of the reaction was monitored by LCMS. After complete Boc-
removal (˜4 hours), the solution was concentrated to dryness with a rotary evaporator, and then
dissolved in 8 m1 water. The resulting solution was stirred for another 2 hour at room temperature at
which time LCMS showed complete removal of the acetonide protecting groups. This mixture was
concentrated and purified by reverse phase liquid chromatography (RPLC) using an Isco
COMBIFLASH® liquid chromatograph eluted with 5% to 40% acetonitrile/water with 0.1% TFA as the
modifier. Yield for three steps 780 mg, 65.0%. Ion(s) found by LCMS: [(M+2H)/2].sup.+=575.8,
[(M+3H)/3].sup.+=384.8.

Example 147. Serial Passage Experiments for Selection of Resistant Influenza Viruses

(1326) To evaluate the potential for development of drug resistant mutant viral strains under selective
pressure with viral inhibitors, serial passage studies were conducted with Conjugates 6 and 33, versus
oseltamivir and baloxavir comparators. Serial passage studies were conducted using either A549 or
MDCK cells. Passages were conducted as follows: 150,000 A549 or MDCK cells were seeded per well
(12-well) in 500 μl DMEM 10% FBS, 1% PS, 1% NaPyr and 1% HEPES and incubated for
approximately 24 hours. Once cells reached approximately 80% confluency, they were washed once
with PBS and incubated for 2 hours in the presence of compounds or PBS alone under normal culture
conditions. Test article concentrations were optimized as required for maximum virus inhibition, while
maintaining enough virus production for subsequent passages. Concentrations of test articles used in
the serial passage experiments are shown in FIGS. 67 and 68. Cells were then infected at an MOI of
0.01 or 0.05 (MDCK and A549 cells, respectively; 150 μl diluted in infection buffer) for 1 hour at room
temperature in a buffer containing PBS, Bovine Albumin 35% and Ca.sup.2+/Mg.sup.2+, followed by
removal of inoculum and washing of cells once with DMEM 1% PS, 1% NaPyr and 1% HEPES (No
FBS). Cells were then Incubated for 24 hours in the presence of test articles diluted in DMEM 1% PS,
1% NaPyr, 1% HEPES and 1 μg/ml TPCK-treated trypsin. After incubation, viral supernatants were
collected, and cells and debris were removed by centrifugation (5 min, 4° C., 1,400×g). S upernatants
were then used to:

(1327) i Quantify viral titer by plaque assay

(1328) ii Conduct a hemagglutination assay to determine if the virus escaped compound inhibition

(1329) iii Re-infect freshly seeded A549 or MDCK cells in presence of compounds.

(1330) The process was repeated for 10 passages, or until resistance was observed for the oseltamivir
and baloxavir controls. Once increased titers in the presence of drugs is detected for two consecutive
passages, the viruses may be plaque purified. Following plaque purification, all 8 genome segments
may be sequenced and compared to PBS treated control virus to detect escape mutations.
Summaries of two different serial passage experiments are shown in FIGS. 67 and 68. In the
experiment summarized in FIG. 67, Conjugate 6 was compared to oseltamivir and baloxavir using
A549 cells infected with A/California/04/09/H1N1 pdm. Conjugate 6, baloxavir and oseltamivir were
used at 0.5 nM, 0.5 nM and 200 nM, respectively. No increases in viral titer were observed for
Conjugate 6 through the course of 11 passages (suggesting no emergence of resistant mutants), while
baloxavir and oseltamivir titers increased to levels similar to those observed in the PBS control after
passages 5 and 11, respectively. In the experiment summarized in FIG. 68, Conjugates 6 and 33 were
compared to oseltamivir and baloxavir using MDCK cells infected with A/WSN/1933 H1N1. Conjugate
6, Conjugate 33, baloxavir and oseltamivir were used at 4 nM, 2 nM, 4 nM, and 50 nM, respectively.
No increases in viral titer were observed for Conjugates 6 or 33 through the course of 10 passages
(suggesting no emergence of resistant mutants), while baloxavir and oseltamivir titers increased to
levels similar to those observed in the PBS control after passages 5 and 10, respectively.

Example 148. Synthesis of Conjugate 6a

(1331) The title conjugate is prepared analogously to Conjugate 6 (Example 20) using Int-7a (Example
100). Maldi TOF analysis of the purified final product gave an average mass of 62063. Da (DAR=2.7).
Yield 175.4 mg, 50% yield.

Example 149. Synthesis of Conjugate 6b

(1332) The title conjugate is prepared analogously to Conjugate 6 (Example 20) using Int-7b (Example
101). Maldi TOF analysis of the purified final product gave an average mass of 62063. Da (DAR=2.8).
Yield 175.4 mg, 50% yield.

Example 150. Synthesis of Conjugate 6c

(1333) The title conjugate is prepared analogously to Conjugate 6 (Example 20) using Int-7c (Example
102). Maldi TOF analysis of the purified final product gave an average mass of 62782. Da (DAR=3.2).
Yield 175.4 mg, 50% yield.

Example 151. Synthesis of Conjugate 44

(1334) This conjugate was prepared analogously to Example 80 (Conjugate 20) by PEG4-azido-Fc
(SEQ ID NO: 73, prepared as in Example 124) and Int-22 (Example 79). Maldi TOF analysis of the
purified final product gave an average mass of 62,882 Da (DAR=5.6).

(1335) The nucleic acid construct encoding the Fc for Conjugate 44 included a nucleic acid encoding
the amino acid sequence of SEQ ID NO: 64, which includes a C-terminal lysine residue. Upon
expression, the C-terminal lysine of the Fc of Conjugate 44 is proteolytically cleaved, resulting in an Fc
having the sequence of SEQ ID NO: 73. The presence or absence of a C-terminal lysine does not alter
the properties of the Fc or the corresponding conjugate.
Example 152. Antibody-Dependent Cellular Cytotoxicity Assay

(1336) Conjugate 6 and Conjugate 33 were tested for antibody-dependent cellular cytotoxicity
(ADCC). A monolayer of MDCK cells was infected with influenza A or influenza B strains at an MOI of
0.001-10, and incubated for 18-24 h at 37° C., in 5% CO.sub.2. ADCC was determined with
commercial report cell line (PROMEGA) according to manufacturer's instructions. Briefly, test articles
were added at concentrations ranging from 1 to 10,000 nM to appropriate wells and incubated for 15
m at 37° C., in 5% CO.sub.2. ADCC was quantified by reading luminescence. Conjugate 6 was tested
against Influenza A/PR/8/1934 (H1N1), showing an MOI-dependent increase in ADCC (FIG. 69), and
a dose dependent increase in ADCC at an MOI of 1 (FIG. 70). Conjugate 33 was tested against
influenza A/PR/8/1934 (H1N1), influenza A/CA/07/2009 (H1N1), and influenza A/HK/1/1968 (H3N2)
FIGS. 71A-71C, and influenza B/Malaysia/2506/2004 (Victoria, FIG. 72), showing an MOI-dependent
increase in ADCC by conjugate 33. Conjugate 33 also showed a dose-dependent increase in ADCC
against influenza A/PR/8/1934 (H1N1) at an MOI of 1 (FIG. 73A) and an MOI of 10 (FIG. 73B). The
monoclonal antibody, Gedivumab (Genentech) was used as a positive control to compare receptor
binding of a full length antibody to that of an Fc conjugate. Gedivumab also showed an MOI-
dependent increase in ADCC when tested against influenza A/PR/8/1934 (H1N1, FIG. 74), and a
dose-dependent increase in ADCC against influenza A/PR/8/1934 (H1N1) at an MOI of 1 (FIG. 75A)
and an MOI of 10 (FIG. 75B).

Example 153. Antibody-Dependent Cellular Phagocytosis Assay

(1337) Conjugate 6 and Conjugate 33 were tested for cellular phagocytosis. A monolayer of MDCK
cells was infected with influenza A or influenza B strains at an MOI of 0.001-10, and incubated for 18-
24 h at 37° C., in 5% CO.sub.2. ADCP was determined with commercial report cell line (PROMEGA)
according to manufacturer's instructions. Briefly, test articles were added at concentrations ranging
from 1 to 10,000 nM to appropriate wells and incubated for 15 m at 37° C., in 5% CO.sub.2. ADCP
was quantified by reading luminescence. Conjugate 6 was tested against Influenza A/PR/8/1934
(H1N1), showing an MOI-dependent increase in ADCP (FIG. 76), and a dose dependent increase in
ADCP at an MOI of 1 (FIG. 77A) and an MOI of 10 (FIG. 77B). Conjugate 33 was tested against
influenza A/PR/8/1934 (H1N1), influenza A/CA/07/2009 (H1N1), and influenza A/HK/1/1968 (H3N2)
(FIGS. 78A-78C, respectively), and influenza B/Malaysia/2506/2004 (Victoria, FIG. 79), showing an
MOI-dependent increase in ADCP by conjugate 33. Conjugate 33 also showed a Dose-dependent
increase in ADCP against influenza A/PR/8/1934 (H1N1) at an MOI of 1 (FIG. 80A) and MOI of 10
(FIG. 80B). Gedivumab (Genentech) was used as a positive control to compare receptor binding of a
full length antibody to that of an Fc conjugate. Gedivumab also showed an MOI-dependent increase in
ADCP when tested against influenza A/PR/8/1934 (H1N1, FIG. 81), and a dose-dependent increase in
ADCP against influenza A/PR/8/1934 (H1N1) at an MOI of 1 (FIG. 82A) and an MOI of 10 (FIG. 82B).

Example 154. Neuraminidase Inhibition with Live Influenza A Virus or Lysates of Neuraminidase
Resistant Mutants

(1338) Neuraminidase inhibition (NAI). Test articles were incubated with neuraminidase (Sino
Biological) or with live viruses for 20 min at 37° C., 5% CO.sub.2. 2′-(4-Methylumbelliferyl)-α-D-N
acetylneuraminic acid substrate was added to appropriate wells and incubated for 1 h at 37° C., 5%
CO.sub.2. NAI was determined by reading fluorescence at 355 nm excitation/460 nm emission.
Neuraminidase inhibition was determined for neuraminidase resistant mutants and for live influenza
virus (Table 66, Table 67, Table 68, and Table 69).

(1339) TABLE-US-00071 TABLE 66 Neuraminidase inhibition with lysates of neuraminidase resistant

mutants H1N1 H1N1 H3N2 H3N2 H3N2 H5N1 H7N9 WT H275Y WT E119V R292K WT WT IC.sub.50
[nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM]
Oseltamivir N/A N/A N/A N/A 3.80 743.90 2.18 455.70 8015.00 15.44 2.95 Zanamivir N/A N/A N/A N/A
2.15 1.93 4.27 112.60 116.50 2.05 11.11 Int-7a N/A N/A 15 N/A 22.58 9.42 21.11 679.40 993.60 17.06
49.04 atoms Int-7b N/A N/A 15 N/A 34.82 13.84 32.07 1079.00 1584.00 29.56 87.54 atoms Int-7b N/A
N/A 15 N/A 2002.00 872.40 2914.00 >10,000 >10,000 2706.00 3325.00 atoms Conjugate 6a Int-7a
2.7 15 SEQ ID 0.37 0.43 4.14 4.66 4.85 0.59 1.18 atoms NO: 18 Conjugate 6b Int-7b 2.8 15 SEQ ID
0.24 0.30 2.05 3.67 7.41 0.54 0.98 atoms NO: 18 Conjugate 6c Int-7c 3.2 15 SEQ ID 0.98 0.66 35.60
4423.00 1451.00 1.25 10.41 atoms NO: 18 Conjugate 33 Int-7a 4.5 15 SEQ ID 1.39 2.61 13.09 13.21
17.69 3.51 5.66 atoms NO: 73

(1340) TABLE-US-00072 TABLE 67 Neuraminidase inhibition with live influenza A virus A/PR/8/1934
A/Ca/7/2009 A/HK/1/1968 B/Brisbane B/Florida/4/2006 (H1N1) (H1N1) (H3N2) 60/2008 (Victoria)
(Yamagata) IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] Oseltamivir
N/A N/A N/A N/A 3.76 0.88 0.16 36.61 24.65 Zanamivir N/A N/A N/A N/A 0.35 0.28 0.43 6.74 3.60 Int-
7a N/A N/A 15 atoms N/A 0.45 2.10 3.62 13.54 25.54 Int-7b N/A N/A 15 atoms N/A 0.98 3.78 5.57
16.03 17.26 Int-7c N/A N/A 15 atoms N/A 150.90 399.40 467.50 1942.00 5512.00 Conjugate 6a Int-7a
2.7 15 atoms SEQ ID 0.22 0.56 0.41 2.24 4.65 NO: 18 Conjugate 6b Int-7b 2.8 15 atoms SEQ ID 0.14
0.25 0.30 1.39 2.30 NO: 18 Conjugate 6c Int-7c 3.2 15 atoms SEQ ID 0.29 0.35 0.60 99.08 44.37 NO:
18 Conjugate 33 Int-7a 4.5 15 atoms SEQ ID 1.12 2.05 1.71 7.75 16.47 NO: 73

(1341) TABLE-US-00073 TABLE 68 Neuraminidase inhibition against H3N2 WT, E119V mutant or live
influenza A/Ca/07/2009 H3N2 WT H3N2 E119V A/Ca/07/2009 pdm Molecule TM DAR Fc Central
linker IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] Oseltamivir N/A N/A N/A N/A 2.873 150.1 0.7411
Zanamivir N/A N/A N/A N/A 6.302 35.68 0.8452 Int-3 N/A N/A N/A 27 atom 31.25 108.3 1.172 Int-4a
N/A N/A N/A N/A 32.94 147.1 7.102 Int-7a N/A N/A N/A 15 atom 33.62 185.9 3.825 Int-7c N/A N/A N/A
15 atom 4428 13972 470.5 Int-22 N/A N/A N/A N/A 13.25 23.23 1.295 Int-73 N/A N/A N/A 14 atom
33.63 174.3 4.605 Int-74 N/A N/A N/A 16 atom 32.89 157.9 1.551 Int-75 N/A N/A N/A 17 atom 34.51
164 1.759 Int-76 N/A N/A N/A 18 atom 32.95 166.4 2.07 Int-80 N/A N/A N/A N/A 80.97 628.9 21.89
Int-91 N/A N/A N/A N/A 95.7 529 46 Conjugate 6 Int-7 3.3 SEQ ID NO: 18 15 atom 206.5 3610 1.366
Conjugate 33 Int-7a 6.1 SEQ ID NO: 73 15 atom 3.051 2.913 0.2656 Conjugate 33 Int-7a 6.8 SEQ ID
NO: 73 15 atom 6.197 86.06 0.1007 Conjugate 43 Int-80 7.0 SEQ ID NO: 73 N/A 11.28 11.64 1.231
Conjugate 44 Int-22 5.6 SEQ ID NO: 73 N/A 6.815 5.816 0.7677

(1342) TABLE-US-00074 TABLE 69 Neuraminidase Inhibition assay [nM] with NA lysate Response
IAV H1N1 NA Lysate Test article TM DAR Central linker 0.1 U/mL 1 U/mL 10 U/mL Oseltamivir N/A
N/A N/A 2.162 19.45 729.9 Zanamivir N/A N/A N/A 0.6719 7.765 76.53 Int-7 N/A N/A 15 atoms 1958
12987 >10,000 Int-23 N/A N/A 14 atoms 0.2684 2.625 27.78 Conjugate 6 Int-7 3.3 15 atoms 16.28
13214 >10,000 Conjugate 21 Int-7 2.2 14 atoms 0.5004 2.65 9.101

Example 155. Cytopathic Effect Assay

(1343) To measure the ability of Conjugate 6 and Conjugate 33 to protect mammalian cells from
infection and destruction by influenza virus, Cytopathic effect (CPE) based microneutralization assays
were conducted as discussed in Example 25, with minor variations. Briefly, a monolayer of MOCK
cells was infected with influenza A or B strains at appropriate MOI varying between 0.001-1. Test
articles were added at concentrations ranging between 0.1-10,000 nM and incubated for 3 days for
influenza A or 5 days for influenza B at 37° C., 5% CO2. CPE was determined by crystal violet staining
by reading absorbance at 595 nm. The results are shown in Table 70, Table 71, Table 72, and Table
73, below.

(1344) TABLE-US-00075 TABLE 70 CPE against influenza A/PR/8/1934 (H1N1) Central MOI 0.01
MOI 0.1 MOI 1 Molecule TM DAR Fc linker EC.sub.50 [nM] Oseltamivir N/A N/A N/A N/A 3005
>10,000 >10,000 Baloxavir N/A N/A N/A N/A 0.4466 5.126 7.993 Conjugate 6 Int-7 3.3 SEQ ID NO: 18
15 atom 0.3561 35.52 1802 Conjugate 33 Int-7a 4.5 SEQ ID NO: 73 15 atom 0.2669 2.458 77.97

(1345) TABLE-US-00076 TABLE 71 CPE against influenza A/Ca/07/2009 (H1N1)pdm Central MOI
0.01 MOI 0.1 MOI 1 Molecule TM DAR Fc linker EC.sub.50 [nM] Oseltamivir N/A N/A N/A N/A 288.2
>10,000 >10,000 Baloxavir N/A N/A N/A N/A 1.439 3.186 11.3 Conjugate 6 Int-7 3.3 SEQ ID NO: 18
15 atom 18.13 513.4 >1,000 Conjugate 33 Int-7a 4.5 SEQ ID NO: 73 15 atom 1.845 39.66 >1,000

(1346) TABLE-US-00077 TABLE 72 CPE against influenza A/WSN/1933 (t = −1 h) Central MOI 0.001
MOI 0.01 MOI 0.1 MOI 1 MOI 10 Molecule TM DAR Fc linker EC.sub.50 [nM] Oseltamivir N/A N/A N/A
N/A 3911 >10,000 >10,000 >10,000 >10,000 Baloxavir N/A N/A N/A N/A 1.261 3.64 8.306 8.266 17.1
Int-7a N/A N/A N/A 15 atom 8.704 13.22 2758 >10,000 >10,000 Conjugate 33 Int-7a 4.5 SEQ ID NO:
73 15 atom 3.097 2.754 8.147 868.1 >10,000

(1347) TABLE-US-00078 TABLE 73 CPE against influenza B (t = −1 h) Central B/Brisbane B/Florida

B/Malaysia Molecule TM DAR Fc linker EC.sub.50 [nM] Oseltamivir N/A N/A N/A N/A 969.1 >10,000
5694 Baloxavir N/A N/A N/A N/A 240.4 30.57 2294 Int-7a N/A N/A N/A 15 atom <1.93 <1.93 0.8237
Conjugate 33 Int-7a 4.5 SEQ ID NO: 73 15 atom 3.699 0.1946 10.48

Example 156. Synthesis of Conjugate 45

(1348) Conjugate 45 was prepared analogously to conjugate 33 (Example 129) using PEG4-azido-Fc
(either SEQ ID NO: 72 (Conjugate 45a) or SEQ ID NO: 73 (Conjugate 45b), prepared as in Example
124) and Int-83 (Example 145). Maldi TOE analysis of a purified preparation of Conjugate 45b gave an
average mass of 62,927 Da (OAR=4.2). The preparation of Conjugate 45a having varying DARs is
also described herein (Example 199). Where the term Conjugate 45 is used, it should not be
considered to be limited to any particular OAR. The resulting conjugate is depicted in FIG. 102.

(1349) The term Conjugate 45, as used herein, is meant to encompass both Conjugate 45a and
Conjugate 45b. Applicant notes that SEQ ID NO: 72 (Conjugate 45a) and SEQ ID NO: 73 (Conjugate
45b) differ only in the Fc allotype, G1m(f) and G1 m(fa), respectively. The differing allotypes are
expected to behave the same with respect the properties described herein.

(1350) The nucleic acid construct encoding the Fc for Conjugate 45a included a nucleic acid encoding
the amino acid sequence of SEQ ID NO: 63, which includes a C-terminal lysine residue. Upon
expression, the C-terminal lysine of the Fc of Conjugate 45a is proteolytically cleaved, resulting in an
Fc having the sequence of SEQ ID NO: 72. Likewise, the nucleic acid construct encoding the Fc for
Conjugate 45b included a nucleic acid encoding the amino acid sequence of SEQ ID NO: 64, whereas
the resulting Fc has the sequence of SEQ ID NO: 73. The presence or absence of a C-terminal lysine
does not alter the properties of the Fc or the corresponding conjugate.

Example 157. Efficacy of Conjugate 45b Against Influenza A (H1N1) in a Lethal Severe Combined
Immunodeficiency (SCID) Mouse Model

(1351) Test articles were evaluated against a lethal Influenza A influenza infection in female BALB/c
scid mice (Jackson Laboratories, 6-8 weeks old). The challenge virus (A/Puerto Rico/8/1934) is a
mouse-adapted isolate capable of causing lethal infections in mice. The experiment comprised 10
groups of 5 mice each. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal
inoculation in a volume of 30 μl (approx. 1 E3 virus per mouse), after being anesthetized with a
mixture of ketamine/xylazine (150 and 10 mg/kg respectively).

(1352) Groups received a single SC treatment of vehicle (PBS), hIgG1 Fc control, or conjugate 45b
two hours post viral challenge. A separate arm of the study consisted of 3 groups of mice treated with
baloxavir marboxil (DC Chemicals, Shanghai, China) orally, twice daily, for 1 day; also starting 2 hours
post viral challenge. The study design is outlined in Table 74. Mice were monitored for 4 weeks and
animals exceeding 20% body weight loss, or were found moribund, were scored as a mortality.

(1353) TABLE-US-00079 TABLE 74 Study design of SCID study Dose Dose N Influenza Test Route/
(mg/ volume (balb/ Group strain Article Schedule kg) (ml/kg) c) 1 A/PR/8/34 PBS SC, T + 2 hrs — 10 5
2 2E2 hIgG1 SC, T + 2 hrs 3 10 5 3 PFU/mouse Baloxavir PO, bid × 1 10 10 5 day 4 Baloxavir PO, bid
× 1 3 10 5 day 5 Conjugate SC, T + 2 hrs 10 10 5 6 45b SC, T + 2 hrs 3 10 5 7 SC, T + 2 hrs 1 10 5 8
SC, T + 2 hrs 0.3 10 5 9 SC, T + 2 hrs 0.1 10 5 10 Uninfected BW control 5

(1354) At study end (Day 28) mice receiving conjugate 45b were fully protected at all dose
concentrations between 10 and 0.3 mg/kg (Table 75). Conjugate 45b only failed to protect against
lethal viral challenge at the lowest tested concentration of 0.1 mg/kg. As expected, groups receiving
vehicle or hIgG1 Fc were not protected. Mice treated with baloxavir were also protected, but at the
significantly higher cumulative doses of 20 mg/kg (80% survival), at a total dose of 6 mg/kg only 60%
of mice survived to Day 28.
(1355) TABLE-US-00080 TABLE 75 Percent survival hIgG1 Baloxavir Baloxavir Conjugate 45b
(mg/kg) Day Vehicle Fc (20) (6) 10 3 1 0.3 0.1 Control 0 100 100 100 100 100 100 100 100 100 100 1
100 100 100 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 100 100 3 100 100
100 100 100 100 100 100 100 100 4 100 100 100 100 100 100 100 100 100 100 5 100 100 100 100
100 100 100 100 100 100 6 80 80 100 100 100 100 100 100 60 100 7 20 20 100 100 100 100 100 100
100 100 8 0 0 100 100 100 100 100 100 100 100 9 0 0 100 100 100 100 100 100 100 100 10 0 0 100
100 100 100 100 100 100 100 11 0 0 100 100 100 100 100 100 100 100 12 0 0 100 100 100 100 100
100 100 100 13 0 0 100 100 100 100 100 100 100 100 14 0 0 100 80 100 100 100 100 100 100 15 0 0
100 80 100 100 100 100 100 100 16 0 0 100 60 100 100 100 100 100 100 17 0 0 100 60 100 100 100
100 100 100 18 0 0 100 60 100 100 100 100 100 100 19 0 0 100 60 100 100 100 100 100 100 20 0 0
100 60 100 100 100 100 100 100 21 0 0 100 60 100 100 100 100 100 100 22 0 0 100 60 100 100 100
100 100 100 23 0 0 100 60 100 100 100 100 80 100 24 0 0 80 60 100 100 100 100 40 100 25 0 0 80
60 100 100 100 100 20 100 26 0 0 80 60 100 100 100 100 20 100 27 0 0 80 60 100 100 100 100 0
100 28 0 0 80 60 100 100 100 100 0 100 Control = Uninfected

(1356) The potency of conjugate 45b in this model of severe immunodeficiency was also evident
based on body weights (Table 76). The lowest concentration of conjugate providing full protection
based on a mortality readout was 0.3 mg/kg. At this dose level, the greatest average weight loss for
the group was transient, and resulted in less than a 3% reduction (occurring on Day 4). Furthermore
the difference in body weight for all fully protective groups was negligible compared to uninfected
mice.

(1357) TABLE-US-00081 TABLE 76 Percent Body Weight hIgG1 Baloxavir Baloxavir Conjugate 45b
(mg/kg) Day Vehicle Fc (20) (6) 10 3 1 0.3 0.1 Control 0 100 100 100 100 100 100 100 100 100 100 1
99.2 97.6 93.8 96.9 95.8 97.4 98 99 98 101.8 2 101.3 98.7 98.3 98.1 96.7 99.5 96.3 98.3 102.6 100.8
3 95.4 94.9 100.3 98.3 100.6 97.9 100.9 99.6 101 101 4 92.3 90.1 103.3 100.8 100.8 101.3 102.4 97.5
99.2 104.6 5 86.5 84.9 103.2 100.5 102.2 102 102.7 99.8 99.2 103.3 6 78.8 78 100.6 99.7 101.5 101.5
101.4 102 99.2 103.3 7 103.6 100 103.5 101.8 100.5 100.7 101.6 103.7 8 104.2 100.2 103.7 103.1
100.4 103.3 102.7 101.7 9 101.4 97.3 103.6 101.5 100.5 103.1 102.9 102.1 10 100.9 96 102.8 100.8
101.8 104.2 102.7 101.7 11 102.3 98.3 104.2 103.7 102.4 104.5 103.3 103.8 12 103 95.3 105.4 105.9
103 105.4 103.8 103.9 13 103.5 93.4 103.6 105.1 104.1 105.2 104 103.9 14 102.3 91.8 103.3 103.6
103.2 104.8 102.6 103.1 15 102.2 104.2 105.8 102.5 104.8 104.7 103.1 16 102.3 102 101.9 104 104.6
100.1 102.1 17 103.3 102 103.3 104.4 103.8 101.4 102.8 18 103.3 106.4 105.3 105.8 105.1 100.4
104.1 19 105.4 106.6 103.6 106.5 105.6 96.7 103.5 20 105.9 105.1 102.1 105.5 106.3 92.9 103.8 21
106.6 108.6 102.8 106.5 107.6 90.6 106 22 103.8 109.1 103.1 105.7 107.3 86.7 102.1 23 102.1 106.3
101.8 105.5 105.3 99.3 24 100 107.9 104 106 104.8 103.7 25 108.6 105.3 104.9 106.1 104.8 26 110
107.3 107.9 105.4 105.2 27 109 106.8 106 102.1 104 28 108.2 106.2 106.7 101.2 104.4 Control =
Uninfected

(1358) Collectively these data demonstrate the potency of conjugate 45b by protecting lethally
challenged mice with single SC doses of conjugate as low as 0.3 mg/kg. This was accomplished in a
severe model of immunodeficiency with mice completely lacking T & B immune cells, which are
essential in clearing influenza infections. These data support the use of conjugate 45b to treat immune
deficient patient populations.

Example 158. Efficacy of Conjugate 45b Subcutaneously Dosed Against Influenza A/Puerto Rico/8/34
(HI NI) in a Lethal Mouse Model

(1359) Conjugate 45b was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/i1934) is a
mouse-adapted isolate capable of causing lethal infections in mice. The experiment comprised 7
groups of 5 mice. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal
inoculation in a volume of 30 μl, after being anesthetized with a mixture of ketamine/xylazine (150 and
10 mg/kg respectively). Mortality and body weights were recorded daily for 14 days and any animal
with a 20% loss of body weight was scored as a death.
(1360) Test groups received a single subcutaneous (SC) treatment of conjugate 45b (1, 0.3, 0.1, 0.03,
or 0.01 mg/kg), hIgG1 Fc control, or vehicle (PBS) 2 hours post viral challenge. The study design is
summarized in Table 77.

(1361) TABLE-US-00082 TABLE 77 Study design for Influenza A/PR/8/34 (H1N1) study Dose Dose
volume # of Group Test article Route/Schedule (mq/kq) (ml/kq) mice 1 Vehicle SC, T + 2 hrs. na 10 5 2
hIgG1 Fc SC, T + 2 hrs. 1 10 5 3 Conjugate 45b SC, T + 2 hrs. 1 10 5 4 SC, T + 2 hrs. 0.3 10 5 5 SC,
T + 2 hrs. 0.1 10 5 6 SC, T + 2 hrs. 0.03 10 5 7 SC, T + 2 hrs. 0.01 10 5

(1362) As expected, mice receiving vehicle or the hIgG1 Fc control succumbed to infection on Day 6
(Table 78). However, mice treated with conjugate 45b were fully protected at 1, 0.3, and 0.1 mg/kg
dose levels. Mortality with conjugate 45b was only seen at the lowest dose concentration of 0.03 and
0.01 mg/kg.

(1363) TABLE-US-00083 TABLE 78 Percent Survival hIgG1 Conjugate 45b (mg/kg) Day Vehicle Fc 1
0.3 0.1 0.03 0.01 0 100 100 100 100 100 100 100 1 100 100 100 100 100 100 100 2 100 100 100 100
100 100 100 3 100 100 100 100 100 100 100 4 100 100 100 100 100 100 100 5 60 40 100 100 100
100 100 6 0 0 100 100 100 100 60 7 0 0 100 100 100 80 0 8 0 0 100 100 100 0 0 9 0 0 100 100 100 0
0 10 0 0 100 100 100 0 0 11 0 0 100 100 100 0 0 12 0 0 100 100 100 0 0 13 0 0 100 100 100 0 0 14 0
0 100 100 100 0 0

(1364) The potency of conjugate 45b was further supported by daily body weight measurements. As
expected, mice treated with vehicle or hIgG1 Fc demonstrated a steady drop in body weight until it
exceeded 20%, at which time they were scored as a mortality (Table 78).

(1365) In contrast to control mice, those groups receiving conjugate 45b at 1, 0.3, and 0.1 mg/kg
maintained healthy body weights throughout the study and never demonstrated more than a transient
body weight drop of less than 7% (0.1 mk/kg dose group, Day 7; Table 79). By both survival and body
weight measurements conjugate 45b demonstrated robust protection from a lethal challenge of
Influenza A/Puerto Rico/8/1934 with a single SC dose as low as 0.1 mg/kg.

(1366) TABLE-US-00084 TABLE 79 Percent Body Weight hIgG1 Conjugate 45b (mg/kg) Day Vehicle
Fc 1 0.3 0.1 0.03 0.01 0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 1 97.0 98.0 97.7 96.7 98.2 98.8
96.2 2 98.6 99.9 100.8 101.9 101.0 100.5 100.4 3 90.7 92.4 98.8 98.9 96.7 93.3 92.0 4 82.1 83.4 97.7
97.5 95.1 87.5 84.7 5 76.2 76.9 98.4 96.3 94.7 87.6 79.3 6 102.1 102.4 99.7 84.1 76.8 7 101.8 103.1
93.8 78.8 8 100.1 102.3 98.3 9 103.4 103.7 104.3 10 103.9 104.6 102.2 11 101.8 102.5 101.7 12
100.9 101.0 102.3 13 106.0 104.8 105.6 14 106.6 104.0 105.0

Example 159. Efficacy of Conjugate 45b Subcutaneously Dosed Against Influenza

A/California/07/2009 (H1N1) Pdm in a Lethal Mouse Model

(1367) Conjugate 45b was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/California/07/2009 (H1N1) pdm)
is a pandemic isolate capable of causing lethal infections in mice. The experiment comprised 5 groups
of 5 mice. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal inoculation in a
volume of 30 μl, after being anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg
respectively). Mortality and body weights were recorded daily and any animal with a 20% loss of body
weight was scored as a death.

(1368) Test groups received a single subcutaneous (SC) treatment of conjugate 45b or vehicle (PBS)
2 hours post viral challenge. The study design and dose levels are summarized in Table 80.

(1369) TABLE-US-00085 TABLE 80 Study design for Influenza A/California/07/2009 (H1N1) pdm
study Dose Dose volume # of Group Test article Route/Schedule (mq/kq) (ml/kq) mice 1 Vehicle SC, T
+ 2 hrs. na 10 5 2 Conjugate 45b SC, T + 2 hrs. 3 10 5 3 Conjugate 45b SC, T + 2 hrs. 1 10 5 4
Conjugate 45b SC, T + 2 hrs. 0.3 10 5 5 Conjugate 45b SC, T + 2 hrs. 0.1 10 5
(1370) As expected, mice receiving vehicle succumbed to infection by Days 7 (Table 81). However,
mice treated with conjugate 45b were fully protected at concentrations as low as 0.3 mg/kg, and
partially so at 0.1 mg/kg (60% survival). Achieving full protection against a highly virulent pandemic
strain with a single dose of less than 1 mg/kg demonstrates the potency of conjugate 45b against
clinically relevant influenza A.

(1371) TABLE-US-00086 TABLE 81 Percent Survival Conjugate 45b (mg/kg) Day Vehicle 3 1 0.3 0.1 0
100 100 100 100 100 1 100 100 100 100 100 2 100 100 100 100 100 3 100 100 100 100 100 4 100
100 100 100 100 5 80 100 100 100 80 6 40 100 100 100 80 7 0 100 100 100 60 8 0 100 100 100 60 9
0 100 100 100 60 10 0 100 100 100 60 11 0 100 100 100 60 12 0 100 100 100 60 13 0 100 100 100
60 14 0 100 100 100 60

(1372) The potency of conjugate 45b was further supported by daily body weight measurements. As
expected, mice treated with vehicle demonstrated a steady drop in body weight until it exceeded 20%,
at which time they were scored as a mortality (Table 82).

(1373) In contrast to control mice, mice receiving conjugate 45b at 3, 1, or 0.3 mg/kg only
demonstrated a transient drop in bodyweight of approximately 10%, peaking on Days 3-5 (Table 82).
By study end (Day 14) these mice largely recovered (or exceeded) their starting weight. By both
survival and body weight measurements conjugate 45b demonstrated robust protection from a lethal
challenge of Influenza A/California/07/2009 (H1N1) pdm with a single 0.3 mg/kg dose administered
SC. Activity against the clinically relevant pandemic strain used in this study supports the utility of
conjugate 45b in treating serious influenza infections.

(1374) TABLE-US-00087 TABLE 82 Percent Body Weight Conjugate 45b (mg/kg) Day Vehicle 3 1 0.3
0.1 0 100 100 100 100 100 1 96.9 95.9 97.1 96.3 96.1 2 99.8 98.2 99.2 100.3 101.5 3 90 94.2 91.1
91.7 90.1 4 81 91.2 89.7 88.1 83.2 5 77.8 94.2 93.2 90.4 82.1 6 97.8 97.4 92.1 7 97.6 96.3 90.3 8 98.6
96.4 91.4 9 101.3 98.9 94 10 99.3 98.9 92.8 11 100.4 101.7 95.3 12 99.5 100.8 96.7 13 100.1 100.9
97.3 14 103.3 103.3 99.9

Example 160. Efficacy of Conjugate 45b Subcutaneously Dosed Against Influenza A/Hong
Kong/1/1968 (H3N2) in a Lethal Mouse Model

(1375) Conjugate 45b was evaluated against a lethal IAV H3N2 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Hong Kong/1/1968) is a mouse-
adapted isolate capable of causing lethal infections in mice. The experiment comprised 3 groups of 5
mice. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal inoculation in a
volume of 30 μl, after being anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg
respectively). Mortality and body weights were recorded daily and any animal with a 20% loss of body
weight was scored as a death.

(1376) Test groups received a single subcutaneous (SC) treatment of conjugate 45b or vehicle (PBS)
2 hours post viral challenge. The study design is summarized in Table 83.

(1377) TABLE-US-00088 TABLE 83 Study design for Influenza A/Hong Kong/1/1968 (H3N2) study
Dose Dose volume # of Group Test article Route/Schedule (mq/kq) (ml/kq) mice 1 Vehicle SC, T + 2
hrs. na 10 5 2 Conjugate 45b SC, T + 2 hrs. 1 10 5 3 Conjugate 45b SC, T + 2 hrs. 0.3 10 5

(1378) As expected, mice receiving vehicle succumbed to infection by Day 7 (Table 84). However,
mice treated with conjugate 45b were fully protected at 1 and 0.3 mg/kg dose levels. In this study
conjugate 45b was not dosed lower than 0.3 mg/kg.

(1379) TABLE-US-00089 TABLE 84 Percent Survival Conjugate 45b (mg/kg) Day Vehicle 1 0.3 0 100
100 100 1 100 100 100 2 100 100 100 3 100 100 100 4 100 100 100 5 40 100 100 6 20 100 100 7 0
100 100 8 0 100 100 9 0 100 100 10 0 100 100 11 0 100 100 12 0 100 100 13 0 100 100 14 0 100 100

(1380) The potency of conjugate 45b was further supported by daily body weight measurements. As
expected, mice treated with vehicle demonstrated a steady drop in body weight until it exceeded 20%,
at which time they were scored as a mortality (Table 85).

(1381) In contrast to control mice, those groups receiving conjugate 45b at 1 and 0.3 mg/kg
maintained nearly healthy body weights throughout the study and never demonstrated more than a
transient body weight drop of less than 11% before regaining nearly their starting body weight by study
end (Table 85). By both survival and body weight measurements conjugate 45b demonstrated robust
protection from a lethal challenge of Influenza A (H3N2) with a single SC dose as low as 0.3 mg/kg.

(1382) TABLE-US-00090 TABLE 85 Percent Body Weight Conjugate 45b (mg/kg) Day Vehicle 1 0.3 0
100 100 100 1 99.2 96 98.1 2 94.1 94.2 94.4 3 89.5 91 89.1 4 88.6 94.1 90.5 5 84.1 93.1 89.1 6 96.3
90.6 7 95 90.2 8 97.3 92.8 9 96.7 93.2 10 97.8 94.6 11 99.9 97.5 12 98.3 97.4 13 98.9 99.5 14 98.6
100.2

Example 161. Efficacy of Conjugate 45b Against Influenza B (Victoria Lineage) in a Lethal Mouse
Model

(1383) Conjugate 45b was evaluated against a lethal Influenza B influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (B/Malaysia/8/1934) is a mouse-
adapted isolate capable of causing lethal infections in mice. The experiment comprised 6 groups of 5
mice. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal inoculation in a
volume of 30 μl (approx. 1 E4 per mouse), after being anesthetized with a mixture of
ketamine/xylazine (150 and 10 mg/kg respectively).

(1384) All groups received a single SC treatment, 2 hours post viral challenge of test article, vehicle
(PBS), or Fc only control (hIgG1 Fc). The study evaluated concentrations of conjugate 45b at 1, 0.3,
0.1, and 0.03 mg/kg. Mice were monitored for 2 weeks and animals exceeding 20% body weight loss,
or were found moribund, were scored as a mortality.

(1385) All mice treated with vehicle or the Fc only control, reached mortality by day 7. In contrast, mice
receiving 1, 0.3, or 0.1 mg/kg of conjugate 45b were fully protected after receiving a single SC dose
(Table 86). The potency of conjugate 45b against Influenza B was further supported by the daily body
measurements (Table 87), which show a less than 5% transient drop at 0.3 mg/kg.

(1386) TABLE-US-00091 TABLE 86 Percent Survival Conjugate 45b (mg/kg) Day Vehicle hIgG1 Fc (1)
1 0.3 0.1 0.03 0 100 100 100 100 100 100 1 100 100 100 100 100 100 2 100 100 100 100 100 100 3
100 100 100 100 100 100 4 100 100 100 100 100 100 5 100 100 100 100 100 100 6 100 80 100 100
100 100 7 0 0 100 100 100 20 8 0 0 100 100 100 0 9 0 0 100 100 100 0 10 0 0 100 100 100 0 11 0 0
100 100 100 0 12 0 0 100 100 100 0 13 0 0 100 100 100 0 14 0 0 100 100 100 0

(1387) TABLE-US-00092 TABLE 87 Percent Body Weight Conjugate 45b (mg/kg) Day Vehicle hIgG1
Fc (1) 1 0.3 0.1 0.03 0 100 100 100 100 100 100 1 98.1 97 98.1 98.2 97.8 98.7 2 98.7 99.7 101.7
100.6 101.5 102.7 3 97.8 97.7 102.1 98.4 101.6 101.3 4 88 87.7 99.8 96.9 96.8 92 5 78.3 80.6 99.6
95.4 94.5 83.8 6 77.9 77.6 102.5 100.1 90.7 80 7 103.5 100.7 86.1 75.3 8 103 100.3 86.3 9 103.2
101.1 91.5 10 102.8 101 94 11 101.2 99.7 94.1 12 101.9 99.9 96.1 13 102.9 101.6 97.6 14 102.5 99.9
98.6

Example 162. Efficacy of Conjugate 45b Against Influenza B (Yamagata Lineage) in a Lethal Mouse
Model

(1388) Conjugate 45b was evaluated against a lethal Influenza B influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (B/Florida/4/2006) is a mouse-
adapted isolate capable of causing lethal infections in mice. The experiment comprised 7 groups of 5
mice. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal inoculation in a
volume of 30 μl (approx. 3E4 per mouse), after being anesthetized with a mixture of ketamine/xylazine
(150 and 10 mg/kg respectively).

(1389) All groups received a single SC treatment, 2 hours post viral challenge of test article, vehicle
(PBS), or Fc only control (hIgG1 Fc). The study evaluated concentrations of conjugate 45b from 3 to
0.03 mg/kg. Mice were monitored for 2 weeks and animals exceeding 20% body weight loss, or were
found moribund, were scored as a mortality.

(1390) All mice treated with vehicle reached mortality by day 8, while the hIgG1 Fc control had 80%
mortality. In contrast, mice receiving conjugate 45b were fully protected after receiving a single SC
dose at all concentrations tested (Table 88).

(1391) TABLE-US-00093 TABLE 88 Percent Survival Conjugate 45b (mg/kg) Day Vehicle hIgG1 Fc (1)
3 1 0.3 0.1 0.03 0 100 100 100 100 100 100 100 1 100 100 100 100 100 100 100 2 100 100 100 100
100 100 100 3 100 100 100 100 100 100 100 4 100 100 100 100 100 100 100 5 100 100 100 100 100
100 100 6 100 100 100 100 100 100 100 7 40 60 100 100 100 100 100 8 0 40 100 100 100 100 100 9
0 20 100 100 100 100 100 10 0 20 100 100 100 100 100 11 0 20 100 100 100 100 100 12 0 20 100
100 100 100 100 13 0 20 100 100 100 100 100 14 0 20 100 100 100 100 100

(1392) The potency of conjugate 45b against Influenza B (Yamagata) was further supported by the
daily body measurements (Table 89), which show a less than 4% transient drop at 0.03 mg/kg, the
lowest tested concentration.

(1393) TABLE-US-00094 TABLE 89 Percent Body Weight Conjugate 45b (mg/kg) Day Vehicle hIgG1
Fc (1) 3 1 0.3 0.1 0.03 0 100 100 100 100 100 100 100 1 98.7 100.5 97.8 99.2 99.1 98.7 96.8 2 99.6
99.8 99.1 98.4 98.9 99.4 98.6 3 98.7 99.3 100.8 101.2 101.2 103.8 100.9 4 89.8 91 100.4 99.3 98.3
98.5 97.3 5 84.6 87.6 99.2 97.5 98 98.2 96.2 6 80.2 86.6 103.4 100.7 100.9 101.8 100.4 7 75.5 79.9
101.3 102.3 100.8 101.4 96.9 8 102.7 103 103 102.7 99.2 9 100.2 101.9 101.7 100 99 10 102.6 102.8
101.7 101.3 100.7 11 103 105.1 104.1 102.3 101.9 12 101 103.2 101.8 100.2 100.1 13 103.3 104.9
103.9 103.7 104.2 14 101.8 104.6 103.1 101.6 102.1

Example 163. Efficacy of Conjugate 45b Against Influenza HI NI, H3N2, and B (Victoria) in a 28-Day
Mouse Prevention Model

(1394) Conjugate 45b was evaluated against lethal challenge by seasonal influenza subtypes (H1N1,
H3N2, and B (Victoria lineage)) in female BALB/c mice (Charles River Laboratories, 6-8 weeks). The
experiment comprised 13 groups of 5 mice, except for group 6 (Vehicle, A/HK/1/68 challenge), which
consisted of 4 animals. On day 0, mice were subcutaneously (SC) administered conjugate 45b at 3, 1,
or 0.3 mg/kg in a single dose. Control mice were also treated by the same route with vehicle (PBS) or
hIgG1 Fc only. Twenty-eight Days after administration of test article, mice were challenged intranasally
with 3× the LD.sub.95 of one of the following seasonal influenza subtypes:

(1395) A/California/07/2009 (H1N1)

(1396) A/Hong Kong/i/i968 (H3N2)

(1397) B/Malaysia/8/1934 (B; Victoria lineage)

(1398) For viral challenge mice were anesthetized with a mixture of ketamine/xylazine (150 and 10
mg/kg respectively), and the virus was given in a volume of 30 μl. Mortality and body weights were
recorded daily and any animal with a 20% loss of body weight was scored as a death.

(1399) For the H1N1 arm of the study all mice treated with vehicle only succumbed to infection by Day
7. The Fc only control was also not protective, with only 20% survival at study end (Day 42, 14 days
after viral challenge). Conjugate 45b however was fully protective for a month against this pandemic
isolate after a single SC dose at 1 mg/kg (P=0.0020; Table 90). Even at the lowest test concentration
(0.3 mg/kg) 80% of mice survived to study end. The daily body weight (BW) percent measurements
further supported the efficacy of conjugate 45b in this model (Table 91), at 1 mg/kg the mice had a
transient loss of 12.1% which is typical against this highly pathogenic strain, which largely recovered
by study end (96.5% of the starting value).

(1400) TABLE-US-00095 TABLE 90 H1N1 Percent Survival Conjugate 45b (mg/kg) Day Vehicle hIgG1
Fc (3) 3 1 0.3 0 100 100 100 100 100 1 100 100 100 100 100 2 100 100 100 100 100 3 100 100 100
100 100 4 100 100 100 100 100 5 100 40 100 100 100 6 20 40 100 100 80 7 0 20 100 100 80 8 0 20
100 100 80 9 0 20 100 100 80 10 0 20 100 100 80 11 0 20 100 100 80 12 0 20 100 100 80 13 0 20
100 100 80 14 0 20 100 100 80

(1401) TABLE-US-00096 TABLE 91 H1N1 Percent Body Weight* Conjugate 45b (mg/kg) Day Vehicle
hlgG1 Fc (3) 3 1 0.3 0 100 100 100 100 100 1 100.2 93.6 99.6 93 97 2 97.1 93 99.5 93 97.7 3 89.1 87
92.7 88.1 90.9 4 82.3 80.6 89.8 89.1 89.4 5 77.7 77.4 89 88 87.2 6 88.9 87.9 87 7 90.9 90.7 8 92.1
90.5 9 92.9 90.4 10 96 92.2 11 95.1 94.5 12 94.7 94.3 13 96.2 96.2 14 97 96.5 *Note, that BWs are
only given until the first death occurs within a group

(1402) For the H3N2 arm of the study (A/Hong Kong/1/1968), all mice treated with vehicle only
succumbed to infection by Day 8. In contrast to vehicle only treatment, all Conjugate 45b
concentrations were fully protective, even at a dose level of 0.3 mg/kg (P=0.0007; Table 92). As seen
with the H1N1 arm of the study, daily BW measurements further supported the efficacy of conjugate
45b against the H3N2 subtype (Table 93), at 0.3 mg/kg the mice had a transient loss of less than 11%,
with BWs largely recovered by study end (98.4% of the starting value).

(1403) TABLE-US-00097 TABLE 92 H3N2 Percent Survival Conjugate 45b (mg/kg) Day Vehicle 3 1
0.3 0 100 100 100 100 1 100 100 100 100 2 100 100 100 100 3 100 100 100 100 4 100 100 100 100 5
100 100 100 100 6 100 100 100 100 7 75 100 100 100 8 0 100 100 100 9 0 100 100 100 10 0 100 100
100 11 0 100 100 100 12 0 100 100 100 13 0 100 100 100 14 0 100 100 100

(1404) TABLE-US-00098 TABLE 93 H3N2 Percent Body Weight Conjugate 45b (mg/kg) Day Vehicle 3
1 0.3 0 100 100 100 100 1 98.4 99.2 99.1 100 2 93.7 98.5 97.7 99.1 3 88.2 96.7 94.6 95.3 4 88.5 97.3
93.4 95.6 5 86.2 95.8 94 92.6 6 81.4 96.8 94.6 89.3 7 77 99.8 97.1 89.6 8 100.3 97.8 91.5 9 98.2 96.9
91.7 10 101.3 101.2 96.8 11 100.5 100.8 97.9 12 98.6 99.7 96.5 13 99.3 98.3 97 14 99.7 100.1 98.4

(1405) The final arm of the study determined the efficacy of conjugate 45b against the Victoria lineage
of influenza B. Typical to what was seen in the other arms of this study, vehicle only treated mice
succumbed to infection by Day 7. Similar to the results against the H1N1 subtype, a single SC dose of
1 mg/kg was fully protective against lethal challenge by influenza B (Table 94; P=0.0031) one month
after conjugate 45b administration. At the lowest test concentration (0.1 mg/kg), 60% of mice survived.
As in the other arms of the study, BW data supports the potency of conjugate 45b. Against the
B/Malaysia strain animals treated at 1 mg/kg showed a less than 6% transient BW loss which had
recovered by study end (Table 95; 100.2%).

(1406) TABLE-US-00099 TABLE 94 Influenza B Percent Survival Conjugate 45b (mg/kg) Day Vehicle
3 1 0.3 0 100 100 100 100 1 100 100 100 100 2 100 100 100 100 3 100 100 100 100 4 100 100 100
100 5 100 100 100 100 6 60 100 100 100 7 0 100 100 100 8 0 100 100 40 9 0 100 100 40 10 0 100
100 40 11 0 100 100 40 12 0 100 100 40 13 0 100 100 40 14 0 100 100 40

(1407) TABLE-US-00100 TABLE 95 Influenza B Percent Body Weight Conjugate 45b (mg/kg) Day
Vehicle 3 1 0.3 0 100 100 100 100 1 97.3 98.7 97.2 98.6 2 97.1 98.8 98.9 99.2 3 96.4 99.2 98.6 98.6 4
88.5 99.6 98.3 96.8 5 80.8 96.2 95.8 90 6 77 96.5 94.5 86.2 7 97.8 95.5 82.8 8 100.5 97 81.6 9 100.1
97.3 10 102.3 99.7 11 102.3 100.3 12 100.7 98.7 13 98.7 98.3 14 101.6 100.2

Example 164. In vitro plaque reduction assay

(1408) Plaque reduction assays were performed in Madin Darby Canine Kidney (MDCK) cells seeded
in 24 well plates. 500,000 MDCK cells were seeded in 0.5 mL of media (DMEM) containing 10% FBS
and incubated for approximately 24 hours. Dilutions of the test articles and the viruses used, H1N1 WT
(A/California/12/2012) and H275Y (A/Texas/23/2012), H3N2 WT (A/Washington/12/2007) and E119V
(A/Texas/12/2007), and B (B/Malaysia/2506/2004), were performed in a buffer containing PBS, Bovine
Albumin 35% and Ca.sup.2+/Mg.sup.2+. Conjugate 45b and baloxavir, zanamivir oseltamivir
comparators were pre-incubated with virus for 30 minutes at room temperature before adding them to
the monolayers of MDCK cells after media removal and one wash with PBS. The MOI for each drug-
virus combination was selected to target 30 plaques in the PBS control well. Adsorption was carried
out for 1 h, the virus-test article mix was removed, and the infected cells were incubated for 48 h in the
presence the test article diluted in a mixture of 1.25% Avicel, DMEM, 0.01% DEAE-dextran and 2
μg/mL of TPCK trypsin. After 48 hours the Avicel mixture was then removed and cells were fixed with
paraformaldehyde and stained with 1% crystal violet to count the plaques. All drugs were tested at six
concentrations, ranging from 0.3 to 100 nM. EC.sub.50 values (nM) were calculated using GraphPad
Prism software. Results are summarized in Table 96.

(1409) TABLE-US-00101 TABLE 96 Summary of conjugate 45b plaque reduction assay EC.sub.50
values for WT and NA mutant influenza strains EC.sub.50 (nM) A/TX/23/ A/TX/12/ A/CA/ 2012
A/WA/12/ 2007 12/2012 (H1N1) 2007 (H3N2) B/Malaysia/ Molecule (H1N1) H275Y (H3N2) E119V
2506/2004 conjugate 1 1.2 ≤0.3 1.2 4.1 45b baloxavir 4 1.6 4.8 3.2 8.4 zanamivir 35 93.8 >100 74
17.3 oseltamivir >100 >100 29.6 >100 22.1

(1410) Conjugate 45b demonstrated its potent activity in plaque reduction assays against all H1N1,
H3N2 and B strains tested, generating EC.sub.50 values lower than those for all three comparator
agents (Table 96). In addition, the activity of conjugate 45b was minimally impacted by the presence of
oseltamivir resistance-conferring NA mutations E119V and H275Y

Example 165. Serial Passage Experiments for Selection of Resistant Influenza Viruses

(1411) To evaluate the potential for development of drug resistant mutant viral strains under selective
pressure with viral inhibitors, serial passage studies were conducted with conjugate 45b, versus
oseltamivir and baloxavir comparators. Serial passage studies were conducted using MDCK cells.
Passages were conducted as follows: 500,000 MDCK cells were seeded per well (24-well) in 500 μl
DMEM 10% FBS, 1% PS, 1% NaPyr and 1% HEPES and incubated for approximately 24 hours. Once
cells reached approximately 80% confluency, they were washed once with PBS and incubated for 2
hours in the presence of compounds or PBS alone under normal culture conditions. Selecting agent
concentrations were optimized as required for maximum virus inhibition, while maintaining enough
virus production for subsequent passages. Concentrations of conjugate 45b, baloxavir, and oseltamivir
used in the serial passage experiments were 4 nM, 4 nM and 200 nM respectively. Cells were infected
at an MOI of 0.01 with A/California/07/2009 H1N1 pdm for 1 hour at room temperature in a buffer
containing PBS, Bovine Albumin 35% and Ca.sup.2+/Mg.sup.2+, followed by removal of inoculum and
washing of cells once with DMEM 1% PS, 1% NaPyr and 1% HEPES (No FBS). Cells were then
Incubated for 24 hours in the presence of selecting agents diluted in DMEM 1% PS, 1% NaPyr, 1%
HEPES and 2 μg/ml TPCK-treated trypsin. After incubation, viral supernatants were collected, and
cells and debris were removed by centrifugation (5 min, 4° C., 1,400×g). Supernatants were then used
to: i Quantify viral titer by plaque assay ii Conduct a hemagglutination assay to determine if the virus
escaped compound inhibition iii Re-infect freshly seeded MDCK cells in presence of compounds

(1412) The process was repeated for 10 passages. Once increased titers in the presence of drugs are
detected the viruses may be plaque purified. Following plaque purification, all 8 genome segments
may be sequenced and compared to PBS treated control virus to detect escape mutations. A
summary of the serial passage is shown in figure FIG. 83. No increases in viral titer were observed for
conjugate 45b through the course of 10 passages (suggesting no emergence of resistant mutants),
while baloxavir and oseltamivirtiters increased to levels similar to those observed in the PBS control
after passages 6 and 8 respectively.

Example 166. Cytopathic Effect Assay

(1413) To measure the ability of Conjugate 45b to protect mammalian cells from infection and
destruction by influenza virus, Cytopathic effect (CPE) based microneutralization assays were
conducted as discussed in Example 25, with minor variations. Briefly, a monolayer of MOCK cells was
infected with influenza A or B strains at appropriate MOI varying between 0.001-1. Test articles were
added at concentrations ranging between 0.1-10,000 nM and incubated for 3 days for influenza A or 5
days for influenza B at 37° C., 5% C02. CPE was determined by crystal violet staining by reading
absorbance at 595 nm. The results are shown in Table 97 and Table 98 below.
(1414) TABLE-US-00102 TABLE 97 CPE of Conjugate 45b against Influenza A H1N1 at MOI 0.01 or
MOI 0.001 (CPE [nM]) Central A/WSN/ A/CA/07/ A/CA/12/ A/Texas/23/2012 Molecule TM DAR Fc
linker 1933 2009pdm 2012 H275 H275Y mutant Oseltamivir N/A N/A N/A N/A >10,000 34.2 107.6
>10,000 Zanamivir N/A N/A N/A N/A N/A 33.16 54.59 327.7 Conjugate Int-83 4.2 SEQ ID 15 atom
3.676 2.964 0.7043 2.101 45b NO: 73 Baloxavir N/A N/A N/A N/A 11.35 2.216 3.718 3.318

(1415) TABLE-US-00103 TABLE 98 CPE of Conjugate 45b against Influenza A H3N2 at MOI 0.01 or
MOI 0.001 (CPE [nM]) A/Bethesda/ A/Texas/12/2007 Central A/HK/ A/Wisconsin/ 956/2006
A/Washington/ E119V Molecule TM DAR Fc linker 1/1968 04/2018 R292K mutant 12/2007 mutant
Oseltamivir N/A N/A N/A N/A 0.7444 151.1 >10,000 1.908 351.4 Zanamivir N/A N/A N/A N/A <0.3 308
9591 1.968 2.096 Conjugate Int-83 4.2 SEQ ID 15 <0.3 8.99 54.01 0.04449 0.6594 45b NO: 73 atom
Baloxavir N/A N/A N/A N/A 2.596 4.916 17.15 0.5252 5.602

Example 167. Neuraminidase Inhibition with Live Influenza A Virus or Lysates of Neuraminidase
Resistant Mutants

(1416) Neuraminidase inhibition (NAI). Test articles were incubated with neuraminidase (Sino
Biological) or with live viruses for 20 min at 37° C., 5% CO.sub.2. 2′-(4-Methylumbelliferyl)-α-D-N
acetylneuraminic acid substrate was added to appropriate wells and incubated for 1 h at 37° C., 5%
CO.sub.2. NAI was determined by reading fluorescence at 355 nm excitation/460 nm emission.
Neuraminidase inhibition was determined for neuraminidase resistant mutants and for live influenza
virus (Table 99, Table100, Table101, Table102, and Table 103).

(1417) TABLE-US-00104 TABLE 99 NAI against live influenza A (H1N1) viruses A/Ca/07/2009
A/PR/8/1934 A/California/12/2012 A/Texas/23/2012 Central (H1N1)pdm (H1N1) (H1N1)pdm09
(H1N1)pdm09 H275Y Molecule TM DAR Fc linker IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM]
IC.sub.50 [nM] Oseltamivir N/A N/A N/A N/A 0.9103 2.427 1.28 367.9 Zanamivir N/A N/A N/A N/A
0.5634 0.6041 0.8891 0.8505 Int-83 N/A N/A N/A 15 atom 28.59 4.231 45.08 26.47 Conjugate Int-83
4.8 SEQ ID 15 atom 0.8124 0.07184 2.527 1.471 45b NO: 73 *All live viruses tested at 1e6 PFU.

(1418) TABLE-US-00105 TABLE 100 NAI against live influenza A (H3N2) viruses A/Wash/1/2007
A/Texas/12/2007 A/Texas/71/2017 A/Bethesda/956/2006 Central (H3N2) (H3N2) E119V (H3N2)
(H3N2) R292K* Molecule TM DAR Fc linker IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50
[nM] Oseltamivir N/A N/A N/A N/A 0.2086 114.2 0.9502 4667 Zanamivir N/A N/A N/A N/A 4.845 3.085
2.321 31.59 Int-83 N/A N/A N/A 15 atom 99.05 50.22 301.1 1147 Conjugate Int-83 4.8 SEQ ID 15
atom 0.0233 2.79 11.3 23.55 45b NO: 73 *All live viruses tested at 1e6 PFU (except R292K which was
tested at 1e5 PFU)

(1419) TABLE-US-00106 TABLE 101 NAI against live influenza A (H1N1)pdm09 viruses
A/Illinois/08/2018 A/Illinois/37/2018 A/Illinois/08/2018 Central (H1N1)pdm09 I38 WT (H1N1)pdm09
I38L (H1N1)pdm09 I38T Molecule TM DAR Fc linker IC.sub.50 [nM] IC.sub.50 [nM] IC.sub.50 [nM]
Oseltamivir N/A N/A N/A N/A 0.43 0.71 0.58 Zanamivir N/A N/A N/A N/A 0.37 0.47 0.50 Int-83 N/A N/A
N/A 15 atom 5.90 6.16 4.81 Conjugate Int-83 4.8 SEQ ID 15 atom 0.04 0.10 0.01 45b NO: 73 *All live
viruses tested at 1e7 PFU.

(1420) TABLE-US-00107 TABLE 102 NAI against live influenza A (H3N2) viruses A/Louisiana/50/2017
A/Louisiana/49/2017 (H3N2) I38 WT (H3N2) I38M Molecule TM DAR Fc Central linker IC.sub.50 [nM]
IC.sub.50 [nM] Oseltamivir N/A N/A N/A N/A 0.26 0.19 Zanamivir N/A N/A N/A N/A 0.48 0.37 Int-83
N/A N/A N/A 15 atom 47.72 26.09 Conjugate Int-83 4.8 SEQ ID 15 atom 4.58 5.13 45b NO: 73 *All live
viruses tested at 1e7 PFU.

(1421) TABLE-US-00108 TABLE 103 NAI against influenza B viruses B/Florida/4/2006

B/Malaysia/2506/2004 B/Colorado/6/2017 Molecule TM DAR Fc Central linker (Yamagata) IC.sub.50
[nM] (Victoria) IC.sub.50 [nM] (Victoria) IC.sub.50 [nM] Oseltamivir N/A N/A N/A N/A 14.37 32.02
35.03 Zanamivir N/A N/A N/A N/A 4.755 4.147 5.112 Int-83 N/A N/A N/A 15 atom 275.3 273.1 124.8
Conjugate Int-83 4.8 SEQ ID 15 atom 2.888 1.514 19.83 45b NO: 73 *All live viruses tested at 1e6
PFU.
Example 168. Antibody-dependent cellular phagocytosis assay

(1422) Conjugate 45b was tested for antibody-dependent cellular phagocytosis (ADCP). A monolayer
of MDCK cells was infected with influenza A or influenza B strains at an MOI of 0.001-10, and
incubated for 18-24 h at 37° C., in 5% CO.sub.2. ADCP was determined with commercial report cell
line (PROMEGA) according to manufacturer's instructions. Briefly, test articles were added at
concentrations ranging from 1 to 10,000 nM to appropriate wells and incubated for 15 m at 37° C., in
5% CO.sub.2. ADCP was quantified by reading luminescence. Conjugate 45b and Fc alone (SEQ ID
NO: 73, prepared analogous to Example 124) were tested against influenza A/PR/8/1934 (H1N1),
influenza A/CA/07/2009 (H1N1), and influenza A/HK/1/1968 (H3N2) (Tables 104-106, respectively),
and influenza B/Malaysia/2506/2004 (Victoria, Table 107), showing an MOI-dependent increase in
ADCP by conjugate 45b. Conjugate 45b also showed a Dose-dependent increase in ADCP against
influenza A/PR/8/1934 (H1N1), influenza A/CA/07/2009 (H1N1), and influenza A/HK/1/1968 (H3N2) at
an MOI of 10 (Tables 108-110, respectively), and influenza B/Malaysia/2506/2004 (Victoria, Table 111).

(1423) TABLE-US-00109 TABLE 104 MOI-dependent increase in ADCP (fold induction) by Conjugate
45b against influenza A Influenza A/PR/8/1934 Conjugate 45b (H1N1) MOI Fc Alone [1 μM] 0 1 1 0.1
1.13 1.8 0.3 1.21 9.95 1 0.96 13.77 3 1.1 14.94 10 1.05 13.76

(1424) TABLE-US-00110 TABLE 105 MOI-dependent increase in ADCP (fold induction) by Conjugate
45b against influenza A Influenza A/CA/7/2009 (H1N1)pdm MOI Fc Alone Conjugate 45b [1 μM] 0
0.98 1.02 0.1 1.14 6.33 0.3 1.03 22.16 1 0.99 18.27 3 0.93 19.61 10 1.05 21.11

(1425) TABLE-US-00111 TABLE 106 MOI-dependent increase in ADCP (fold induction) by Conjugate
45b against influenza A Influenza A/HK/1/1968 Conjugate 45b (H3N2) MOI Fc Alone [1 μM] 0 1.02
0.98 0.1 1.07 1.48 0.3 1.05 2.63 1 1.18 2.74 3 1.01 4.3 10 1.06 4.39

(1426) TABLE-US-00112 TABLE 107 MOI-dependent increase in ADCP (fold induction) by Conjugate
45b against influenza B Influenza B/Malaysia/2506/ Conjugate 45b 2004 MOI Fc Alone [1 μM] 0 1.07
0.93 0.1 0.98 1.67 0.3 0.99 6.93 1 1.14 25.86 3 1.11 34.29 10 1 30.38

(1427) TABLE-US-00113 TABLE 108 Dose-dependent increase in ADCP (fold induction) by Conjugate
45b against influenza A at MOI 10 Influenza A/PR/8/1934 (H1N1) [nM] Fc Alone Conjugate 45b 0 0.96
1.04 100 1.22 3.44 300 1.56 7 1000 1.91 17.04 3000 2.22 28.54 10000 3.08 44.93

(1428) TABLE-US-00114 TABLE 109 Dose-dependent increase in ADCP (fold induction) by Conjugate
45b against influenza A at MOI 10 Influenza A/CA/7/2009 (H1N1)pdm [nM] Fc Alone Conjugate 45b 0
0.98 1.02 100 1.64 2.32 300 1.74 5.41 1000 2.15 10.04 3000 2.13 24.28 10000 2.24 33.87

(1429) TABLE-US-00115 TABLE 110 Dose-dependent increase in ADCP (fold induction) by Conjugate
45b against influenza A at MOI 10 Influenza A/HK/1/1968 (H3N2) [nM] Fc Alone Conjugate 45b 0 1.01
0.99 100 1.03 1.81 300 1.48 3.33 1000 2.29 6.09 3000 2.87 9.01 10000 2.11 9.96

(1430) TABLE-US-00116 TABLE 111 Dose-dependent increase in ADCP (fold induction) by Conjugate
45b against influenza B/Malaysia/2506/2004 at MOI 10 Influenza B/Malaysia/2506/2004 MOI Fc Alone
Conjugate 45b 0 1.15 0.85 100 1.3 6.67 300 2.09 26.21 1000 2.32 61.43 3000 3.26 96.95 10000 3.91
104.49

Example 169. Antibody-Dependent Cellular Cytotoxicity Assay

(1431) Conjugate 45b was tested for antibody-dependent cellular cytotoxicity (ADCC). A monolayer of
MDCK cells was infected with influenza A or influenza B strains at an MOI of 0.001-10, and incubated
for 18-24 h at 37° C., in 5% CO.sub.2. ADCC was determined with commercial report cell line
(PROMEGA) according to manufacturer's instructions. Briefly, test articles were added at
concentrations ranging from 1 to 10,000 nM to appropriate wells and incubated for 15 m at 37° C., in
5% CO.sub.2. ADCC was quantified by reading luminescence. Conjugate 45b and Fc alone (SEQ ID
NO: 73, prepared analogous to Example 124) were tested against influenza A/PR/8/1934 (H1N1),
influenza A/CA/07/2009 (H1N1), and influenza A/HK/1/1968 (H3N2) (Tables 112-114, respectively),
and influenza B/Malaysia/2506/2004 (Victoria, Table 115), showing an MOI-dependent increase in
ADCC by conjugate 45b. Conjugate 45b also showed a Dose-dependent increase in ADCC against
influenza A/PR/8/1934 (H1N1), influenza A/CA/07/2009 (H1N1), and influenza A/HK/1/1968 (H3N2) at
an MOI of 10 (Tables 116-118, respectively), and influenza B/Malaysia/2506/2004 (Victoria, Table 119).

(1432) TABLE-US-00117 TABLE 112 MOI-dependent increase in ADCC (fold induction) by Conjugate
45b against influenza A Influenza A/PR/8/1934 (H1N1) MOI Fc Alone Conjugate 45b [1 μM] 0 0.97
1.03 0.1 1.22 1.54 0.3 1.27 2.27 1 1.47 4.03 3 1.92 6.14 10 2.34 11.46

(1433) TABLE-US-00118 TABLE 113 MOI-dependent increase in ADCC by Conjugate 45b against
influenza A Influenza A/CA/7/2009 (H1N1)pdm Conjugate 45b MOI Fc Alone [1 μM] 0 1.02 0.98 0.1
1.23 1.48 0.3 1.4 2.21 1 1.62 3.74 3 1.74 6.08 10 2.14 9.59

(1434) TABLE-US-00119 TABLE 114 MOI-dependent increase in ADCC (fold induction) by Conjugate
45b against influenza A Influenza A/HK/1/1968 (H3N2) Conjugate 45b MOI Fc Alone [1 μM] 0 0.99
1.01 0.1 1.31 1.46 0.3 1.66 2.17 1 2.48 3.4 3 1.43 2.74 10 0.89 2.34

(1435) TABLE-US-00120 TABLE 115 MOI-dependent increase in ADCC (fold induction) by Conjugate
45b against influenza B Influenza B/Malaysia/2506/2004 Conjugate 45b MOI Fc Alone [1 μM] 0 0.97
1.03 0.1 1.27 3.6 0.3 1.64 9.38 1 2.36 22.09 3 2.65 26.21 10 2.87 34.46

(1436) TABLE-US-00121 TABLE 116 Dose-dependent increase in ADCC (fold induction) by Conjugate
45b against influenza A at MOI 10 Influenza A/PR/8/1934 (H1N1) [nM] Fc Alone Conjugate 45b 0 0.93
1.07 100 1.12 1.78 300 1.25 3.8 1000 1.4 5.16 3000 1.19 7.09 10000 1.03 5.68

(1437) TABLE-US-00122 TABLE 117 Dose-dependent increase in ADCC (fold induction) by Conjugate
45b against influenza A at MOI 10 Influenza A/CA/7/2009 (H1N1)pdm [nM] Fc Alone Conjugate 45b 0
0.91 1.09 100 1.16 3.44 300 1.02 5.53 1000 1.21 6.18 3000 1.17 6.05 10000 1.15 5.48

(1438) TABLE-US-00123 TABLE 118 Dose-dependent increase in ADCC (fold induction) by Conjugate
45b against influenza A at MOI 10 Influenza A/HK/1/1968 (H3N2) [nM] Fc Alone Conjugate 45 0 0.97
1.03 100 1.11 1.42 300 1.15 2.24 1000 1.11 2.93 3000 1.11 3.08 10000 1.09 2.67

(1439) TABLE-US-00124 TABLE 119 Dose-dependent increase in ADCC (fold induction) by Conjugate
45b against influenza B/Malaysia/2506/2004 at MOI 10 Influenza B/Malaysia/2506/2004 MOI Fc Alone
Conjugate 45 0 0.96 1.04 100 1.18 4.16 300 1.11 5.34 1000 1.18 15.21 3000 1.04 14.84 10000 1.02
12.11

Example 170. Efficacy of Conjugate 45b in Lethal Mouse Influenza Model: Study #58

(1440) Conjugate 45b was evaluated against a lethal influenza A/PR/8/1934 (3E2 PFU) in female
BALB/c mice (6-8 weeks old, n=5/group). The study design is shown in Table 120. Mice were treated
with test articles at t=+2 h (SC) post-infection with a single dose for Conjugate 45b or twice daily for 4
days for control agents. All mice were monitored for body weight daily. Mice were sacrificed on day 4
post-infection and both lung lobes were harvested. Lungs were homogenized to determine the viral
burden and immune response. Data are shown in tables 121-123.

(1441) TABLE-US-00125 TABLE 120 Efficacy Study #58 design Test article Dose Dose/day Group
(DAR) Route/Schedule [mg/kg] [mg/kg] 1 PBS SC, T + 2 h N/A N/A 2 hlgG1 Fc SC, T + 2 h 3 3 3
Oseltamivir PO, BID × 4 5 10 4 Oseltamivir PO, BID × 4 50 100 5 Baloxavir PO, BID × 4 15 30 6
Conjugate 45b (4.8) SC, T + 2 h 0.1 0.1 7 Conjugate 45b (4.8) SC, T + 2 h 0.3 0.3 8 Conjugate 45b
(4.8) SC, T + 2 h 1 1 9 Conjugate 45b (4.8) SC, T + 2 h 3 3 10 uninfected N/A N/A N/A

(1442) TABLE-US-00126 TABLE 121 Viral burden on day 4 post-infection in PFU/g Log reduction
Group Test article [mg/kg] PFU/g PFU/g 1 PBS [0] 1.92E+07 0.00 2 hlgG1 Fc [3] 5.12E+07 −0.43 3
Oseltamivir [5] 2.86E+06 0.83 4 Oseltamivir [50] 2.66E+06 0.86 5 Baloxavir [15] BLD (<10) N/A (>6) 6
Conjugate 45b [0.1] 1.50E+06 1.11 7 Conjugate 45b [0.3] 1.26E+05 2.18 8 Conjugate 45b [1]
1.32E+04 3.16 9 Conjugate 45b [3] 5.00E+03 3.58 10 Uninfected BLD N/A
(1443) TABLE-US-00127 TABLE 122 Cytokine levels on day 4 post-infection Test article TNF-α IL-6
MCP-1 MIP-1α KC [mg/kg] [pg/mL] [pg/mL] [pg/mL] [pg/mL] [pg/mL] PBS [0] 1077.2 1446.6 21219.9
3750.8 8090.4 hlgG1 Fc [3] 958.6 1129.5 20534.7 3545.1 7730.8 Oseltamivir [5] 861.6 714.2 5301.6
1439.8 3466.4 Oseltamivir [50] 501.7 467.1 4572.8 948.2 2705.5 Baloxavir [15] 522.5 308.7 338.7
185.6 392.0 Conjugate 45b [0.1] 515.3 470.5 6807.3 1506.5 2954.1 Conjugate 45b [0.3] 521.7 424.9
2957.5 820.1 1788.5 Conjugate 45b [1] 460.0 329.8 2005.2 451.7 1262.8 Conjugate 45b [3] 464.8
309.2 739.4 246.4 678.8 Uninfected 488.0 312.6 423.9 177.8 444.6

(1444) TABLE-US-00128 TABLE 123 Cytokine levels on day 4 post-infection in fold-change as

compared to uninfected control Test article [mg/kg] TNF-α IL-6 MCP-1 MIP-1α KC PBS [0] 2.21 4.63
50.06 21.10 18.20 hlgG1 Fc [3] 1.96 3.61 48.44 19.94 17.39 Oseltamivir [5] 1.77 2.28 12.51 8.10 7.80
Oseltamivir [50] 1.03 1.49 10.79 5.33 6.09 Baloxavir [15] 1.07 0.99 0.80 1.04 0.88 Conjugate 45b [0.1]
1.06 1.51 16.06 8.48 6.65 Conjugate 45b [0.3] 1.07 1.36 6.98 4.61 4.02 Conjugate 45b [1] 0.94 1.05
4.73 2.54 2.84 Conjugate 45b [3] 0.95 0.99 1.74 1.39 1.53 Uninfected 1.00 1.00 1.00 1.00 1.00

Example 171. Efficacy of Conjugate 45b in Lethal Mouse Influenza Model: Study #55

(1445) Conjugate 45b was evaluated against a lethal influenza A/CA/07/2009 (3E4 PFU) in female
BALB/c mice (6-8 weeks old, n=5/group). The study design is shown in Table 124. Mice were treated
with test articles at t=+2 h (SC) post-infection with a single dose for Conjugate 45b or twice daily for 4
days for control agents. All mice were monitored for body weight daily. Mice were sacrificed on day 4
post-infection and both lung lobes were harvested. Lungs were homogenized to determine the viral
burden and immune response. Data are shown in Tables 125 and 126A-126B.

(1446) TABLE-US-00129 TABLE 124 Efficacy study #58: Study design Test article Route/ Dose
Dose/day Group (DAR) Schedule [mg/kg] [mg/kg] 1 PBS SC, T + 2 h N/A N/A 2 hlgG1 Fc SC, T + 2 h
30 30 3 Oseltamivir PO, BID × 4 5 10 4 Baloxavir PO, BID × 4 15 30 4 Conjugate 45b (4.8) SC, T + 2
h 0.3 0.3 6 Conjugate 45b (4.8) SC, T + 2 h 1 1 7 Conjugate 45b (4.8) SC, T + 2 h 3 3 8 Conjugate
45b (4.8) SC, T + 2 h 30 30 9 uninfected N/A N/A N/A

(1447) TABLE-US-00130 TABLE 125 Viral burden on day 4 post-infection in PFU/g Log reduction
Group Test article [mg/kg] PFU/g PFU/g 1 PBS [0] 1.71E+07 0.00 2 hlgG1 Fc [30] 1.08E+07 0.20 3
Oseltamivir [5] 2.05E+07 −0.08 4 Baloxavir [15] 1.51E+02 5.06 5 Conjugate 45b [0.1] 8.81E+06 0.29 6
Conjugate 45b [0.3] 4.25E+06 0.60 7 Conjugate 45b [1] 1.24E+05 2.14 8 Conjugate 45b [3] 3.72E+03
3.66 9 uninfected 0.00 0.00

(1448) TABLE-US-00131 TABLE 126A Cytokine levels on day 4 post-infection TNF-α IL-6 MCP-1 MIP-
1α KC Test article [mg/kg] [pg/mL] [pg/mL] [pg/mL] [pg/mL] [pg/mL] PBS [0] 1409.1 728.1 10139.0
2439.6 2542.4 hlgG1 Fc [30] 1489.6 709.6 9414.3 2245.0 2212.8 Oseltamivir [5] 1260.1 714.2 8891.9
1454.1 2327.7 Baloxavir [15] 586.5 238.5 378.5 175.0 247.7 Conjugate 45b [0.3] 1211.4 426.2 4059.8
886.7 1530.2 Conjugate 45b [1] 1069.7 378.9 3874.0 765.3 1759.6 Conjugate 45b [3] 1069.6 427.6
2457.6 720.0 1478.5 Conjugate 45b [30] 662.5 284.2 1185.6 406.4 1069.3 Uninfected 523.1 227.3
247.5 179.6 293.1

(1449) TABLE-US-00132 TABLE 126B Cytokine level on day 4 pose-infection in fold-change as

compared to uninfected control Test article [mg/kg] INF-γ TNF-α IL-6 MCP-1 MIP-1α KC PBS [0] 3.39
2.69 3.20 40.96 13.59 8.67 hIgG1 Fc [3] 3.06 2.85 3.12 38.03 12.50 7.55 Oseltamivir [5] 2.44 2.41
2.59 35.92 8.10 7.94 Baloxavir [15] 0.94 1.12 1.05 1.53 0.97 0.85 Conjugate 45b [0.3] 1.98 2.32 1.87
16.40 4.94 5.22 Conjugate 45b [1] 1.87 2.04 1.67 15.65 4.26 6.00 Conjugate 45b [3] 1.85 2.04 1.88
9.93 4.01 5.04 Conjugate 45b [30] 1.18 1.27 1.25 4.79 2.26 3.65 Uninfected 1.00 1.00 1.00 1.00 1.00
1.00

Example 172. In Vitro Cross-Species Fc Receptor Binding of Conjugate 45b

(1450) Binding of Conjugate 45b to Fc gamma receptors from multiple species was performed using
the ELISA method described below. Nunc Maxisorp 96-well plates were coated overnight at 4° C. with
1 μg (100 μL/well) of Conjugate 45b in carbonate buffer. Unconjugated human IgG1 Fc and isotype
control antibodies were also coated overnight under the same conditions. The next day, plates were
washed 5× with 300 μL/well 1×PBS pH 7.4 supplemented with 0.05% Tween 20 (PBST), then blocked
with 200 μL/well of 1% BSA in PBST for 1 hr at room temperature on an orbital plate shaker. The
plates were washed 5× with 300 μL/well PBST, then incubated with duplicate 2-fold serial dilutions of
recombinant His-tagged Fc gamma receptor (0.5-1,000 ng; 100 μL/well) in diluent (0.5% BSA in PBS
0.025% Tween 20) for 2 hr at room temperature with shaking. Human and cynomolgus monkey
FcγR1/CD64 were screened at a starting concentration of 25 ng/100 μL/well. The plates were washed
5× with 300 μL/well PBST, then incubated with 100 μL/well of mouse anti-His HRP antibody (cat no.
MAB050H, R&D Systems) diluted 1:1,000 in diluent for 1 hr at room temperature with shaking. The
plates were washed 8× with 300 μL/well PBST, with 1 min incubation between washes, and developed
with 100 μL/well TMB substrate reagent for 5-10 min. The reaction was stopped with 100 μL/well 1 N
H.sub.2SO.sub.4 and the absorbance read at 450 nm with an EnSpire multimode plate reader
(PerkinElmer). Half maximal effective concentration (EC.sub.50) was calculated with GraphPad Prism
version 8 using nonlinear regression analysis (Sigmoidal, 4PL) of binding curves. Binding of Conjugate
45b to FcRn receptors from multiple species was performed essentially as described for the Fc
gamma binding ELISA with the following modification. Following the blocking step, binding assays
were performed in duplicate at pH 5.8, the optimal pH for FcRn binding, and pH 7.4.

(1451) Conjugate 45b bound to Fc gamma receptors from mouse, human, rat and cynomolgus
monkey with comparable avidity to human IgG1 Fc (Table 127). Conjugate 45b bound immune
activating Fc gamma receptors with higher avidity compared to immune inhibitory Fc gamma
receptors. At pH 5.8, Conjugate 45b bound FcRn receptors from all species with equivalent avidity to
human IgG1 Fc. As expected, conjugate 45b FcRn binding was reduced at pH 7.4 (Table 127).

(1452) TABLE-US-00133 TABLE 127 Cross-species Fc receptor binding for Conjugate 45b EC.sub.50
[nM] Conjugate hIgG1 Species Fc receptor Signal/function 45 Fc hIgG1 Mouse FcγR1 /CD64
Activating 12 9 3 FcγR2B/CD32b Inhibitory 228 135 153 FcγR3/CD16 Activating >455 >455 >455
FcγR4/CD16-2 Activating 3 0.5 6E−07 FcRn pH 5.8 IgG, albumin recycling 6 6 5 FcRn pH 7.4 IgG,
albumin recycling 80 14 40 Human FcγR1/CD64 Activating 12 12 7 FcγR2A/CD32a Inhibitory 334 193
144 (R167) FcγR3A/CD16a Activating 20 9 2 FcγR3B/CD16b Activating >455 >455 >455 FcRn pH 5.8
IgG, albumin recycling 49 49 44 FcRn pH 7.4 IgG, albumin recycling >455 >455 >455 Rat
FcγR1/CD64 Activating 7 3 2 FcγR2A/CD32a Inhibitory >455 >455 1 FcγR2B/CD32b Inhibitory 561 49
208 FcγR3A/CD16a Activating 6 3 0.008 FcRn pH 5.8 IgG, albumin recycling 25 15 13 FcRn pH 7.4
IgG, albumin recycling 104 >230 >230 Cynomolgus FcγR1/CD64 Activating 24 18 0.37 monkey
FcγR2A/CD32a Inhibitory 1995 688 693 FγR2B/CD32b Inhibitory 11 6 1 FcγR3/CD16 Activating >320
>320 >320 FcRn pH 5.8 IgG, albumin recycling 31 17 28 FcRn pH 7.4 IgG, albumin recycling 88 32 78

Example 173. 7-Day Mouse PK Study Comparing IV, SC and IM Administration of Conjugate 45b

(1453) Mouse PK studies were performed using male CD-i mice 6 weeks of age. Mice were injected
IV, SC and IM with 5 mg/kg of test article (5 ml/kg dose volume). Animals were housed under standard
IACUC approved housing conditions. At appropriate times animals were non-terminally bled (retro-
orbital, cheek, or by tail vein) with blood collected in K.sub.2EDTA tubes to prevent coagulation.
Collected blood was centrifuged (2,000×g, for 10 minutes) and plasma withdrawn for analysis of test
article concentrations over time. The plasma concentrations for Conjugate 45b at each time point were
measured by sandwich ELISA as follows: Conjugate 45b molecules were captured on neuraminidase
coated plates and then detected using an HRP-conjugated anti-human IgG-Fc antibody. Protein
concentration was calculated in GraphPad Prism using 4PL non-linear regression of Conjugate 45b (or
hIgG1 Fc) standard curves. A more detailed method description is provided above. The curves
comparing Conjugate 45b are shown in FIG. 84, and the curves comparing hIgG1 Fc (SEQ ID NO: 73)
are shown in FIG. 85. The Conjugate 45b plasma exposure levels for SC and IM were comparable
and resulting bioavailability from either SC or IM routes were approximately 77% compared to IV
administration (Tables 128-130). Conjugate 45b plasma concentrations were equivalent for all routes
at approximately 24 h post-injection.

(1454) TABLE-US-00134 TABLE 128 Mouse PK 5 mg/kg IV administration Time (hr) Mouse 0.0833 1
3 5 24 48 72 96 168 Dose Conc (mg/kg) Route Animal (ug/mL) 5 IV Mean 35.8 30.1 20.8 23 11.1 11.3
6.9 8.08 5.21 Mouse AUClast Half- CI_obs Dose Tmax C0 Cmax (hr * AUCINF_obs life (mL/ Vss_obs
Vz_obs (mg/kg) Route Animal (hr) (ug/mL) (ug/mL) ug/mL) (hr * ug/mL) (hr) min/kg) (mL/kg) (mL/kg) 5
IV Mean 0.0833 36.4 35.8 1600 2560 128 0.0325 336 361

(1455) TABLE-US-00135 TABLE 129 Mouse PK 5 mg/kg IM administration Time (hr) 0.25 1 3 5 24 48
72 96 168 Dose Conc Tmax Cmax AUClast AUCINF_obs (mg/kg) Route Animal (ug/mL) (hr) (ug/mL)
(hr * ug/mL) (hr * ug/mL) 5 IM Mean 0.195 1.44 3.69 3.83 8.52 10.9 7.94 7.35 5.56 48 10.9 1240 2720

(1456) TABLE-US-00136 TABLE 130 Mouse PK 5 mg/kg SC administration Time (hr) 0.25 1 3 5 24 48
72 96 168 Dose Conc Tmax Cmax AUClast AUCINF_pred (mg/kg) Route Animal (ug/mL) (hr) (ug/mL)
(hr * ug/mL) (hr * ug/mL) 5 SC Mean 0.148 0.387 3.23 2.55 8.89 9.29 9.13 7.49 5.46 48 9.29 1220
2280

Example 174. 7-Day Mouse PK Study Comparing Dose Linearity of Conjugate 45b

(1457) Mouse PK studies were performed using male CD-1 mice 6 weeks of age. Mice were injected
SC with 1, 3, 10, 30, or 100 mg/kg of test article (5 m1/kg dose volume). Animals were housed under
standard IACUC approved housing conditions. At appropriate times animals were non-terminally bled
(retro-orbital, cheek, or by tail vein) with blood collected in K2EDTA tubes to prevent coagulation.
Collected blood was centrifuged (2,000×g, for 10 minutes) and plasma withdrawn for analysis of test
article concentrations over time. The plasma concentrations for Conjugate 45b at each time point were
measured by sandwich ELISA as follows: Conjugate 45b molecules were captured on neuraminidase
coated plates and then detected using an HRP-conjugated anti-human IgG-Fc antibody. Protein
concentration was calculated in GraphPad Prism using 4PL non-linear regression of Conjugate 45b (or
hIgG1 Fc) standard curves. A more detailed method description is provided above. The curves
comparing Conjugate 45b detected by NA and Fc capture are shown in FIGS. 86 and 87, respectively.
In mouse, reasonable linear dose proportionality from 1 to 100 mg/kg SC administration of Conjugate
45b was observed (Table 131).

(1458) TABLE-US-00137 TABLE 131 Mouse PK dose proportional from 1-100 mg/kg Time (hr) 0.25
0.5 1 2 4 24 96 120 168 Dose Conc (mg/kg) Route Animal (ug/mL) 1 SC 1 0.83 Missing 5.9 0.764
9.39 4.11 1.1 2.13 0.317 3 SC 3 0.355 0.0874 1.02 0.52 5.16 11.4 9.84 8.32 6.84 10 SC 10
1.01 0.879 1.81 4.65 15.5 41.3 18.4 26.7 15 30 SC 30 0.179 0.616 5.71 7.6 56 125 75 83.7 49.7
100 SC 100 22.4 72.3 313 446 733 657 183 246 160 Dose Dose Tmax Cmax AUClast AUCINF_pred
(mg/kg) D Route (mg) (hr) (hr * ug/mL) (hr * ug/mL) (hr * ug/mL) 1 1 SC 1 4 9.39 436 465 3
3 SC 3 24 11.4 1520 2900 10 10 SC 10 24 41.3 4280 6650 30 30 SC 30 24 125 14200
22300 100 100 SC 100 4 733 60700 75300

Example 175. 14-Day Rat PK Study Following IV or SC Administration of Conjugate 45b

(1459) Rat PK studies were performed by Seventh Wave Laboratories (Maryland Heights, Mo.) using
male Sprague Dawley Rats 46-49 days of age. Rats were injected IV via the tail vein with 5 mg/kg
Conjugate 45b or SC with 5 or 50 mg/kg of test article (5 m1/kg dose volume) (FIGS. 88-89). Animals
were housed under standard IACUC approved housing conditions. At appropriate times animals were
non-terminally bled (retro-orbital, cheek, or by tail vein) with blood collected in K2EDTA tubes to
prevent coagulation. Collected blood was centrifuged (2,000×g, for 10 minutes) and plasma withdrawn
for analysis of test article concentrations over time (Table 132). The plasma concentrations for
Conjugate 45b at each time point were measured by sandwich ELISA as follows: Conjugate 45b
molecules were captured either on neuraminidase coated plates and then detected using an HRP-
conjugated anti-human IgG-Fc antibody (Tables 133-134). hIgG1 was captured using anti-hIgG1 Fc
antibody. Protein concentration was calculated in GraphPad Prism using 4PL non-linear regression of
Conjugate 45b standard curves. A more detailed method description is provided above. The curves
comparing Conjugate 45b shown in FIG. 88 demonstrate linear dose proportionality. Conjugate 45b
plasma levels for IV and SC administration converge at 24 hr post injection (FIG. 89). Data are shown
in Tables 135 and 136.
(1460) TABLE-US-00138 TABLE 132 Rat PK Study Design Plasma Collection Time Points Dose/ 10
min IV; 1 4 8 24 72 120 168 336 Dose path 30 min SC h h h h h h h h Group 1 5 mpk IV X X X X X X
X X X (n = 3) Group 2 5 mpk SC X X X X X X X X X (n = 3) Group 3 50 mpk SC X X X X X X X X X (n
= 3)

(1461) TABLE-US-00139 TABLE 133 H1N1 (A/CA/04/2009) NA Capture 5 mpk IV 5 mpk SC Time
g1.1 g1.2 g1.3 g2.1 g2.2 (hr) n1 n2 n1 n2 n1 n2 n1 n2 n1 10 m/30 m 139.0184 116.434 128.9835
126.3728 99.90003 96.30608 0.336397 0.352108 0.26567 1 103.0288 107.5882 108.6186 112.708
80.42177 83.55724 0.601405 0.600861 0.50378 4 67.18744 70.49817 65.26259 59.96993 52.09719
60.13481 3.331236 3.577401 3.291326 8 49.96849 56.19489 56.94676 57.68125 48.90448 55.23444
9.120017 8.164349 6.224308 24 33.99514 33.83988 34.76042 33.60106 33.04519 32.52448
24.64741 22.57097 19.34957 72 24.03197 23.99513 26.1422 25.50985 23.92523 24.53129 26.81972
23.95593 19.1652 120 17.28743 17.88652 18.16672 17.61109 17.14011 16.81636 18.13665
18.07425 15.58847 168 16.37894 14.18949 15.0406 14.28349 12.29568 13.27299 16.36903
16.31503 13.37669 336 8.706758 9.475766 8.069783 8.240227 7.314977 7.120923 8.57262
8.694249 6.866837 5 mpk SC 50 mpk SC Time g2.2 g2.3 g3.1 g3.2 g3.3 (hr) n2 n1 n2 n1 n2 n1 n2 n1
n2 10 m/30 m 0.24961 0.259523 0.205043 1.111022 1.021191 2.951729 3.184274 3.78442 3.30374 1
0.53983 0.680512 0.48924 2.858977 6.907325 4.617704 7.575 8.21763 4 3.31843 3.942251
3.945549 19.50094 17.82466 17.83342 19.84081 27.2367 27.0064 8 6.16066 7.856424 7.047854
43.56345 44.30664 55.25942 53.41513 58.2069 56.4707 24 21.3297 20.90625 23.61215 156.8759
159.158 210.1323 209.9947 208.024 181.09 72 23.1289 18.91189 16.60657 142.6993 137.0533
152.6497 155.1297 149.881 142.385 120 19.193 16.70847 16.4339 117.7776 112.064 136.8244
141.3221 137.704 132.748 168 14.9629 12.9219 12.3945 100.8302 116.8275 99.00073 118.0093
97.4236 96.2287 336 7.0874 7.635397 7.876635 53.87703 51.16129 57.02179 55.22173 54.121
59.193

(1462) TABLE-US-00140 TABLE 134 hIgG Fc Capture 5 mpk IV 5 mpk SC Time g1.1 g1.2 g1.3 g2.1
g2.2 (hr) n1 n2 n1 n2 n1 n2 n1 n2 n1 10 m/30 m 131.8211 132.2644 138.5205 124.2897 92.28536
93.95777 0.301641 0.272994 0.3302 1 105.1676 118.2516 111.4847 110.7111 83.06342 83.13157
0.492532 0.510306 0.372862 4 61.3296 59.41234 59.52775 47.35522 53.9032 61.12485 3.615572
3.792592 3.297844 8 50.28953 48.15108 52.90292 48.1017 48.46983 8.261891 9.08554 6.598579 24
29.91591 30.67022 30.25926 31.29131 28.39885 29.88579 23.96117 19.31521 16.86928 72
19.38442 21.26014 20.06189 20.38354 17.46987 18.56471 19.96086 21.67188 15.66477 120
12.13442 13.49033 12.39415 11.80685 13.47915 12.68468 14.71684 15.61295 11.56458 168
14.61786 14.48309 13.2966 13.12374 12.4622 13.06133 13.73621 9.73 11.21421 336 8.33093
8.680053 7.88281 7.805351 6.912784 6.784823 7.736292 7.661658 6.466908 5 mpk SC 50 mpk SC
Time g2.2 g2.3 g3.1 g3.2 g3.3 (hr) n2 n1 n2 n1 n2 n1 n2 n1 n2 10 m/30 m 0.58892 0.071346
0.209457 0.85958 2.600291 2.362582 2.417453 3.26701 3.24606 1 0.35967 0.267192 0.28284
2.328096 2.360296 3.618227 3.5931 7.33632 7.4207 4 3.46322 4.157923 4.501974 20.52563
19.67262 19.46794 19.46851 28.6829 31.5564 8 5.92739 8.778635 7.990053 45.29742 44.30181
54.38999 63.46517 61.1055 70.8567 24 17.6982 17.00515 18.2297 155.7401 138.399 200.935
236.9577 239.536 249.133 72 16.3864 19.15136 19.56853 152.6623 171.8249 181.134 173.9026
186.293 194.744 120 11.8629 14.66388 14.79211 113.865 103.0581 131.7811 129.7576 153.859
149.209 168 12.2178 14.54814 12.79694 94.56107 112.0218 105.5593 101.004 104.147 113.431 336
6.00365 7.32696 6.554063 49.96583 43.11898 58.15043 57.84531 54.516 49.5564

(1463) TABLE-US-00141 TABLE 135 Rat PK 5 mg/kg IV administration Time (hr) Rat 0.167 1 4 8 24
72 120 168 336 Dose Conc (mg/kg) (ug/mL) 5 118 99.3 62.5 54.2 33.6 24.7 17.5 14.2 8.15 Rat Dose
Tmax C0 Cmas AUClast AUCINF_obs Half-life CI_obs Vss_obs Vz_obs (mg/kg) (hr) (ug/mL) (ug/mL)
(hr * ug/mL) (hr * ug/mL) (hr) (mL/min/kg) (mL/kg) (mL/kg) 5 0.167 122 118 6340 8690 199 0.00959
144 166

(1464) TABLE-US-00142 TABLE 136 Rat PK 5 mg/kg and 50 mg/kg SC administration Time (hr) 0.5 1
4 8 24 72 120 168 336 Rat Conc Tmax Cmax AUClast AUCINF_obs Dose (mg/kg) Route Animal
(ug/mL) (hr) (ug/mL) (hr * ug/mL) (hr * ug/mL) 5 SC Mean 0.278 0.569 3.57 7.43 22.1 21.4 17.4 14.4
7.79 24 22.1 4860 6970 50 SC Mean 2.56 6.04 21.5 51.9 188 147 130 105 55.1 24 188 35800 49800
Example 176. 14-Day Non-Human Primate PK Study Following SC Administration of Conjugate 45b

(1465) Non-human primate (NHP) PK studies were performed by Charles River using male and
female Cynomolgus monkeys 4.5-8 years old with body weights ranging from 2.5-6.5 kg. NHPs were
injected SC with 5 or 20 mg/kg of test article (5 m1/kg dose volume) on day 1 and 8. Animals were
housed under standard IACUC approved housing conditions. At appropriate times animals were non-
terminally bled (via femoral or cephalic veins) with blood collected in K2EDTA tubes to prevent
coagulation. Collected blood was centrifuged (2,000×g, for 10 minutes) and plasma withdrawn for
analysis of test article concentrations over time (Table 137). The plasma concentrations for Conjugate
45b at each time point were measured by sandwich ELISA as follows: Conjugate 45b molecules were
captured on neuraminidase coated plates and then detected using an HRP-conjugated anti-human
IgG-Fc antibody (FIG. 90-91). Protein concentration was calculated in GraphPad Prism using 4PL
non-linear regression of Conjugate 45b standard curves. A more detailed method description is
provided above. The curves comparing Conjugate 45b are shown in FIG. 92. The dose response is
linear between 5 and 20 mg/kg SC. Accumulation of Conjugate 45b was observed following the
second administration of Conjugate 45b on day 8.

(1466) TABLE-US-00143 TABLE 137 Monkey PK high exposures after second weekly dose Time (hr)
0.0833 1 2 4 8 24 72 120 168 Dose Conc Tmax Cmax AUClast (mg/kg) Route Group Animal (ug/mL)
(hr) (hr * ug/mL) (hr * ug/mL) 5 SC D1 Mean blq 1.05 3.62 9.91 17.9 22.2 26.1 23.8 20 72 26.1 3800
D8 Mean 24.3 19.4 25.7 31 61 47.3 54.1 42.8 33.3 8 61 7740 20 SC D1 Mean blq 10.9 6.77 28.8 61.9
101 107 71.6 85.1 72 107 14600 D8 Mean 63.4 59.5 70.4 107 150 197 167 139 99.1 24 197 25400

Example 177. Efficacy of Conjugate 45b Against an Oseltamivir-Resistant Isolate in a Lethal Mouse
Influenza Model

(1467) Conjugate 45b was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Perth/261/2009) is a mouse-
adapted isolate that carries the H275Y mutation resulting in resistance to the neuraminidase inhibitor
oseltamivir.

(1468) The experiment comprised 9 groups of 5 mice. At day 0, all mice were challenged with
A/Perth/261/2009 (H1N1) at 2× the LD90 by intranasal inoculation in a volume of 50 μl, to mice lightly
anesthetized with isoflurane. Groups 1-8 received a single treatment by SC, 2 hours post challenge. In
addition to the vehicle (PBS) only group, human IgG1 (Fc alone) was included as an additional
negative control. Group 9 received Oseltamivir phosphate via oral delivery, starting 8 hours post
infection twice daily for 5 days (Table 138). All mice were monitored for survival (Table 139) and weight
loss (Table 140) for 14 days after challenge.

(1469) Mice treated with Conjugate 45b showed 100% survival against challenge by influenza
(A/Perth/261/2009) with single doses at 10, 3, 1, and 0.3 mg/kg. Furthermore, despite the small group
size (n=5) these results were statistically significant relative to the vehicle control (Table 139). No mice
survived to the end of the study if dosed with vehicle (PBS), and only 20% survived if treated with
hIgG1 Fc only. The oseltamivir group had no survivors despite treatment with a dose shown to be
protective against oseltamivir-sensitive isolates previously (20 mg/kg, bid×5 days). These results
confirm that the challenge virus is resistant to oseltamivir, and sensitive to Conjugate 45b.

(1470) The potency of Conjugate 45b against influenza containing the H275Y mutation was further
supported by body weight data (Table 140). Groups receiving a single dose of Conjugate 45b at
concentrations of 1 mg/kg or more demonstrated 3%, or less, transient weight loss which was
recovered by study end.

(1471) TABLE-US-00144 TABLE 138 Study design Group Challenge Dose Treatment n = 5 Day 0
Compound (mg/kg) Route/Schedule 1 Influenza A virus Vehicle (PBS) N/A SC, q.d. 2 hours 2 (H1N1)
Fc alone 10 post-challenge 3 A/Perth/261/2009 Conjugate 45b 10 4 via IN route. Conjugate 45b 3 5
Conjugate 45b 1 6 Conjugate 45b 0.3 7 Conjugate 45b 0.1 8 Conjugate 45b 0.03 9 Oseltamivir 20 PO,
b.i.d. (Tamiflu ™) 8 hours after challenge for 5 days
(1472) TABLE-US-00145 TABLE 139 Percent Survival Test agent (mg/kg) hIgG1 Fc oseltamivir
Conjugate 45b (mg/kg) Day Vehicle (10) (200) 10 3 1 0.3 0.1 0.03 0 100 100 100 100 100 100 100
100 100 1 100 100 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 100 3 100 100
100 100 100 100 100 100 100 4 100 80 80 100 100 100 100 100 80 5 100 60 60 100 100 100 100 80
40 6 80 60 0 100 100 100 100 0 0 7 0 40 0 100 100 100 100 0 0 8 0 20 0 100 100 100 100 0 0 9 0 20
0 100 100 100 100 0 0 10 0 20 0 100 100 100 100 0 0 11 0 20 0 100 100 100 100 0 0 12 0 20 0 100
100 100 100 0 0 13 0 20 0 100 100 100 100 0 0 14 0 20 0 100 100 100 100 0 0 Significance na 0.665
0.0035 0.0035 0.0035 0.0035 0.0035 ns ns relative to vehicle

(1473) TABLE-US-00146 TABLE 140 Percent Body Weight (grams) Test agent (mg/kg) hIgG1 Fc
oseltamivir Conjugate 45b (mg/kg) Day Vehicle (10) (200) 10 3 1 0.3 0.1 0 100 100 100 100 100 100
100 100 1 101 101 102 99 97 98 96 97 2 98 100 99 101 99 102 99 98 3 91 93 89 101 95 97 94 89 4
89 88 84 101 95 97 95 84 5 89 89 82 104 100 101 98 83 6 79 85 74 100 94 97 93 74 7 78 83 105 100
102 97 8 84 105 100 103 98 9 85 101 98 101 96 10 90 103 98 101 98 11 92 102 98 101 97 12 99 105
103 103 102 13 101 106 103 103 102 14 98 104 101 101 101

Example 178. Efficacy of Conjugate 45b Against a Second Oseltamivir-Resistant Isolate in a Lethal
Mouse Influenza Model

(1474) In a study similar to that shown in Example 177, conjugate 45b was tested for activity against a
second influenza A (H1 N1) isolate carrying the H275Y mutation conferring resistance to oseltamivir.
For this study the mutation was carried in A/Texas/23/2012. As before, BALB/c mice (Charles River; 6-
8 weeks) were used and challenged intranasally with 2× the LD95 (5E4 pfu/mouse). Oseltamivir was
dosed orally at 20 mg/kg, bid for 5 days starting 2 hours after viral challenge. All other test articles
were dosed SC, as listed in Table 141. Animals were monitored for 14 days, and body weights (BW)
were recorded daily. If an animal reached 20% BW loss it was recorded as a mortality.

(1475) TABLE-US-00147 TABLE 141 Study design Influenza Test Route/ Dose N Group strain Article
Schedule (mg/kg) (balb/c) 1 A/Texas/23/2012 PBS SC, T + 2 hrs — 5 2 pdm (H275Y) hIgG1 SC, T + 2
hrs 3 5 3 (H1N1) Oseltamivir PO, bid × 5 (T + 2 h) 20 5 4 5E4 PFU/ Conjugate 45b SC, T + 2 hrs 3 5 5
mouse Conjugate 45b SC, T + 2 hrs 1 5 6 Conjugate 45b SC, T + 2 hrs 0.3 5 7 Conjugate 45b SC, T +
2 hrs 0.1 5

(1476) Similar to the previous study, neither vehicle nor the hIgG1 Fc (SEQ ID NO: 73) only controls
afforded protection from lethal challenge (20 and 0% survival respectively; Table 142). In contrast,
groups treated with Conjugate 45b at concentrations ranging from 0.3 to 3.0 mg/kg were fully
protected from lethal challenge. Furthermore, conjugate 45b at 0.1 mg/kg demonstrated partial
protection, with a 60% survival rate over the 14 days of the study. However a 200 mg total dose of
oseltamivir failed to protect mice from viral challenge as expected. BW data (Table 143; recorded until
the first death within a group) mirrored the survival results with only transient loss of weight seen in
protected groups (Conjugate 45, 0.3 to 3.0 mg/kg). The oseltamivir mutation (H275Y) tested in this
study is a clinically relevant mutation which is shown by this study to be highly susceptible to
Conjugate 45b.

(1477) TABLE-US-00148 TABLE 142 Percent Survival Test agent (mg/kg) hIgG1 oseltamivir
Conjugate 45b (mg/kg) Day Vehicle Fc (1) (200) 3 1 0.3 0.1 0 100 100 100 100 100 100 100 1 100
100 100 100 100 100 100 2 100 100 100 100 100 100 100 3 100 100 100 100 100 100 100 4 100 100
100 100 100 100 100 5 20 60 20 100 100 100 60 6 20 0 0 100 100 100 60 7 20 0 0 100 100 100 60 8
20 0 0 100 100 100 60 9 20 0 0 100 100 100 60 10 20 0 0 100 100 100 60 11 20 0 0 100 100 100 60
12 20 0 0 100 100 100 60 13 20 0 0 100 100 100 60 14 20 0 0 100 100 100 60

(1478) TABLE-US-00149 TABLE 143 Percent Body Weight (grams) Test agent (mg/kg) hIgG1
oseltamivir Conjugate 45b (mg/kg) Day Vehicle Fc (1) (200) 3 1 0.3 0.1 0 100 100 100 100 100 100
100 1 100 99 97 99 98 97 98 2 95 93 92 96 96 93 94 3 88 85 84 93 90 85 86 4 82 80 78 95 90 84 80 5
78 75 94 90 84 79 6 96 94 85 7 101 97 89 8 100 96 88 9 101 99 91 10 99 99 91 11 100 99 92 12 101
99 94 13 100 98 94 14 103 101 99
Example 179. Efficacy of Conjugate 45b Subcutaneously Dosed Against Influenza A/WSN/1933
(H1N1) in a Lethal Mouse Model

(1479) Conjugate 45b was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/WSN/1933) is a mouse-adapted
isolate capable of causing lethal infections in mice. The experiment comprised 7 groups of 5 mice. At
day 0, all mice were challenged with virus at 3× the LD95 by intranasal inoculation in a volume of 30 μl
(2E3 virus/mouse), after being anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg
respectively). Mortality and body weights were recorded daily for 21 days and any animal with a 20%
loss of body weight was scored as a death.

(1480) Test groups received a single subcutaneous (SC) treatment of conjugate 45b at 3, 1, 0.3, 0.1,
or 0.03 mg/kg, or hIgG1 Fc control, or vehicle (PBS) 2 hours post viral challenge in a dose volume of
10 ml/kg. The study design is summarized in Table 144.

(1481) TABLE-US-00150 TABLE 144 Study Design Influenza Test Route/ Dose N Group strain Article
Schedule (mg/kg) (balb/c) 1 A/WSN/1933 PBS SC, T + 2 hrs — 5 2 (H1N1) hIgG1 SC, T + 2 hrs 1 5 3
~2E3 PFU/ Conjugate 45b SC, T + 2 hrs 3 5 4 mouse Conjugate 45b SC, T + 2 hrs 1 5 5 Conjugate
45b SC, T + 2 hrs 0.3 5 6 Conjugate 45b SC, T + 2 hrs 0.1 5 7 Conjugate 45b SC, T + 2 hrs 0.03 5

(1482) As expected, mice receiving vehicle or the hIgG1 Fc control succumbed to infection on Day 7
or 8, respectively (Table i45). However, mice treated with conjugate 45b were fully protected at
concentrations down to 0.3 mg/kg, and 80% protection at 0.1. Complete loss of protection by
conjugate 45b was only seen at the lowest dose concentration of 0.03 mg/kg.

(1483) TABLE-US-00151 TABLE 145 Percent Survival Conjugate 45b (mg/kg) Day Vehicle hIgG1 Fc 3
1 0.3 0.1 0.03 0 100 100 100 100 100 100 100 1 100 100 100 100 100 100 100 2 100 100 100 100
100 100 100 3 100 100 100 100 100 100 100 4 100 100 100 100 100 100 100 5 100 100 100 100 100
100 100 6 40 60 100 100 100 100 100 7 0 20 100 100 100 100 100 8 0 0 100 100 100 100 40 9 0 0
100 100 100 80 0 10 0 0 100 100 100 80 0 11 0 0 100 100 100 80 0 12 0 0 100 100 100 80 0 13 0 0
100 100 100 80 0 14 0 0 100 100 100 80 0

(1484) The daily body weight measurements were consistent with survival observations. As expected,
mice treated with vehicle or hIgG1 Fc demonstrated a steady drop in body weight until it exceeded
20%, at which time they were scored as a mortality (Table 146).

(1485) In contrast to control mice, those groups receiving conjugate 45b at 3.1, and 0.3 mg/kg
maintained healthy body weights throughout the study and never demonstrated more than a transient
body weight drop of less than 3% (1 ink/kg dose group, Day 18; Table 146). By both survival and body
weight measurements conjugate 45b demonstrated robust protection from a lethal challenge of
Influenza A/WSN/1933 with a single SC dose as low as 0.3 mg/kg.

(1486) TABLE-US-00152 TABLE 146 Percent Body Weight Conjugate 45b (mg/kg) Day Vehicle hIgG1
Fc 3 1 0.3 0.1 0.03 0 100 100 100 100 100 100 100 1 96.8 96.6 98.5 98.9 99 96.9 99.4 2 99.9 98.1
100.9 99.9 100.5 101.2 100.7 3 92 90.8 101.1 100.6 99 97 94.8 4 83.5 85.3 98.4 98.2 96 94.6 92.4 5
79.7 82.3 99 102.5 99.2 94.2 91.5 6 76.1 78.8 101.2 105.4 102.1 90.9 87.3 7 100 102.8 101.6 84.8
82.3 8 98.4 101.9 100.8 81.9 76 9 102.2 102.8 101.6 84.3 10 101.9 103 101.7 11 101.3 102.1 99.5 12
99.5 99.9 99.3 13 100 101.8 101.7 14 102.1 102.3 102.7 15 99.8 101.6 101.6 16 100.1 102.1 103.1 17
98.7 98.7 100.5 18 99.3 97.8 99.4 19 98.8 98.3 100.2 20 100.1 100.1 100.8 21 102.3 100.5 101.1

Example 180. Efficacy of Conjugate 45b Intravenously Dosed Against Influenza A/Texas/36/91 (H1N1)
in a Lethal Mouse Model of Delayed Treatment

(1487) Conjugate 45b was evaluated against a lethal influenza A (H1N1) infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Texas/36/91) is a mouse-
adapted isolate capable of causing lethal infections in mice. At day 0, all mice were challenged with
virus at 2× the LD95 (˜75 virus/mouse) by intranasal inoculation in a volume of 50 μl, after being
anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg respectively). Mortality, clinical
signs, and body weight were recorded daily for 15 days and any animal with a 20% loss of body
weight was scored as a death.

(1488) The study design is detailed in Table 147, and consists of multiple arms. The control arm
comprises vehicle (PBS) and hIgG1 Fc only groups, dosed 24 hours after viral challenge (an
uninfected group was also part of this arm). The second arm consisted of oseltamivir dosed at 4× its
humanized dose, with initiation of treatment delayed for 24, 48, 72, or 96 hours. The final arms
consisted of conjugate 45b administered as a single IV doses at 1 or 3 mg/kg; each being dosed on
the same schedule as the oseltamivir arm above.

(1489) As expected, vehicle and hIgG1 Fc were not protective when dosed 24 hours after viral
challenge and resulted in complete mortality by Day 6. In this study, oseltamivir, at 4× the humanized
dose (200 mg/kg cumulative dose) was only partially efficacious when dosing was delayed 24 hours
(Table 148; 80% survival). At 48 hours post-challenge the efficacy of oseltamivir dropped even lower,
with only 40% of mice surviving to study end. If oseltamivir treatment was delayed until 72 or 96 hours
there was no protection.

(1490) In contrast, conjugate 45b was fully protective at 1 and 3 mg/kg when dosing was delayed 24
hours. At 48 hours post challenge the 3 mg/kg group was still fully protected, and the 1 mg/kg was
nearly so, with 80% survival. At 72 hours the 1 mg/kg also showed 80% protection however, the 3
mg/kg demonstrated only 20% protection. Collectively both the 1 and 3 mg/kg conjugate 45b dose
groups show superior potency to oseltamivir in this delayed treatment model as measured by survival.
Notably as well, the total dose of conjugate 45b was 1 or 3 mg/kg while oseltamivir was given at 200
mg/kg.

(1491) As expected, the weight data supports the survival findings (Table 149) and in general shows
conjugate 45b treated mice to have superior retention of body weight. Although not statistically
significant, weight is better retained in conjugate 45b treated mice, even though the oseltamivir-treated
groups received a much higher dose.

(1492) TABLE-US-00153 TABLE 147 Study design Treatment Group Challenge Dose Schedule (n = 5)
(Day 0) Test Article (mg/kg) (pi) Route Readout 1 Influenza A Vehicle N/A 24-hours Single Daily weight
virus, H1N1 (PBS) treatment and health 2 strain hIgG1 Fc 3 24-hours IV score 3 A/TX/36/91 via
Conjugate 3 24-hours monitoring for 4 IN route 45b 48-hours 15 days total. 5 72-hours (Day 0 − 6 96-
hours Day 14 pi) 7 1 24-hours % Survival 8 48-hours 9 72-hours 10 96-hours 11 Oseltamivir 20 24-
hours PO, bid for 12 phosphate 48-hours 5 days 13 72-hours 14 96-hours 15 Naïve mice: untreated
and uninfected

(1493) TABLE-US-00154 TABLE 148 Percent Survival Conjugate 45b (mg/kg) Conjugate 45b (mg/kg)
Oseltamivir (mg/kg) 1 3 20 Day Vehicle hIgG1 Fc 24 hrs 48 hrs 72 hrs 96 hrs 24 hrs 48 hrs 72 hrs 96
hrs 24 hrs 48 hrs 72 hrs 96 hrs 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 1 100
100 100 100 100 100 100 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 100 100
100 100 100 100 3 100 100 100 100 100 100 100 100 100 100 100 100 100 100 4 80 100 100 80 100
100 100 100 100 100 100 100 100 100 5 20 60 100 80 80 40 100 100 20 60 100 100 60 0 6 0 0 100
80 80 0 100 100 20 0 100 100 40 0 7 0 0 100 80 80 0 100 100 20 0 100 60 0 0 8 0 0 100 80 80 0 100
100 20 0 80 40 0 0 9 0 0 100 80 80 0 100 100 20 0 80 40 0 0 10 0 0 100 80 80 0 100 100 20 0 80 40 0
0 11 0 0 100 80 80 0 100 100 20 0 80 40 0 0 12 0 0 100 80 80 0 100 100 20 0 80 40 0 0 13 0 0 100 80
80 0 100 100 20 0 80 40 0 0 14 0 0 100 80 80 0 100 100 20 0 80 40 0 0

(1494) TABLE-US-00155 TABLE 149 Percent Body Weight Conjugate 45b (mg/kg) Conjugate 45b
(mg/kg) Oseltamivir (mg/kg) 1 3 20 Day Vehicle hIgG2 Fc 24 hrs 48 hrs 72 hrs 96 hrs 24 hrs 48 hrs 72
hrs 96 hrs 24 hrs 48 hrs 72 hrs 96 hrs Na ve 0 100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 1 98 100 98 98 98 100 103 102 100 101 98 101 99 99 99 2 96 102 97 97 99 99 104 103
100 100 99 99 99 99 102 3 88 93 92 90 91 90 99 95 92 92 94 93 89 89 103 4 83 87 93 88 85 85 99 92
85 85 95 92 86 84 101 5 78 81 95 91 83 79 103 96 79 80 94 91 82 78 101 6 73 79 98 93 87 76 105 97
91 80 91 88 83 103 7 100 95 87 106 102 92 88 84 81 103 8 101 99 93 108 105 99 91 88 104 9 98 98
96 103 103 100 97 91 104 10 100 99 98 105 105 100 100 93 105 11 100 98 98 103 105 100 101 91
104 12 101 100 99 104 107 99 102 93 106 13 100 99 100 105 106 99 101 94 105 14 101 100 101 107
107 102 103 96 105

Example 181. Safety of Conjugate 45b Evaluated in a 14-Day Cynomolgus Monkey Dose-Range
Finder Toxicity Study

(1495) Cynomolgus monkeys were administered either 5 mpk or 20 mpk, or 50 mpk of Conjugate 45b
by subcutaneous injection on days 0 and 7 of the study. Compared with vehicle controls, no significant
effects on body weight gain, organ weights, food consumption were observed at any dose tested.
Plasma exposures (measured by AUC) increased proportionally with dose. These preclinical safety
results are consistent with a high therapeutic index (54×), based on AUC ratios from the highest dose
in the toxicology study versus AUCs required for 28-day protection from influenza infection in lethal
mouse influenza models. No test article related adverse effects were observed at any dose tested. A
summary of observations is provided in Table 150.

(1496) TABLE-US-00156 TABLE 150 Summary of 14 day dose-range finder toxicity study Findings at
highest dose (50 mpk), Parameter compared to vehicle Clinical observations No findings Hematology
No change from vehicle Clinical Chemistry No change from vehicle Coagulation No change from
vehicle Urinalysis No change from vehicle lmmunophenoytyping No change from vehicle Cytokines No
change from vehicle Histopathology No findings

Example 182. Efficacy of Conjugate 45b Subcutaneously Dosed Against Influenza

A/California/07/2009 (H1N1) Pdm in a Non-Lethal Ferret Model

(1497) Influenza A infections in ferrets is generally considered to have similar pathogenesis to that
seen in humans. Therefore, conjugate 45b was tested in a non-lethal ferret model challenged with
influenza A/CA/07/09, and H1N1 pandemic strain of clinical relevance. Briefly, male ferrets (3-5
months old) were obtained from Tripe F farms and verified to lack antibodies against influenza A
H1N1. Each ferret received either Conjugate 45b, Vehicle (PBS), or hIgG1 Fc in a single intravenous
(IV) injection 24±2.0 hours prior to challenge or oseltamivir phosphate (20 mg/kg), by oral gavage
starting 4±0.5 hours prior to influenza virus inoculation. Administration of oseltamivir continued twice
daily (12±1.5 hours apart) for 5 days after virus inoculation. Test concentrations and the experimental
design are detailed in Table 151.

(1498) Ferrets were challenged intranasally with 1 E6 infectious particles of influenza

A/California/07/2009 (H1N1) in a volume of 0.5 mL after being anesthetized with ketamine (25 mg/kg)
and xylazine (2 mg/kg). Animals were also anesthetized as above for administration of test articles.
The duration of the study was 14 days and readouts consisted of daily body weights (BW), clinical
signs, temperature, and nasal washes (0.5 mL) or throat swabs at the times listed in Table 151. Viral
burden in nasal washes was determined using standard plaque assays with MDCK cells. Viral burden
of throat swabs was performed using standard qPCR methods. Plasma samples were also obtained
from group 6 animals (dosed with 3 mg/kg of conjugate 45b) at 5 minutes, 2, 24, 72, and 120 hours.
As detailed in Table 152, control animals treated with vehicle (PBS) or Fc only began losing BW
starting on Day 1 which peaked on Day 6 (10.8 and 10.1% respectively). A similar drop in BW was
also seen at the lowest conjugate 45b dose (0.3 mg/kg). In contrast, ferrets treated with conjugate
between 1 and 30 mg/kg showed an approximate 50% reduction in BW loss.

(1499) As predicted based on BW, conjugate 45b also demonstrated a reduction in viral burden from
nasal washes, the primary site of influenza infection in ferrets (Table 153 and Table 154). The efficacy
of conjugate 45b is most notably demonstrated in the Day 2 burdens in which a close to 2-Log
reduction is achieved at the highest dose relative to vehicle treated animals. Furthermore, the
reduction in viral titer with conjugate 45b is dose-responsive between 30 and 1 mg/kg. This trend is
largely repeated in the Day 4 titers, although slightly muted because the immune system plays a
greater role in non-lethal ferret models (as evident by the reduction in vehicle-treated titers between
Day 2 and 4).
(1500) Based on the two primary study readouts (nasal titers and BW), collectively these data
demonstrate the ability of conjugate 45b to reach the upper respiratory tract at therapeutic
concentrations in an important model mirroring human disease.

(1501) TABLE-US-00157 TABLE 151 Study design and dosing schedule Test Route/ Dose Nasal
Throat Group N Material Schedule (mg/kg) Washes Swabs 1 5 PBS IV, single @ — 2, 4, 6, 8 2, 4, 6, 8
(vehicle) T-24 hrs. 2 5 hIgG1 Fc IV, single @ 30 2, 4, 6, 8 — T-24 hrs. 3 5 Oseltamivir.sup.1 PO, bid ×
20 2, 4, 6, 8 — 5 days, @ −4 hrs. 4 5 Conjugate IV, single @ 30 2, 4, 6, 8 2, 4, 6, 8 45b T-24 hrs. 5 5
Conjugate IV, single @ 10 2, 4, 6, 8 2, 4, 6, 8 45b T-24 hrs. 6 5 Conjugate IV, single @ 3 2, 4, 6, 8 2, 4,
6, 8 45b T-24 hrs. 7 5 Conjugate IV, single @ 1 2, 4, 6, 8 2, 4, 6, 8 45b T-24 hrs. 8 5 Conjugate IV,
single @ 0.3 2, 4, 6, 8 — 45b T-24 hrs.

(1502) TABLE-US-00158 TABLE 152 Average Body Weight Changes by Day (% Initial) Day Group 1 2
3 4 5 6 7 8 9 10 11 12 13 14 G1 0.08 −4.34 −5.60 −6.15 −7.31 −10.80 −9.77 −7.61 −7.05 −5.67 −5.20
−5.53 −4.36 −4.23 G2 −1.90 −6.35 −7.19 −7.60 −8.02 −10.06 −9.71 −9.54 −7.71 −6.66 −5.94 −6.38
−7.32 −4.99 G3 1.78 −0.39 −0.90 −0.14 −0.90 −1.78 −1.00 −1.19 −2.57 0.04 0.39 0.10 0.22 2.15 G4
−1.59 −4.47 −3.26 −3.10 −3.81 −5.23 −5.34 −5.47 −5.51 −5.14 −5.12 −4.56 −5.23 −4.56 G5 0.41
−3.45 −2.95 −3.33 −2.90 −3.65 −4.03 −4.00 −4.46 −3.29 −3.29 −3.89 −3.06 −2.69 G6 −0.51 −6.03
−4.55 −3.43 −4.19 −5.72 −4.99 −5.90 −4.81 −4.09 −4.23 −2.51 −3.33 −2.69 G7 −0.47 −4.76 −5.29
−4.43 −5.97 −6.10 −5.95 −5.33 −4.98 −4.27 −4.17 −2.85 −4.17 −3.39 G8 0.10 −5.50 −7.74 −8.98
−9.76 −11.52 −9.16 −8.35 −7.73 −6.06 −4.63 −4.84 −4.53 −4.24

(1503) TABLE-US-00159 TABLE 153 Viral Burden in Nasal Washes (Log10) Day 2 Day 4 Day 6 Day 8
G1 6.12 4.36 Below Below LOD LOD G2 6.31 4.42 Below Below LOD LOD G3 5.80 — Below Below
LOD LOD G4 4.26 3.12 Below Below LOD LOD G5 4.34 3.37 Below Below LOD LOD G6 5.11 3.44
Below Below LOD LOD G7 5.62 3.84 Below Below LOD LOD G8 6.07 3.56 Below Below LOD LOD

(1504) TABLE-US-00160 TABLE 154 Change in Viral Burden Relative to Vehicle (G1) (Log 10) Day 2
Day 4 Day 6 Day 8 G1 0.00 0.00 na na G2 0.19 0.06 na na G3 −0.32 — na na G4 −1.86 −1.24 na na
G5 −1.79 −0.99 na na G6 −1.01 −0.92 na na G7 −0.50 −0.52 na na G8 −0.06 −0.80 na na

(1505) Temperature changes were also recorded (AM & PM) and are listed in Table 155. Temperature
changes over the course of the study largely support the efficacy of conjugate 45b seen with nasal
burdens and body weights. Most notably is the overall reduction in time animals show an elevated
temperature upon treatment with conjugate 45b or oseltamivir, relative to vehicle treated ferrets.

(1506) In this study animals were observed daily and scored for clinical symptoms of influenza, and
their severity. In this non-lethal model, sneezing was the dominant sign of illness recorded by
technicians (Table 156). Relative to vehicle treated ferrets, animals treated with conjugate 45b or the
positive control showed fewer instances of sneezing, which resolved quicker relative to group 1. For
clarity, the 0.3 mg/kg treatment group alongside vehicle and oseltamivir groups is graphed in FIG. 96.

(1507) In this study conjugate 45b demonstrated potent activity relative to vehicle treated animals by
all readouts (nasal burden, body weight, temperature, & clinical score). Importantly, conjugate 45b was
more potent than significantly higher doses of oseltamivir at reducing viral burden in nasal washes, the
primary readout for this study. The observed efficacy of conjugate 45b in an important model
recognized as mimicking human disease supports the therapeutic potential of this candidate.

(1508) TABLE-US-00161 TABLE 155 Temperature changes over the study Average Change in
Degrees (° C.) by Day and time (AM/PM) 1 1 2 2 3 3 4 4 5 5 6 7 8 9 10 11 12 13 14 Group (AM) (PM)
(AM) (PM) (AM) (PM) (AM) (PM) (AM) (PM) (AM) (AM) (AM) (AM) (AM) (AM) (AM) (AM) (AM) G1 0.8
0.9 0.2 0.5 0.8 0.2 0.1 −1.1 0.5 −0.1 0.5 0.6 0.0 0.6 0.2 0.4 0.5 0.3 −0.4 G2 1.2 0.8 −0.3 0.1 −1.1 0.7
0.5 −0.2 0.0 −0.4 0.8 0.6 0.5 0.5 0.0 0.8 0.5 0.5 −0.4 G3 −0.4 −0.1 0.8 0.0 −0.6 −0.3 0.5 −1.7 −0.3
−0.8 0.2 −0.1 −0.2 0.1 −1.2 −0.1 0.4 0.1 −0.1 G4 −0.9 −0.3 0.6 −0.8 −1.4 −0.5 0.2 −1.4 −0.9 −1.2 0.3
0.0 −0.4 0.5 0.3 0.1 0.1 0.3 −0.8 G5 −0.2 −0.1 0.7 −0.7 −0.8 −0.7 −0.4 −1.7 −0.1 −1.1 0.2 0.0 −0.2 0.1
−0.2 0.2 0.1 0.1 −0.4 G6 0.1 0.6 1.1 −0.1 −1.2 −0.8 0.7 −0.5 −0.7 −0.7 0.8 0.3 −0.4 0.2 0.4 0.1 0.1 0.5
−0.5 G7 −0.3 0.0 0.8 −0.5 −1.8 −0.4 −0.3 −1.1 −0.6 −0.9 −0.4 0.6 −0.1 −0.7 −0.2 0.1 0.3 0.5 0.1 G8
−0.1 0.6 0.8 0.1 −1.6 −0.4 0.3 −0.6 −0.2 −1.0 0.0 −0.3 0.1 −0.4 −0.2 0.2 0.1 0.2 −0.1

(1509) TABLE-US-00162 TABLE 156 Clinical Scores Number of animals with clinical signs per day
Group 1 2 3 4 5 6 7 8 9 10 11 12 13 14 G1 0 3 2 2 3 2 4 4 2 3 3 0 2 0 G2 0 1 2 1 1 3 2 2 3 3 0 0 0 0 G3
0 0 2 2 1 1 2 1 0 1 0 0 1 0 G4 0 2 1 2 3 1 1 1 1 2 1 0 0 0 G5 0 2 2 2 2 0 0 1 1 1 0 0 0 0 G6 0 1 1 2 1 0
2 0 0 3 0 0 0 0 G7 0 2 2 1 1 2 3 1 1 1 0 1 0 0 G8 0 1 2 1 1 3 1 1 1 0 1 0 0 0

Example 183. 5-Day PK Analysis of Conjugate 45b in Ferrets Following a Single IV Injection 3 mg/kg

(1510) Ferret PK studies were performed by IIT Research Institute (IITRI) using male ferrets 3-5
months old. Ferrets (n=5) were administered conjugate 45b by a single intravenous (IV) injection at 3
mg/kg (2 mL dose volume) 24 h prior to intranasal challenge with 1×10.sup.6 plaque forming units
(PFU) of A/California/07/2009 (H1N1) influenza virus. Ferrets were anesthetized priorto dosing and
virus challenge using a ketamine (25 mg/kg) and xylazine (2 mg/kg) mixture. Blood (˜0.5-1 mL) was
collected at 5 min, 2, 24, 72 and 120 hr post dosing and processed for plasma. Nasal washed (0.5 mL
in PBS) were collected at days 2, 4, 6, and 8. Conjugate 45b molecules were captured on
neuraminidase (NA) or Fc coated plates and then detected using an HRP-conjugated anti-human IgG-
Fc antibody. Protein concentration was calculated in GraphPad Prism using 4PL non-linear regression
of conjugate 45b standard curves. Mean plasma concentrations were used to calculate
pharmacokinetic parameters using Phoenix WinNonlin 7.0. A more detailed method description is
provided below.

(1511) In the NA-capture ELISA, Nunc Maxisorp 96-well plates (cat no. 12-565-136, ThermoFisher)
were coated with 0.1 U/well NA from A/California/04/2009 (H1N1) (cat no. 11058-VNAHC, Sino
Biological) in 1×KPL coating buffer (cat no 5150-0041, SeraCare). Plates were incubated at room
temperature for 1 hr on an orbital plate shaker (500 rpm). Serial dilutions of plasma samples were
plated and incubated at room temperature for 2 hrs with shaking (sample diluent: 0.5% BSA in PBS
0.025% Tween 20+nai{umlaut over (v)}e ferret plasma final concentration of 1:100; plasma was
excluded from nasal wash samples). Conjugate 45b standard curves ranging from 0.230 to 500 ng/mL
were run on each plate in duplicate. Following the 2 hr incubation, plates were washed 5× in 300 μL
PBS with 0.05% Tween 20. Conjugate bound to NA on the plates was then probed with an HRP
conjugated anti-human IgG Fc F(ab′)2 (cat no. 709-036-098, Jackson) diluted 1:2,000 in sample
diluent for 1 hr at room temp. Plates were then washed 8× in 300 μL PBS with 0.05% Tween 20 and
developed with TMB substrate for 7-8 minutes. The reaction was stopped with 1 N H.sub.2SO.sub.4.
Absorbance was read at 450 nm with an EnSpire multimode plate reader (PerkinElmer). Conjugate
45b in plasma samples was interpolated using GraphPad Prism Version 8 following nonlinear
regression analysis (Sigmoidal, 4PL analysis) of the standard curves. The resulting mean plasma
concentrations were then used to calculate pharmacokinetic parameters by non-compartmental
analysis using Phoenix WinNonlin 7.0.

(1512) In the Fc-capture ELISA, Nunc Maxisorp 96-well plates (cat no. 12-565-136, ThermoFisher)
were coated overnight at 4° C. with 0.1 μg/100 μL/well of mouse anti-human IgG (CH.sub.2 domain)
clone R.sub.10Z8E9 (cat no. MCA5748G, BioRad) in carbonate buffer (cat no. C3041,
MilliporeSigma). Plates were washed 5× with 300 μL/well PBST and blocked with 200 μL/well 5% non-
fat dry milk (cat no. 9999S, Cell Signaling Technology) in PBST for 1 hr at room temperature with
shaking on an orbital plate shaker (500 rpm). Serial dilutions of plasma samples were plated and
incubated at room temperature for 2 hrs with shaking (sample diluent: 2.5% non-fat dry milk in PBS
0.025% Tween 20+naïve ferret plasma final concentration of 1:100; plasma was excluded from nasal
wash samples). Conjugate 45b standard curves ranging from 0.03 to 55 ng/mL were run on each plate
in duplicate. Following the 2 hr incubation, plates were washed 5× with 300 μL/well PBST. Conjugate
bound to Fc on the plates was then probed with 100 μL/well of HRP conjugated anti-human IgG Fc
F(ab′)2 (cat no. 709-036-098, Jackson Immunoresearch) diluted 1:2,000 in sample diluent for 1 hr at
room temp with shaking. Plates were then washed 8× in 300 μL/well PBST and developed with 100
μL/well TMB substrate reagent (cat no. 555214, BD) for 7-8 minutes. The reaction was stopped with
100 μL/well 1N H.sub.2SO.sub.4 and the absorbance read at 450 nm with an EnSpire multimode plate
reader (PerkinElmer). Conjugate 45b in plasma samples was interpolated using nonlinear regression
analysis and PK parameters calculated as described above.

(1513) The PK profiles comparing conjugate 45b levels in plasma and nasal washes are shown in
FIGS. 93 and 94, respectively. Similar conjugate 45b plasma exposure levels were observed for the
NA- and Fc-capture ELISAs. The mean conjugate 45b ferret plasma concentration was ˜10 μg/mL at
day 5 following a single IV dose at 3 mg/kg. Levels in nasal washes were approximately 3-5% of levels
in plasma at matched timepoints.

Example 184. Efficacy of Conjugate 45b Subcutaneously Dosed Against Influenza A/PR/8/34 (H1N1)
in a High Viral Challenge Lethal Mouse Model

(1514) Conjugate 45b was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/PR/8/34) is a mouse-adapted
isolate capable of causing lethal infections in mice. The experiment comprised 10 groups of 5 mice in
2 experimental arms. At day 0, mice were challenged with virus at 2× the LD95 (1×; groups 1-5) by
intranasal inoculation in a volume of 30 μl or with 50× the LD95 (25×; groups 6-10), after being
anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg respectively). Mortality and body
weights were recorded daily for 14 days and any animal with a 20% loss of body weight was scored as
a death. Test groups received a single subcutaneous (SC) treatment of conjugate 45b at 3, 1, 0.3, or
0.1 mg/kg 2 hours post viral challenge in a dose volume of 10 m1/kg. The study design is summarized
in Table 157.

(1515) In the 1× study arm vehicle treated mice succumbed to the challenge virus on Day 6.
Conjugate 45b in contrast, demonstrated exceptional potency and fully protected 1× virally challenged
mice at the lowest dose (Table 158A). Body weight data (Table 159A) showed a transient loss of
weight which was largely recovered by study end for all conjugate 45b treated mice.

(1516) In the groups challenged with 25× more virus (50× the LD95) mice succumbed to infection
faster with 80% reaching mortality by Day 5, and 100% mortality by Day 6 (Table 158B). Mice treated
with conjugate 45b were fully protected at 1 or 3 mg/kg, while the 0.3 mg/kg group had a 60% survival
rate. Surprisingly, even with the greater viral challenge the 1 and 3 mg/kg treatment only showed a
modest drop in body weight and ended the study with a net weight gain (Table 159B).

(1517) This study demonstrates the exceptional potency of conjugate 45b even with challenge by very
high viral titers.

(1518) TABLE-US-00163 TABLE 157 General study design Influenza A Test Route, Schedule Dose
Group strain Article (T + 2 hours) (mg/kg) N 1 A/PR/8/34 Vehicle (PBS) SC, single — 5 2 (H1N1)
Conjugate 45b SC, single 3 5 3 3E2 Conjugate 45b SC, single 1 5 4 PFU/mouse Conjugate 45b SC,
single 0.3 5 5 via IN Conjugate 45b SC, single 0.1 5 6 A/PR/8/34 Vehicle (PBS) SC, single — 5 7
(H1N1) Conjugate 45b SC, single 3 5 8 7.5E3 Conjugate 45b SC, single 1 5 9 PFU/mouse Conjugate
45b SC, single 0.3 5 10 via IN Conjugate 45b SC, single 0.1 5

(1519) TABLE-US-00164 TABLE 158A Percent Survival (1x challenge) Conjugate 45b (mg/kg) Day
Vehicle 3 1 0.3 0.1 0 100 100 100 100 100 1 100 100 100 100 100 2 100 100 100 100 100 3 100
100 100 100 100 4 100 100 100 100 100 5 100 100 100 100 100 6 0 100 100 100 100 7 0
100 100 100 100 8 0 100 100 100 100 9 0 100 100 100 100 10 0 100 100 100 100 11 0
100 100 100 100 12 0 100 100 100 100 13 0 100 100 100 100 14 0 100 100 100 100

(1520) TABLE-US-00165 TABLE 158B Percent Survival (25x challenge) Conjugate 45b (mg/kg) Day
Vehicle 3 1 0.3 0.1 0 100 100 100 100 100 1 100 100 100 100 100 2 100 100 100 100 100 3 100
100 100 100 100 4 100 100 100 100 100 5 20 100 100 80 60 6 0 100 100 60 0 7 0 100
100 60 0 8 0 100 100 60 0 9 0 100 100 60 0 10 0 100 100 60 0 11 0 100 100
60 0 12 0 100 100 60 0 13 0 100 100 60 0 14 0 100 100 60 0

(1521) TABLE-US-00166 TABLE 159A Percent Body Weight (1x challenge) Conjugate 45b (mg/kg)
Day Vehicle 3 1 0.3 0.1 0 100 100 100 100 100 1 98.5 97.5 96.1 96.6 98.2 2 99.6 98.2 99.9 97.5
100 3 92.8 97.4 98.6 95.8 96.8 4 85.7 98.7 98.9 93.7 90.4 5 79 98.4 100.1 95.6 92.4 6 99.5 99.2
96.7 91.4 7 100.7 101.3 98.9 88.5 8 97.8 99.2 96.9 nd 9 102.9 103.2 100.7 92.1 10 101.4 101.4
99.7 95.9 11 100.9 101.5 99.6 96.7 12 102.1 104.5 100.9 98.8 13 102.7 102.6 101.8 99.3 14 101.9
103.4 101.1 98.6 nd = not done

(1522) TABLE-US-00167 TABLE 159B Percent Body Weight (25x challenge) Conjugate 45b (mg/kg)
Day Vehicle 3 1 0.3 0.1 0 100 100 100 100 100 1 97.2 96.6 97.4 97.6 98.6 2 95 99.6 100.9 98 98.7
3 nd nd nd nd nd 4 79.9 98.5 90.3 82.8 82.5 5 75.7 100.3 91.5 82.7 76.7 6 100.2 96.4 7 102.2
99.1 8 99.5 97.5 9 104.3 103 10 103.5 102.2 11 102.9 103.4 12 105.5 106.1 13 105.8 105.1 14
103.9 106 nd = not done

Example 185. Activity of Conjugate 45b Against High Path Influenza A (H5N1, H7N9) in a Cytopathic
Effects (CPE) Assay

(1523) An in vitro assay to determine the potency of conjugate 45b was conducted against BSL-3
(high path) influenza A, and generally followed standard procedures. Briefly, different concentrations of
test articles were mixed with virus (approximately 250 TC.sub.ID50) and allowed to incubate at 35° C.
for one hour. After incubation, the mixture was added to an 80-90% confluent monolayer of MDCK
cells. After a 90 minute incubation, cells were washed and the test article was re-applied. The
monolayer was subsequently overlaid with carboxymethylcellulose to minimize viral spreading and
was allowed to incubate for two days. After two days of culture, cells were washed with PBS and fixed
with 10% formalin. After fixation the MDCK monolayer was permeabilized with Triton X-100 and
immunostained with a mouse mAb against influenza nucleoprotein. Monolayers were read, and the
stained area per well was calculated to determine EC.sub.50/100 values.

(1524) The results of the study are summarized in Table 160 and demonstrate the potency of
conjugate 45b against highly pathogenic strains with pandemic potential. Importantly, conjugate 45b
generated IC.sub.50 values at, or below, 17 nM against four H5N1 and one H7N9 isolate. In contrast,
oseltamivir and zanamivir demonstrated incomplete coverage of the high path strains in this panel.
Most notably was their lack of activity against the important H7N9 isolate. Conjugate 45b, however,
was highly potent against this isolate (0.5 nM). These results suggest that the potential of conjugate
45b to treat pandemics caused by highly-virulent influenza to be superior to that of oseltamivir and
zanamivir, and roughly equal to baloxavir.

(1525) TABLE-US-00168 TABLE 160 In vitro activity of conjugate 45b against high path influenza
isolates. IC50 for viral replication (in nM) Conju- Conju- HA/NA gate gate Osel- Balox- Zanam-
Influenza type Type 45b 6 tamivir avir ivir A/Vietnam/ H5N1 5.3 0.5 168.7 1.7 16.9 1194/2004 (clade 1)
A/Indonesia/ H5N1 16.9 0.5 ≥300 1.7 16.9 05/2005 (clade 2.1) A/Turkey/ H5N1 1.7 0.5 168.7 1.7 5.3
turkey/1/2005 (clade 2.2) A/Hong Kong/ H5N1 0.5 1.7 ≥300 1.7 53.3 156/ (clade 0) A/Anhui/1/ H7N9
0.5 0.2 ≥300 5.3 ≥300 2013 A/Netherlands/ H1N1* 1.7 0.5 ≥300 1.7 ≥300 602/2009 *Seasonal
comparator

Example 186. 8-Day PK Analysis of Conjugate 45b in Ferret Nasal Washes Following a Single IV
Injection at 30, 10, 3, 1 or 0.3 mg/kg

(1526) Ferret PK studies were performed by IIT Research Institute (IITRI) using male ferrets 3-5
months old. Ferrets (n=5) were administered conjugate 45b by a single intravenous (IV) injection at
30, 10, 3, 1 or 0.3 mg/kg (2 mL dose volume) 24 h prior to intranasal challenge with 1×10.sup.6 plaque
forming units (PFU) of A/California/07/2009 (H1N1) influenza virus. Ferrets were anesthetized prior to
dosing and virus challenge using a ketamine (25 mg/kg) and xylazine (2 mg/kg) mixture. Nasal
washes were collected at days 2, 4, 6 and 8 post challenge by anesthetizing animals with ketamine
(25 mg/kg) and xylazine (2 mg/kg), injecting 0.5 mL of sterile PBS supplemented with penicillin (100
U/mL), streptomycin (100 μg/mL) and gentamycin (50 μg/mL) into each nostril, and collecting the
expelled fluid into a specimen cup. The volume of recovered nasal wash was recorded and the
concentration of conjugate 45b determined by Fc capture ELISA. Conjugate 45b molecules were
captured either on neuraminidase coated plates or anti-hIgG1 antibody coated plates and then
detected using an HRP-conjugated anti-human IgG-Fc antibody. hIgG1 was captured using anti-hIgG1
Fc antibody.

(1527) Briefly, Nunc Maxisorp 96-well plates (cat no. 12-565-136, ThermoFisher) were coated
overnight at 4° C. with 0.1 μg/100 μL/well of mouse anti-human IgG (CH.sub.2 domain) clone
R.sub.10Z8E9 (cat no. MCA5748G, BioRad) in carbonate buffer (cat no. C3041, MilliporeSigma).
Plates were washed 5× with 300 μL/well PBST and blocked with 200 μL/well 5% non-fat dry milk (cat
no. 9999S, Cell Signaling Technology) in PBST for 1 hr at room temperature with shaking on an orbital
plate shaker (500 rpm). Nasal wash samples were initially diluted 1:10 in matrix-matched diluent (1:10
dilution of nasal wash from naïve animals in PBS with 0.5% BSA and 0.025% Tween 20), then 3-fold
serially diluted and 100 μL/well added to plates and incubated at room temperature for 2 hr with
shaking. Standard curves for conjugate 45b, ranging from 0.03 to 55 ng/mL, were run on each plate in
duplicate. Following the 2 hr incubation, plates were washed 5× with 300 μL/well PBST. Conjugate 45b
bound to Fc on the plates was then probed with 100 μL/well of HRP conjugated anti-human IgG Fc
F(ab′)2 (cat no. 709-036-098, Jackson Immunoresearch) diluted 1:2,000 in sample diluent for 1 hr at
room temp with shaking. Plates were then washed 8× in 300 μL/well PBST and developed with 100
μL/well TMB substrate reagent (cat no. 555214, BD) for 7-8 minutes. The reaction was stopped with
100 μL/well 1 N H.sub.2SO.sub.4 and the absorbance read at 450 nm with an ENSPIRE® multimode
plate reader (PerkinElmer). Conjugate 45b in ferret nasal wash samples was interpolated using
GraphPad Prism Version 8 following nonlinear regression analysis (Sigmoidal, 4PL analysis) of the
standard curves. The PK profiles comparing conjugate 45b in ferret nasal washes are shown in FIG.
94 and FIG. 95. A dose proportional increase in conjugate 45b ferret nasal wash levels was observed
between 0.3 and 30 mg/kg IV. The 8-day time point for the 0.3 mg/kg cohort was below the limit of Fc
capture detection (1 ng/mL).

Example 187. Efficacy of Conjugate 45b Against Influenza A (HI NI) in a Lethal Mouse Influenza
Model, Dosed by Three Different Routes

(1528) Conjugate 45b was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/California/07/2009) is a
pandemic isolate capable of causing lethal infections in mice. The experiment comprised 10 groups of
5 mice. At day 0, all mice were challenged with virus at 3× the LD.sub.95 (˜3E4 virus/mouse) by
intranasal inoculation in a volume of 30 μl after being lightly anesthetized with ketamine/xylazine (100
and 10 mg/kg respectively). Groups received a single treatment of conjugate 45b or vehicle (PBS), 2
hours after challenge, either intravenously (IV), intramuscular (IM), or subcutaneously (SC) (Table
160). Mortality and body weight (BW) were monitored daily for 14 days. Any mouse losing more than
20% BW was scored as a mortality.

(1529) TABLE-US-00169 TABLE 160 General study design Route, Influenza A Test Schedule Dose
Group strain Article (T + 2 hrs.) (mg/kg) N 1 A/CA/07/09 Vehicle (PBS) IV, single — 5 2 (H1N1)
Conjugate 45b IV, single 1 5 3 3E4 PFU/ Conjugate 45b IV, single 0.3 5 4 mouse Conjugate 45b IV,
single 0.1 5 5 via IN Conjugate 45b IM, single 1 5 6 Conjugate 45b IM, single 0.3 5 7 Conjugate
45b IM, single 0.1 5 8 Conjugate 45b SC, single 1 5 9 Conjugate 45b SC, single 0.3 5 10 Conjugate
45b SC, single 0.1 5

(1530) In order to determine the potency of Conjugate 45b by different dose routes, matching
concentrations of conjugate (1, 0.3, and 0.1 mg/kg) were dosed either IV, IM, or SC. As measured by
survival, all dose routes were 100% efficacious by any dose route at 0.1 mg/kg, which previously was
determined to be the lowest effective dose when conjugate 45b was dosed SC (Table 161). However,
mice treated with vehicle reached 80% mortality by Day 6. When measured by survival, these data
demonstrate the equivalent potency of conjugate 45b regardless of dose route.

(1531) TABLE-US-00170 TABLE 161 Percent survival Conjugate 45b (Dose Route) (mg/kg) IV IM SC
Day Vehicle 1 0.3 0.1 1 0.3 0.1 1 0.3 0.1 0 100 100 100 100 100 100 100 100 100 100 1 100 100 100
100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 100 100 3 100 100 100 100 100
100 100 100 100 100 4 100 100 100 100 100 100 100 100 100 100 5 100 100 100 100 100 100 100
100 100 100 6 20 100 100 100 100 100 100 100 100 100 7 20 100 100 100 100 100 100 100 100 100
8 20 100 100 100 100 100 100 100 100 100 9 20 100 100 100 100 100 100 100 100 100 10 20 100
100 100 100 100 100 100 100 100 11 20 100 100 100 100 100 100 100 100 100 12 20 100 100 100
100 100 100 100 100 100 13 20 100 100 100 100 100 100 100 100 100 14 20 100 100 100 100 100
100 100 100 100

(1532) Body weight data strongly supports the survival data demonstrating that conjugate 45b is highly
potent by either dose route (Table 162). As typical for this highly virulent pandemic isolate, mice
showed a BW loss of approximately 10% around Day 3 or 4. Strikingly, the difference in BW between
the same dose concentrations varied only minimally by dose route (see italics, Table 162). BW data
from this study further supports the conclusion that conjugate 45b is efficacious regardless of dose
route.

(1533) TABLE-US-00171 TABLE 162 Percent body weight* Conjugate 45b (Dose Route) (mg/kg) IV
IM SC Day Vehicle 1 0.3 0.1 1 0.3 0.1 1 0.3 0.1 0 100 100 100 100 100 100 100 100 100 100 1 96.3
96.6 98.5 96.9 97.6 98 100 100.3 99.9 101.1 2 96.8 100.7 100.7 100.3 101.8 100.2 100.9 101.9 101.3
101.4 3 87.7 91.4 90.8 91.9 92.3 91.3 90.4 93.1 90.8 90.7 4 82.8 94.3 91.1 89.8 94.5 91.4 89 91.3 88
88.4 5 80.8 97.5 94.2 92.2 96.6 93.5 93.1 95.1 90 89.5 6 78.9 97.4 95.5 93.2 96.8 95.2 94.8 96.8 90.6
90.1 7 100.1 97.5 96.8 99.7 96.7 96.7 96.9 95 92.9 8 98.5 97 95.1 98.4 94.9 96.4 97.1 94.8 93.2 9
99.8 96.8 95.7 98.5 94.8 95.7 96.6 93.8 93.3 10 99.3 97.3 95.5 98.7 94.8 97.7 95.2 94.8 93.9 11 103.4
100.8 101.4 103.3 100.1 101.1 100.2 99.4 98.1 12 102.3 100.4 100.3 101.7 99.4 100.5 99.1 98.2 98
13 102.2 100 99.8 101.4 99.5 101.1 99 98.7 98.4 14 103.6 101.8 100.4 102.5 101.2 102.7 101.9 100.4
99.9 *Data are group average until first mortality in group

(1534) By two different readouts conjugate 45b was found to be equally efficacious if dosed by IV, SC,
or IM dose routes. The dose route flexibility of conjugate 45b is a significant advantage, allowing for
different formulations and dose routes for hospital and outpatient settings if necessary.

Example 188 Synthesis of Conjugate 46

(1535) Preparation of the Click reagent solution: 0.0050M CuSO.sub.4 in PBS buffer solution: 10.0 mg
CuSO.sub.4 was dissolved in 12.53 mL PBS, then took 5.00 mL this CuSO.sub.4 solution and added
43.1 mg BTTAA (CAS #1334179-85-9) and 247.5 mg sodium ascorbate to give the Click reagent
solution (0.0050M CuSO.sub.4, 0.020M BTTAA and 0.25M sodium ascorbate).

(1536) To a solution of azido functionalized Fc (65.5 mg, 10.0 mL, 1.13 μmol, azido DAR-5.9, SEQ ID
NO: 76) in a 15 mL centrifuge tube was added to alkyne derivatized small molecule (22.7 mg, 15.2
μmol, described in Int-83. Example 145, 3.0 equivalents per each azido of the Fc). After gently
agitating to dissolve all solids, the mixture was treated with the Click reagent solution (1.80 mL). The
resulting mixture was gently rotated for 12 hours at ambient temperature. It was purified by affinity
chromatography over a protein A column, followed size exclusion chromatography (see general
conjugate purification protocol). Maldi TOF analysis of the purified final product gave an average mass
of 66,420 Da (DAR=5.8). Yield 57 mg with 98% purity. The resulting conjugate is depicted in FIG. 102.

(1537) Applicant notes that Conjugate 46 may alternately be prepared using an Fc domain having the
amino acid sequence of SEQ ID NO: 77, corresponding to a difference in Fc allotype. The differing
allotypes are expected to behave the same with respect the properties described herein.

(1538) Applicant further notes that the nucleic acid construct encoding the Fc for Conjugate 46
included a nucleic acid encoding the amino acid sequence of SEQ ID NO: 67, which includes a C-
terminal lysine residue. Upon expression, the C-terminal lysine of the Fc of Conjugate 46 is
proteolytically cleaved, resulting in an Fc having the sequence of SEQ ID NO: 76. The presence or
absence of a C-terminal lysine does not alter the properties of the Fc or the corresponding conjugate.

Example 189. 30-Day Comparative Non-Human Primate PK Study Following IV Administration of

Conjugate 45b and Conjugate 46

(1539) Non-human primate (NHP) PK studies were performed by BTS Research (San Diego, Calif.)
using male and female cynomolgus monkeys 5-9 years old with body weights ranging from 3.5-8.5 kg.
NHPs were injected IV with 2 mg/kg of test article (0.4 mL/kg dose volume). Animals were housed
under standard IACUC approved housing conditions. At appropriate times animals were non-terminally
bled (via femoral or cephalic veins) with blood collected in K2EDTA tubes to prevent coagulation.
Collected blood was centrifuged (2,000×g, for 10 minutes) and plasma withdrawn for analysis of test
article concentrations over time (Table 163). The plasma concentrations for Conjugate 45b and
Conjugate 46 at each time point were measured by sandwich ELISA. Briefly, test articles were
captured on Fc-coated plates and then detected using a HRP-conjugated anti-human IgG-Fc antibody.
Protein concentrations were calculated in GraphPad Prism using 4PL non-linear regression of
Conjugate 45b or Conjugate 46 standard curves. A more detailed method description is provided
above. The curves comparing Conjugate 45b and Conjugate 46 are shown in FIG. 97. Conjugate 46
demonstrates a significantly improved terminal half-life of ˜45 days compared with ˜10 days for
Conjugate 45b. AUCs for Conjugate 46 are 2× greater than the AUCs for Conjugate 45b (Table 163).

(1540) TABLE-US-00172 TABLE 163 Monkey PK, Conjugate 45b vs. Conjugate 46 Time (hr) 0.25 4 8
24 72 120 168 240 336 672 AUClast Half− Dose Conc Tmax Cmax (hr * life (mg/kg) Route Conjugate
(ug/mL) (hr) (ug/mL) ug/mL) (hr) 2 IV Conjugate 45b Mean 32.6 24.8 20.1 14.1 9.97 7.61 6.33 4.47
3.62 1.47 0.25 32.6 3450 249 2 IV Conjugate 46 Mean 35.4 29 25.7 20.5 15.1 13 11.2 10.4 8.71 7.97
0.25 35.4 7210 1080

Example 190. Activity of Conjugate 45b and Standard of Care Comparators Against Influenza A
Seasonal, Pandemic, and Drug-Resistant Strains in a Cytopathic Effects (CPE) Assay

(1541) An in vitro assay to determine the potency of conjugate 45b compared to controls of
Oseltamivir, Zanamivir, and Baloxavir was conducted as performed in Example 166, and generally
followed standard procedures. Data are shown in Tables 164-167.

(1542) TABLE-US-00173 TABLE 164 CPE against influenza A/CA/07/2009 (H1N1) pdm in MDCK
SIAT1 cells MOI 0.001 MOI 0.01 Molecule EC.sub.50 [nM] EC.sub.50 [nM] Oseltamivir 18.54 34.2
Zanamivir 16.321 33.16 Conjugate 45b 0.8355 2.964 Baloxavir <0.3 2.216

(1543) TABLE-US-00174 TABLE 165 CPE against influenza A WT and H275Y mutant (H1N1) in
MDCK SIAT1 cells Influenza A/ Influenza A/ California/12/2012 Texas/23/2012 EC.sub.50 [nM] (H1N1)
(H1N1) H275Y Molecule MOI 0.001 MOI 0.01 MOI 0.001 MOI 0.01 Oseltamivir 107.6 1653 >10,000
>10,000 Zanamivir 54.59 558.1 327.7 >10,000 Conjugate 45b 0.7043 20.1 2.101 81.86 Baloxavir
3.718 4.924 3.318 21.24

(1544) TABLE-US-00175 TABLE 166 CPE against influenza A WT and E119V mutant (H3N2) in
MDCK SIAT1 cells Cytopathic Effect Influenza A/Washington/ Influenza A/Texas/ EC.sub.50 [nM]
12/2007 (H3N2) 12/2007 (H3N2) E119V Molecule MOI 0.001 MOI 0.01 MOI 0.001 MOI 0.01
Oseltamivir N/A 12.39 96.04 >10,000 Zanamivir N/A 29.57 50.17 940.1 Conjugate 45b N/A 0.0637
1.193 43.12 Baloxavir N/A 0.9758 5.196 27.87

(1545) TABLE-US-00176 TABLE 167 CPE [nM] against influenza B at MOI 0.01 Molecule TM DAR Fc
Central linker B/Florida/4/2006 B/Brisbane/60/2008 B/Malaysia/2506/2004 B/Colorado/6/2017
Oseltamivir N/A N/A N/A N/A 1203 1568 735.7 >10,000 Zanamivir N/A N/A N/A N/A 61.57 607.9 360.7
2746 Conjugate Int−83 4.2 SEQ ID 15 atom 10.78 7.291 6.252 25.93 45b NO: 73 Baloxavir N/A N/A
N/A N/A 20.95 46.84 61.33 100.9

Example 191. 14-Day Mouse PK Study Comparing Plasma and Epithelial Lining Fluid (ELF)
Concentrations of Conjugate 45b

(1546) Female BALB/c mice from Charles River Laboratories were allowed to acclimate for 5 days
prior to study commencement. Animals were housed 3-6 per cage with free access to food and water.
All procedures were performed to NeoSome IACUC policies and guidelines. Mice were injected
subcutaneously (SC) with 20 mg/kg of test article (10 mL/kg dose volume). At selected time points, 3
mice were euthanized by CO.sub.2 inhalation. Blood was collected through cardiac puncture into
K2EDTA tubes for plasma retention. Following blood collection, a bronchoalveolar lavage (BAL) was
performed by exposing the trachea, inserting a 23G tubing adaptor, and performing 2×0.5 mL flushes
with sterile 1×PBS pH 7.4. The recovered fluid volume was recorded and retained. Once the BAL
procedure was complete, the lungs were removed, weighed and stored at −80° C. Aliquots of the
plasma and BAL fluid (BALF) were decanted prior to −80° C. storage of the samples for use in a urea
quantification assay. The collected BALF was centrifuged at 12,000 RPM for 5 minutes at room
temperature to pellet the alveolar macrophages with both the pellet and supernatant stored at −80° C.
until shipment to sponsor. The plasma concentrations for conjugate 45b at each time point were
measured by indirect ELISA as described in detail above. Briefly, conjugate 45b molecules were
captured on neuraminidase (NA) coated plates and then detected using a HRP-conjugated anti-human
IgG Fcγ specific F(ab′)2. The same ELISA was performed on BALF harvested as described above.
Conjugate 45b plasma concentrations were calculated in GraphPad Prism using 4PL non-linear
regression of conjugate 45b standard curves. ELF volume and conjugate 45b concentration in ELF
was determined using urea as a dilution marker as described previously (Rennard et al., 1986 J Appl
Physiol 60:532-538). The curves comparing conjugate 45b to ELF levels are shown in FIG. 98. By 2 h
post injection, conjugate 45b epithelial lining fluid (ELF) levels are ˜60% of plasma exposure levels
(AUCs) across the rest of the time course indicating nearly immediate partitioning of conjugate 45b
from plasma to the ELF in the lung (FIG. 98, Table 168).

(1547) TABLE-US-00177 TABLE 168 Conjugate 45b plasma and ELF levels in mice over 2 weeks.
Time (hr) 1 2 4 8 24 48 72 120 168 336 Conc Tmax Cmax AUClast Group (ug/mL) (hr) (ug/mL) (hr *
ug/mL) ELF 5.61 29.9 70.6 98.4 149 105 94.2 49.5 47.4 16.1 24 149 19000 Plasma 30.7 63.9 110 180
197 178 144 104 87 29.4 24 197 32500

Example 192. 7-Day Mouse PK Study Comparing IV, SC and IM Administration of Conjugate 45b in
SCID Mice

(1548) Mouse PK studies were performed using male severe combined immunodeficient (SCID) mice
6 weeks of age which lack an adaptive immune system. Mice were injected IV, SC or IM with 5 mg/kg
of test article (10 mL/kg dose volume). Animals were housed under standard IACUC approved
housing conditions. At appropriate times animals were non-terminally bled (retro-orbital, cheek, or by
tail vein) with blood collected in K2EDTA tubes to prevent coagulation. Collected blood was
centrifuged (2,000×g, for 10 minutes) and plasma withdrawn for analysis of test article concentrations
overtime. The plasma concentrations for conjugate 45b at each time point were measured by ELISA
as described in detail above. Briefly, conjugate 45b molecules were captured on neuraminidase (NA)
coated plates and then detected using a HRP-conjugated anti-human IgG Fcγ specific F(ab′).sub.2.
For the Fc capture ELISA, hIgG1 was captured using an anti-hIgG1 Fc antibody and then detected
using a HRP-conjugated anti-human IgG Fcγ specific F(ab′).sub.2. Protein concentration was
calculated in GraphPad Prism using 4PL non-linear regression of conjugate 45b standard curves. The
curves comparing conjugate 45b PK profiles in SCID mice over 7 days are shown in FIGS. 99-100.
The conjugate 45b plasma exposure levels for SC and IM were comparable and the resulting
bioavailability from either SC or IM routes were approximately 77% compared to IV administration.
Conjugate 45b plasma concentrations were equivalent for all dose routes at approximately 24 h post-
injection.

Example 193. 7-Day Mouse PK Study Comparing SC Administration of Conjugate 45b Vs Conjugate
46

(1549) Mouse PK studies were performed using male CD-1 mice 6 weeks of age. Mice were injected
SC with 10 mg/kg of test article (10 mL/kg dose volume). Animals were housed under standard IACUC
approved housing conditions. At appropriate times animals were non-terminally bled (retro-orbital,
cheek, or by tail vein) with blood collected in K2EDTA tubes to prevent coagulation. Collected blood
was centrifuged (2,000×g, for 10 minutes) and plasma withdrawn for analysis of test article
concentrations over time. The plasma concentrations for conjugate 45b at each time point were
measured by indirect ELISA as described in detail above. Briefly, conjugate 45b molecules were
captured on neuraminidase (NA) coated plates and then detected using a HRP-conjugated anti-human
IgG Fcγ specific F(ab′).sub.2. Protein concentration was calculated in GraphPad Prism using 4PL non-
linear regression of conjugate 45b standard curves. The curves comparing the 7-day PK profiles of
conjugate 45b and conjugate 46 are shown in FIG. 101. The plasma exposure levels for conjugate 46
were approximately 50% greater than for conjugate 46. Compared to WT human IgG1, the half-life of
human IgG1 YTE Fc variant is known to be reduced in mice due to enhanced mouse FcRn binding at
neutral pH, which negates the improved binding to mouse FcRn at acidic pH (Dall'Acqua et al. 2002 J
Immunol 169:5171-5180).

Example 194. Combination Treatment of Conjugate 45b and Baloxavir

(1550) Cytopathic effect (CPE). A monolayer of MOCK Siat1 cells was infected with influenza A
subtypes at appropriate MOI varying between 0.01-1. Conjugate 45b was tested alone or in
combination with standard of care agent, e.g. baloxavir, at concentrations ranging between 1-1,000 nM
and incubated for 3 days for influenza A at 37° C., 5% CO.sub.2. CPE was determined by crystal violet
staining by reading absorbance at 595 nm. Data are shown in Tables 169-172. When used in
combination with baloxavir, conjugate 45b is effective at inhibiting viral replication at significantly lower
concentrations than when used alone (with reductions in EC.sub.50S of >10-fold), even when
baloxavir is present at concentrations below its EC.sub.50.

(1551) TABLE-US-00178 TABLE 169 Variation in Conjugate 45b EC.sub.50 in the presence of fixed
concentrations of baloxavir in CPE versus influenza A/CA/07/2009(H1N1) pdm in MDCK SIAT cells at
MOI 0.01 Molecule Baloxavir [nM] MOI 0.01 EC.sub.50 [nM] Conjugate 45 0 7.369 1 0.7347 2 <0.39 4
<0.39 8 <0.39 16 <0.39 32 <0.39 64 <0.39 EC.sub.50 of Baloxavir alone = 2.41 nM

(1552) TABLE-US-00179 TABLE 170 Variation in Conjugate 45b EC.sub.50 in the presence of fixed
concentrations of baloxavir in CPE assays versus influenza A/CA/07/2009(H1N1) pdm in MDCK SIAT
cells at MOI 0.1 Molecule Baloxavir [nM] MOI 0.1 EC.sub.50 [nM] Conjugate 45b 0 >100 (405.5) 1
9.169 2 4.671 4 <0.39 8 <0.39 16 <0.39 32 <0.39 64 <0.39 EC.sub.50 of Baloxavir alone = 7.95 nM

(1553) TABLE-US-00180 TABLE 171 Variation in Conjugate 45b EC.sub.50 in the presence of fixed
concentrations of baloxavir in CPE assays influenza A/Texas/71/2017 (H3N2) pdm in MDCK SIAT
cells at MOI 0.01 Molecule Baloxavir [nM] MOI 0.01 EC.sub.50 [nM] Conjugate 45b 0 45.93 1 5.242 2
2.429 4 0.8822 8 0.2175 16 0.02432 32 0.04907 64 0.1346 EC.sub.50 of Baloxavir alone = 8.238 nM

(1554) TABLE-US-00181 TABLE 172 Variation in Conjugate 45b EC50 in the presence of fixed
concentrations of baloxavir in CPE assays versus influenza A/Texas/71/2017 (H3N2) pdm in MDCK
SIAT cells at MOI 0.1 Molecule Baloxavir [nM] MOI 0.1 EC.sub.50 [nM] Conjugate 45b 0 30015 1 105
2 100.9 4 10.17 8 <0.39 16 <0.39 32 <0.39 64 <0.39 EC.sub.50 of Baloxavir alone = 17.31 nM

Example 195. Synthesis of Int-91

(1555) ##STR00799## ##STR00800## ##STR00801##

(1556) To a 0° C. stirring solution of the previously prepared-zanamivir derivative (1.993 g, 2.362

mmol, Int-22 described in Example 79) in methanol (30 mL) it was added DBU (2.4 mL). The
temperature was raised to ambient and after 1 h the reaction evolved exclusively to the desired
product. All the volatiles were removed by rotatory evaporation. The residue was purified by silica
column using an Isco CombiFlash liquid chromatography eluted with 0% to 30% methanol and
dichloromethane. Yield 1.584 g, 82%. Ions found by LCMS: [(M+H+Na)].sup.+=840.0,
[(M+H)].sup.+=818.2.

(1557) Step b.

(1558) ##STR00802##

(1559) To a 0° C. stirring solution of step a product (500 mg, 0.611 mmol) in pyridine (15.0 mL) it was
added tosyl chloride (146 mg, 0.764 mmol) 5.0 mL of dichloromethane) over 1 hour with the aid of a
syringe pump. Upon full consumption of tosyl chloride (HPLC monitoring) an equal aliquot of tosyl
chloride was added in the same fashion, and this addition was repeated an additional two times (total
equivalents of tosyl chloride at the end of the reaction was 5). The reaction was quenched with
methanol (0.5 mL), and all the volatiles were removed by rotatory evaporation. The residue was
purified by silica column using an Isco COMBIFLASH® liquid chromatography eluted with 0% to 30%
methanol and dichloromethane. Yield 0.429 g, 72%. Ions found by LCMS: [(M+H)].sup.+=972.2.

(1560) Step c.

(1561) ##STR00803##

(1562) To a stirring solution of step b product (547 mg, 0.563 mmol) in DMF (5.0 mL) was added 18-
crown-6 (59 mg, 0.225 mmol), and sodium azide (183 mg, 2.814 mmol), then the temperature was
raised to 50° C. Upon completion of the reaction by LCMS, all volatiles were removed by rotatory
evaporation. The residue was purified by silica column using an Isco COMBIFLASH® liquid
chromatography eluted with 0% to 30% methanol and dichloromethane. Yield 0.333 g, 70%. Ions
found by LCMS: [(M+H)].sup.+=843.2.

(1563) Step d.

(1564) ##STR00804##

(1565) To a stirring solution of step c product (548 mg, 0.650 mmol) in THE (8.0 mL) it was added N-
hydroxysuccinimidyl ester of cyclopropane acid (237 mg, 1.300 mmol) and trimethylphosphine (133
μL, 1.300 mmol). Upon completion by LCMS, all the volatiles were removed by rotatory evaporation.
The residue was purified by silica column using an Isco COMBIFLASH® liquid chromatography eluted
with 0% to 50% methanol and dichloromethane. Yield 0.488 g, 85%. Ions found by LCMS:
[(M+H)].sup.+=885.2.

(1566) Step e.

(1567) ##STR00805##

(1568) To a 0° C. stirring solution of step d product (488 mg, 0.551 mmol) in tetrahydrofuran and water
(2.0 mL) was added lithium hydroxide (14 mg, 0.606 mmol). Upon completion by LCMS, amberlite
IRN-77 was added until pH was found acidic. The mixture was filtered with the aid of ethyl acetate,
and the resin was discarded. All the volatiles were removed by rotatory evaporation and the crude
material was dissolved in dichloromethane (4.0 mL) and TFA (2.0 mL). Upon completion, all the
volatiles were evaporated by rotatory evaporation. The residue was purified by HPLC (0 to 40%
methanol and water, using 0.1% TFA as modifier). Yield 370 mg, 86%. Ions found by LCMS:
[(M+H)].sup.+=671.2.

Example 196. Synthesis of Conjugate 47

(1569) A solution of azido functionalized Fc (50 mg, 5.0 mL, 0.862 μmol, SEQ ID NO: 73, DAR-7.0)
was added to a 40 mL centrifuge tube containing alkyne functionalized small molecule (6.1 mg, 7.760
μmol, Int-91, prepared as described in Example 195). After gently shaking to dissolve all solids, it was
added a solution of L-ascorbic acid sodium (12.3 mg, 62.08 μmol), copper (II) sulfate (2.5 mg, 15.52
μmol), and BTTAA (26.7 mg, 62.08 μmol) in PBS 7.4 buffer (6.984 mL). The resulting mixture was
gently shaken overnight. It was purified by affinity chromatography over a protein A column, followed
size exclusion chromatography (see general conjugate purification protocol provided herein). Maldi
TOF analysis of the purified final product gave an average mass of 64,423 Da (DAR=6.9). Yield 35.3
mg, 70% yield.

(1570) The nucleic acid construct encoding the Fc for Conjugate 47 included a nucleic acid encoding
the amino acid sequence of SEQ ID NO: 64, which includes a C-terminal lysine residue. Upon
expression, the C-terminal lysine of the Fc of Conjugate 47 is proteolytically cleaved, resulting in an Fc
having the sequence of SEQ ID NO: 73. The presence or absence of a C-terminal lysine does not alter
the properties of the Fc or the corresponding conjugate.

Example 197. Synthesis of carbamate Int-92

(1571) ##STR00806## ##STR00807## ##STR00808##

(1572) Intermediate prepared as described at Example 21 (5.15 g, 12.74 mmol) was dissolved in
acetone (100 mL). Amberlite IRN-77 acidic resin was added, bringing the pH to ˜4, when measured
with pH paper. The reaction was heated until all starting material was consumed. Upon cooling, the
reaction was filtered, and the filtrate was concentrated by rotatory evaporation. The residue was
purified by silica column using an Isco COMBIFLASH® liquid chromatography eluted with 0% to 30%
methanol and dichloromethane. Yield 3.535 g, 62%. Ions found by LCMS: [(M+H+Na)].sup.+=467.2,
[(M+H-t-Bu)].sup.+=389.2, [(M+H-Boc)].sup.+=345.2.

(1573) Step b.

(1574) ##STR00809##

(1575) To a stirring solution of step a product (3.258 g, 7.391 mmol) and p-nitrophenol chloroformate
(2.979 g, 14.78 mmol) in pyridine (80 mL), was added DMAP (1.806 g, 14.78 mmol). After 18 h,
additional p-nitrophenol chloroformate (1.490 g, 7.391 mmol) and DMAP (0.903 g, 7.391 mmol) were
added. Upon reaction completion, all the volatiles were removed by rotatory evaporation. The residue
was taken up in DCM (250 mL), and filtered. The filtrate was washed with a 2 N solution of sulfuric
acid (3×100 mL), then a saturated solution of sodium bicarbonate (3×100 mL). The organics were
dried with brine (200 mL), then magnesium sulfate, filtered, and concentrated. The residue was
purified by silica column using an Isco COMBIFLASH® liquid chromatography eluted with 0% to 100%
acetone and hexanes. Yield 4.122 g, 91%. Ions found by LCMS: [(M+H+Na)].sup.+=632.1,
[(M+H)].sup.+=554.0, [(M+H-Boc)].sup.+=510.2.

(1576) Step c.

(1577) ##STR00810##

(1578) To a 0° C. stirring solution of step b product (4.00 g, 6.562 mmol) and DIPEA (2.400 mL, 13.78
mmol) in DCM (40 mL), was added propargyl-PEG4-amine. The temperature was raised to ambient
and stirring was continued until complete by LCMS. All the volatiles were removed by rotatory
evaporation. The residue was purified by silica column using an Isco COMBIFLASH® liquid
chromatography eluted with 0% to 50% methanol and dichloromethane. Yield 4.130 g, 90%. Ions
found by LCMS: [(M+H+Na)].sup.+=724.1, [(M+H-Boc)].sup.+=602.2.

(1579) Step d.

(1580) ##STR00811##

(1581) The product from step c (4.13 g, 5.885 mmol) was dissolved in acetic acid (24 mL) and water
(12 mL), then stirred until complete conversion was observed by LCMS. The reaction was
concentrated by rotatory evaporation. To a stirring solution of this residue (4.60 g, 5.340 mmol) in
pyridine (150 mL) was slowly added a solution of Tosyl-Cl (1.392 g, 7.299 mmol) in DCM (10 mL) by
syringe pump. Upon consumption of the Tos-Cl, and additional amount (1.392 g, 7.299 mmol) was
added. Upon complete conversion of the starting material, all the volatiles were removed by rotatory
evaporation. The residue was purified by silica column using an Isco COMBIFLASH® liquid
chromatography eluted with 0% to 50% methanol and dichloromethane. Yield 3.380 g, 71%. Ions
found by LCMS: [(M+H+Na)].sup.+=838.0, [(M+H-Boc)].sup.+=716.2.

(1582) Step e.

(1583) ##STR00812##

(1584) To a stirring solution of the step d tosylate (3.280 g, 4.020 mmol) in DMF (30 mL) was added
18-crown-6 (425 mg, 1.608 mmol), and sodium azide (1.307 g, 20.10 mmol), then the temperature
was raised to 50° C. Upon completion by LCMS, all the volatiles were evaporated by rotatory
evaporation. The residue was purified by silica column using an Isco COMBIFLASH® liquid
chromatography eluted with 0% to 50% methanol and dichloromethane. Yield 2.651 g, 96%. Ions
found by LCMS: [(M+H+Na)].sup.+=709.2, [(M+H-Boc)].sup.+=587.2.

(1585) Step f.

(1586) ##STR00813##

(1587) To a stirring solution of step e product (2.649 mg, 3.858 mmol) in THE (20 mL) was added N-
hydroxysuccinimidyl ester of cyclopropane acid (1.413 g, 7.715 mmol) and trimethylphosphine (795
μL, 7.715 mmol). Upon completion of the reaction by LCMS, all the volatiles were evaporated by
rotatory evaporation. The residue was purified by silica column using an Isco COMBIFLASH® liquid
chromatography eluted with 0% to 50% methanol and dichloromethane. Yield 1.744 g, 62%. Ions
found by LCMS: [(M+H)].sup.+=729.2.

(1588) Step g.

(1589) ##STR00814##

(1590) To a 0° C. stirring solution of step f product (1.744 g, 2.393 mmol) in DCM (15 mL) was added
TFA (15 mL), then the temperature was raised to ambient. Upon consumption of all starting material,
volatiles were removed by rotatory evaporation. LCMS analysis of this crude showed
[(M+H)].sup.+=629.2. This material was taken up in a 0° C. stirring solution of DIPEA (7.502 mL, 43.07
mmol) in DCM (15 mL), and treated with bis-Boc-p-nitrophenol chloroformate (817 mg, 2.632 mmol).
Upon consumption of the starting material, all the volatiles were removed by rotatory evaporation. The
residue was purified by silica column using an Isco COMBIFLASH® liquid chromatography eluted with
0% to 50% methanol and dichloromethane. Yield 631 mg, 30%. Ions found by LCMS:
[(M+H)].sup.+=871.2.

(1591) Step h.

(1592) ##STR00815##

(1593) To a 0° C. stirring solution of step g product (315 mg, 0.362 mmol) in acetonitrile (4.0 mL) and
1.0 M solution of NaCl in water (8 mL), it was added a 1.0 M solution of NaOH in water (15 mg, 380
μL, 0.380 mmol). Stirring was continued overnight, while the temperature was gently allowed to reach
ambient. The reaction was quenched with acetic acid (100 μL) and all the volatiles were removed by
rotatory evaporation. The residue was suspended in DCM:MeOH=9:1 and filtered. All the volatiles
were removed by rotatory evaporation. LCMS analysis of this crude material showed a major peak
with the desired [(M+H)].sup.+=857.2. The residue was dissolved in DCM (3.0 mL) and TFA (3.0 mL)
to remove the boc groups. Upon completion by LCMS, all the volatiles were removed by rotatory
evaporation. The residue was purified by HPLC (0 to 40% methanol and water, using 0.1% TFA as
modifier). Yield 370 mg, 86%. Ions found by LCMS: [(M+H)].sup.+=657.2.

Example 198. Synthesis of Conjugate 48

(1594) A solution of azido functionalized Fc (33.3 mg, 3.33 mL, 0.576 μmol, SEQ ID NO: 73; DAR-7.0)
was added to a 50 mL centrifuge tube containing alkyne functionalized small molecule (6.1 mg, 7.760
μmol, Int-92 prepared as described in Example 197). After gently shaking to dissolve all solids, this
solution was added to a solution of L-ascorbic acid sodium (8.6 mg, 11.21 μmol), copper (II) sulfate
(2.5 mg, 15.52 μmol), and BTTAA (26.7 mg, 62.08 μmol) in PBS 7.4 buffer (6.984 mL). The resulting
solution was gently rotated overnight. It was purified by affinity chromatography over a protein A
column, followed size exclusion chromatography (see general conjugate purification protocol
described herein). Maldi TOF analysis of the purified final product gave an average mass of 63,734 Da
(DAR=6.2). Yield 35.3 mg, 63% yield.

(1595) The nucleic acid construct encoding the Fc for conjugate 48 included a nucleic acid encoding
the amino acid sequence of SEQ ID NO: 64, which includes a C-terminal lysine residue. Upon
expression, the C-terminal lysine of the Fc of conjugate 48 is proteolytically cleaved, resulting in an Fc
having the sequence of SEQ ID NO: 73. The presence or absence of a C-terminal lysine does not alter
the properties of the Fc or the corresponding conjugate.

Example 199. The Effects of DAR on Neuraminidase Inhibition

(1596) Neuraminidase Inhibition (NAI)

(1597) Test articles were incubated with live viruses at designated PFU/mL for 20 min at 37° C., 5%
CO.sub.2. NA-Fluor substrate was added to appropriate wells and incubated for 1 h at 37° C., 5%
CO.sub.2. NAI was determined by reading fluorescence at 355 nm excitation/460 nm emission. The %
NAI was calculated using the formula: % NAI=(1−(FI.sub.virus with TA−FI.sub.no virus))/(FI.sub.virus
only−FI.sub.no virus)×100. IC.sub.50 was calculated using non-linear regression analysis software in
GraphPad Prism.

(1598) Conjugate 45a demonstrated DAR-dependent increased activity reaching maximal activity at
DAR 3.3 or greater against influenza A/PR/8/1934 (H1N1) (Table 173).

(1599) The A/Bethesda/956/2006 (H3N2) R.sub.292K mutant showed resistance to oseltamivir and
showed decreased susceptibility against zanamivir. Conjugate 45a did not shift against this mutant
and demonstrated DAR-dependent increased activity reaching maximal activity at DAR 3.3 or greater
against influenza (Table 173).

(1600) TABLE-US-00182 TABLE 173 Summary IC.sub.50 for Conjugate 110 against influenza A
(H1N1) and (H3N2) subtype IC.sub.50 [nM] IC.sub.50 [nM] Test A/PR/8/1934 A/Bethesda/956/2006
article DAR (H1N1) (H3N2) R292K Oseltamivir N/A 5.276 >1,000 Zanamivir N/A 1.072 20.79 Int-83
N/A 11.48 510.6 Conjugate 45a 0.4 23.61 78.79 Conjugate 45a 1 12.25 29.31 Conjugate 45a 3.3
2.772 4.166 Conjugate 45a 4.7 2.706 3.13 Conjugate 45a 5.1 3.259 3.583 Conjugate 45a 7.3 3.098
2.509

Example 200. Secondary Infection Mouse Models

(1601) Secondary Bacterial Infection Model with MRSA.

(1602) Efficacy studies were conducted in 6-8 week old female BALB/c mice (Charles River)
challenged intranasally with 3E3 PFU/mouse (sub-lethal) of A/CA/07/2009 (H1N1)pdm (Virapur, Lot
#E1020A1). Conjugate 45b or human IgG1 Fc controls was administered as a single subcutaneous
(SC) dose 2 h post-challenge at 0.3-3 mg/kg. On day 6 post-infection, mice were challenged
intranasally with sub-lethal dose of methicillin-resistant Staphylococcus aureus (MRSA) TCH1516 at
5E7 colony forming units (CFU) (FIG. 104).

(1603) For bacterial lung burden determination, mice were sacrificed by CO.sub.2 and both lung lobes
were harvested to determine bacterial burden at 24 h post-infection with MRSA. Lungs were
homogenized with 1 mm silica beads in 1 mL PBS using a MagNA Lyser (Roche). Homogenization
was carried out at 6,000 rpm for 60 s and chilled on ice for 5 min in-between runs. For CFU
determination, lung homogenates were serially 10-fold diluted in PBS and plated onto LA plates. CFU
were calculated relative to weight of the lung (CFU/g lung). For survival studies the general health
status of animals was monitored and body weights (BW) recorded daily for 14 days. Moribund or
animals exhibiting >20% of body weight (BW) loss, were recorded as a mortality. Survival, BW curves,
and statistical analysis were performed with GraphPad Prism (version 6.07).

(1604) Conjugate 45b demonstrated protection against bacterial superinfection mouse model with sub-
lethal challenge of influenza A/CA/07/2009 (H1N1)pdm followed by sub-lethal infection with MRSA.
Cumulatively, the two sublethal infections resulted in 100% mortality in mice that were treated with
hIgG1 Fc (FIG. 105A). Conjugate 45b treatment at 0.3 or 3 mg/kg—to prevent disease with influenza
—resulted in 100% survival of mice. Conjugate 45b demonstrated reduction of bacterial burden in the
lung as compared to hIgG1 Fc at 24 h after challenge with MRSA (FIG. 105B). The bacterial burden
following conjugate 45b treatment was comparable to the burden observed in mice that were only
challenged with MRSA.
(1605) These data demonstrate the potential for conjugate 45b in mitigating complications from
infection with influenza such as S. aureus superinfection that contribute to mortality associated with
influenza infection.

(1606) Secondary Bacterial Infection Model with Streptococcus pneumoniae

(1607) Efficacy studies were conducted in 6-8 week old female BALB/c mice (Charles River)
challenged intranasally with 3E1 PFU/mouse (sub-lethal) of A/CA/07/2009 (H1N1)pdm (Virapur, Lot
#1512B4) (FIG. 106). Conjugate 45b or human IgG1 Fc controls was administered as a single
subcutaneously (SC) dose 2 h post-challenge at 0.3 mg/kg. On day 6 post-infection, mice were
challenged intranasally with sub-lethal dose of Streptococcus pneumoniae (SPN) strain 6301 at 1 E5
CFU. The general health of animals was monitored and BW recorded daily for 14 days. Moribund, or
animals exhibiting >20% or more of BW loss, were recorded as a mortality. Survival, BW curves, and
statistical analysis were performed with GraphPad Prism (version 6.07).

(1608) Conjugate 45b demonstrated protection against bacterial superinfection mouse model with sub-
lethal challenge of influenza A/CA/07/2009 (H1N1)pdm followed by sub-lethal infection with SPN.
Cumulatively, the two sublethal infections resulted in 100% death in mice that were treated with hIgG1
Fc. Conjugate 45b treatment at 0.3 mg/kg—to prevent disease with influenza—resulted in 100%
survival of mice demonstrating the potential for conjugate 45b in mitigating complications from
infection with influenza against the most common cause of bacterial superinfection, S. pneumoniae
(FIG. 107). Bacterial superinfection significantly contributes to mortality associated with influenza.

Example 201. Dosing Optimization Study for Conjugate 45a Treatment of an A/Vietnam/1203/2004
(H5N1) Virus Infection in BALB/c Mice

(1609) Female 18-20 g BALB/c mice were obtained from Charles River Laboratories (Wilmington,
Mass.) for this experiment. The mice were quarantined for 6 days before use and maintained on
Teklad Rodent Diet (Harlan Teklad) and tap water at the Laboratory Animal Research Center of Utah
State University.

(1610) Highly pathogenic avian influenza A/Vietnam/1203/2004 (H5N1), was obtained from the
Centers for Disease Control (Atlanta, Ga.). Viral propagation was done in Madin-Darby canine kidney
(MDCK) cells (American Type Culture Collection, Manassas, Va.). Parent virus was passaged once to
prepare a challenge pool. The challenge pool was then titrated in MDCK cells before use.

(1611) Animal numbers and study groups are described in Table 174. Groups of mice were treated by
the sub-cutaneous (sc) injection with 0.3, 1, 3, and 10 mg/kg doses of conjugate 45a on a single
occasion, either 7 days before virus infection, or 4 hours after virus infection. Oseltamivir (10 mg/kg),
used as a positive control for the virus challenge dose, was administered by oral gavage twice daily,
beginning 4 hours post-infection. Unconjugated Fc, used as placebo, was administered in a similar
manner to conjugate 45a. In addition, three non-treated control mice were maintained for weight
comparison.

(1612) For influenza virus challenge, mice were anesthetized by i.p. injection of ketamine/xylazine (50
mg/kg/5 mg/kg) prior to challenge by the intranasal route with approximately 5 plaque forming units
(1× LD.sub.90) of virus per mouse in a 90 μm inoculum volume.

(1613) Mice were weighed prior to treatment and then every other day thereafter to assess the effects
of treatment on ameliorating weight loss due to virus infection. All mice were observed for morbidity
and mortality through day 21.

(1614) Kaplan-Meier survival curves were generated and compared by the Log-rank (Mantel-Cox) test
followed by pairwise comparison using the Gehan-Breslow-Wilcoxon test in Prism 8.3 (GraphPad
Software Inc.). Mean body weights were analyzed by one-way analysis of variance (ANOVA) followed
by Tukey's multiple comparison tests using Prism 8.3.
(1615) TABLE-US-00183 TABLE 174 Dosing Optimization Study for Efficacy No./ Group Infected
Treatment Observations/ Cage No. Y or N Compound Dosage Schedule Testing 10 1 Yes conjugate
45a 10 mg/kg Once, Observed for 10 3 Yes conjugate 45a 3 mg/kg 7 days pre- weight loss and 10 5
Yes conjugate 45a 1 mg/kg infection mortality through 10 7 Yes conjugate 45a 0.3 mg/kg day 21 10 9
Yes Placebo — Once, 4 h (unconjugated Fc, post- SEQ ID NO: 72) infection 10 11 Yes conjugate 45a
10 mg/kg 10 13 Yes conjugate 45a 3 mg/kg 10 15 Yes conjugate 45a 1 mg/kg 10 17 Yes conjugate
45a 0.3 mg/kg 10 19 Yes Oseltamivir 30 b.i.d. × 5 d, mg/kg/day beg 4 h post- infection 3 2 No
Untreated mice observed for normal weight gain

(1616) Following treatment with conjugate 45a at 7 days before virus infection, 100% protection was
observed for the 10 mg/kg dose, 80% protection for the 0.3 and 3 mg/kg doses, and 70% protection
for the 1 mg/kg dose (FIG. 108A). 30% survival in the placebo group is unusual, so the 1 mg/kg dose
did not provide significant protection. In addition, all doses provided significant protection from weight
loss (FIG. 108B).

(1617) Following treatment with conjugate 45a at 4 hours after virus infection, 100% protection was
observed for the 1, 3, and 10 mg/kg doses, and 70% protection for the 0.3 mg/kg dose (FIG. 109A).
Due to the 30% survival observed in the placebo group, the 0.3 mg/kg dose did not provide significant
protection. In addition, all doses provided significant protection from weight loss (FIG. 109B).

Example 202. Enzyme-Linked Lectin Assay (ELLA)

(1618) The ELLA assay was conducted as reported in Gao et al. (J. Vis. Exp. Doi:10.3791/54573,
2016) with minor modifications. Briefly, Nunc Maxisorp 96-well plates (ThermoFisher) were coated with
2.5 μg fetuin (Sigma-Aldrich) in 1×KPL coating buffer (SeraCare) overnight at 4° C. The next day,
plates were washed with PBS at pH 7.4 supplemented with 0.05% Tween 20 (PBST). Test articles
were tested at 0.001-1000 nM and added in 50 μL/well. Influenza virus was added at 1 e5-1e6
PFU/well in 50 μL/well. Plates were incubated for 16-18 h at 37° C., 5% CO.sub.2. After washing
plates, peanut agglutinin conjugated to HRP (PNA-HRP) at 0.13 μg for 2 h, washed again and
developed with 100 μL/well TMB substrate (BD) for 3-5 min. The reaction was stopped with 100
μL/well 1 N H.sub.2SO.sub.4. Absorbance was read at 450 nm with an EnSpire multimode plate
reader. IC.sub.50 was calculated with GraphPad Prism version 8 using nonlinear regression analysis
(Dose-response (Inhibition)).

(1619) Conjugate 45b demonstrated increased potency as compared to oseltamivir or zanamivir in

ELLA with IC.sub.50 between against representative influenza A and B strains (Table 175).

(1620) TABLE-US-00184 TABLE 175 Activity of conjugate 45b in enzyme-linked lectin assay (ELLA)
against influenza A and B (IC.sub.50) Influenza Subtype/ Conjugate hlgG1 Oseltamivir Zanamivir
Strain lineage 45b [nM] Fc [nM] [nM] [nM] A/PR/8/1934 H1N1 0.11 >1,000 25.4 3.8 A/CA/7/2009
H1N1pdm 0.02 >1,000 0.99 0.09 A/ H3N2 0.003 >1,000 0.07 0.24 Hong Kong/ 1/1968 B/Florida/
Yamagata 0.02 >1,000 1.00 0.09 4/2006 B/Malaysia/ Victoria 0.15 >1,000 3.2 9.3 2506/2004

Example 203. Conjugate 45b Attributes that Allow for Potential Multivalent Binding to Influenza Virus
Neuraminidase

(1621) Conjugate 45b comprises a chemically and biologically stable conjugate of Int-83 with an N-
terminally extended hIgG1 Fc domain (SEQ ID NO: 73). As shown in FIG. 110B, Int-83 includes
zanamivir dimers that are symmetrically fused through methyl carbamate moieties on the C7-hydroxyl
of zanamivir to a flexible 15 A central-linker that spaces the zanamivir monomers by approximately 18
Å. Multiple copies of the Int-83 are conjugated to surface exposed lysine residues on the Fc domain
through a 32-atom flexible polyethylene glycol (PEG)-based cross-linker, which projects them out from
the surface of the Fc by 35-40 Å when the linker is fully extended (FIG. 1101B). Specific solvent-
exposed lysine residues were sites for conjugation (FIG. 110B). The average ratio of Int-83 to Fc used
in Conjugate 45b was 4.5:1. Synthesis of conjugate 45b is described in Example 156. The spatial
distribution of preferred sites for conjugation (FIG. 110B) coupled with the length and flexibility of the
cross-linker allow, in principle, for conjugate 45b to simultaneously interact with multiple NA active
sites within a tetramer (ca. or 70 Å, respectively, FIG. 110A). Additionally, the spacing of Int-83 in
conjugate 45b allows for bridging of NA active sites across neighboring NA tetramers on the same
virion (25-30 Å), or across two virions (16 Å).

(1622) Universal Activity, and Low Resistance Potential of Conjugate 45b In Vitro

(1623) We investigated the intrinsic antiviral activity of conjugate 45b in the absence of immune
engagement using a standard neuraminidase inhibition (NAI) assay that employs a small molecule
substrate. Conjugate 45b demonstrated potent activity against influenza A and B with median
IC.sub.50 of 1.7 nM against influenza A H1N1 strains (n=7, ranging from 1.1 nM to 4.8 nM), 4.2 nM
against influenza A H3N2 strains (n=6, ranging from 0.3 nM to 14.9 nM) and 4.4 nM against influenza
B strains (n=6, ranging from 1.0 nM to 20.7 nM), respectively (Table 176). In general, conjugate 45b
performed similarly to zanamivir and oseltamivir, but was more potent than oseltamivir versus
influenza B strains (Table 176). Importantly, conjugate 45b IC.sub.50s did not shift against clinically
relevant variants with reduced susceptibility to approved neuraminidase inhibitors. The activity of
conjugate 45b was 1.7 nM against H275Y in H1N1 subtype, 2.6 nM E119V, 1.2 nM R.sub.292K in
H3N2 subtype or 6.7 nM R.sub.152K in B type. Oseltamivir or zanamivir lost up to >100-fold or >10-
fold in potency, respectively, against these variants (Table 177). Conjugate 45b retained potent activity
against cell lysates from H5N1 and H7N9 viruses, with IC.sub.50s of 1.9 nM and 1.2 nM, respectively
(Table 178). Oseltamivir and zanamivir had comparable activity versus H5N1, but oseltamivir and
zanamivir lost activity against H7N9 with IC.sub.50s of >1 μM or >100 nM, respectively (Table 178).

(1624) TABLE-US-00185 TABLE 176 Universal, broad-spectrum activity of conjugate 45b against
influenza A (H1N1), (H3N2) and B (Yamagata and Victoria lineage) in neuraminidase inhibition
(median IC.sub.50) and cell-based cytopathic effect assay (median EC.sub.50). Molecule type/subtype
IC.sub.50 [nM] EC.sub.50 [nM] Conjugate A (H1N1) 1.7 1.9 45b A (H3N2) 4.2 0.9 B 4.4 7.4
Oseltamivir A (H1N1) 2.2 2414 A (H3N2) 0.4 8185 B 37.7 758.5 Zanamivir A (H1N1) 1.0 1093 A
(H3N2) 1.3 >10,000 B 6.3 80.2

(1625) TABLE-US-00186 TABLE 177 Activity of conjugate 45b against variants with reduced
susceptibility in neuraminidase inhibition (IC.sub.50) Live Zana- influenza subtype/ Conjugate
Oseltamivir mivir strain Variant lineage 45b [nM] [nM] [nM] A/Texas/ H275Y H1N1pdm09 1.7 507.1 1.1
23/2012 A/Texas/ E119V H3N2 2.6 148.9 3.8 12/2007 A/Bethesda/ R292K H3N2 1.2 >1000 22.18
956/2006 B/Memphis/ R152K Yamagata 6.7 >1000 125.5 20/1996

(1626) TABLE-US-00187 TABLE 178 Activity of conjugate 45b against cell lysate from influenza A
H5N1 and H7N9 in neuraminidase inhibition (IC.sub.50) NA cell subtype/ Conjugate Oseltamivir
Zanamivir lysate from lineage 45b [nM] [nM] [nM] A/Anhui/1/2005 H5N1 1.9 3.7 0.6
A/Shanghai/1/2013 H7N9 1.2 >1,000 100.4

(1627) Next, we tested the activity of conjugate 45b in an enzyme-linked lectin assay (ELLA) in which
a large glycoprotein is used as the substrate. The presentation of sialic acid (Sia) in the ELLA assay is
more similar to NA substrate presentation on the surface of a cell. As a result, access to the substrate
is more limited and NA activity in the ELLA assay has been shown to be influenced by factors that
block access to the NA active site (Chen Y. Q., et al., J. Virol. 93: doi.1i0.1128/JVI.01526-18, 2019).
Interestingly, compared with the NA inhibition results using a small molecule substrate as described
above, the activity of conjugate 45b was enhanced (>1 Ox) versus zanamivir and oseltamivir against
influenza A/PR/8/1934 (H1N1) (Table 179). With conjugate 45b, steric hindrance of adjacent intra- or
inter-NA tetramer NA active sites by the Fc domain, or multivalent target engagement causing viral
aggregation may have contributed to the enhancements in potency observed in the ELLA assay.

(1628) TABLE-US-00188 TABLE 179 Broad-spectrum activity of conjugate 45b against influenza A
and B in CPE (EC.sub.50). Conjugate Zana- Influenza subtype/ 45b Oseltamivir mivir strain lineage
[nM] [nM] [nM] A/WSN/1933 H1N1 0.2059 51.36 29.12 A/PR/8/1934 H1N1 0.782 1461 7581
A/PR/8/1934 H1N1 32.49 >10,000 not tested (mouse-adapted) A/CA/7/2009 H1N1pdm 2.964 34.2
33.16 A/CA/12/2012 H1N1pdm09 0.4931 >10,000 1093 A/Texas/23/2012 H1N1pdm09 2.886 >10,000
>10,000 H275Y A/Illinois/08/ H1N1pdm09 9.584 3366 4278 2018 A/Illinois/37/ H1N1pdm09 0.4304
200.4 179.1 2018 I38L A/Illinois/08/ H1N1pdm09 1.35 >10,000 >10,000 2018 I38T A/Hong Kong/
H3N2 36.57 6369 not tested 1/1968 (mouse-adapted) A/Texas/71/2017 H3N2 0.3748 >10,000
>10,000 A/Washington/ H3N2 0.04449 1.908 1.968 01/2007 A/Texas/12/ H3N2 0.6594 351.4 2.096
2007 E119V A/Louisiana/ H3N2 1.41 >10,000 >10,000 50/2017 A/Louisiana/49/ H3N2 0.606 >10,000
4815 2017 I38M A/Bethesda/956/ H3N2 20.1 1653 558.1 2006 R292K B/Florida/4/2006 Yamagata
12.8 1030 76.89 B/Brisbane/ Victoria 6.288 487 254.2 60/2008 B/Malaysia/ Victoria 1.512 471.2 83.52
2506/2004 B/Colorado/ Victoria 8.485 4480 56.98 6/2017

(1629) Even greater discrimination between conjugate 45b and zanamivir and oseltamivir was
observed in cell-based cytopathic effect (CPE) assays. Conjugate 45b demonstrated potent activity
against influenza A and B types with median EC.sub.50s of 1.3 nM for influenza A H1N1 strains (n=6,
ranging from 0.43 nM to 32.4 nM), 0.9 nM for influenza A H3N2 strains (n=4, ranging from 0.04 nM to
36.6 nM) and 7.4 nM for influenza B strains (n=4, ranging from 1.5 nM to 12.8 nM), respectively in
MOCK SIAT1 or for mouse-adapted influenza strains in MOCK cells (Table 179). Notably, conjugate
45b was 3 logs more active than oseltamivir and zanamivir against some of the influenza strains
tested (Table 176). When conjugate 45b was tested against high-pathogenicity strains in cell-based
microneutralization assays, EC.sub.50s for conjugate 45b were 1.7 nM versus H5N1 strains (n=4) and
5.3 nM versus an H7N9 strain (n=1) (Table 180). The CC.sub.50 for conjugate 45b in MDCK-SIAT1 or
MOCK cells was >10,000 nM (data not shown). Therefore, the calculated selectivity index (SI) for
conjugate 45b in cell-based assay was >1,000× for influenza A and B types.

(1630) TABLE-US-00189 TABLE 180 Broad-spectrum activity of conjugate 45b against high-
pathogenic influenza A (H5N1) and (H7N9) in microneutralization (IC.sub.50). subtype/ Conjugate
Oseltamivir Zanamivir Influenza strain lineage 45b [nM] [nM] [nM] A/Vietnam/1194/ H5N1 1.7 168.7
16.9 2004 A/Indonesia/05/ H5N1 1.7 >300 16.9 2005 A/turkey/Turkey/ H5N1 1.7 168.7 5.3 1/2005
A/Hong Kong/ H5N1 1.7 >300 53.3 156/97 A/Anhui/1/2013 H7N9 5.3 >300 >300 A/Netherlands/
H1N1pdm09 1.7 >300 >300 602/2009* *Pandemic control influenza A strain

(1631) In addition to CPE assays, serial passage experiments were conducted using MDCK cells
infected with influenza A/CA/07/2009 (H1N1)pdm to compare the resistance potential of conjugate 45b
to the two commercially dominant influenza antivirals, oseltamivir and baloxivir. Conjugate 45b had a
superior resistance profile to both comparators and demonstrated no reduction in antiviral activity
throughout the 10 passage of the experiment. Baloxivir and oseltamivir antiviral activity was reduced to
that of the drug free control after 6 and 8 passages, respectively. Sequencing of the viral genomes
from drug-resistant viral plaques in the later oseltamivir passages showed that resistance was
conferred by an NA active-site mutation (E 119K) that has been observed in the clinic (REF).

(1632) Immune Cell Engagement by Conjugate 45b

(1633) Human IgG1 Fc domain was selected as the protein carrier for conjugate 45b, because it is the
most activating human antibody IgG isotype with the longest circulating half-life. Despite utilizing a
heterogeneous lysine conjugation strategy with conjugate 45b, we observed similar Fcγ receptor
binding to all human and murine Fcγ receptors tested with conjugate 45b compared to the
unconjugated Fc control and human IgG1 (FIGS. 111A-111H). In functional assays, conjugate 45b
induced potent antibody-dependent cellular cytotoxicity (ADCC) in MOI (FIG. 111I) and dose-
dependency against influenza A/PR/8/1934 (H1N1) infected MDCK Slat1 cells (FIG. 111J). Thus,
conjugate 45b can bind to Fey receptors and functionally engage immune cells.

(1634) Conjugate 45b is Highly Effective in Multiple Lethal Influenza Challenge Models

(1635) The potency and antiviral spectrum of conjugate 45b observed in vitro translated to efficacy in
animal infection models. Single, 0.3 mg/kg or lower subcutaneous (SC) doses of conjugate 45b were
fully protective in lethal mouse challenge models against A/CA/07/2009 (H1N1)pdm, A/WSN/1933
(H1N1), A/CA/12/2012 (H1 Ni)pdm09, A/Texas/23/2012 (H1 Ni)pdm09 H275Y variant, A/Hong
Kong/1/1968 (H3N2), B/Florida/4/2006 (Yamagata) and B/Malaysia/2506/2004 (Victoria) (FIGS. 112A-
112H). Treatment with conjugate 45b often resulted in only limited, transient body weight loss following
minimal protective dose (FIGS. 113A-113H). When tested against the avian pandemic strain
A/Vietnam/1203/2004 (H5N1) in mice, conjugate 45b demonstrated 70% protection at 0.3 mg/kg and
100% protection at 1 mg/kg or higher (FIG. 112I). Oseltamivir at 6× the humanized dose in mice was
only 90% protective (FIG. 112I).

(1636) To better quantify conjugate 45b efficacy, and to compare its performance with oseltamivir, viral
load and cytokine levels in lung were measured 4 days post-infection following a lethal challenge
model with mouse-adapted influenza A/PR/8/1934 (H1N1). In this model, the minimal protective dose
of conjugate 45b was 0.1 mg/kg (SC) (FIG. 112A) and was accompanied by transient body-weight
(BW) loss of ca. 5% (FIG. 113A). Conjugate 45b demonstrated dose-dependent reduction in lung viral
burden (PFU/g in lung tissue) of 1.06 logs at 0.1 mg/kg, 2.12 logs 0.3 mg/kg and 3.17 logs at 1 mg/kg
and 3.63 logs at 3 mg/kg as determined by plaque assay. By comparison, oseltamivir dosed at the
human equivalent (5 mg/kg) dose (BID x 4 days) or at 10× the human equivalent dose (50 mg/kg)
(BID×4 days) showed no dose response, and resulted in moderate viral load reductions of ca. 0.8 log.
Interestingly, the two oseltamivir doses tested led to different survival outcomes. Oseltamivir at 5
mg/kg or 50 mg/kg dosed for 5 days BID resulted in 0% survival or 80%, respectively (FIGS. 114A-
114B).

(1637) To understand the effectiveness of conjugate 45b in reducing a potentially harmful immune
response that could lead to lung damage, levels of specific inflammatory and lung injury cytokines
were measured at the same time point. Conjugate 45b effectively reduced levels in a dose-dependent
manner, with all cytokines measured approaching levels similar to uninfected controls at the 3 mg/kg
dose (FIGS. 112K-112L and FIGS. 115A-115C. Overall, the magnitude of cytokine reduction induced
by conjugate 45b was more pronounced than afforded by treatment with the human equivalent dose of
oseltamivir for all cytokines tested and was superior for KC, MIP-1a, MCP-1 when compared to mice
treated at 10× human equivalent dose of oseltamivir (FIGS. 112K-112L and FIGS. 115A-115C). These
data demonstrate that conjugate 45b induces potent, dose-dependent reduction in viral burden that
correlated with reductions in cytokine levels in the lung by conjugate 45b that was not observed with
oseltamivir.

(1638) The impact of dosing route on efficacy was evaluated for conjugate 45b. In a lethal challenge
model using mice against A/CA/07/2009 (H1N1)pdm, conjugate 45b was fully protective at 0.1 mg/kg
following intravenous (IV), intramuscular (IM) or SC administration (FIGS. 116A-116B). When
comparing plasma levels in mice following IV, IM or SC dosing, the plasma levels of conjugate 45b
were comparable after 24 h as determined by an enzyme-linked immunosorbent (ELISA) assay. Of
note, two different ELISA capture methods were used to measure conjugate 45b levels in plasma—
one that utilized an anti-human Fc capture antibody to measure Fc levels, and one that utilized viral
NA for capture to detect intact conjugate (FIGS. 116C-116D). The plasma levels of conjugate 45b
measured by both methods were identical within experimental error, demonstrating that conjugate 45b
is a stable as an intact conjugate in vivo.

(1639) Conjugate 45b is Highly Effective in Immune-Compromised Hosts

(1640) High-risk groups for severe infections with influenza and complications from influenza infection
include the elderly and immune-compromised. To determine the effectiveness of conjugate 45b in an
immune-compromised background, we determined the efficacy of conjugate 45b in severely combined
immune-deficiency (SCID) mice, which lack mature T-cells and B-cells and are complement-deficient.
Conjugate 45b demonstrated full protection with single 0.3 mg/kg dose in SCID mice infected with
A/CA/07/2009 (H1N1)pdm (FIG. 112M), which is the same protective dose for immune-competent
mice.

(1641) Taken together, these results highlight the potential of conjugate 45b to provide universal
influenza protection in healthy and high-risk populations.

(1642) Conjugate 45b Demonstrates a Long-Duration of Action in Prevention Models

(1643) Pharmacokinetic (PK) profiling, prophylactic efficacy and preclinical toxicology studies further
highlight the potential for use of conjugate 45b as a durable, long-acting agent for universal influenza
prevention. The half-life of conjugate 45b was determined after a single IV administration in mouse
and cynomolgus monkey and ranges from 5-10 days in 1- and 4-week PK studies, respectively. These
results are typical for mABs and is supported by comparable binding curves of conjugate 45b, hIgG1
Fc and full-length human IgG1 isotype control antibody to murine, cynomolgus monkey, and human
FcRn in pH-dependency (FIGS. 117A-117C). Conjugate 45b exhibits dose-linear PK in mice across a
wide dose range from 1-100 mg/kg (FIG. 118A). In mouse model, conjugate 45b establishes a rapid
equilibrium between plasma and lung, allowing for a rapid onset of action for treatment indications,
and distributes to epithelial lining fluid (ELF) at high levels (FIG. 118B). High levels of conjugate 45b in
ELF appeared rapidly and were sustained at ca. 60% of plasma levels (FIG. 118B).

(1644) To assess conjugate 45b efficacy in a prevention setting, and determine target plasma levels
necessary for protection in lethal influenza challenge models, mice were dosed 28 days prior to
infection. A single SC dose of 1 mg/kg conferred full protection against influenza A/CA/07/2009
(H1N1)pdm, A/HK/1/1968 (H3N2), B/Malaysia/2506/2004 (Victoria) and B/Florida/4/2006 (Yamagata)
(FIGS. 118C-118F). The corresponding conjugate 45b plasma level necessary for protection,
measured at the time of infection, was 0.5 μg/mL (8 nM, data not shown). Multi-species PK and the
target plasma level of conjugate 45b required for protection will be used to estimate human doses
necessary for long-term prevention in humans, and was used to estimate therapeutic margins for
conjugate 45b in preclinical toxicology studies. In a two-week dose-range finding toxicity study
conducted in cynomolgus monkeys no adverse events were observed at highest dose tested (20
mg/kg). The therapeutic index (TI) based on plasma exposure ratios in the 20 mg/kg dosed monkeys
and the 1 mg/kg dosed mice is >50 (Tables 181 and 182). This safety margin, coupled with allometric
scaling incorporating the high potency and sustained exposures of conjugate 45b (data not shown),
suggest that one to two doses, protection for an entire influenza season with conjugate 45b is
achievable in humans.

(1645) TABLE-US-00190 TABLE 181 Dose-proportionality of conjugate 45b in mice Dose T.sub.max
C.sub.max AUC Test article [mg/kg] [h] [μg/mL] [h × μg/mL] Conjugate 45b 1 4 9.39 436 3 24 11.4
1520 10 24 41.3 4280 30 24 125 14200 100 4 733 60700

(1646) TABLE-US-00191 TABLE 182 Dose-proportionality of conjugate 45b in cynomolgus monkeys

Test Dose T.sub.max C.sub.max AUC article Day [mg/kg] [h] [μg/mL] [h × μg/mL] Conjugate 1 5 72
26.1 3800 45b 8 5 8 61 7740 1 20 72 107 14600 8 20 24 197 25400

(1647) In the cynomolgus monkey toxicology study, following 2 weekly SC doses at 5 or 20 mg/kg, no
adverse effect on BW, clinical chemistry, hematology, coagulation, cytokines, or urinalysis were
observed (data not shown).

Example 204. Determination of Optimal Drug-to-Antibody Ratio (DAR) of Conjugate 45a Against
Influenza A (H1N1) in a Lethal Mouse Influenza Model

(1648) DAR variants of Conjugate 45a were evaluated in a lethal H1N1 influenza infection model in
female BALB/c mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto
Rico/8/1934) is a mouse-adapted isolate capable of causing lethal infections in mice. The experiment
comprised 2 study arms with a total of 25 groups of 5 mice. At day 0, all mice were challenged with
virus at 3× the LD.sub.95 (˜2E4 virus/mouse) by intranasal inoculation in a volume of 30 μl after being
lightly anesthetized with ketamine/xylazine(100 and 10 mg/kg respectively). Groups received a single
treatment of conjugate 45a, DAR variant of conjugate 45a, Fc only, or vehicle (PBS), 2 hours after viral
challenge by intramuscular (IM) administration. Conjugate 45a variants were tested with average
DARs of 0.4, 1.0, 3.3, 4.7, 5.1, or 7.3. Mortality and body weight (BW) were monitored daily for 14
days. Any mouse losing more than 20% BW was scored as a mortality. The experimental outline for
each study arm is summarized in Tables 183 and 184.

(1649) TABLE-US-00192 TABLE 183 Low average DAR arm study design Cmpd prep Influenza A
Dose Vol strain (IN Dose volume needed Group challenge) Test Article DAR Route, Schedule (mg/kg)
ml/kg mg/ml (ml) N 1 A/PR/8/34 Vehicle (PBS) — IM, single, T + 2 h — 5 — 1 5 2 (H1N1) Fc alone —
IM, single, T + 2 h 0.3 5 0.06 1 5 3 3E2 Conjugate 45a 0.4 IM, single, T + 2 h 0.3 5 0.06 1 5 4
PFU/mouse Conjugate 45a 0.4 IM, single, T + 2 h 0.1 5 0.02 1 5 5 Conjugate 45a 0.4 IM, single, T + 2
h 0.03 5 0.006 1 5 6 Conjugate 45a 1 IM, single, T + 2 h 0.3 5 0.06 1 5 7 Conjugate 45a 1 IM, single, T
+ 2 h 0.1 5 0.02 1 5 8 Conjugate 45a 1 IM, single, T + 2 h 0.03 5 0.006 1 5 9 Conjugate 45a 3.3 IM,
single, T + 2 h 0.3 5 0.06 1 5 10 Conjugate 45a 3.3 IM, single, T + 2 h 0.1 5 0.02 1 5 11 Conjugate 45a
3.3 IM, single, T + 2 h 0.03 5 0.006 1 5 12 Conjugate 45a 4.7 IM, single, T + 2 h 0.3 5 0.06 1 5 13
Conjugate 45a 4.7 IM, single, T + 2 h 0.1 5 0.02 1 5 14 Conjugate 45a 4.7 IM, single, T + 2 h 0.03 5
0.006 1 5

(1650) TABLE-US-00193 TABLE 184 High DAR arm study design Cmpd prep Influenza A Dose strain
(IN Dose volume Vol needed Group challenge) Test Article DAR Route, Schedule (mg/kg) ml/kg mg/ml
(ml) N 1 A/PR/8/34 Vehicle (PBS) — IM, single, T + 2 h — 5 — 1 5 2 (H1N1) Fc alone — IM, single, T
+ 2 h 0.3 5 0.06 1 5 3 3E2 Conjugate 45a 4.7 IM, single, T + 2 h 0.3 5 0.06 1 5 4 PFU/mouse
Conjugate 45a 4.7 IM, single, T + 2 h 0.1 5 0.02 1 5 5 Conjugate 45a 4.7 IM, single, T + 2 h 0.03 5
0.006 1 5 6 Conjugate 45a 5.1 IM, single, T + 2 h 0.3 5 0.06 1 5 7 Conjugate 45a 5.1 IM, single, T + 2
h 0.1 5 0.02 1 5 8 Conjugate 45a 5.1 IM, single, T + 2 h 0.03 5 0.006 1 5 9 Conjugate 45a 7.3 IM,
single, T + 2 h 0.3 5 0.06 1 5 10 Conjugate 45a 7.3 IM, single, T + 2 h 0.1 5 0.02 1 5 11 Conjugate 45a
7.3 IM, single, T + 2 h 0.03 5 0.006 1 5

(1651) In the first study arm, variants with an average DAR of 0.4, 1.0, 3.3, or 4.7 were run at
concentrations of 0.03, 0.1, and 0.3 mg/kg (single IM administration at T+2 h relative to viral
challenge). In this study vehicle (PBS) treated mice succumbed to infection by Day 7, and those
treated with Fc only (SEQ ID NO: 72) reached mortality on Day 6 (Table 185). In contrast, conjugate
45a variants with average DARs of 1.0, 3.3, and 4.7 were fully protected at the highest dose
concentration (0.3 mg/kg). Only the 0.4 average DAR conjugate failed to offer protection, with mice
reaching mortality by Day 9, indicating it had the lowest potency average DAR of the conjugates
tested. At the next lowest conjugate dose (0.1 mg/kg) only mice treated with conjugates having an
average DAR of 3.3 or 4.7 were fully protected. The two lower average DAR conjugates (0.4 and 1.0)
reached mortality on Day 7 and 9, respectively, at this dose. In the final dose group (0.03 mg/kg) only
the conjugate 45a variant with an average DAR of 4.7 demonstrated any potency (80% survival).
Based on mortality data a clear trend was evident in this study arm, with increasing average DAR
resulting in increasing potency.

(1652) BW data for arm 1 study groups supported the trends seen in the mortality data (Table 186).
For example, in the 0.3 mg/kg dose groups, conjugate 45a variants with average DARs of 1.0, 3.3,
and 4.7 were all protective based on mortality, but mice receiving the 1.0 average DAR variant had
greater BW loss than the higher average DAR constructs. This resulted in the loss of protection by the
1.0 average DAR variant when the dose was lowered to 0.1 mg/kg. Similarly, at 0.1 mg/kg mice
receiving the 3.3 average DAR variant were protected from lethal challenge based on mortality, but
had greater BW loss than the animals treated with the 4.7 average DAR variant. By both study
readouts (mortality and BW), greater potency was seen with increasing average DAR.

(1653) TABLE-US-00194 TABLE 185 Mortality data (% survival) for arm 1. Average DAR 0.4 1.0 3.3
4.7 Day post Vehicle Fc alone (0.3 (0.1 (0.03 (0.3 (0.1 (0.03 (0.3 (0.1 (0.03 (0.3 (0.1 (0.03 challenge
(PBS) (0.3 mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) 0 100 100 100
100 100 100 100 100 100 100 100 100 100 100 1 100 100 100 100 100 100 100 100 100 100 100 100
100 100 2 100 100 100 100 100 100 100 100 100 100 100 100 100 100 3 100 100 100 100 100 100
100 100 100 100 100 100 100 100 4 100 100 100 100 100 100 100 100 100 100 100 100 100 100 5
80 100 100 100 100 100 100 100 100 100 100 100 100 100 6 40 0 100 60 80 100 100 80 100 100 100
100 100 100 7 0 40 0 0 100 80 0 100 100 80 100 100 100 8 20 100 20 100 100 60 100 100 100 9 0
100 0 100 100 20 100 100 80 10 100 100 100 0 100 100 80 11 100 100 100 100 100 80 12 100 100
100 100 100 80 13 100 100 100 100 100 80 14 100 100 100 100 100 80

(1654) TABLE-US-00195 TABLE 186 BW data for arm 1. Average group BW until the first death within
a group. Fc Average DAR alone 0.4 1.0 3.3 4.7 Day post Vehicle (0.3 (0.3 (0.1 (0.03 (0.3 (0.1 (0.03
(0.3 (0.1 (0.03 (0.3 (0.1 (0.03 challenge (PBS) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk)
mpk) mpk) mpk) 0 100 100 100 100 100 100 100 100 100 100 100 100 100 100 1 96.9 98.8 98.2 97.9
97.7 98 97.6 97.9 97 97.1 99.9 97.7 97.8 99.8 2 100 101.9 101.2 100.3 99.8 99.9 99.6 100.9 101.8
99.2 102.9 102.4 100.9 100.7 3 93.1 93.4 96.8 95 98.3 96.3 96.6 94.3 100.1 97.7 98.1 101.3 98.2 99.4
4 83.6 83.3 89 84.7 88.1 91.6 88.8 86.3 99.7 93.9 92 100.9 95 97.2 5 79.9 80 86.3 81.8 84.2 95.6 89.1
82.9 101.1 96.3 91.8 100.4 97.4 98.9 6 81.8 76.9 77.5 95.7 84.5 77.4 102.3 96.6 88.8 103.5 99.9 97.5
7 76.8 91.4 78.9 103.6 92.3 82.4 105 100.8 91.3 8 95.7 104.5 92.9 103.9 101.3 85.3 9 97.2 104.2 96.4
103.9 103.4 83.9 10 98.5 105 98.2 107.8 104.3 11 98.2 104.7 99.8 106.8 103.5 12 98.9 105.1 102.9
108.7 104.5 13 99.7 107.2 104.3 110.5 106.1 14 99.9 105.8 103.6 109 104.7

(1655) In arm 2 of the study, conjugate 45a variants with average DARs of 4.7, 5.1, and 7.3 were
evaluated at the same dose levels as arm 1 (0.3, 0.1, and 0.03 mg/kg). In contrast to the results seen
in arm 1, increasing average DAR above 4.7 did not increase the potency of conjugate 45a. All three
DAR variants were of approximate equal potency based on mortality and BW (Tables 187 and 188,
respectively). Only slight differences were seen in these higher DAR constructs which were within the
normal experimental error seen in efficacy models (note the slightly faster time to death for vehicle
treated animals in arm 2). The later point includes the observation that the 4.7 average DAR conjugate
was protective in the first arm at 0.03 mg/kg, but not in arm 2. Collectively the results of both study
arms show a distinct increase in potency up to an average DAR of 4.7, but then further increases in
average DAR do not translate into greater potency. Therefore, an average DAR of 4.7 is the minimum
average DAR which achieves the maximum potency of conjugate 45a.

(1656) TABLE-US-00196 TABLE 187 Mortality data (% survival) for arm 2 Fc Average DAR Alone 4.7
5.1 7.3 Day post Vehicle (0.3 (0.3 (0.1 (0.03 (0.3 (0.1 (0.3 (0.03 (0.1 (0.3 challenge (PBS) mpk) mpk)
mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) 0 100 100 100 100 100 100 100 100 100 100 100 1 100
100 100 100 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 100 100 100 3 100
100 100 100 100 100 100 100 100 100 100 4 100 100 100 100 100 100 100 100 100 100 100 5 40
100 100 100 100 100 100 100 100 100 100 6 0 40 100 100 100 100 100 100 100 100 100 7 0 100 100
60 100 100 80 100 100 100 8 100 100 60 100 100 40 100 100 80 9 100 100 40 100 100 20 100 100
40 10 100 100 0 100 80 0 100 100 0 11 100 100 100 80 100 100 12 100 100 100 80 100 100 13 100
100 100 80 100 100 14 100 100 100 80 100 100

(1657) TABLE-US-00197 TABLE 188 BW data for arm 2: Average group BW until the first death within
a group Fc Average DAR Alone 4.7 5.1 7.3 Day post Vehicle (0.3 (0.3 (0.1 (0.03 (0.3 (0.1 (0.03 (0.3
(0.1 (0.03 challenge (PBS) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) mpk) 0 100 100 100
100 100 100 100 100 100 100 100 1 99.5 98.4 98.4 98.3 98.2 98.1 98.5 98.6 99.4 96.5 99.4 2 98.7
100.3 99.9 100.5 99.3 97.7 98.5 101.3 100.2 102 99.1 3 93.7 95.8 99.8 100.7 99.3 101.1 99.7 100.8
100.7 100.9 100.3 4 81 84.9 99.6 99 93.2 98 95.4 93.5 100.1 99.9 97.4 5 77.7 81.2 100 98.8 91.5 98.5
97.1 91 96.7 99.7 93.7 6 76.9 100 98.9 86 99.9 95.6 85.9 100.3 99.2 88.6 7 102.5 100.5 81.6 102 88.8
82.9 103.4 97.8 85.3 8 102.8 100.8 103.3 87.8 103.5 98.8 9 101.9 103.3 104.9 89.4 104.5 102.3 10
103.6 105.4 105.6 95.3 106.4 105 11 103.4 104 105.1 106.2 103.9 12 104 104 104.5 105.4 104.2 13
103.8 104 105.1 104.9 103.1 14 102.3 102.5 102.8 103.1 103.1

Example 205. Determination of Conjugate 45a Potency Against an Influenza A Pandemic Strain
(A/California/12/2012; H1N1) in a Lethal Mouse Model

(1658) Conjugate 45a was evaluated in a lethal H1N1 influenza infection model in female BALB/c mice
(Charles River Laboratories, 6-8 weeks). The challenge virus (A/CA/12/2012) is a pandemic isolate
capable of causing lethal infections in mice. The experiment was comprised of 7 groups, with 5 mice
per group. At day 0, all mice were challenged with virus at 3× the LD.sub.95 (3E4 virus/mouse) by
intranasal inoculation in a volume of 30 μl after being lightly anesthetized with ketamine/xylazine(100
and 10 mg/kg respectively). Groups received a single treatment of conjugate 45a, hIgG1 Fc, or vehicle
(PBS), 2 hours after viral challenge by intramuscular (IM) administration (right flank). Conjugate 45a
was tested at concentrations of 3, 1, 0.3, 0.1, and 0.03 mg/kg. Mortality and body weight (BW) were
monitored daily for 21 days. Any mouse losing more than 20% BW was scored as a mortality. The
experimental outline is summarized in Table 189.

(1659) TABLE-US-00198 TABLE 189 Study outline Dose Dose Volume N Test Route/ (mg/ volume
needed (balb/ Group Article Schedule kg) (ml/kg) mg/ml (ml) c) 1 PBS IM, T + 2 hrs — 5 — 1 5 2 Fc
alone IM, T + 2 hrs 3 5 0.6 1 5 3 Conjugate IM, T + 2 hrs 3 5 0.6 1 5 4 45 a IM, T + 2 hrs 1 5 0.2 1 5 5
IM, T + 2 hrs 0.3 5 0.06 1 5 6 IM, T + 2 hrs 0.1 5 0.02 1 5 7 IM, T + 2 hrs 0.03 5 0.006 1 5

(1660) In this study a single IM administration of Conjugate 45a fully protected mice from lethal
challenge by a pandemic seasonal (H1N1) influenza isolate at 0.3, 1, and 3 mg/kg (Table 190).
Additionally, partial protection was seen (relative to vehicle) in the two lowest dose groups (0.1 and
0.03 mg/kg) with 60 and 20% survival, respectively. In contrast, vehicle treated animals succumbed to
infection by Day 8, while hIgG1 Fc only treated animals reached 80% mortality. The potency of
Conjugate 45a was also supported by BW data. Mice treated with fully protective doses (3, 1, and 0.3
mg/kg) displayed transient BW loss around Day 3-5, before steadily recovering BW through the end of
the study (Day 21) (Table 191). Collectively this study demonstrated the potency of Conjugate 45a, by
both mortality and BW readouts, against an important pandemic influenza isolate.

(1661) TABLE-US-00199 TABLE 190 Percent survival hIgG1 Conjugate 45 a Day Post Vehicle Fc (3
(1 (0.3 (0.1 (0.03 Challenge (PBS) (3 mpk) mpk) mpk) mpk) mpk) mpk) 0 100 100 100 100 100 100
100 1 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 3 100 100 100 100 100 100 100
4 100 100 100 100 100 100 100 5 40 60 100 100 100 60 20 6 20 20 100 100 100 60 20 7 20 20 100
100 100 60 20 8 0 20 100 100 100 60 20 9 20 100 100 100 60 20 10 20 100 100 100 60 20 11 20 100
100 100 60 20 12 20 100 100 100 60 20 13 20 100 100 100 60 20 14 20 100 100 100 60 20 15 20 100
100 100 60 20 16 20 100 100 100 60 20 17 20 100 100 100 60 20 18 20 100 100 100 60 20 19 20 100
100 100 60 20 20 20 100 100 100 60 20 21 20 100 100 100 60 20

(1662) TABLE-US-00200 TABLE 191 Average body weight loss (%), until first death within a group
Day hIgG1 Post Fc Conjugate 45 a Chal- Vehicle (3 (3 (1 (0.3 (0.1 (0.03 lenge (PBS) mpk) mpk) mpk)
mpk) mpk) mpk) 0 100 100 100 100 100 100 100 1 97.8 99 96.8 97.9 97.1 99.8 98.4 2 91.6 92 96 93.8
91.8 90.8 91 3 84.5 86.9 93.5 92.2 87.1 84.7 84.3 4 80.4 82.5 95.8 92.3 88.1 80.1 80.3 5 76.5 77.6
95.2 90.6 86.9 78.2 76.6 6 98.2 94.5 89.7 7 97.7 92.8 89.9 8 98.6 94.1 91.4 9 98.3 96.8 92.7 10 99.7
98.2 95 11 102.8 100.3 98.7 12 99.6 99 96.2 13 101.4 101.8 97.5 14 101.5 100.8 96.4 15 100.8 99.1
96.6 16 100.4 101.4 97.7 17 102.7 103.9 100.1 18 102.4 102.5 98.6 19 103.9 103.9 100.9 20 104.6
104.1 100.3 21 104 103.9 99.5

Example 206. Efficacy of Conjugate 45b in Combination with Baloxavir Against Influenza A (H1N1) in
a Lethal Mouse Influenza Model

(1663) Conjugate 45b was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/California/07/2009) is a
pandemic isolate capable of causing lethal infections in mice. The experiment comprised 11 groups of
5 mice. At day 0, all mice were challenged with virus at 3× the LD.sub.95 (˜3E4 virus/mouse) by
intranasal inoculation in a volume of 30 μl after being lightly anesthetized with ketamine/xylazine (100
and 10 mg/kg respectively). Groups received treatment with vehicle, conjugate 45b, baloxavir marboxil
(DC Chemicals, cat no DC111056; suspended in 0.5% methyl cellulose), or a combination (2 hours
after challenge). Baloxavir was dosed orally (PO), twice daily for 3 days and conjugate 45b was
administered subcutaneously (SC) as a single dose (Table 192). Mortality and body weight (BW) were
monitored daily for 14 days. Any mouse losing more than 20% BW was scored as a mortality.

(1664) TABLE-US-00201 TABLE 192 Study Design Route, Schedule Dose Influenza Test (T + (mg/
Group A strain Article 2 hours) kg) N 1 A/CA/07/09 Vehicle (PBS) SC, single — 5 2 (H1N1) conjugate
45b SC, single 0.01 5 3 3E4 PFU/ conjugate 45b SC, single 0.03 5 4 mouse conjugate 45b SC, single
0.1 5 5 via IN conjugate 45b SC, single 0.3 5 6 Baloxavir PO, bid × 3 days 1 5 7 Baloxavir PO, bid × 3
days 3 5 8 Baloxavir PO, bid × 3 days 10 5 9 Baloxavir PO, bid × 3 days 3 5 conjugate 45b SC, single
0.01 5 10 Baloxavir PO, bid × 3 days 3 5 conjugate 45b SC, single 0.03 5 11 Baloxavir PO, bid × 3
days 3 5 conjugate 45b SC, single 0.1 5

(1665) In order to determine the potency of conjugate 45b and baloxavir separately the dose range
was determined for each molecule. For conjugate 45b doses of 0.1 and 0.3 mg/kg were protective
while 0.01 and 0.03 were not based on survival (Table 193). For baloxavir doses of 1 and 3 mg/kg
were able to delay, but not prevent death by Day 14. In contrast, a 10 mg/kg dose of baloxavir was
80% protective.

(1666) For combination studies the mid-range dose of baloxavir (3 mg/kg) was dosed in conjunction
with conjugate 45b at 0.01, 0.03, and 0.1 mg/kg (groups 9-11). As shown in Table 193, neither
conjugate 45b at 0.03 mg/kg or baloxavir at 3 mg/kg are protective individually. However, significantly,
in combination they demonstrated 80% protection over the course of the study (group 10).

(1667) TABLE-US-00202 TABLE 193 % Survival conjugate 45 b Baloxavir (mg/ conjugate 45 b /

(mg/kg) kg) (PO, bid Baloxavir Combo (SC, single) x3 days) (mg/kg) Day Vehicle 0.01 0.03 0.1 0.3 1 3
10 0.01 / 3 0.03 / 3 0.1 / 3 0 100 100 100 100 100 100 100 100 100 100 100 1 100 100 100 100 100
100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 100 100 100 3 100 100 100 100 100
100 100 100 100 100 100 4 100 100 100 100 100 100 100 100 100 100 100 5 20 60 60 100 100 100
100 100 100 100 100 6 0 20 40 100 100 100 100 100 100 100 100 7 0 20 20 100 100 100 100 100
100 100 100 8 0 0 0 100 100 100 80 100 100 100 100 9 0 0 0 100 100 40 0 100 0 80 100 10 0 0 0 100
100 0 0 80 0 80 100 11 0 0 0 100 100 0 0 80 0 80 100 12 0 0 0 100 100 0 0 80 0 80 100 13 0 0 0 100
100 0 0 80 0 80 100 14 0 0 0 100 100 0 0 80 0 80 100

(1668) Body weight data further illustrates the enhanced active of the two molecules administered in
combination (Table 194). When dosed at 0.1/3 mg/kg (conjugate 45b/baloxavir) a dramatic reduction in
BW loss is observed. Conjugate 45b dosed at 0.1 mg/kg by itself is protective but animals
demonstrate an 18.8% drop in BW on Day 4. However, this dose administered with a non-protective
dose of baloxavir (3 mg/kg) reduces the maximum BW loss to 4.5% (group 11).

(1669) TABLE-US-00203 TABLE 194 % Body Weight conjugate Baloxavir conjugate 45 b (mg/kg)
(mg/kg) (PO 45 b / Baloxavir (SC, single) bid x3 days) Combo (mg/kg) Day Vehicle 0.01 0.03 0.1 0.3 1
3 10 0.01 / 3 0.03 / 3 0.1 / 3 0 100 100 100 100 100 100 100 100 100 100 100 1 96.9 97.9 97 97.7
99.8 99.2 97.1 95.9 97.5 96.7 96.8 2 94.3 96.8 95.2 97.3 99.3 99.8 99 101.8 98.9 95.4 98 3 85.4 86.3
85.5 85.5 88.4 90.3 92.1 97.4 94 93.2 95.5 4 78.9 79.8 79.8 81.2 86 90.5 92.5 98 94.2 92.7 95.5 5
77.2 78.7 79.3 81.6 95.3 94.6 97.3 100.8 99.1 96.9 100 6 83.9 98.6 91.1 89.9 98.9 93.3 94.3 99.2 7
85.8 99.1 84.6 83.3 91.1 85.6 87.8 100.4 8 87.9 99.6 79.5 77.9 86.9 79.4 83.9 99.8 9 90 99.1 75.7
86.4 74.8 85.2 102.4 10 89.2 99.9 88.2 100.4 11 92.8 99.9 101 12 97.7 100.8 100.9 13 98.7 101.8
100.3 14 101 101.4 102.1

(1670) In this important study two different groups demonstrated enhanced potency of conjugate 45b
with the co-administration of baloxavir. Also, not to be overlooked is the critical observation that the
two compounds do not inhibit the activity of each other. The observation that the reverse is true, and
the combination is more effective is a benefit that would likely translate into the clinic.

Example 207. Alternate Synthesis of Conjugate 45a

(1671) ##STR00816## ##STR00817## ##STR00818##

(1672) A solution of azido-PEG4-TFP ester (0.1 g, 0.067 mmol) and alkyne functionalized dimer
(0.0383 g, 0.0871 mmol) in DMF (2.0 mL), were treated with a solution of copper(II)sulfate (0.0027 g,
0.0168 mmol), sodium ascorbate (0.0133 g, 0.067 mmol), and THPTA (0.0116 g, 0.027 mmol) at room
temperature, in water (1.5 mL). The reaction was then vacuum flushed with nitrogen 3× and stirred
under an atmosphere of nitrogen. LCMS after 30 min shows complete consumption of starting
material. The reaction was acidified with 400 μL of acetic acid, and then purified directly by reverse
phase chromatography eluting with a gradient of 5% to 100% acetonitrile/water with 0.1% TFA. The
product containing fractions were combined, frozen, and lyophilized overnight. Yield of triple TFA salt
was 69%. Ion(s) found by LCMS: (M+2H).sup.+2=795.4, (M+3H).sup.+3=530.8,
(M+4H).sup.+4=398.4.

(1673) Step b.

(1674) ##STR00819##
(1675) A solution of Fc (0.100 g in 5.2 mL, 1.717 μmol, MW=58,218, SEQ ID NO: 72) in pH=7.4 PBS
buffer was treated with solid TFP ester (0.0273 g, 17.17 μmol) from the previous step. The pH was
adjusted to ˜7.0 with borate buffer (120 μL, 1 M, pH 8.5) then was gently rocked at room temperature.
Maldi TOF after 1.5 hr shows an average DAR of 3.3, which did not change upon further mixing. After
24 hr additional TFP ester (0.0073 g, 4.6 μmol) was added and rocking was continued for another 3 h.
The crude conjugate was purified Protein A and SEC according to general purification methods. Total
yield after Protein A was ˜83%, and after SEC ˜77%. Maldi TOF of the purified conjugate showed an
average mass of 63,574, which equates to an average DAR of 4.0.

(1676) The alternate synthesis described in Example 207 is advantageous at it avoids exposing the Fc
to copper+2 and sodium ascorbate, leading to a cleaner crude conjugate that is 98.9% pure by
analytical SEC after protein A purification alone. At this level of purity it may be possible to eliminate
the SEC purification which is time very consuming and costly. Initial by attempts with an azido-PEG4-
NHS ester were only partially successful because the NHS ester is too reactive to be purified, and the
crude click reaction mixture had to be mixed with the Fc, thus necessitating copper removal and high
molecular weight aggregate removal (from exposure to sodium ascorbate). Also this approach did not
generate DAR's greater than 2. Subsequent attempts using a less reactive active ester (TFP
tetrafluorophenol) that is stable enough to withstand reverse phase purification and lyophilization,
allows the click reaction with azido TFP ester to be done separate from the Fc, purified, and then
mixed with the Fc. In Example 207 an average DAR of 4.0 was achieved and higher DARs are
possible by adding more of the TFP ester.

(1677) The nucleic acid construct encoding the Fc for Conjugate 45a included a nucleic acid encoding
the amino acid sequence of SEQ ID NO: 63, which includes a C-terminal lysine residue. Upon
expression, the C-terminal lysine of the Fc of Conjugate 45a is proteolytically cleaved, resulting in an
Fc having the sequence of SEQ ID NO: 72. The presence or absence of a C-terminal lysine does not
alter the properties of the Fc or the corresponding conjugate.

Example 208. Efficacy of Conjugate 45a in Combination with Baloxavir Against Influenza A (H1N1) in
a Lethal Mouse Influenza Model (Confirmation Study)

(1678) Conjugate 45a was evaluated in combination with baloxavir marboxil (BXM) against a lethal
IAV H1N1 influenza infection in female BALB/c mice (Charles River Laboratories, 6-8 weeks). This
was a follow up to Example 206 which tested the same combination in an initial experiment, but used
a subcutaneous dose route for conjugate 45a instead of intramuscular (IM), which was used in the
present study. The challenge virus (A/California/07/2009) is a pandemic isolate capable of causing
lethal infections in mice. The experiment comprised 8 groups of 5 mice. At day 0, all mice were
challenged with virus at 3× the LD.sub.95 (3E4 virus/mouse) by intranasal inoculation in a volume of
30 μl after being lightly anesthetized with ketamine/xylazine(100 and 10 mg/kg respectively). Groups
received treatment with vehicle, hIgG1 Fc only, conjugate 45a, BXM (DC Chemicals, cat no DC11056;
suspended in 0.5% methyl cellulose), or a combination (2 hours after challenge). BXM was dosed
orally (PO), twice daily for 3 days and conjugate 45a was administered by IM as a single dose (Table
195). Mortality and body weight (BW) was monitored daily for 21 days. Any mouse losing more than
20% BW on 2 consecutive days was scored as a mortality.

(1679) TABLE-US-00204 TABLE 195 Study design for conjugate 45a and BXM combination study
Influenza A Route, Schedule Dose Group strain Test Article (T + 2 hours) (mg/kg) N 1 A/CA/07/09
Vehicle (PBS) IM, single — 5 2 (H1N1) hIgG1 Fc IM, single 0.1 5 3 3E4 Conjugate 45a IM, single 0.03
5 4 PFU/mouse via Conjugate 45a IM, single 0.1 5 5 IN BXM PO, bid × 3 days 3 5 6 BXM PO, bid × 3
days 10 5 7 BXM PO, bid × 3 days 3 5 Conjugate 45a IM, single 0.03 5 8 BXM PO, bid × 3 days 3 5
Conjugate 45a IM, single 0.1 5

(1680) In order to determine the potency of conjugate 45a and BXM separately the dose range was
determined for each molecule. For conjugate 45a a dose of 0.1 mg/kg was protective, while a 0.03
mg/kg dose was not based on survival (Table 196). For BXM, the group receiving 10 mg/kg (bid×3
days) was protected, while those receiving 3 mg/kg with the same dose schedule were not. Groups
administered vehicle or hIgG1 Fc only were not protected. The combination of conjugate 45a (0.1
mg/kg) with BXM (3 mg/kg) was also fully protected as expected based on the above results.

(1681) Group 7 animals received sub-efficacious levels of both conjugate 45a (0.03 mg/kg) and BXM
(3 mg/kg) in combination. Although neither test article was significantly protective when administered
individually, in combination they were fully protective (Table 196). Animals in group 7 also only
displayed transient BW lose which did not exceed 5.2% (Day 3) (Table 197). By study end this group
had exceeded its starting BW reaching 106.3% of initial BW on Day 21. In addition to not exhibiting
any antagonistic effects between conjugate 45a and BXM, the opposite was found, suggesting an
added benefit of co-treatment with these two therapeutics.

(1682) TABLE-US-00205 TABLE 196 % Survival for Study Groups Conjugate Baloxavir Conjugate
Controls 45 a (mg/kg) 45 a / hIgG1 (mg/kg) (PO, Baloxavir Fc (IM, bid Combo (0.1 single) x3 days)
(mg/kg) Day Vehicle mg/kg) 0.03 0.1 3 10 0.03 / 3 0.1 / 3 0 100 100 100 100 100 100 100 100 1 100
100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 3 100 100 100 100 100 100 100
100 4 100 100 80 100 100 100 100 100 5 80 60 60 100 100 100 100 100 6 80 20 20 100 100 100 100
100 7 60 0 20 100 100 100 100 100 8 0 20 100 60 100 100 100 9 20 100 0 100 100 100 10 20 100
100 100 100 11 20 100 100 100 100 12 20 100 100 100 100 13 20 100 100 100 100 14 20 100 100
100 100 15 20 100 100 100 100 16 20 100 100 100 100 17 20 100 100 100 100 18 20 100 100 100
100 19 20 100 100 100 100 20 20 100 100 100 100 21 20 100 100 100 100

(1683) TABLE-US-00206 TABLE 197 Average Body Weight by Day (group average; until 1st death in
a group) Conjugate Controls Conjugate Baloxavir 45 a / hIgG1 45 a (mg/ (mg/kg) Baloxavir Fc kg) (IM,
(PO bid Combo (0.1 single) x3 days) (mg/kg) Day Vehicle mg/kg) 0.03 0.1 3 10 0.03 / 3 0.1 / 3 0 100
100 100 100 100 100 100 100 1 102.1 100.2 99.9 99.6 97.9 97.9 100.3 100 2 94.9 94.3 93.3 95 90.5
98.7 99.9 99.6 3 85 84.2 83.1 83.7 88.5 94.6 94.8 94.3 4 81.3 79.6 79.7 81.4 90.7 95.8 96.9 96.1 5
82.4 80.1 82.3 88.3 95.3 99.4 100.9 100.4 6 91.1 87.7 96.7 99 99.2 7 91.7 81 89.6 96.8 98.5 8 96.9
76.7 90.9 101.6 101.9 9 95.7 92.6 99.1 99 10 99.4 98.2 101.7 102.3 11 98.6 95.8 98.7 100.3 12 98.4
96.8 100.7 99.9 13 101 99.5 102.9 101.1 14 102.3 99.2 103.7 103.6 15 101.2 97.8 102.1 100.2 16
102.8 99.5 103.1 102.8 17 101.9 98.3 102.7 101.1 18 102.3 97.1 101.7 100.1 19 103.9 98.9 101.6
100.6 20 103.9 98.9 104.5 102.6 21 107 101.2 106.3 103.8

Example 209. Determination of Conjugate 45a Potency Against a Component of the 2020-2021
Northern Hemisphere Quadrivalent Vaccine (A/Hawaii/70/2019Pdm; H1N1) in a Lethal Mouse Model

(1684) Conjugate 45a was evaluated in a lethal H1N1 influenza infection model in female BALB/c mice
(Charles River Laboratories, 6-8 weeks). The challenge virus (A/Hawaii/70/2019) is a pandemic H1N1
isolate capable of causing lethal infections in mice. It is also a recommended component of the 2020-
2021 northern hemisphere quadrivalent vaccine.

(1685) The experiment was comprised of 9 groups, with 5 mice per group. At day 0, all mice were
challenged with virus at 2× the LD.sub.95 (2E3 virus/mouse) by intranasal inoculation in a volume of
30 μl after being anesthetized with ketamine/xylazine (100 and 10 mg/kg respectively). Groups
received a single treatment of Conjugate 45a, hIgG1 Fc, or vehicle (PBS), 2 hours after viral challenge
by intramuscular (IM, 5 m1/kg dose volume) administration (right flank). Conjugate 45a was tested at
concentrations of 3, 1, 0.3, 0.1, 0.03, and 0.01 mg/kg. An uninfected group was also included in the
study as a control. Mortality and body weight (BW) was monitored daily for 21 days. Any mouse
maintaining a 20% BW loss on two consecutive days was scored as a mortality. The experimental
outline is summarized in Table 198.

(1686) In this study a single IM administration of conjugate 45a fully protected mice from lethal
challenge by A/HA/70/2019 (H1N1) at 0.3, 1, and 3 mg/kg (Table 199). The potency of Conjugate 45a
at these dose levels was significant compared to vehicle treated animals (P=0.0031). Vehicle and
hIgG1 Fc treated animals in contrast reached 100% mortality on Day 5. Partial protection was also
seen in the 0.1 and 0.03 mg/kg dose groups (60 and 20% survival respectively), but was not
statistically significant compared to vehicle treated animals. Collectively the survival data
demonstrated that conjugate 45a is potent against this important influenza isolate and a clear dose
response was evident in the lower dose groups (FIG. 119).

(1687) The potency of conjugate 45a was also supported by BW data. Mice treated with fully
protective doses (3, 1, and 0.3 mg/kg) displayed transient BW loss on Day 3 (Maximum of 12% BW
loss at a conjugate 45a dose of 0.3 mg/kg), before steadily recovering BW through the end of the
study (Day 21) (Table 200). BW trends mirrored the survival outcome of the study and supported the
efficacy of the conjugate against this H1N1 isolate.

(1688) Collectively this study demonstrated the potency of conjugate 45a, by both mortality and BW
readouts, against an important pandemic influenza isolate. As A/HA/70/2019 has been selected for
inclusion in the 2020 northern hemisphere vaccine it is considered a clinically relevant isolate and a
threat to public health. The potency of conjugate 45a against this pandemic strain with a single IM
dose of 0.3 mg/kg supports its continued development as a therapeutic against influenza.

(1689) TABLE-US-00207 TABLE 198 Outline of conjugate 45 a potency against A/HA/70/2019 study
Influenza A Dose strain (IN Test Route, Dose volume Number Group challenge) Article Schedule
(mg/kg) (ml/kg) of mice 1 Uninfected Vehicle IM, single, — 5 5 (PBS) T + 2 h 2 A/HA/ Vehicle IM,
single, — 5 5 70/2019 (PBS) T + 2 h 3 (H1N1) hIgG1 Fc IM, single, 0.3 5 5 ~2E3 (SEQ ID T + 2 h
pfu/ml NO: 72) 4 Conjugate IM, single, 3 5 5 45a T + 2 h 5 Conjugate IM, single, 1 5 5 45a T + 2 h 6
Conjugate IM, single, 0.3 5 5 45a T + 2 h 7 Conjugate IM, single, 0.1 5 5 45a T + 2 h 8 Conjugate IM,
single, 0.03 5 5 45a T + 2 h 9 Conjugate IM, single, 0.01 5 5 45a T + 2 h

(1690) TABLE-US-00208 TABLE 199 % Survival of animals by group and date Controls hIgG1 Fc (0.3
Conjugate 45 a mg/kg) (SEQ (mg/kg) (IM, single) Day Uninfected Vehicle ID NO: 72) 3 1 0.3 0.1 0.03
0.01 0 100 100 100 100 100 100 100 100 100 1 100 100 100 100 100 100 100 100 100 2 100 100 100
100 100 100 100 100 100 3 100 100 100 100 100 100 100 100 100 4 100 60 60 100 100 100 100 100
80 5 100 0 0 100 100 100 60 20 60 6 100 100 100 100 60 20 60 7 100 100 100 100 60 20 40 8 100
100 100 100 60 20 0 9 100 100 100 100 60 20 10 100 100 100 100 60 20 11 100 100 100 100 60 20
12 100 100 100 100 60 20 13 100 100 100 100 60 20 14 100 100 100 100 60 20 15 100 100 100 100
60 20 16 100 100 100 100 60 20 17 100 100 100 100 60 20 18 100 100 100 100 60 20 19 100 100
100 100 60 20 20 100 100 100 100 60 20 21 100 100 100 100 60 20

(1691) TABLE-US-00209 TABLE 200 % Average Body Weight by Day (group average; until 1.sup.st
death within a group) Controls Conjugate 45 a hIgG1 Fc (mg/kg) (IM, single) Day Uninfected Vehicle
(0.3 mg/kg) 3 1 0.3 0.1 0.03 0.01 0 100 100 100 100 100 100 100 100 100 1 100.8 97.9 97.2 98.6 98.4
98.1 98.5 98.4 99.2 2 103.2 93.9 94.3 100.3 98.9 96.5 96.3 95.9 95.5 3 100.9 84.3 84.8 94.1 92.6 88
85.8 86.2 84.1 4 103.4 78.2 95.1 95.3 88.2 84.1 81.3 81.2 5 103.1 97.5 96.2 91.5 82.7 77 6 105.5
100.3 99.2 96.3 7 104.4 99.6 99.1 95.6 8 105.1 102.2 102.4 100.5 9 104.7 100.7 100.7 99.9 10 103.9
101.6 100.9 100.2 11 106.8 102.3 101.7 100.9 12 108.7 102.8 103.4 101.9 13 105.7 102.1 102.3
101.5 14 108.7 102.9 104.7 103.8 15 110.15 103.1 104 102.8 16 106.5 100.9 101.5 99.5 17 105.5
100.7 101 100.3 18 106.1 100.6 101.6 100.5 19 107.7 100.9 103 101.5 20 107.2 99.9 102.5 101.5 21
107.6 102.5 102.8 104.2

Example 210. Efficacy of Conjugate 45a Against Influenza A/Puerto Rico/8/1934 (H1N1) in a 4-Week
Lethal Mouse Model

(1692) Conjugate 45a was evaluated against a lethal IAV H1N1 influenza infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/1934) is a mouse-
adapted isolate capable of causing lethal infections in mice. The experiment comprised 7 groups of 5
mice. At day 0, all mice were challenged with virus at 3× the LD95 by intranasal (IN) inoculation in a
volume of 30 μl, after being anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg
respectively). The only exception was group 1 which consisted of uninfected mice used as a body
weight (BW) control. Mortality and BW were recorded daily for 28 days and any animal with a
cumulative 20% loss of weight for 2 consecutive days was scored as a death.
(1693) Test groups received a single intramuscular (IM) injection, 2 hours post viral challenge of
conjugate 45a (0.01 to 0.3 mg/kg), hIgG1 Fc only (0.3 mg/kg, SEQ ID NO: 72), or vehicle (PBS).
Injections were in the thigh muscle of the right hind limb at a dose volume of 5 m1/kg.

(1694) TABLE-US-00210 TABLE 201 Study outline Dose Group Influenza A Test Article Route,
Schedule (mg/kg) N 1 Uninfected Vehicle (PBS) IM, single, T + 2 h — 5 2 A/PR/8/34 Vehicle (PBS) IM,
single, T + 2 h — 5 3 (H1N1) hIgG1 Fc IM, single, T + 2 h 0.3 5 4 3E2 Conjugate 45a IM, single, T + 2
h 0.3 5 5 PFU/mouse Conjugate 45a IM, single, T + 2 h 0.1 5 6 Conjugate 45a IM, single, T + 2 h 0.03
5 7 Conjugate 45a IM, single, T + 2 h 0.01 5

(1695) Animals treated with vehicle only began to reach mortality on Day 5, with 100% mortality by
Day 7. Similarly, mice treated with hIgG1 Fc only (which lacks the antiviral moiety of the conjugate)
had fully succumbed to infection on Day 7 (FIG. 120 and Table 202). In contrast, groups receiving
conjugate 45a at 0.1 and 0.3 mg/kg were fully protected until study end on Day 28. Relative to vehicle
only treated mice this difference in survival was statistically significant (P=0.0020 for both groups by
the Log-rank (Mantel-Cox) test). Conjugate 45a administered at dose levels of 0.01 and 0.03 mg/kg
however were sub-efficacious, with mice reaching 100% mortality on Days 8 and 7 respectively (FIG.
120 and Table 202).

(1696) TABLE-US-00211 TABLE 202 % Survival of Study Groups by Day Controls hIgG1 Fc
Conjugate (0.3 45 a (mg/kg) Day Uninfected Vehicle mg/kg) 0.3 0.1 0.03 0.01 0 100 100 100 100 100
100 100 1 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 3 100 100 100 100 100 100
100 4 100 100 100 100 100 100 100 5 100 80 100 100 100 100 100 6 100 20 40 100 100 80 80 7 100
0 0 100 100 0 20 8 100 100 100 0 9 100 100 100 10 100 100 100 11 100 100 100 12 100 100 100 13
100 100 100 14 100 100 100 15 100 100 100 16 100 100 100 17 100 100 100 18 100 100 100 19 100
100 100 20 100 100 100 21 100 100 100 22 100 100 100 23 100 100 100 24 100 100 100 25 100 100
100 26 100 100 100 27 100 100 100 28 100 100 100

(1697) In addition to survival, BW was also monitored daily throughout the study. Uninfected mice
(group 1) steadily gained BW reaching 108.7% of their starting BW by study end, indicating the
general good health of test animals (Table 203). In contrast, negative control groups (2 and 3) began
losing BW on Day 1 until the first animal in each group reached mortality on Day 5 and 6 respectively.
After the first death within a group BW values were not recorded because the group becomes a biased
population.

(1698) Mice treated with a protective dose of conjugate 45a (groups 4 and 5) only displayed a
transient BW loss of less than 5%. By study end both of these groups exceeded their starting BW
(105.9 and 108.7% respectively). As expected, mice receiving a sub-protective dose of conjugate 45a
(groups 6 and 7) showed a steady loss of weight until the animals began reaching mortality.

(1699) Collectively this data shows the potency of conjugate 45a against lethal challenge by influenza
A (H1N1). Significantly, protection was achieved by a single IM injection of less than 1 mg/kg. Lastly,
animals were followed for a full 4-weeks after viral challenge indicating that protected animals had
most likely full cleared the infection.

(1700) TABLE-US-00212 TABLE 203 % Average Body Weight by Day (group average; until 1st death
within a group) Controls hIgG1 Conjugate 45 a Fc (0.3 (mg/kg) (IM, single) Day Uninfected Vehicle
mg/kg) 0.3 0.1 0.03 0.01 0 100 100 100 100 100 100 100 1 100.5 98.1 97.7 96 98.7 98.5 98.2 2 101.3
97.8 98.3 96.5 99 100.4 98.4 3 102 92.1 92.7 97.2 98.3 94.6 94.4 4 103.2 84.3 85.1 97.4 95.3 89.2
87.7 5 106.5 80.8 101.2 102.3 89.1 83.9 6 104.9 74.7 99.7 98.6 81.4 77.9 7 106.8 102.6 101.3 8 108.3
102.3 102.7 9 103.6 99.6 101.7 10 108.4 103.9 105.4 11 105.1 100.5 102.4 12 108 103.8 107.1 13
107.7 102.9 105.1 14 106.3 102.9 105.3 15 106.6 103.4 105.9 16 104.2 101.2 102.9 17 104.9 101.2
103.9 18 107 103.9 106.9 19 107.3 104.3 106.1 20 105.6 103 105.7 21 104.7 103 105.6 22 108.5
106.8 107.8 23 106.8 103.5 106 24 106.4 101.4 107.7 25 106.1 103.2 107 26 109.3 106.1 109.4 27
108 105.1 107.8 28 108.7 105.9 108.7
Example 211. Efficacy of Conjugate 45a and Oseltamivir Against Influenza H1N1 A/CA/12/2012 and
A/PR/8/1934 in a Lethal Mouse Model

(1701) Conjugate 45a and oseltamivir were evaluated against lethal challenge by two influenza H1N1
subtypes (A/PR/8/34 and A/CA12/12) in female BALB/c mice (Charles River Laboratories, 6-8 weeks).
The experiment comprised 10 groups of 5 mice split into two experimental arms (Table 204). At T+2 h,
mice were intramuscularly (IM) administered conjugate 45a at 0.3 mg/kg in a single dose. Also at T+2
h mice were orally administered oseltamivir at 5 or 50 mg/kg, twice daily for 5 days. Control mice were
treated IM with vehicle (PBS) or hIgG1 Fc only. Two hours prior to compound administration mice were
intranasally challenged with 3× the LD95 of A/PR/8/34 (3E2 pfu) or A/CA/12/12 (3E4 pfu). For viral
challenge mice were anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg
respectively), and the virus was given in a volume of 30 μl. Mortality and body weights (BW) were
recorded daily for 14 days and any animal with a 20% loss of BW was scored as a death.

(1702) Against A/PR/8/34 challenge mice treated with vehicle or Fc only (SEQ ID NO: 72) succumbed
to infection by day 7, while those administered Conjugate 45a at 0.3 mg/kg were fully protected (Table
205). For oseltamivir treated animals, a 50 mg/kg dose was 80% protective while a dose of 5 mg/kg
only delayed death until day 10. Against A/CA/12/12 vehicle and Fc control animals reached 100%
mortality by day 6, and once again, a 0.3 mg/kg dose of Conjugate 45a was fully protective. In
contrast, oseltamivir was unprotective at 5 mg/kg, and even the 50 mg/kg dose group only had 40%
survival. BW data for both study arms supported the mortality findings (Tables 205-208).

(1703) Collectively this data demonstrates superior activity by a single 0.3 mg/kg dose of conjugate is
superior to oseltamivir, even when the latter is dosed at 5 or 50 mg/kg twice daily for 5 days.

(1704) TABLE-US-00213 TABLE 204 Study design Cmpd prep Influenza A Dose strain (IN Test Route,
Dose volume Vol needed Group challenge) Article DAR Schedule (mg/kg) ml/kg mg/ml (ml) N 1
A/PR/8/34 Vehicle — IM, single, — 5 — 1 5 (H1N1) (PBS) T + 2 h 2 3E2 PFU/mouse Fc only — IM,
single, 0.3 5 0.06 1 5 T + 2 h 3 Conjugate 4.6 IM, single, 0.3 5 0.06 1 5 45a T + 2 h 4 Oseltamivir —
PO, bid × 5 days 5 10 0.5 14 5 5 Oseltamivir — PO, bid × 5 days 50 10 5 14 5 Arm II 6 A/CA/12/12
Vehicle — IM, single, — 5 — 1 5 (H1N1) (PBS) T + 2 h 7 3E4 PFU/mouse Fc only — IM, single, 0.3 5
0.06 1 5 T + 2 h 8 Conjugate 4.6 IM, single, 0.3 5 0.06 1 5 45a T + 2 h 9 Oseltamivir — PO, bid × 5
days 5 10 0.5 14 5 10 Oseltamivir — PO, bid × 5 days 50 10 5 14 5

(1705) TABLE-US-00214 TABLE 205 % Survival of A/PR/8/34 Conjugate Day post Vehicle Fc only 45a
Oseltamivir Oseltamivir challenge (PBS) (0.3 mpk) (0.3 mpk) (5 mpk) (50 mpk) 0 100 100 100 100 100
1 100 100 100 100 100 2 100 100 100 100 100 3 100 100 100 100 100 4 100 100 100 100 100 5 100
100 100 100 100 6 0 60 100 100 100 7 0 100 100 100 8 100 60 100 9 100 40 100 10 100 0 80 11 100
80 12 100 80 13 100 80 14 100 80

(1706) TABLE-US-00215 TABLE 206 % Survival of A/CA/12/2012 challenged mice. Conjugate Day
post Vehicle Fc only 45a Oseltamivir Oseltamivir challenge (PBS) (0.3 mpk) (0.3 mpk) (5 mpk) (50
mpk) 0 100 100 100 100 100 1 100 100 100 100 100 2 100 100 100 100 100 3 100 100 100 100 100 4
80 100 100 100 100 5 0 20 100 20 40 6 0 100 20 40 7 100 20 40 8 100 0 40 9 100 40 10 100 40 11
100 40 12 100 40 13 100 40 14 100 40

(1707) TABLE-US-00216 TABLE 207 Average % BW of A/PR/8/34 challenged mice, until the first
death within a group Conjugate Day post Vehicle Fc only 45a Oseltamivir Oseltamivir challenge (PBS)
(0.3 mpk) (0.3 mpk) (5 mpk) (50 mpk) 0 100 100 100 100 100 1 98.9 100.2 99.6 98.5 98.6 2 102 104.2
102.5 100.7 99.2 3 94.7 97.1 100.6 99.4 100.3 4 85.8 88.8 100.9 93.5 97.9 5 78.9 81.5 99 88.5 90.5 6
74.9 77.6 102 90 96 7 104 84.5 89.7 8 103.9 79.4 84.2 9 104.6 86.3 10 105.4 90.3 11 103.4 12 103.9
13 104.3 14 106.3

(1708) TABLE-US-00217 TABLE 208 Average % BW of A/CA/12/2012 challenged mice, until the first
death within a group Conjugate Day post Vehicle Fc only 45a Oseltamivir Oseltamivir challenge (PBS)
(0.3 mpk) (0.3 mpk) (5 mpk) (50 mpk) 0 100 100 100 100 100 1 99.6 97.6 100.2 98.3 98.9 2 92.2 90.6
91.2 92.9 93.5 3 85.4 83.5 86.7 86.4 86.3 4 78 77.9 85.5 80.9 82.2 5 87.2 76.2 78.7 6 90.7 7 94.2 8
96.3 9 97.8 10 100 11 98.6 12 98.1 13 98.5 14 100.4

Example 212. Efficacy of Conjugate 45a Against Influenza B/Florida/4/2006 (Yamagata) in a 28-Day
Mouse Prevention Model

(1709) Conjugate 45a was evaluated against lethal challenge by an influenza B seasonal influenza
subtype (B/Florida/4/2006) in female BALB/c mice (Charles River Laboratories, 6-8 weeks). The
experiment comprised 7 groups of 5 mice. On day 0, mice were subcutaneously (SC) administered
conjugate 45a at 3, 1, 0.3, 0.01, or 0.3 mg/kg in a single dose. Control mice were also treated by the
same route with vehicle (PBS) or hIgG1 Fc only. Twenty-eight Days after administration of test article,
mice were challenged intranasally with 3× the LD.sub.95 of B/Florida. A summary of the experimental
design is provided in Table 209. For viral challenge mice were anesthetized with a mixture of
ketamine/xylazine (150 and 10 mg/kg respectively), and the virus was given in a volume of 30 μl.
Mortality and body weights (BW) were recorded daily for 21 days and any animal with a 20% loss of
body weight was scored as a death.

(1710) Mice treated with vehicle only reached mortality by day 7, while those treated with the Fc only
negative control succumbed to infection by day 8 (Table 210). In contrast, those receiving conjugate
45a at 1 or 3 mg/kg were fully protected over the course of the study. Group 4 animals which were
treated at 0.3 mg/kg also demonstrated 80% survival. At a dose concentration of 0.1 mg/kg survival
dropped to 40%, and at 0.03 mg/kg no protection was afforded by conjugate 45a. BW data (Table 211)
mirrored the mortality results and mice receiving a fully protective dose (1 and 3 mg/kg) showed less
than a transient 5% drop in BW on day 4, before recovering their starting BW.

(1711) Collectively, these data support the robust potency of conjugate 45a against in important
seasonal subtype of influenza. It also demonstrates the utility of conjugate 45a as a long-term
preventative against influenza.

(1712) TABLE-US-00218 TABLE 209 General study design Cmpd prep Influenza B Dose Vol strain (IN
Route, Dose Dose volume needed Group challenge) Test Article DAR Schedule (mg/kg) timing ml/kg
mg/ml (ml) N 1 B/FL/4/2006 Vehicle (PBS) — SC, single — T − 28 Days 10 — 1.5 5 2 (Yamagata) Fc
only — SC, single 3 T − 28 Days 10 0.3 1.5 5 3 5E4 Conjugate 45a 4.7 SC, single 3 T − 28 Days 10
0.3 1.5 5 4 PFU/mouse Conjugate 45a 4.7 SC, single 1 T − 28 Days 10 0.1 1.5 5 5 Conjugate 45a 4.7
SC, single 0.3 T − 28 Days 10 0.03 1.5 5 6 Conjugate 45a 4.7 SC, single 0.1 T − 28 Days 10 0.01 1.5
5 7 Conjugate 45a 4.7 SC, single 0.03 T − 28 Days 10 0.003 1.5 5

(1713) TABLE-US-00219 TABLE 210 % Survival for study groups Day Post Vehicle Fc only Conjugate
45a Conjugate 45a Conjugate 45a Conjugate 45a Conjugate 45a Infection (PBS) (3 mpk) (3 mpk) (1
mpk) (0.3 mpk) (0.1 mpk) (0.03 mpk) 0 100 100 100 100 100 100 100 1 100 100 100 100 100 100 100
2 100 100 100 100 100 100 100 3 100 100 100 100 100 100 100 4 100 100 100 100 100 100 100 5
100 100 100 100 100 100 100 6 60 100 100 100 100 100 100 7 0 20 100 100 80 80 80 8 0 100 100 80
40 0 9 100 100 80 40 10 100 100 80 40 11 100 100 80 40 12 100 100 80 40 13 100 100 80 40 14 100
100 80 40 15 100 100 80 40 16 100 100 80 40 17 100 100 80 40 18 100 100 80 40 19 100 100 80 40
20 100 100 80 40 21 100 100 80 40

(1714) TABLE-US-00220 TABLE 211 Average % daily BWs until the first death within a group Day
Post Vehicle Fc only Conjugate 45a Conjugate 45a Conjugate 45a Conjugate 45a Conjugate 45a
Infection (PBS) (3 mpk) (3 mpk) (1 mpk) (0.3 mpk) (0.1 mpk) (0.03 mpk) 0 100 100 100 100 100 100
100 1 97 94.9 97.1 98.3 96.9 96.9 97.2 2 97.8 96.3 97.7 98.5 99.5 97.9 98.9 3 93.9 94.3 99.9 100.9
99.7 97.6 98.8 4 84.5 85.3 95.8 95.8 90.9 88.9 89.3 5 81.4 83.1 102 99.4 95.1 90.6 87.5 6 76.5 78.2
101.1 99.8 91.9 85.9 81.9 7 98.7 97.9 87 79.5 76 8 99.6 97.9 88 9 99.6 99 10 99.9 100.2 11 101.9
100.7 12 99.5 99.4 13 100 99.4 14 99.4 101 15 98 97 16 100 99.4 17 97.5 98.2 18 99.4 100.4 19 98.8
100.9 20 100.8 101.9 21 100 100.1

Example 213. Fc-Mediated Immune Contribution of Conjugate 45a

(1715) Efficacy studies were conducted in 6-8 weeks female BALB/c mice (Charles River) or
Fcer1g.sup.−/− model 583 (Taconic) challenged intranasally with 3E2 PFU/mouse (3× the LD9s) of
mouse-adapted influenza A/Puerto Rico/8/1934 (H1N1) or with 3e4 PFU/mouse of influenza
A/CA/07/2009 (H1N1)pdm. AVCs or controls—human IgG1 Fc or PBS—were administered as a single
dose administered subcutaneously (SC) or intramuscular (IM) as indicated 2 h post-challenge at 0.03-
3 mg/kg. Body weights (BW) were recorded daily. Mortality is defined as body weight loss of greater
20% for two consecutive days or when animals are moribund.

(1716) For viral burden and cytokine analysis, at 4 days post-infection, mice were sacrificed by
CO.sub.2 and both lung lobes were harvested. Lungs were homogenized with 1 mm silica beads in 1
mL PBS using a MagNA Lyser (Roche). Homogenization was carried out at 6,000 rpm for 60 s and
chilled on ice for 5 min in-between runs. After lung homogenization tubes were centrifuged for 10 min
at 600×g and supernatant was transferred into new tube. For PFU determination, supernatants of lung
homogenate were diluted in infection buffer ranging from 10.sup.−1 to 10.sup.−6. 100 μL of virus
dilutions were added to confluent monolayer of MDCK cells in 24 well plates and incubated for 1 h at
room temperature with rocking every 15 min. After removing the virus, liquid overlay media containing
Avicel was added to MDCK cells. Cells were incubation at 37° C., 5% CO.sub.2 for 40 h. After
incubation, the media was removed and cells were stained with crystal violet to enumerate plaques.
and plaque forming units (PFU) were calculated relative to weight of the lung (PFU/g lung). For
cytokine analysis, supernatants of lung homogenate were serially diluted 2-fold in 96 well plate.
Cytokine levels for IL-6, MIP-1ca and MCP-1 were determined by ELISA according to manufacturer's
instructions (R&D Systems).

(1717) Conjugate 45a at 0.03 mg/kg or higher dose was fully protective against a lethal challenge with
influenza A/CA/07/2009 (H1N1)pdm in Balb/C (WT) and Fcer1g.sup.−/− (KO) mice (see Table 212)
suggesting that the protection of conjugate 45a is independent of Fc-mediated immune contribution.

(1718) TABLE-US-00221 TABLE 212 Efficacy of Conjugate 45a in Balb/C mice (WT) or Fcer1g.sup.−/
− (KO) mice against influenza A/CA/07/2009 (H1N1)pdm. Mouse Test article [mg/kg] Dose route
background % Survival hIgG1 Fc [1] IM WT 0 conjugate 45a [0.03] IM WT 100 conjugate 45a [0.1] IM
WT 100 conjugate 45a [0.3] IM WT 100 conjugate 45a [1] IM WT 100 hIgG1 Fc [1] IM KO 0 conjugate
45a [0.03] IM KO 100 conjugate 45a [0.1] IM KO 100 conjugate 45a [0.3] IM KO 100 conjugate 45a [1]
IM KO 100

(1719) Conjugate 45a at 0.1 mg/kg or higher dose was fully protective against a lethal challenge with
influenza A/PR/8/1934 (H1N1) in Balb/C mice (see Table 213). To determine the immune contribution
in WT Balb/c mice, a version of conjugate 45a on an aglycosylated Fc mutant (N297A), conjugate 49
(e.g., an Fc comprising SEQ ID NO: 72 further comprising an N297A mutation), was made. This Fc
mutant is known to result in largely abrogated immune effector function. conjugate 49 was also
protective at 0.1 mg/kg suggesting that the protection conferred by conjugate 45a is independent of
Fc-mediated immune contribution. To determine if the efficacy of conjugate 45a can be enhanced by
increased Fc-mediated immune effector function, a conjugate 45a version was conjugated to an Fc
comprising a DE (S239D/1332E) mutation (e.g., an Fc comprising SEQ ID NO: 72 further comprising
an S239D/1332E mutation), conjugate 50. Conjugate 50 was fully protective at 0.1 mg/kg or higher
dose, which is comparable to activity of conjugate 45a. However, following treatment with conjugate 50
at 0.03 mg/kg, one mouse survived. Thus, the activity of conjugate 45a at DAR 4.5 is independent of
Fc-mediated immune contribution.

(1720) TABLE-US-00222 TABLE 213 Efficacy of AVCs against influenza A/PR/8/1934 (H1N1) in
Balb/C mice (WT). Test article [mg/kg] Dose route Fc % Survival hIgG1 Fc [1] IM WT 0 conjugate 45a
[0.01] IM WT 0 conjugate 45a [0.03] IM WT 0 conjugate 45a [0.1] IM WT 100 conjugate 45a [0.3] IM
WT 100 conjugate 49 [0.01] IM N297A 0 conjugate 49 [0.03] IM N297A 0 conjugate 49 [0.1] IM N297A
100 conjugate 49 [0.3] IM N297A 100 conjugate 49 [1] IM N297A 100 conjugate 50 [0.01] IM
S239D/I332E 0 conjugate 50 [0.03] IM S239D/I332E 20 conjugate 50 [0.1] IM S239D/I332E 100
conjugate 50 [0.3] IM S239D/I332E 100
(1721) After a lethal challenge with influenza in a mouse model, lung PFU burden and lung cytokine
levels were determined on day 4 post-infection. Conjugate 45a and conjugate 49 demonstrated a
dose-dependent log reduction in viral burden to comparable levels (see table 214). Conjugate 50
demonstrated higher reduction in viral burden at 0.1 mg/kg between 0.96 or 0.83 logs higher as for
conjugate 45a or conjugate 49, respectively. No biological relevant difference was observed between
negative controls, PBS and hIgG1 Fc as expected.

(1722) TABLE-US-00223 TABLE 214 Viral burden reduction by conjugates on day 4 post-infection
challenged with influenza A/PR/8/1934 (H1N1) in a mouse model. Test article [mg/kg] Dose route
PFU/g Log reduction PBS SC 2.19E + 07 0.00 hIgG1 Fc [3] SC 2.85E + 07 −0.11 conjugate 45a [0.03]
SC 1.84E + 07 0.07 conjugate 45a [0.1] SC 6.34E + 06 0.54 conjugate 45a [0.3] SC 2.63E + 05 1.92
conjugate 45a [1] SC 9.99E + 03 3.34 conjugate 45a [3] SC 1.53E + 03 4.16 conjugate 49 [0.03] SC
9.06E + 06 0.38 conjugate 49 [0.1] SC 4.68E + 06 0.67 conjugate 49 [0.3] SC 1.36E + 05 2.21
conjugate 49 [1] SC 4.57E + 04 2.68 conjugate 49 [3] SC 5.89E + 03 3.57 conjugate 50 [0.1] SC 6.40E
+ 05 1.53

(1723) Similarly, conjugate 45a and conjugate 49 reduced cytokines, IL-6, MIP-1a, MOP-1, in dose-
dependency to similar levels (see table 215). As for conjugate 50 at 0.1 mg/kg slightly lower levels for
IL-6 and MIP-1a, but comparable levels were observed, conjugate 50 at 0.1 mg/kg showed markedly
increased MOP-1 levels as compared to conjugate 45a and conjugate 49. No biological relevant
difference was observed between negative controls, PBS and hIgG1 Fc as expected. Table 215:
Cytokine levels in fold-change versus uninfected control for IL-6, MIP-1ac and MOP-i on dy 4 post-
infection challenged with influenza A in a mouse model.

(1724) TABLE-US-00224 TABLE 215 Cytokine levels in fold-change versus uninfected control for IL-6,
MIP-1α and MCP-1 on day 4 post-infection challenged with influenza A in a mouse model. Test article
[mg/kg] IL-6 MCP-1 MIP-1α PBS 4.4 30.0 13.7 hIgG1 Fc [3] 4.6 31.9 15.4 conjugate 45a [0.03] 3.1
14.8 14.3 conjugate 45a [0.1] 2.5 10.2 7.1 conjugate 45a [0.3] 2.1 3.2 3.7 conjugate 45a [1] 2.0 1.2 2.2
conjugate 45a [3] 2.0 1.2 2.1 conjugate 49 [0.03] 2.3 19.4 10.6 conjugate 49 [0.1] 2.4 11.1 5.7
conjugate 49 [0.3] 2.9 10.7 5.0 conjugate 49 [1] 2.5 4.7 2.4 conjugate 49 [3] 2.3 4.5 2.2 conjugate 50
[0.1] 1.9 25.6 4.9 Uninfected 1.0 1.0 1.0

Example 214. Efficacy of Conjugate 45a Against Influenza 2020-2021 Vaccine Strains Neuraminidase
Inhibition (NAI)

(1725) NAI activity was determined using the commercial NA-Fluor kit. Briefly, live viruses were
adjusted to 1e5 PFU/mL and added to appropriate wells in 96 well plate (black). Test articles were
added at concentrations ranging from 0.001 to 1,000 nM to appropriate wells. Virus and test article
were incubated for 20-30 min at 37° C., 5% CO.sub.2. Next, NA substrate was added to each well and
incubated for 1 h at 37° C., 5% CO.sub.2. NAI was determined by reading fluorescence at 355 nm
excitation/460 nm emission. IC.sub.50 was calculated with GraphPad Prism version 8 using nonlinear
regression analysis (Dose-response (Inhibition)).

(1726) Conjugate 45a demonstrated comparable potency by IC.sub.50 as compared to oseltamivir or

zanamivir in NAI against influenza vaccine strains 2020-21 for Northern Hemisphere (Table 216).

(1727) TABLE-US-00225 TABLE 216 Activity of conjugate 45a in neuraminidase inhibition (NAI) assay
against influenza vaccine strains 2020-21 for Northern Hemisphere (IC.sub.50) subtype/ conjugate
Oseltamivir Zanamivir influenza strain lineage 45a [nM] [nM] [nM] A/Hawaii/70/2019 H1N1pdm09 0.21
0.32 0.26 A/Hong H3N2 4.8 0.76 1.05 Kong/2671/2019 B/Phuket/3073/ Yamagata 4.58 24.44 2.78
2013 B/Washington/ Victoria 3.11 16.71 2.3 02/2019

Enzyme-Linked Lectin Assay (ELLA).

(1728) Nunc Maxisorp 96-well plates (ThermoFisher) were coated with 2.5 μg fetuin (Sigma-Aldrich) in
1×KPL coating buffer (SeraCare) overnight at 4° C. The next day, plates were washed with PBS at pH
7.4 supplemented with 0.05% Tween 20 (PBST). Test articles were tested at 0.001-1000 nM and
added in 50 μL/well. Influenza virus was added at 5e4-5e5 PFU/mL in 50 μL/well. Plates were
incubated for 16-18 h at 37° C., 5% CO.sub.2. After washing plates, peanut agglutinin conjugated to
HRP (PNA-HRP) was added at 0.13 μg in 100 uL buffer for 2 h, washed again and developed with 100
μL/well TMB substrate (BD) for 3-5 min. The reaction was stopped with 100 μL/well 1 N
H.sub.2SO.sub.4. Absorbance was read at 450 nm with an EnSpire multimode plate reader. IC.sub.50
was calculated with GraphPad Prism version 8 using nonlinear regression analysis (Dose-response
(Inhibition)).

(1729) Conjugate 45a demonstrated increased potency by IC.sub.50 as compared to oseltamivir or

zanamivir in NAI against influenza vaccine strains 2020-21 for Northern Hemisphere (Table 217).

(1730) TABLE-US-00226 TABLE 217 Activity of conjugate 45a in enzyme-linked lectin assay (ELLA)
against influenza vaccine strains 2020-21 for Northern Hemisphere (IC.sub.50) subtype/ conjugate
hIgG1 Fc Oseltamivir Zanamivir influenza strain lineage 45a [nM] [nM] [nM] [nM] A/Hawaii/70/2019
H1N1pdm09 0.2 >1,000 5.9 2.7 A/Hong Kong/2671/2019 H3N2 0.1 >1,000 0.3 0.4
B/Phuket/3073/2013 Yamagata 2.8 >1,000 28.8 9 B/Washington/02/2019 Victoria 3.9 >1,000 47.9 6.6

Plaque Reduction Assay (PRA).

(1731) Test articles ranging from 0.3 to 100 nM were incubated with virus were pre-incubated in buffer
containing 0.28% bovine serum albumin and Ca.sup.2+/Mg.sup.2+ in PBS for 30 min at room
temperature (RT). A confluent monolayers of Madin Darby Canine Kidney (MDCK) cells in 24 well
plate were washed once with PBS. After incubation of test article and virus, both were added to MDCK
cells. The MOI for each drug-virus combination was selected to target 30 plaques in the PBS control
well. Virus and test article were removed after 1 h and the infected cells were incubated for 48 h at 35°
C. in the presence the test article diluted in a mixture of 1.25% Avicel, DMEM, 0.01% DEAE- dextran
and 2 μg/mL of TPCK trypsin. After 48 h the Avicel mixture was removed, cells were fixed with
paraformaldehyde and stained with 1% crystal violet to count the plaques. EC.sub.50 was calculated
with GraphPad Prism version 8 using nonlinear regression analysis (Dose-response (Inhibition)).

(1732) Conjugate 45a demonstrated increased potency by EC.sub.50 as compared to oseltamivir,

zanamivir or baloxavir in PRA against influenza vaccine strains 2020-21 for Northern Hemisphere
(Table 218).

(1733) TABLE-US-00227 TABLE 218 Activity of conjugate 45a in plaque reduction assay (PRA)
against influenza vaccine strains 2020-21 for Northern Hemisphere (EC.sub.50) subtype/ conjugate
45a Oseltamivir Zanamivir Baloxavir influenza strain lineage [nM] [nM] [nM] [nM] A/Hawaii/70/2019
H1N1pdm09 0.14 7.08 3.69 10.3 B/Phuket/3073/2013 Yamagata 2.61 28.5 13.9 30.96
B/Washington/02/2019 Victoria 7.46 28.72 9.77 18.27

Cytopathic Effect (CPE).

(1734) A confluent monolayer of MDCK Siat1 cells in 96 well plate was incubated with test articles at
concentrations ranging between 0.01-10,000 nM. After 1 h incubation at RT, influenza virus was added
at MOI 0.01. After an additional 1 h incubation at RT, plates were incubated for 3 days for influenza A
or 5 days for influenza B at 37° C., 5% CO.sub.2. CPE was determined by crystal violet staining by
reading absorbance at 595 nm. EC.sub.50 was calculated with GraphPad Prism version 8 using
nonlinear regression analysis (Dose-response (Inhibition)).

(1735) Conjugate 45a demonstrated increased potency by EC.sub.50 as compared to oseltamivir,

zanamivir or baloxavir in PRA against influenza vaccine strains 2020-21 for Northern Hemisphere
(Table 219).

(1736) TABLE-US-00228 TABLE 219 Activity of conjugate 45a in cytopathic effect (CPE) assay
against influenza vaccine strains 2020-21 for Northern Hemisphere (EC.sub.50) subtype/ conjugate
Oseltamivir Zanamivir Baloxavir influenza strain lineage 45a [nM] [nM] [nM] [nM] A/Hawaii/70/2019
H1N1pdm09 1.65 326.6 686.2 11.1 A/Hong Kong/2671/2019 H3N2 0.38 7.7 170 1
B/Phuket/3073/2013 Yamagata 0.33 27.6 5.2 10.01 B/Washington/02/2019 Victoria 1.06 1028 1.93
9.98

Example 215. Efficacy of Conjugate 45a (*Protein a Column Purified and *Protein a Column Flow-
Through) Against Influenza A/Puerto Rico/8/1934 (H1N1) in a Lethal Mouse Model

(1737) Conjugate 45a (protein A purified) and *conjugate 45a (protein A column flow-through) were
evaluated against a lethal IAV H1N1 influenza infection in female BALB/c mice (Charles River
Laboratories, 6-8 weeks). The challenge virus (A/Puerto Rico/8/1934) is a mouse-adapted isolate
capable of causing lethal infections in mice. The experiment comprised 13 groups of 5 mice and the
purpose was to evaluate the relative potency between the three test agents. At day 0, all mice were
challenged with virus at 3× the LD95 (5E2 pfu) by intranasal (IN) inoculation in a volume of 30 μl, after
being anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg respectively). Group 1
consisted of control mice treated with vehicle only, the remaining groups received one of the
conjugates at doses ranging from 0.01 to 0.3 mg/kg. Mortality and body weights (BW) were recorded
daily for 14 days and any animal with a cumulative 20% loss of weight for 2 consecutive days was
scored as a death.

(1738) Test groups received a single intramuscular (IM) injection, 2 hours post viral challenge of test
article (study outline is detailed in Table 220). Injections were in the thigh muscle of the right hind limb
at a dose volume of 5 m1/kg.

(1739) TABLE-US-00229 TABLE 220 Study outline for Conjugate 45a process development study
Influenza A strain Dose Group (IN challenge) Test Article Route, Schedule (mg/kg) N 1 A/PR/8/34
Vehicle (PBS) IM, single, T + 2 h — 5 2 (H1N1) Conjugate 45a IM, single, T + 2 h 0.3 5 3 5E2 (protein
A IM, single, T + 2 h 0.1 5 4 PFU/mouse column IM, single, T + 2 h 0.03 5 5 purified) IM, single, T + 2
h 0.01 5 6 *Conjugate 45a IM, single, T + 2 h 0.3 5 7 (protein A IM, single, T + 2 h 0.1 5 8 column IM,
single, T + 2 h 0.03 5 9 flow-through) IM, single, T + 2 h 0.01 5

(1740) Animals treated with vehicle only began to reach mortality on Day 6, with 100% mortality by
Day 8 (FIG. 121 and Table 221). In contrast, both conjugates (e.g., protein A column purified conjugate
45a and protein A column flow-through *conjugate 45a) were fully protective at 0.1 and 0.3 mg/kg
through study end (Day 14). At the two lowest test concentrations (0.03 and 0.01 mg/kg), no, or partial
protection was seen (40 and 0% for Conjugate 45a, respectively; 80 and 40% for *Conjugate 45a).
However the differences between conjugates at 0.03 and 0.01 mg/kg was not statistically significant
(Log-rank Mantel-Cox test) and probably represents normal experimental variation from very low test
concentrations.

(1741) TABLE-US-00230 TABLE 221 % survival by group and day (mg/kg) Vehicle Conjugate
Conjugate Conjugate Conjugate *Conjugate *Conjugate *Conjugate *Conjugate Day (PBS) 45a (0.3)
45a (0.1) 45a (0.03) 45a (0.01) 45a (0.3) 45a (0.1) 45a (0.03) 45a (0.01) 0 100 100 100 100 100 100
100 100 100 1 100 100 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 100 3 100
100 100 100 100 100 100 100 100 4 100 100 100 100 100 100 100 100 100 5 100 100 100 100 100
100 100 100 100 6 80 100 100 100 100 100 100 100 100 7 20 100 100 100 100 100 100 100 100 8 0
100 100 100 40 100 100 100 80 9 100 100 40 0 100 100 80 40 10 100 100 40 100 100 80 40 11 100
100 40 100 100 80 40 12 100 100 40 100 100 80 40 13 100 100 40 100 100 80 40 14 100 100 40 100
100 80 40 Conjugate 45a purified using a protein A column; *Conjugate 45a from protein A column
flowthrough (e.g., conjugate that did not bind to protein A)

(1742) Body weight (BW) data (Table 222) was also evaluated for all dose groups. As expected, BW
data largely reflected survival data with all fully protective doses with any conjugate demonstrating
transient loss before recovery by study end. In these dose groups the average BW on Day 14 was
100% or greater. Collectively this data demonstrates the potency of all 3 conjugates against lethal
challenge by influenza A (H1N1). Significantly, protection was achieved by a single IM injection of 0.1
mg/kg. By the conditions evaluated in this study Conjugates 45a and *Conjugate 45a (e.g., conjugate
that did not bind to protein A column) demonstrated comparable potency.
(1743) TABLE-US-00231 TABLE 222 % BW of animals by group and day (mg/kg) (Data shown until
the first death within a group) Vehicle Conjugate Conjugate Conjugate Conjugate *Conjugate 45a
*Conjugate 45a *Conjugate 45a *Conjugate 45a Day (PBS) 45a (0.3) 45a (0.1) 45a (0.03) 45a (0.01)
(0.3) (0.1) (0.03) (0.01) 0 100 100 100 100 100 100 100 100 100 1 99.5 100.5 99 100.1 99.2 99 98
99.3 99.3 2 98.4 101.8 99.8 98.6 101 101.4 97.1 100.7 99 3 93.3 103.1 99.4 98 98.6 101.9 98.3 100.8
99.2 4 87.7 101.5 94.4 94.1 92.7 103 97.9 98.8 93.5 5 82.2 97.8 96 92.3 90 99.6 95.2 97 89.4 6 78.6
101.6 99.2 91.8 86.5 99.8 97.7 99.1 91.9 7 102.7 99.6 86.7 81.1 102.1 98.7 93.3 84 8 104.3 103.1
83.9 76.4 104.6 101.7 92.4 79.9 9 102.2 100.3 80.4 102.4 97.7 93.1 10 99.2 100.8 102.5 98.4 11 105
103.2 104.8 100.1 12 105.1 101.8 103.8 101.1 13 103.8 102 102.9 100.8 14 105.1 103.8 102.4 101.1
Conjugate 45a purified using a protein A column; *Conjugate 45a from protein A column flow-through
(e.g., conjugate that did not bind to protein A)

Example 216. Comparison of Conjugates 45a and 46 in a Humanized Mouse Model (FcRn) Against
Influenza A/California/07/2009Pdm (H1N1) in a Lethal Infection Study

(1744) Conjugates 45a and 46 were evaluated against a lethal IAV H1N1 influenza infection in female
B6.Cg-Fcgrttm1 Dcr Tg(FCGRT)32Dcr/DcrJ mice (6-8 weeks old; Jackson Labs #014565). These
mice express the human neonatal receptor (FcRn) which is an essential factor in the prolonged half-
life of antibodies or Fc containing therapeutics. Conjugate 46 contains the YTE Fc mutation which has
been shown to extend half-life in humans and cynomolgus monkeys. Although the YTE mutation is
silent in wild-type mice, it is permissive in transgenic murine species expressing FcRn. Therefore we
evaluated the relative efficacy of Conjugates 45a and 46 which are identical except for the presence of
the YTE mutation in the latter, to determine if prolonged half-life offered a potency advantage in a Day
−7 prevention model.

(1745) The challenge virus (A/California/07/2009) is an H1N1 pandemic isolate capable of causing
lethal infections in mice. The experiment comprised 10 groups of 5 mice (Table 223). Seven days prior
to viral challenge mice were administered a single dose of test article by intramuscular (IM) injection
into the thigh muscle of the right hind limb at a dose volume of 5 m1/kg. At day 0, all mice were
challenged with virus at 3× the LD95 (3E4 pfu) by intranasal (IN) inoculation in a volume of 30 μl, after
being anesthetized with a mixture of ketamine/xylazine (150 and 10 mg/kg respectively). Group 1
consisted of control mice treated with vehicle only, and group 2 animals were dosed with Fc only
(hIgG1 Fc), the remaining groups received one of the conjugates at doses ranging from 1 to 0.03
mg/kg. Mortality and body weights (BW) were recorded daily for 14 days and any animal with a
cumulative 20% loss of weight for 2 consecutive days was scored as a death.

(1746) Survival data is presented in Table 224 and shows control animals (groups 1 and 2) reached
full mortality on Day 6 as expected. In contrast, mice receiving either conjugate at 1.0 or 0.3 mg/kg
were fully protected through study end (Day 21). At the lowest dose concentration (0.03 mg/kg) no
significant protection was evident for either conjugate. However, at 0.1 mg/kg a relative difference in
potency between Conjugates 45a and 46 was apparent. Conjugate 45a containing a wild-type Fc
sequence was not significantly more potent than vehicle or Fc only dosed animals, while Conjugate 46
treated animals were (80% survival; P=0.0016 relative to vehicle) (FIG. 122). These data suggest the
YTE mutation of Conjugate 46 offers a potency advantage over Conjugate 45a, likely due to prolonged
half-life. BW data for all study groups is listed in Table 225 and supports the survival data.

(1747) Collectively this study demonstrates an increase in potency of conjugate 46, relative to
conjugate 45a. As both conjugates have identical targeting moieties it follows that the improvement in
efficacy of conjugate 46 is due to the YTE mutation. This conclusion is supported by plasma levels of
both conjugates collected from study animals (0.1 mg/kg dose groups) the day prior to viral challenge
(Conjugate 45a, 0.09 μg/ml; Conjugate 46, 0.48 μg/ml). The higher plasma levels of Conjugate 46 at
the time of viral challenge resulted in superior protection relative to Conjugate 45a.

(1748) TABLE-US-00232 TABLE 223 General protocol outline for FcRn mouse study Influenza A strain
(IN Route, Dose Dose Group challenge) Test Article Schedule (mg/kg) timing N 1 A/CA/−7/09
Vehicle(PBS) IM, single — T − 5 2 ~3E4 hIgG1 Fc 1 7 Days 3 PFU/mouse Conjugate 45a 1 4
Conjugate 45a 0.3 5 Conjugate 45a 0.1 6 Conjugate 45a 0.03 7 Conjugate 46 1 8 Conjugate 46 0.3 9
Conjugate 46 0.1 10 Conjugate 46 0.03

(1749) TABLE-US-00233 TABLE 224 % survival by group and day (mg/kg) Vehicle Conjugate
Conjugate Conjugate Conjugate Conjugate 46 Conjugate 46 Conjugate 46 Conjugate 46 Day (PBS)
hIgG1 Fc 45a (1) 45a (0.3) 45a (0.1) 45a (0.03) (1) (0.3) (0.1) (0.03) 0 100 100 100 100 100 100 100
100 100 100 1 100 100 100 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 100 100
100 3 100 100 100 100 100 100 100 100 100 100 4 100 80 100 100 100 100 100 100 100 100 5 20 60
100 100 100 100 100 100 100 100 6 0 0 100 100 80 80 100 100 100 20 7 100 100 60 20 100 100 100
0 8 100 100 20 20 100 100 80 9 100 100 20 20 100 100 80 10 100 100 20 20 100 100 80 11 100 100
20 20 100 100 80 12 100 100 20 20 100 100 80 13 100 100 20 20 100 100 80 14 100 100 20 20 100
100 80 15 100 100 20 20 100 100 80 16 100 100 20 20 100 100 80 17 100 100 20 20 100 100 80 18
100 100 20 20 100 100 80 19 100 100 20 20 100 100 80 20 100 100 20 20 100 100 80 21 100 100 20
20 100 100 80

(1750) TABLE-US-00234 TABLE 225 % BW of animals by group and day (mg/kg). *Data shown until
the first death within a group Day Post Vehicle Conjugate Conjugate Conjugate Conjugate Conjugate
Conjugate Conjugate Conjugate Infection (PBS) hIgG1 Fc 45a (1) 45a (0.3) 45a (0.1) 45a (0.03) 46 (1)
46 (0.3) 46 (0.1) 46 (0.03) 0 100 100 100 100 100 100 100 100 100 100 1 97.2 97.7 97.8 100.4 99.4
98 96.9 100.8 99.4 98.6 2 89.5 90.7 96.7 95.6 94 92.8 96.3 100.7 94.3 91.8 3 82.9 83.8 98.7 91.9 86.8
88.3 97.8 95.2 86.5 85.4 4 79 80.1 100.9 93.3 84.4 85.3 97.7 97.1 86.5 83.5 5 75.1 98.5 91.9 81.8 84
96.7 97.3 84.6 79.3 6 102 93.7 80.4 82.2 97.8 101.4 84.4 76.7 7 99.5 94.1 97.2 99.9 82.5 8 99.8 94.5
98.3 100.8 84.8 9 98.8 98.3 102.4 10 99.3 97.2 103.1 11 103.9 101.4 103.5 12 103.8 99.8 103.2 13
102.1 100.2 102.8 14 103.8 104.3 104.9 15 103.8 102.1 106.2 16 101.9 100 101.9 17 101.2 102.3
103.6 18 101.9 103.6 104.5 19 102.3 104.3 104 20 102.4 102.3 105.5 21 104.2 102 106.9

Example 217. Efficacy of Conjugate 45a in an Influenza A (H1N1) Mouse Model Designed to Mimic
the Early-Stage Pathogenies of Human Infections

(1751) Previous examples provided in this patent have utilized a model in which mice are heavily
sedated with ketamine/xylazine (150 and 10 mg/kg respectively) during viral challenge. After intranasal
(IN) viral challenge (3× the LD9s in 30 μl) mice are kept on their backs until recovery (approx. 30 min).
This enhances drainage of virus into the lungs (lower respiratory tract or LRT) generating a robust and
very reproducible screening model for anti-influenza therapeutics. However, it does not replicate the
natural infection process in which generally fewer viral particles are seeded into the upper respiratory
tract (URT) of humans. This study was designed to more closely mimic the natural infection process
by seeding virus into the URT, and investigating two different challenge inoculums.

(1752) This study utilized female BALB/c mice (Charles River Laboratories, 6-8 weeks) which were
challenged with influenza A/California/07/2009 (H1N1), a pandemic isolate capable of causing lethal
infections in mice, at 3E4 (3× the LD.sub.95) and 3E3 pfu/mouse. The viral challenge was done on
isoflurane (3%) anesthetized mice by IN inoculation with a volume of 30 μl. The experiment comprised
13 groups of mice and the general study design is shown in Table 226. Three days prior to viral
challenge mice were administered a single IM (thigh muscle) dose of Conjugate 45a between 0.001
and 0.3 mg/kg. Negative controls animals were treated with PBS only. One group was also “sham
infected” using PBS instead of virus. Mortality and BW were recorded daily for 14 days and any animal
with a cumulative 20% loss of weight for 2 consecutive days was scored as a death.

(1753) Survival results from the study are summarized in Table 227. At both viral concentrations (3E3
and 3E4 pfu/mouse) vehicle treated mice were not fully protected from mortality as expected. The 3E3
PBS group reached 100% mortality compared to 60% at 3E4. Although counter-intuitive this is likely
the result of normal experimental variation in URT models. Importantly, Conjugate 45a was fully
protective against challenge with 3E4 pfu down to a single IM dose of 0.03 mg/kg. In the 3E3 arm,
Conjugate 45a offered full protection with a single 0.01 mg/kg dose (P=0.0031 to vehicle). The
remarkable protection afforded by conjugate 45a based on a survival endpoint indicates superior PK
and activity in the URT of mice.
(1754) BW data for study animals is tabulated in Table 228 and mirrors the results of the survival data.
The lowest fully protective dose (0.03 mg/kg) group only displayed transient weight loss compared to
sham (PBS) infected mice and terminal BWs (Day 14) were within 1.5% of each other in the 3E4
challenge arm. For the 3E3 arm the lowest protective dose (0.01 mg/kg) was within ˜5% of sham
infected animals at study end.

(1755) Collectively this study indicates Conjugate 45a has exceptional exposure in the URTs of mice
and is extremely effective at preventing the spread of virus into the LRT, where lethal infections occur.
Furthermore, conservation of BW in treated mice and lack of obvious clinical symptoms indicates
Conjugate 45a has the potential to act as a superior preventative against Influenza A.

(1756) TABLE-US-00235 TABLE 226 General study outline Virus Dose Test challenge Dose volume
Group Influenza A strain Article (IN) Route, Schedule (mg/kg) (ml/kg) N 1 Sham infection Vehicle
(PBS) PBS IM, single, T − 72 h — — 5 2 A/CA/07/09 (H1N1) Vehicle (PBS) 3.00E+04 IM, single, T −
72 h — 5 3 Conjugate 45a IM, single, T − 72 h 0.3 4 IM, single, T − 72 h 0.1 5 IM, single, T − 72 h 0.03
6 IM, single, T − 72 h 0.01 7 IM, single, T − 72 h 0.003 8 Vehicle (PBS) 3.00E+03 IM, single, T − 72 h
— 9 Conjugate 45a IM, single, T − 72 h 0.1 10 IM, single, T − 72 h 0.03 11 IM, single, T − 72 h 0.01 12
IM, single, T − 72 h 0.003 13 IM, single, T − 72 h 0.001

(1757) TABLE-US-00236 TABLE 227 Percent survival of groups by day Conjugate 45a (mg/kg) .Math.
Conjugate 45a (mg/kg) .Math. Sham 3E4 pfu/mouse challenge groups 3E3 pfu/mouse challenge
groups Day Post (PBS) Vehicle 0.3 0.1 0.03 0.01 0.003 Vehicle 0.1 0.03 0.01 0.003 0.001 Infection
infection (PBS) mg/kg mg/kg mg/kg mg/kg mg/kg (PBS) mg/kg mg/kg mg/kg mg/kg mg/kg 0 100 100
100 100 100 100 100 100 100 100 100 100 100 1 100 100 100 100 100 100 100 100 100 100 100 100
100 2 100 100 100 100 100 100 100 100 100 100 100 100 100 3 100 100 100 100 100 100 100 100
100 100 100 100 100 4 100 100 100 100 100 100 100 100 100 100 100 100 100 5 100 100 100 100
100 100 100 100 100 100 100 100 100 6 100 60 100 100 100 60 60 100 100 100 100 100 100 7 100
60 100 100 100 60 20 60 100 100 100 100 80 8 100 60 100 100 100 60 20 0 100 100 100 80 80 9 100
60 100 100 100 60 20 100 100 100 80 80 10 100 60 100 100 100 60 20 100 100 100 80 80

(1758) TABLE-US-00237 TABLE 228 Percent starting BW of animals by group and day. *Data shown
until the first death within a group Conjugate 45a (mg/kg) .Math. Conjugate 45a (mg/kg) .Math. 3E4
pfu/mouse challenge groups 3E3 pfu/mouse challenge groups Day Sham Vehicle Vehicle Post (PBS)
(PBS) 0.3 0.1 0.03 0.01 0.003 (PBS) 0.1 0.03 0.01 0.003 0.001 Infection infection (3E4) mg/kg mg/kg
mg/kg mg/kg mg/kg (3E3) mg/kg mg/kg mg/kg mg/kg mg/kg 0 100 100 100 100 100 100 100 100 100
100 100 100 100 1 101.5 103.4 103.2 102.2 104.6 101.3 102.3 104.7 100.7 102.8 103.9 99.8 101.9 2
98.6 99.1 100.5 100.6 102.3 97.2 100 100.8 98.5 100.9 101.7 96.8 99.4 3 101.1 92.4 99.5 98.9 97.4
92.3 92.6 95.4 99.1 99.8 97.7 94.6 97.2 4 104.2 89 100.3 99.2 96.2 88.1 87.3 89.6 101.6 97.9 97.9
88.8 94 5 104.2 87.3 102.1 101.6 95.7 86 82.4 84.7 101.7 98.2 96.2 86.4 90.9 6 104.6 85.2 104.3
103.9 94.6 83 81 102.5 97.1 92.9 84 84.3 7 105.1 82.1 103.5 104.7 97.2 76.1 102.7 94.5 87.4 79.5
81.8 8 105.2 106.9 100.8 98.7 102.7 99.2 90 83 9 103.9 104.3 104.1 99.8 101.2 98.8 93.3 10 105.4
106.6 105.9 103.9 104.8 100.9 99.8

Example 218. Efficacy of Conjugate 45a Against Influenza A/California/07/2009 (H1N1) by Dose
Route in a Delayed Treatment Mouse Model

(1759) Conjugate 45a was evaluated against a lethal influenza A (H1N1) infection in female BALB/c
mice (Charles River Laboratories, 6-8 weeks). The challenge virus (A/California/07/2009) is a
pandemic isolate capable of causing lethal infections in mice. The experiment comprised 7 groups of 5
mice. The general study design is shown in Table 229, briefly: On day 0 all mice were challenged with
virus at 2-3× the LD95 by intranasal (IN) inoculation in a volume of 30 μl, after being anesthetized with
a mixture of ketamine/xylazine (150 and 10 mg/kg respectively). Test groups received a single
intramuscular (IM) or intravenous (IV) injection, 24 hours post viral challenge of conjugate 45a (0.03 to
0.3 mg/kg) or vehicle (PBS). IM Injections were in the thigh muscle of the right hind limb at a dose
volume of 10 m1/kg. IV injections were administered into the tail vein at the same dose volume.
Mortality and BW were recorded daily for 14 days and any animal with a cumulative 20% loss of
weight for 2 consecutive days was scored as a death.
(1760) Survival results from the study are summarized in Table 230. Mice treated with vehicle (PBS)
rapidly began to lose BW after challenge, reaching 100% mortality on Day 5. Animals receiving
Conjugate 45a at a dose of 0.3 mg/kg by IV (group 2) or IM (group 5) were fully protected through
study end (Day 14). Both groups also displayed similar BW trends, with a transient loss of weight
before recovering, and eventually exceeding, their initial BW by study end (Table 231).

(1761) At the lowest Conjugate 45a dose tested (0.03 mg/kg) all mice succumbed to infection by Day
6 regardless of dose route. The intermediate dose groups (0.1 mg/kg) demonstrated 40% survival
when dosed by IV, and was not protective (0% survival) when dosed by IM. This difference however
was not statistically significant (P=0.0993). Collectively this study demonstrated the potency of
Conjugate 45a against an important pandemic isolate of influenza A (H1N1) with a single IV or IM
dose of less than 1 mg/kg. Importantly, efficacy was comparable between both dose routes indicating
the ability of Conjugate 45a to be used in an outpatient setting (IM dosing). Lastly, Conjugate 45a was
fully protective even though dosing was delayed for 24 hours, allowing the initial viral inoculum time to
establish/propagate. This suggests Conjugate 45a may have therapeutic use as both a preventative
and a therapeutic.

(1762) TABLE-US-00238 TABLE 229 General study outline Route, Influenza Schedule A Test (T + 24
Dose Group strain Article hours) (mg/kg) ml/kg N 1 A/CA/07/09 Vehicle (PBS) IV, single, — 10 5 (3E4
pfu/mouse) T + 24 2 Conjugate 45a IV, single, 0.3 10 5 T + 24 3 IV, single, 0.1 10 5 T + 24 4 IV, single,
0.03 10 5 T + 24 5 IM, single, 0.3 10 5 T + 24 6 IM, single, 0.1 10 5 T + 24 7 IM, single, 0.03 10 5 T +
24

(1763) TABLE-US-00239 TABLE 230 Percent survival by group and day (mg/kg) (dose route)
Conjugate 45a Conjugate 45a Conjugate 45a Conjugate 45a Conjugate 45a Conjugate 45a Day
Vehicle (PBS) (0.3) (IV) (0.1) (IV) (0.03) (IV) (0.3) (IM) (0.1) (IM) (0.03) (IM) 0 100 100 100 100 100
100 100 1 100 100 100 100 100 100 100 2 100 100 100 100 100 100 100 3 100 100 100 100 100 100
100 4 100 100 100 100 100 100 100 5 0 100 80 20 100 40 60 6 100 40 0 100 0 0 7 100 40 100 8 100
40 100 9 100 40 100 10 100 40 100 11 100 40 100 12 100 40 100 13 100 40 100 14 100 40 100

(1764) TABLE-US-00240 TABLE 231 Percent Starting BW of animals by group and day (mg/kg) (dose
route). *Data shown until the first death within a group. Day Post Conjugate 45a Conjugate 45a
Conjugate 45a Conjugate 45a Conjugate 45a Conjugate 45a Challenge Vehicle (PBS) (0.3) (IV) (0.1)
(IV) (0.03) (IV) (0.3) (IM) (0.1) (IM) (0.03) (IM) 0 100 100 100 100 100 100 100 1 97.5 99.5 96.9 99.1
98.6 97.2 97.6 2 95.1 99.4 96 94 97.1 95.5 95 3 86.6 94.5 89.9 86.8 89.7 88.8 89.1 4 79.9 93 82.6
79.2 89.1 80.8 81.2 5 74.6 94.7 81 74.6 92.1 76.4 75.7 6 97.4 94.3 7 99.3 97.1 8 101.9 98.7 9 102
101.7 10 105 103.6 11 103.3 100.9 12 102.4 101.2 13 105.9 102.8 14 104.2 101.6

Example 219. A Single-Dose Subcutaneous Rangefinding Study and Single-Dose Toxicity and
Toxicokinetic (2-Week Exposure) Study of Conjugate 45a in Sprague-Dawley Rats

(1765) The purpose of this study, was to evaluate the tolerability in male and female rats then toxicity
and toxicokinetics (TK) of conjugate 45a when administered as a single subcutaneous (SC) dose to
male Sprague Dawley rats.

(1766) This study consisted of two phases; Phase 1 tolerability (Groups 1-3) and Phase 2 toxicology
(Groups 4-7) and satellite TK (Groups 8-10). Each treatment group in Phase 1 was comprised of one
female and one male Sprague Dawley rat. Phase 1 rats were administered either 100 mg/kg conjugate
45a(Group 1), 200 mg/kg conjugate 45a(Group 2), or 400 mg/kg conjugate 45a(Group 3) on a single
day via SC injection at a dose volume of 5 mL/kg (5 mL×2 sites for Group 3, mid-scapular and dorsal
lumbar). Each Phase 2 toxicology group (Groups 4-7) was comprised of five male Sprague Dawley
rats. Each Phase 2 satellite TK group (Groups 8-10) was comprised of four male Sprague Dawley rats.
Based on the tolerability in Phase 1, Phase 2 rats were administered either PBS (Group 4), 50 mg/kg
conjugate 45a(Groups 5 and 8), 150 mg/kg conjugate 45a(Groups 6 and 9), or 400 mg/kg conjugate
45a (Groups 7 and 10) on a single day via SC injection at a dose volume of 5 mL/kg (5 mL×2 sites for
Group 3, mid-scapular and dorsal lumbar for Groups 7 and 10).
(1767) Clinical observations for Phase 1 were recorded twice daily on Days 1-3 and detailed
observations were recorded at randomization. Clinical observations for Phase 2 were recorded twice
daily on Days 1-15 and detailed observations were recorded on Day 1 (prior to dosing) and on Day 14.
Body weight measurements were recorded at randomization and pre-dose for Phase 1. Body weight
measurements for Phase 2 were recorded at randomization, on Day 1, Day 3, Day 7, Day 14, and on
Day prior to necropsy. Food consumption measurements for Phase 2 were recorded on Days 1, 4, 7,
10, 12, and 14. Plasma samples were collected from satellite TK rats (Groups 8-10) at 0.5, 1, 2, 4, 8,
24, 72, 120, 168, 240, and 336 hours post-dose for analysis of systemic exposure to conjugate 45a.
Urine samples were collected from toxicology rats (Groups 4-7) for 24 hours from Day 14 to Day 15.
Blood samples for the evaluation of hematology, chemistry, and coagulation endpoints were collected
on Day from toxicology rats (Groups 4-7). Following blood sample collections, necropsy was
conducted on toxicology rats (Groups 4-7). Protocol-specified tissues were collected and evaluated
grossly, select organs were weighed, and tissues were fixed for microscopic evaluation. Tissues were
subsequently processed and evaluated microscopically.

(1768) Tolerability:

(1769) Based on the absence of test article-related abnormal observations, administration of conjugate
45aat <400 mg/kg as a single subcutaneous injection (at either one or two sites on each dosing day)
was well tolerated for up to 3 days in male and female Sprague Dawley rats.

(1770) Toxicokinetics:

(1771) Conjugate 45aplasma levels were maintained over the 2-week exposure period and were
comparable between the neuraminidase (NA)-capture and the Fc-capture assays. This observation
suggested that the intact molecule (containing at least 1 target moiety linked to the Fc group)
remained stable after dose administration in vivo. Mean plasma exposures appeared to increase
approximately dose-proportionally from 50 to 400 mg/kg.

(1772) Toxicology:

(1773) Based on the absence of test article-related changes in body weight, food consumption, clinical
observations, organ weights, hematologic parameters, clinical chemistry, macroscopic findings and
microscopic findings, administration of conjugate 45a at <400 mg/kg as a single subcutaneous
injection (high dose administered across two sites) was well tolerated for up to 14 days in male
Sprague Dawley rats. This dose corresponded to mean AUC0-inf values of 312,000 and 319,000
μg.Math.hr/mL and mean Cmax values of 1150 and 974 μg/mL for NA-capture and Fc-capture assays,
respectively.

Example 220. A Single-Dose Subcutaneous Rangefinding Study and Single-Dose Toxicity and
Toxicokinetic (2-Week Exposure) Study of Conjugate 45a in Sprague-Dawley Rats

(1774) The purpose of this study was to evaluate the tolerability in male and female rats, then toxicity
and toxicokinetics of Conjugate 45a when administered as a single subcutaneous (SC) dose to male
Sprague Dawley Rats. Male animals in Phase 2 were administered 50, 150, or 400 mg/kg/dose
conjugate 45a.

(1775) All study animals survived to scheduled sacrifice. There were no test article-related changes in
organ weights, hematologic parameters, clinical chemistry, macroscopic findings and microscopic
findings. Recorded microscopic findings were present at a similar incidence in control animals and test
article exposed groups, or were considered to represent incidental “background” findings that are seen
commonly in rats of this strain and age.

NUMBERED EMBODIMENTS

(1776) 1. A conjugate described by any one of formulas (D-I), (M-I), (1), or (2):

(1777) ##STR00820##
(1778) wherein each A.sub.1 and each A.sub.2 is independently described by formula (A-I)-(A-XII):

(1779) ##STR00821## ##STR00822## ##STR00823##

(1780) wherein R.sub.1 is selected from —OH, —NH.sub.2, —NHC(═NH)NH.sub.2, and —

NHC(═NH)NHR.sub.6;

(1781) R.sub.2 and R.sub.3 are each independently selected from —H, —OH, —F, —Cl, and —Br;

(1782) R.sub.4 is selected from —CO.sub.2H, —P(═O)(OH).sub.2, —SO.sub.3H;

(1783) R.sub.5 is selected from —COCH.sub.3, —COCF.sub.3, —SO.sub.2CH.sub.3;

(1784) X is selected from —O— and —S—;

(1785) Y is selected from:

(1786) ##STR00824##

R.sub.6 is selected from

(1787) ##STR00825## ##STR00826##

(1788) R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15
aryl, and C2-C15 heteroaryl;

(1789) R.sub.8 is selected from C3-C20 heterocycloalkyl, C5-C15 aryl, and C2-C15 heteroaryl;

(1790) n is 1 or 2;

(1791) each E comprises an Fc domain monomer, an albumin protein, an albumin protein-binding

peptide, or an Fc-binding peptide;

(1792) L is a linker covalently attached to E and to each Y of each A.sub.1 or each A.sub.1 and
A.sub.2;

(1793) T is an integer from 1 to 20, and

(1794) each squiggly line in formulas (D-I), (M-I), (1), or (2) indicates that L is covalently attached to
each E;

(1795) or a pharmaceutically acceptable salt thereof.

(1796) 2. The conjugate of embodiment 1, wherein the conjugate is described by formula (D-I):
(A.sub.1-L A.sub.2) T (D-I)

(1797) ##STR00827##

(1798) wherein each A.sub.1 and each A.sub.2 is independently selected from any one of formulas (A-
I)-(A-XII);

(1799) each E comprises an Fc domain monomer, an albumin protein, an albumin protein-binding

peptide, or an Fc-binding peptide;

(1800) n is 1 or 2;

(1801) T is an integer from 1 to 20; and

(1802) the squiggly line connected to the E indicates that each A.sub.1-L-A.sub.2 is covalently
attached to E, or a pharmaceutically acceptable salt thereof.

(1803) 3. The conjugate of embodiment 2, wherein each A.sub.1 and each A.sub.2 is independently
selected from any one of formulas (A-I), (A-II), (A-VI), or (A-VII);

(1804) each E comprises an Fc domain monomer, an albumin protein, an albumin protein-binding

peptide, or an Fc-binding peptide;

(1805) n is 1 or 2;

(1806) T is an integer from 1 to 20; and

(1807) the squiggly line connected to the E indicates that each A.sub.1-L-A.sub.2 is covalently
attached to E,

(1808) or a pharmaceutically acceptable salt thereof.

(1809) 4. The conjugate of embodiment 3, wherein each A.sub.1 and each A.sub.2 is described by
formula (A-I) or a pharmaceutically acceptable salt thereof.

(1810) 5. The conjugate of any one of embodiments 1-4, wherein the conjugate is described by
formula (D-II):

(1811) ##STR00828##

(1812) or a pharmaceutically acceptable salt thereof.

(1813) 6. The conjugate of embodiment 5, wherein the conjugate is described by formula (D-I1-1):

(1814) ##STR00829##

(1815) or a pharmaceutically acceptable salt thereof.

(1816) 7. The conjugate of embodiment 6, wherein the conjugate is described by formula (D-II-2):

(1817) ##STR00830##

(1818) or a pharmaceutically acceptable salt thereof.

(1819) 8. The conjugate of embodiment 7, wherein the conjugate is described by formula (D-II-3):

(1820) ##STR00831##

(1821) wherein L′ is the remainder of L, and

(1822) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1823) or a pharmaceutically acceptable salt thereof.

(1824) 9. The conjugate of embodiment 8, wherein L′ is a nitrogen atom.

(1825) 10. The conjugate of embodiment 9 wherein the conjugate has the structure selected from

(1826) ##STR00832##

11. The conjugate of embodiment 6, wherein the conjugate is described by formula (D-II-4):

(1827) ##STR00833##
(1828) or a pharmaceutically acceptable salt thereof.

(1829) 12. The conjugate of embodiment 11, wherein the conjugate is described by formula (D-II-5):

(1830) ##STR00834##

(1831) wherein L′ is the remainder of L, and

(1832) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1833) or a pharmaceutically acceptable salt thereof.

(1834) 13. The conjugate of embodiment 12, wherein L′ is a nitrogen atom.

(1835) 14. The conjugate of embodiment 13, wherein the conjugate has the structure selected from

(1836) ##STR00835##

15. The conjugate of embodiment 11, wherein the conjugate has the structure of

(1837) ##STR00836##

(1838) or a pharmaceutically acceptable salt thereof.

(1839) 16. The conjugate of embodiment 6, wherein the conjugate is described by formula (D-II-6):

(1840) ##STR00837##

(1841) wherein R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl;
C5-C15 aryl, and C2-C15 heteroaryl;

(1842) or a pharmaceutically acceptable salt thereof.

(1843) 17. The conjugate of embodiment 16, wherein R.sub.7 is selected from C1-C20 alkyl, C3-C20
cycloalkyl, C3-C20 heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl.

(1844) 18. The conjugate of embodiment 16 or 17, wherein R.sub.7 is selected from methyl, ethyl,
propyl, or butyl.

(1845) 19. The conjugate of any one of embodiments 16-18, wherein the conjugate is described by
formula (D-II-7):

(1846) ##STR00838##

(1847) or a pharmaceutically acceptable salt thereof.

(1848) 20. The conjugate of embodiment 19, wherein the conjugate is described by formula (D-II-8):

(1849) ##STR00839##

(1850) wherein L′ is the remainder of L, and

(1851) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1852) or a pharmaceutically acceptable salt thereof.

(1853) 21. The conjugate of embodiment 20, wherein the conjugate has the structure of:

(1854) ##STR00840##
(1855) or a pharmaceutically acceptable salt thereof.

(1856) 22. The conjugate of embodiment 21, wherein the conjugate is described by

(1857) ##STR00841##

(1858) or a pharmaceutically acceptable salt thereof.

(1859) 23. The conjugate of any one of embodiments 16-18, wherein the conjugate is described by
formula (D-II-9):

(1860) ##STR00842##

(1861) or a pharmaceutically acceptable salt thereof.

(1862) 24. The conjugate of embodiment 23, wherein the conjugate is described by formula (D-I1-10):

(1863) ##STR00843##

(1864) wherein L′ is the remainder of L, and

(1865) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1866) or a pharmaceutically acceptable salt thereof.

(1867) 25. The conjugate of embodiment 24, wherein the conjugate has the structure of

(1868) ##STR00844##

(1869) or a pharmaceutically acceptable salt thereof.

(1870) 26. The conjugate of embodiment 2 or 3, wherein the conjugate is described by formula (D-III):

(1871) ##STR00845##

(1872) or a pharmaceutically acceptable salt thereof.

(1873) 27. The conjugate of embodiment 26, wherein the conjugate is described by formula (D-III-1):

(1874) ##STR00846##

(1875) or a pharmaceutically acceptable salt thereof.

(1876) 28. The conjugate of embodiment 27, wherein the conjugate is described by formula (D-III-2):

(1877) ##STR00847##

(1878) or a pharmaceutically acceptable salt thereof.

(1879) 29. The conjugate of embodiment 28, wherein the conjugate is described by formula (D-III-3):

(1880) ##STR00848##

(1881) wherein L′ is the remainder of L, and

(1882) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1883) or a pharmaceutically acceptable salt thereof.

(1884) 30. The conjugate of embodiment 27, wherein the conjugate is described by formula (D-III-4):
(1885) ##STR00849##

(1886) or a pharmaceutically acceptable salt thereof.

(1887) 31. The conjugate of embodiment 30, wherein the conjugate is described by formula (D-III-5):

(1888) ##STR00850##

(1889) wherein L′ is the remainder of L, and

(1890) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1891) or a pharmaceutically acceptable salt thereof.

(1892) 32. The conjugate of embodiment 27, wherein the conjugate is described by formula (D-III-6):

(1893) ##STR00851##

(1894) or a pharmaceutically acceptable salt thereof.

(1895) 33. The conjugate of embodiment 32, wherein the conjugate is described by formula (D-III-7):

(1896) ##STR00852##

(1897) wherein L′ is the remainder of L, and

(1898) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1899) or a pharmaceutically acceptable salt thereof.

(1900) 34. The conjugate of embodiment 27, wherein the conjugate is described by formula (D-III-8):

(1901) ##STR00853##

(1902) or a pharmaceutically acceptable salt thereof.

(1903) 35. The conjugate of embodiment 34, wherein the conjugate is described by formula (D-III-9):

(1904) ##STR00854##

(1905) wherein L′ is the remainder of L, and 3

(1906) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1907) or a pharmaceutically acceptable salt thereof.

(1908) 36. The conjugate of embodiment 2, wherein the conjugate is described by formula (D-IV):

(1909) ##STR00855##

(1910) or a pharmaceutically acceptable salt thereof.

(1911) 37. The conjugate of embodiment 36, wherein the conjugate is described by formula (D-IV-1):

(1912) ##STR00856##

(1913) or a pharmaceutically acceptable salt thereof.

(1914) 38. The conjugate of embodiment 37, wherein the conjugate is described by formula (D-IV-2):
(1915) ##STR00857##

(1916) wherein L′ is the remainder of L, and

(1917) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1918) or a pharmaceutically acceptable salt thereof.

(1919) 39. The conjugate of embodiment 2 or 3, wherein the conjugate is described by formula (D-V):
Y-L-Y.

(1920) ##STR00858##

(1921) or a pharmaceutically acceptable salt thereof.

(1922) 40. The conjugate of embodiment 39, wherein the conjugate is described by formula (D-V-1):

(1923) ##STR00859##

(1924) or a pharmaceutically acceptable salt thereof.

(1925) 41. The conjugate of embodiment 40, wherein the conjugate is described by formula (D-V-2):

(1926) ##STR00860##

(1927) or a pharmaceutically acceptable salt thereof.

(1928) 42. The conjugate of embodiment 41, wherein the conjugate is described by formula (D-V-3):

(1929) ##STR00861##

(1930) wherein L′ is the remainder of L, and

(1931) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1932) or a pharmaceutically acceptable salt thereof.

(1933) 43. The conjugate of embodiment 42, wherein L′ is a nitrogen atom.

(1934) 44. The conjugate of embodiment 42, wherein y.sub.1 and y.sub.2 are each 1, y.sub.1 and
y.sub.2 are each 2, or y.sub.1 and y.sub.2 are each 3.

(1935) 45. The conjugate of embodiment 40, wherein the conjugate is described by formula (D-V-4):

(1936) ##STR00862##

(1937) or a pharmaceutically acceptable salt thereof.

(1938) 46. The conjugate of embodiment 45, wherein the conjugate is described by formula (D-V-5):

(1939) ##STR00863##

(1940) wherein L′ is the remainder of L, and

(1941) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1942) or a pharmaceutically acceptable salt thereof.

(1943) 47. The conjugate of embodiment 46, wherein L′ is a nitrogen atom.

(1944) 48. The conjugate of embodiment 46, wherein y.sub.1 and y.sub.2 are each 1, y.sub.1 and
y.sub.2 are each 2, or y.sub.1 and y.sub.2 are each 3.

(1945) 49. The conjugate of embodiment 39, wherein the conjugate is described by formula (D-V-6):

(1946) ##STR00864##

(1947) or a pharmaceutically acceptable salt thereof.

(1948) 50. The conjugate of embodiment 49, wherein the conjugate is described by formula (D-V-7):

(1949) ##STR00865##

(1950) or a pharmaceutically acceptable salt thereof.

(1951) 51. The conjugate of embodiment 50, wherein the conjugate is described by formula (D-V-8):

(1952) ##STR00866##

(1953) wherein L′ is the remainder of L, and

(1954) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1955) or a pharmaceutically acceptable salt thereof.

(1956) 52. The conjugate of embodiment 51, wherein L′ is a nitrogen atom.

(1957) 53. The conjugate of embodiment 51, wherein y.sub.1 and y.sub.2 are each 1, y.sub.1 and
y.sub.2 are each 2, or y.sub.1 and y.sub.2 are each 3.

(1958) 54. The conjugate of embodiment 49, wherein the conjugate is described by formula (D-V-9):

(1959) ##STR00867##

(1960) or a pharmaceutically acceptable salt thereof.

(1961) 55. The conjugate of embodiment 51, wherein the conjugate is described by formula (D-V-10):

(1962) ##STR00868##

(1963) wherein L′ is the remainder of L, and

(1964) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1965) or a pharmaceutically acceptable salt thereof.

(1966) 56. The conjugate of embodiment 55, wherein L′ is a nitrogen atom.

(1967) 57. The conjugate of embodiment 56, wherein y.sub.1 and y.sub.2 are each 1, y.sub.1 and
y.sub.2 are each 2, or y.sub.1 and y.sub.2 are each 3.

(1968) 58. The conjugate of embodiment 2 or 3, wherein the conjugate is described by formula (D-VI):

(1969) ##STR00869##

(1970) or a pharmaceutically acceptable salt thereof.

(1971) 59. The conjugate of embodiment 58, wherein the conjugate is described by formula (D-VI-1):

(1972) ##STR00870##
(1973) or a pharmaceutically acceptable salt thereof.

(1974) 60. The conjugate of embodiment 59, wherein the conjugate is described by formula (D-VI-2):

(1975) ##STR00871##

(1976) or a pharmaceutically acceptable salt thereof.

(1977) 61. The conjugate of embodiment 60, wherein the conjugate is described by formula (D-VI-3):

(1978) ##STR00872##

(1979) wherein L′ is the remainder of L, and

(1980) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1981) or a pharmaceutically acceptable salt thereof.

(1982) 62. The conjugate of embodiment 59, wherein the conjugate is described by formula (D-VI-4):

(1983) or a pharmaceutically acceptable salt thereof.

(1984) ##STR00873##

63. The conjugate of embodiment 62, wherein the conjugate is described by formula (D-VI-5):

(1985) ##STR00874##

(1986) wherein L′ is the remainder of L, and

(1987) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1988) or a pharmaceutically acceptable salt thereof.

(1989) 64. The conjugate of embodiment 59, wherein the conjugate is described by formula (D-VI-6):

(1990) ##STR00875##

(1991) or a pharmaceutically acceptable salt thereof.

(1992) 65. The conjugate of embodiment 64, wherein the conjugate is described by formula (D-VI-7):

(1993) ##STR00876##

(1994) wherein L′ is the remainder of L, and

(1995) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(1996) or a pharmaceutically acceptable salt thereof.

(1997) 66. The conjugate of embodiment 59, wherein the conjugate is described by formula (D-VI-8):

(1998) ##STR00877##

(1999) or a pharmaceutically acceptable salt thereof.

(2000) 67. The conjugate of embodiment 66, wherein the conjugate is described by formula (D-VI-9):

(2001) ##STR00878##
(2002) wherein L′ is the remainder of L, and 3

(2003) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2004) or a pharmaceutically acceptable salt thereof.

(2005) 68. The conjugate of embodiment 2 or 3, wherein the conjugate is described by formula (D-VII):

(2006) ##STR00879##

(2007) or a pharmaceutically acceptable salt thereof.

(2008) 69. The conjugate of any one of embodiments 39-68 wherein R.sub.1 is —OH.

(2009) 70. The conjugate of any one of embodiments 39-68 wherein R.sub.1 is —NH.sub.2.

(2010) 71. The conjugate of any one of embodiments 39-68 wherein R.sub.1 is —
NHC(═NH)NH.sub.2.

(2011) 72. The conjugate of embodiment 2, wherein the conjugate is described by formula (D-VIII):

(2012) ##STR00880##

(2013) or a pharmaceutically acceptable salt thereof.

(2014) 73. The conjugate of embodiment 72, wherein the conjugate is described by formula (D-VIII-1):

(2015) ##STR00881##

(2016) or a pharmaceutically acceptable salt thereof.

(2017) 74. The conjugate of embodiment 73, wherein the conjugate is described by formula (D-VIII-2):

(2018) ##STR00882##

(2019) or a pharmaceutically acceptable salt thereof.

(2020) 75. The conjugate of embodiment 74, wherein the conjugate is described by formula (D-VIII-3):

(2021) ##STR00883##

(2022) wherein L′ is the remainder of L, and

(2023) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2024) or a pharmaceutically acceptable salt thereof.

(2025) 76. The conjugate of embodiment 75, wherein L′ is a nitrogen atom.

(2026) 77. The conjugate of embodiment 75, wherein the conjugate has the structure selected from

(2027) ##STR00884##

78. The conjugate of embodiment 73, wherein the conjugate is described by formula (D-VIII-4):

(2028) ##STR00885##

(2029) or a pharmaceutically acceptable salt thereof.

(2030) 79. The conjugate of embodiment 78, wherein the conjugate is described by formula (D-VIII-5):
(2031) ##STR00886##

(2032) wherein L′ is the remainder of L, and

(2033) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2034) or a pharmaceutically acceptable salt thereof.

(2035) 80. The conjugate of embodiment 79, wherein L′ is a nitrogen atom.

(2036) 81. The conjugate of embodiment 79, wherein the conjugate has the structure selected from

(2037) ##STR00887##

82. The conjugate of embodiment 78, wherein the conjugate is described by the structure of

(2038) ##STR00888##

83. The conjugate of embodiment 73, wherein the conjugate is described by formula (D-VIII-6):

(2039) ##STR00889##

(2040) or a pharmaceutically acceptable salt thereof.

(2041) 84. The conjugate of embodiment 83, wherein the conjugate is described by formula (D-VIII-7):

(2042) ##STR00890##

(2043) wherein L′ is the remainder of L, and

(2044) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2045) or a pharmaceutically acceptable salt thereof.

(2046) 85. The conjugate of embodiment 73, wherein the conjugate is described by formula (D-VIII-8):

(2047) ##STR00891##

(2048) or a pharmaceutically acceptable salt thereof.

(2049) 86. The conjugate of embodiment 85, wherein the conjugate is described by formula (D-VIII-9):

(2050) ##STR00892##

(2051) wherein L′ is the remainder of L, and

(2052) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2053) or a pharmaceutically acceptable salt thereof.

(2054) 87. The conjugate of embodiment 72, wherein the conjugate is described by formula (D-VIII-
10):

(2055) ##STR00893##

(2056) or a pharmaceutically acceptable salt thereof.

(2057) 88. The conjugate of embodiment 87, wherein the conjugate is described by formula (D-VIII-
11):
(2058) ##STR00894##

(2059) wherein L′ is the remainder of L, and

(2060) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2061) or a pharmaceutically acceptable salt thereof.

(2062) 89. The conjugate of embodiment 2, wherein the conjugate is described by formula (D-IX):

(2063) ##STR00895##

(2064) or a pharmaceutically acceptable salt thereof.

(2065) 90. The conjugate of embodiment 89, wherein the conjugate is described by formula (D-IX-1):

(2066) ##STR00896##

(2067) or a pharmaceutically acceptable salt thereof.

(2068) 91. The conjugate of embodiment 90, wherein the conjugate is described by formula (D-IX-2):

(2069) ##STR00897##

(2070) or a pharmaceutically acceptable salt thereof.

(2071) 92. The conjugate of embodiment 90, wherein the conjugate is described by formula (D-IX-3):

(2072) ##STR00898##

(2073) or a pharmaceutically acceptable salt thereof.

(2074) 93. The conjugate of embodiment 90, wherein the conjugate is described by formula (D-IX-4):

(2075) ##STR00899##

(2076) or a pharmaceutically acceptable salt thereof.

(2077) 94. The conjugate of embodiment 90, wherein the conjugate is described by formula (D-IX-5):

(2078) ##STR00900##

(2079) or a pharmaceutically acceptable salt thereof.

(2080) 95. The conjugate of embodiment 90, wherein the conjugate is described by formula (D-IX-6):

(2081) ##STR00901##

(2082) or a pharmaceutically acceptable salt thereof.

(2083) 96. The conjugate of embodiment 2, wherein the conjugate is described by formula (D-X):

(2084) ##STR00902##

(2085) or a pharmaceutically acceptable salt thereof.

(2086) 97. The conjugate of embodiment 96, wherein the conjugate is described by formula (D-X-1):

(2087) ##STR00903##
(2088) or a pharmaceutically acceptable salt thereof.

(2089) 98. The conjugate of embodiment 97, wherein the conjugate is described by formula (D-X-2):

(2090) ##STR00904##

(2091) or a pharmaceutically acceptable salt thereof.

(2092) 99. The conjugate of embodiment 97, wherein the conjugate is described by formula (D-X-3):

(2093) ##STR00905##

(2094) or a pharmaceutically acceptable salt thereof.

(2095) 100. The conjugate of any one of embodiments 1-99, wherein L or L′ comprises one or more
optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally
substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted
C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20
cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20
cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20
cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15
arylene, optionally substituted C2-C15 heteroarylene, O, S, NR.sup.i, P, carbonyl, thiocarbonyl,
sulfonyl, phosphate, phosphoryl, or imino, wherein R.sup.i is H, optionally substituted C1-C20 alkyl,
optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted
C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20
heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl,
optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl,
optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl,
optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl.

101. The conjugate of embodiment 100, wherein the backbone of L or L′ consists of one or more
optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally
substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted
C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20
cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20
cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20
cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15
arylene, optionally substituted C2-C15 heteroarylene, O, S, NR.sup.i, P, carbonyl, thiocarbonyl,
sulfonyl, phosphate, phosphoryl, or imino, wherein R.sup.i is H, optionally substituted C1-C20 alkyl,
optionally substituted C1-C20 heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted
C2-C20 heteroalkenyl, optionally substituted C2-C20 alkynyl, optionally substituted C2-C20
heteroalkynyl, optionally substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl,
optionally substituted C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl,
optionally substituted C8-C20 cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl,
optionally substituted C5-C15 aryl, or optionally substituted C2-C15 heteroaryl.

102. The conjugate of embodiment 100 or 101, wherein L or L′ is oxo substituted.

103. The conjugate of any one of embodiments 1-102, wherein the backbone of L or L′ comprises no
more than 250 atoms.

104. The conjugate of any one of embodiments 1-103, wherein L or L′ is capable of forming an amide,
a carbamate, a sulfonyl, or a urea linkage.

105. The conjugate of any one of embodiments 1-99, wherein L or L′ is a bond.

106. The conjugate of any one of embodiments 1-99, wherein L or L′ is an atom.

107. The conjugate of any one of embodiments 1-106, wherein each L is described by formula (D-L-I):

(2096) ##STR00906##

(2097) wherein L.sup.A is described by formula G.sup.A1-(Z.sup.A1).sub.g1—(Y.sup.A1).sub.h1—

(Z.sup.A2).sub.i1—(Y.sup.A2).sub.j1—(Z.sup.A3).sub.k1—(Y.sup.A3).sub.l1—(Z.sup.A4).sub.m1—
(Y.sup.A4).sub.n1—(Z.sup.A5).sub.o1-G.sup.A2;

(2098) L.sup.B is described by formula G.sup.B1-(Z.sup.B1).sub.g2—(Y.sup.B1).sub.h2—

(Z.sup.B2).sub.i2—(Y.sup.B2).sub.j2—(Z.sup.B3).sub.k2—(Y.sup.B3).sub.l2—(Z.sup.B4).sub.m2—
(Y.sup.B4).sub.n2—(Z.sup.B5).sub.o2-G.sup.B2;

(2099) L.sup.C is described by formula G.sup.C1-(Z.sup.C1).sub.g3—(Y.sup.C1).sub.h3—

(Z.sup.C2).sub.i3—(Y.sup.C2).sub.j3—(Z.sup.C3).sub.k3—(Y.sup.C3).sub.l3—(Z.sup.C4).sub.m3—
(Y.sup.C4).sub.n3—(Z.sup.C5).sub.o3-G.sup.C2;

(2100) G.sup.A1 is a bond attached to Q;

(2101) G.sup.A2 is a bond attached to A1;

(2102) G.sup.B1 is a bond attached to Q;

(2103) G.sup.B2 is a bond attached to A2;

(2104) G.sup.G1 is a bond attached to Q;

(2105) G.sup.C2 is a bond attached to E or a functional group capable of reacting with a functional
group conjugated to E (e.g., maleimide and cysteine, amine and activated carboxylic acid, thiol and
maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene
and tetrazine);

(2106) each of Z.sup.A1, Z.sup.A2, Z.sup.A3, Z.sup.A4, Z.sup.A5, Z.sup.B1, Z.sup.B2, Z.sup.B3,
Z.sup.B4, Z.sup.B5, Z.sup.C1 Z.sup.C2, Z.sup.C3, Z.sup.C4, and Z.sup.C5 is, independently,
optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally
substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted
C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20
cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20
cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20
cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15
arylene, or optionally substituted C2-C15 heteroarylene;

(2107) each of Y.sup.A1, Y.sup.A2, Y.sup.A3, Y.sup.A4, Y.sup.B1, Y.sup.B2, Y.sup.B3, Y.sup.B4,
Y.sup.C1, Y.sup.C2, Y.sup.C3 and Y.sup.C4 is, independently, O, S, NR.sup.i, P, carbonyl,
thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino;

(2108) R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl,
optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally
substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20
cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl,
optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl,
optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally
substituted C2-C15 heteroaryl; each of g1, h1, i1, j1, k1, l1, m1, n1, o1, g2, h2, i2, j2, k2, l2, m2, n2,
o2, g3, h3, i3, j3, k3, l3, m3, n3, and o3 is, independently, 0 or 1;

(2109) Q is a nitrogen atom, optionally substituted C1-C20 alkylene, optionally substituted C1-C20
heteroalkylene, optionally substituted C2-C20 alkenylene, optionally substituted C2-C20
heteroalkenylene, optionally substituted C2-C20 alkynylene, optionally substituted C2-C20
heteroalkynylene, optionally substituted C3-C20 cycloalkylene, optionally substituted C3-C20
heterocycloalkylene, optionally substituted C4-C20 cycloalkenylene, optionally substituted C4-C20
heterocycloalkenylene, optionally substituted C8-C20 cycloalkynylene, optionally substituted C8-C20
heterocycloalkynylene, optionally substituted C5-C15 arylene, or optionally substituted C2-C15
heteroarylene.

(2110) 108. The conjugate of embodiment 107, wherein L is selected from

(2111) ##STR00907## ##STR00908## ##STR00909## ##STR00910## ##STR00911##

##STR00912## ##STR00913## ##STR00914## ##STR00915## ##STR00916## ##STR00917##

wherein z.sub.1 and z.sub.2 are each, independently, and integer from 1 to 20; and

(2112) R.sub.9 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15
aryl, and C2-C15 heteroaryl.

(2113) 109. The conjugate of embodiment 108, wherein Y is:

(2114) ##STR00918##

(—NH(C═O)O—) and L is:

(2115) ##STR00919##

110. The conjugate of embodiment 108, wherein Y is:

(2116) ##STR00920##

(—NH(C═O)O—) and L is:

(2117) ##STR00921##

111. The conjugate of embodiment 108, wherein Y is:

(2118) ##STR00922##

(—NH(C═O)O—) and L is:

(2119) ##STR00923##

112. The conjugate of embodiment 108, wherein Y is:

(2120) ##STR00924##

and L is:

(2121) ##STR00925##

113. The conjugate of embodiment 1, wherein the conjugate is described by formula (M-I):

(2122) ##STR00926##

wherein each A.sub.1 is independently selected from any one of formulas (A-I)-(A-XII);

(2123) each E comprises an Fc domain monomer, an albumin protein, an albumin protein-binding

peptide, or an Fc-binding peptide;

(2124) n is 1 or 2;

(2125) T is an integer from 1 to 20; and

(2126) L is a linker covalently attached to each of E and A.sub.1,

(2127) the squiggly line connected to the E indicates that each A.sub.1-L is covalently attached to E;

(2128) or a pharmaceutically acceptable salt thereof.

(2129) 114. The conjugate of embodiment 113, wherein each A.sub.1 is independently selected from
any one of formulas (A-I), (A-II), (A-VI), or (A-VII);

(2130) each E comprises an Fc domain monomer, an albumin protein, an albumin

(2131) protein-binding peptide, or an Fc-binding peptide, and

(2132) the squiggly line connected to the E indicates that each A.sub.1-L is covalently attached to E;

(2133) or a pharmaceutically acceptable salt thereof.

(2134) 115. The conjugate of embodiment 114, wherein each A.sub.1 is independently selected from
formula (A-I).

(2135) 116. The conjugate of embodiment 115, wherein the conjugate is described by formula (M-II):

(2136) ##STR00927##

(2137) or a pharmaceutically acceptable salt thereof.

(2138) 117. The conjugate of embodiment 116, wherein the conjugate is described by formula (M-II-1):

(2139) ##STR00928##

(2140) or a pharmaceutically acceptable salt thereof.

(2141) 118. The conjugate of embodiment 117, wherein the conjugate is described by formula (M-II-2):

(2142) ##STR00929##

(2143) or a pharmaceutically acceptable salt thereof.

(2144) 119. The conjugate of embodiment 118, wherein the conjugate is described by formula (M-II-3):

(2145) ##STR00930##

(2146) wherein L′ is the remainder of L, and

(2147) y.sub.1 is an integer from 1-20,

(2148) or a pharmaceutically acceptable salt thereof.

(2149) 120. The conjugate of embodiment 119, wherein the conjugate is described by formula (M-II-4):

(2150) ##STR00931##

(2151) or a pharmaceutically acceptable salt thereof.

(2152) 121. The conjugate of embodiment 120, wherein the conjugate is described by formula (M-II-5):

(2153) ##STR00932##

(2154) wherein L′ is the remainder of L, and

(2155) y.sub.1 is an integer from 1-20,

(2156) or a pharmaceutically acceptable salt thereof.

(2157) 122. The conjugate of embodiment 121, wherein the conjugate has the structure of

(2158) ##STR00933##

(2159) or a pharmaceutically acceptable salt thereof.

(2160) 123. The conjugate of embodiment 116, wherein the conjugate is described by formula (M-II-6):

(2161) ##STR00934##

(2162) wherein R.sub.7 is selected from H, C1-C20 alkyl, C3-C20 cycloalkyl, C3-C20 heterocycloalkyl;
C5-C15 aryl, and C2-C15 heteroaryl;

(2163) or a pharmaceutically acceptable salt thereof.

(2164) 124. The conjugate of embodiment 123, wherein R.sub.7 is selected from C1-C20 alkyl, C3-
C20 cycloalkyl, C3-C20 heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl.

(2165) 125. The conjugate of embodiment 123 or 124, wherein R.sub.7 is selected from methyl, ethyl,
propyl, or butyl.

(2166) 126. The conjugate of any one of embodiments 123-125, wherein the conjugate is described by
formula (M-II-7):

(2167) ##STR00935##

(2168) or a pharmaceutically acceptable salt thereof.

(2169) 127. The conjugate of embodiment 126, wherein the conjugate is described by formula (M-II-8):

(2170) ##STR00936##

(2171) wherein L′ is the remainder of L, and

(2172) y.sub.1 is an integer from 1-20,

(2173) or a pharmaceutically acceptable salt thereof.

(2174) 128. The conjugate of embodiment 127, wherein the conjugate has the structure of

(2175) ##STR00937##

(2176) or a pharmaceutically acceptable salt thereof.

(2177) 129. The conjugate of embodiment 127, wherein the conjugate is described by the formula (M-
II-9):

(2178) ##STR00938##

(2179) or a pharmaceutically acceptable salt thereof.

(2180) 130. The conjugate of embodiment 129, wherein the conjugate is described by the formula (M-
II-10):

(2181) ##STR00939##
(2182) wherein L′ is the remainder of L, and

(2183) y.sub.1 is an integer from 1-20,

(2184) or a pharmaceutically acceptable salt thereof.

(2185) 131. The conjugate of embodiment 130, wherein the conjugate has the structure

(2186) ##STR00940##

(2187) or a pharmaceutically acceptable salt thereof.

(2188) 132. The conjugate of embodiment 113 or 114, wherein the conjugate is described by formula
(M-III):

(2189) ##STR00941##

(2190) or a pharmaceutically acceptable salt thereof.

(2191) 133. The conjugate of embodiment 132, wherein the conjugate is described by formula (M-III-
1):

(2192) ##STR00942##

(2193) or a pharmaceutically acceptable salt thereof.

(2194) 134. The conjugate of embodiment 133, wherein the conjugate is described by formula (M-III-
2):

(2195) ##STR00943##

(2196) or a pharmaceutically acceptable salt thereof.

(2197) 135. The conjugate of embodiment 134, wherein the conjugate is described by formula (M-III-
3):

(2198) ##STR00944##

(2199) wherein L′ is the remainder of L, and

(2200) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2201) or a pharmaceutically acceptable salt thereof.

(2202) 136. The conjugate of embodiment 133, wherein the conjugate is described by formula (M-III-
4):

(2203) ##STR00945##

(2204) or a pharmaceutically acceptable salt thereof.

(2205) 137. The conjugate of embodiment 136, wherein the conjugate is described by formula (M-III-
5):

(2206) ##STR00946##

(2207) wherein L′ is the remainder of L, and

(2208) y.sub.1 and y.sub.2 are each independently an integer from 1-20,
(2209) or a pharmaceutically acceptable salt thereof.

(2210) 138. The conjugate of embodiment 133, wherein the conjugate is described by formula (M-III-
6):

(2211) ##STR00947##

(2212) or a pharmaceutically acceptable salt thereof.

(2213) 139. The conjugate of embodiment 138, wherein the conjugate is described by formula (M-III-
7):

(2214) ##STR00948##

(2215) wherein L′ is the remainder of L, and

(2216) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2217) or a pharmaceutically acceptable salt thereof.

(2218) 140. The conjugate of embodiment 133, wherein the conjugate is described by formula (M-III-
8):

(2219) ##STR00949##

(2220) or a pharmaceutically acceptable salt thereof.

(2221) 141. The conjugate of embodiment 140, wherein the conjugate is described by formula (M-III-
9):

(2222) ##STR00950##

(2223) wherein L′ is the remainder of L, and 3

(2224) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2225) or a pharmaceutically acceptable salt thereof.

(2226) 142. The conjugate of embodiment 113, wherein the conjugate is described by formula (M-IV):

(2227) ##STR00951##

(2228) or a pharmaceutically acceptable salt thereof.

(2229) 143. The conjugate of embodiment 142, wherein the conjugate is described by formula (M-IV-
1):

(2230) ##STR00952##

(2231) or a pharmaceutically acceptable salt thereof.

(2232) 144. The conjugate of embodiment 143, wherein the conjugate is described by formula (M-IV-
2):

(2233) ##STR00953##

(2234) wherein L′ is the remainder of L, and

(2235) y.sub.1 and y.sub.2 are each independently an integer from 1-20,
(2236) or a pharmaceutically acceptable salt thereof.

(2237) 145. The conjugate of embodiment 113 or 114, wherein the conjugate is described by formula
(M-V):

(2238) ##STR00954##

(2239) or a pharmaceutically acceptable salt thereof.

(2240) 146. The conjugate of embodiment 145, wherein the conjugate is described by formula (M-V-1):

(2241) ##STR00955##

(2242) or a pharmaceutically acceptable salt thereof.

(2243) 147. The conjugate of embodiment 146, wherein the conjugate is described by formula (M-V-2):

(2244) or a pharmaceutically acceptable salt thereof.

(2245) ##STR00956##

148. The conjugate of embodiment 147, wherein the conjugate is described by formula (M-V-3):

(2246) ##STR00957##

(2247) wherein L′ is the remainder of L, and

(2248) y.sub.1 is an integer from 1-20,

(2249) or a pharmaceutically acceptable salt thereof.

(2250) 149. The conjugate of embodiment 148, wherein the conjugate is described by formula (M-V-4):

(2251) ##STR00958##

(2252) or a pharmaceutically acceptable salt thereof.

(2253) 150. The conjugate of embodiment 149, wherein the conjugate is described by formula (M-V-5):

(2254) ##STR00959##

(2255) wherein L′ is the remainder of L, and

(2256) y.sub.1 is an integer from 1-20,

(2257) or a pharmaceutically acceptable salt thereof.

(2258) 151. The conjugate of embodiment 145, wherein the conjugate is described by formula (M-V-6):

(2259) ##STR00960##

(2260) or a pharmaceutically acceptable salt thereof.

(2261) 152. The conjugate of embodiment 151, wherein the conjugate is described by formula (M-V-7):

(2262) ##STR00961##

(2263) or a pharmaceutically acceptable salt thereof.

(2264) 153. The conjugate of embodiment 152, wherein the conjugate is described by formula (M-V-8):
(2265) ##STR00962##

(2266) wherein L′ is the remainder of L, and

(2267) y.sub.1 is an integer from 1-20,

(2268) or a pharmaceutically acceptable salt thereof.

(2269) 154. The conjugate of embodiment 151, wherein the conjugate is described by formula (M-V-9):

(2270) ##STR00963##

(2271) or a pharmaceutically acceptable salt thereof.

(2272) 155. The conjugate of embodiment 154, wherein the conjugate is described by formula (M-V-
10):

(2273) ##STR00964##

(2274) wherein L′ is the remainder of L, and

(2275) y.sub.1 is an integer from 1-20,

(2276) or a pharmaceutically acceptable salt thereof.

(2277) 156. The conjugate of embodiment 113 or 114, wherein the conjugate is described by formula
(M-VI):

(2278) ##STR00965##

(2279) or a pharmaceutically acceptable salt thereof.

(2280) 157. The conjugate of embodiment 156, wherein the conjugate is described by formula (M-VI-
1):

(2281) ##STR00966##

(2282) or a pharmaceutically acceptable salt thereof.

(2283) 158. The conjugate of embodiment 157, wherein the conjugate is described by formula (M-VI-
2):

(2284) ##STR00967##

(2285) or a pharmaceutically acceptable salt thereof.

(2286) 159. The conjugate of embodiment 158, wherein the conjugate is described by formula (M-VI-
3):

(2287) ##STR00968##

(2288) wherein L′ is the remainder of L, and

(2289) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2290) or a pharmaceutically acceptable salt thereof.

(2291) 160. The conjugate of embodiment 157, wherein the conjugate is described by formula (M-VI-
4):
(2292) ##STR00969##

(2293) or a pharmaceutically acceptable salt thereof.

(2294) 161. The conjugate of embodiment 160, wherein the conjugate is described by formula (M-VI-
5):

(2295) ##STR00970##

(2296) wherein L′ is the remainder of L, and

(2297) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2298) or a pharmaceutically acceptable salt thereof.

(2299) 162. The conjugate of embodiment 157, wherein the conjugate is described by formula (M-VI-
6):

(2300) ##STR00971##

(2301) or a pharmaceutically acceptable salt thereof.

(2302) 163. The conjugate of embodiment 162, wherein the conjugate is described by formula (M-VI-
7):

(2303) ##STR00972##

(2304) wherein L′ is the remainder of L, and

(2305) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2306) or a pharmaceutically acceptable salt thereof.

(2307) 164. The conjugate of embodiment 157, wherein the conjugate is described by formula (M-VI-
8):

(2308) ##STR00973##

(2309) or a pharmaceutically acceptable salt thereof.

(2310) 165. The conjugate of embodiment 164, wherein the conjugate is described by formula (M-VI-
9):

(2311) ##STR00974##

(2312) wherein L′ is the remainder of L, and 3

(2313) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2314) or a pharmaceutically acceptable salt thereof.

(2315) 166. The conjugate of embodiment 113, wherein the conjugate is described by formula (M-VII):

(2316) ##STR00975##

(2317) or a pharmaceutically acceptable salt thereof.

(2318) 167. The conjugate of any one of embodiments 113-166 wherein R.sub.1 is OH.
(2319) 168. The conjugate of any one of embodiments 113-166 wherein R, is NH.sub.2.

(2320) 169. The conjugate of any one of embodiments 113-166 wherein R.sub.1 is —
NHC(═NH)NH.sub.2.

(2321) 170. The conjugate of embodiment 113, wherein the conjugate is described by formula (M-VIII):

(2322) ##STR00976##

(2323) or a pharmaceutically acceptable salt thereof.

(2324) 171. The conjugate of embodiment 170, wherein the conjugate is described by formula (M-VIII-
1):

(2325) or a pharmaceutically acceptable salt thereof.

(2326) ##STR00977##

172. The conjugate of embodiment 171, wherein the conjugate is described by formula (M-VIII-2):

(2327) ##STR00978##

(2328) or a pharmaceutically acceptable salt thereof.

(2329) 173. The conjugate of embodiment 172, wherein the conjugate is described by formula (M-VIII-
3):

(2330) ##STR00979##

(2331) wherein L′ is the remainder of L, and

(2332) y.sub.1 is an integer from 1-20,

(2333) or a pharmaceutically acceptable salt thereof.

(2334) 174. The conjugate of embodiment 173, wherein the conjugate is described by formula (M-VIII-
4):

(2335) ##STR00980##

(2336) or a pharmaceutically acceptable salt thereof.

(2337) 175. The conjugate of embodiment 174, wherein the conjugate is described by formula (M-VIII-
5):

(2338) ##STR00981##

(2339) wherein L′ is the remainder of L, and

(2340) y.sub.1 is an integer from 1-20,

(2341) or a pharmaceutically acceptable salt thereof.

(2342) 176. The conjugate of embodiment 175, wherein the conjugate has the structure of:

(2343) ##STR00982##

(2344) or a pharmaceutically acceptable salt thereof.

(2345) 177. The conjugate of embodiment 171, wherein the conjugate is described by formula (M-VIII-
6):

(2346) ##STR00983##

(2347) or a pharmaceutically acceptable salt thereof.

(2348) 178. The conjugate of embodiment 177, wherein the conjugate is described by formula (M-VIII-
7):

(2349) ##STR00984##

(2350) wherein L′ is the remainder of L, and

(2351) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2352) or a pharmaceutically acceptable salt thereof.

(2353) 179. The conjugate of embodiment 171, wherein the conjugate is described by formula (M-VIII-
8):

(2354) ##STR00985##

(2355) or a pharmaceutically acceptable salt thereof.

(2356) 180. The conjugate of embodiment 179, wherein the conjugate is described by formula (M-VIII-
9):

(2357) ##STR00986##

(2358) wherein L′ is the remainder of L, and

(2359) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2360) or a pharmaceutically acceptable salt thereof.

(2361) 181. The conjugate of embodiment 180, wherein the conjugate is described by formula (M-VIII-
10):

(2362) ##STR00987##

(2363) or a pharmaceutically acceptable salt thereof.

(2364) 182. The conjugate of embodiment 181, wherein the conjugate is described by formula (M-VIII-
11):

(2365) ##STR00988##

(2366) wherein L′ is the remainder of L, and

(2367) y.sub.1 and y.sub.2 are each independently an integer from 1-20,

(2368) or a pharmaceutically acceptable salt thereof.

(2369) 183. The conjugate of embodiment 113, wherein the conjugate is described by formula (M-IX):

(2370) ##STR00989##

(2371) or a pharmaceutically acceptable salt thereof.

(2372) 184. The conjugate of embodiment 183, wherein the conjugate is described by formula (M-IX-
1):

(2373) ##STR00990##

(2374) or a pharmaceutically acceptable salt thereof.

(2375) 185. The conjugate of embodiment 184, wherein the conjugate is described by formula (M-IX-
2):

(2376) ##STR00991##

(2377) or a pharmaceutically acceptable salt thereof.

(2378) 186. The conjugate of embodiment 184, wherein the conjugate is described by formula (M-IX-
3):

(2379) ##STR00992##

(2380) or a pharmaceutically acceptable salt thereof.

(2381) 187. The conjugate of embodiment 184, wherein the conjugate is described by formula (M-IX-
4):

(2382) ##STR00993##

(2383) or a pharmaceutically acceptable salt thereof.

(2384) 188. The conjugate of embodiment 184, wherein the conjugate is described by formula (M-IX-
5):

(2385) ##STR00994##

(2386) or a pharmaceutically acceptable salt thereof.

(2387) 189. The conjugate of embodiment 184, wherein the conjugate is described by formula (M-IX-
6):

(2388) ##STR00995##

(2389) or a pharmaceutically acceptable salt thereof.

(2390) 190. The conjugate of embodiment 113, wherein the conjugate is described by formula (M-X):

(2391) ##STR00996##

(2392) or a pharmaceutically acceptable salt thereof.

(2393) 191. The conjugate of embodiment 190, wherein the conjugate is described by formula (M-X-
1):

(2394) ##STR00997##

(2395) or a pharmaceutically acceptable salt thereof.

(2396) 192. The conjugate of embodiment 191, wherein the conjugate is described by formula (M-X-
2):

(2397) ##STR00998##
(2398) or a pharmaceutically acceptable salt thereof.

(2399) 193. The conjugate of embodiment 190, wherein the conjugate is described by formula (M-X-
3):

(2400) ##STR00999##

(2401) or a pharmaceutically acceptable salt thereof.

(2402) 194. The conjugate of any one of embodiments 113-193, wherein L or L′ comprises one or
more optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally
substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted
C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20
cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20
cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20
cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15
arylene, optionally substituted C2-C15 heteroarylene, O, S, NR.sup.i, P, carbonyl, thiocarbonyl,
sulfonyl, phosphate, phosphoryl, or imino,

(2403) wherein R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20
heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl,
optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally
substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted
C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20
cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or
optionally substituted C2-C15 heteroaryl.

(2404) 195. The conjugate of embodiment 194, wherein the backbone of L or L′ consists of one or
more optionally substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally
substituted C2-C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted
C2-C20 alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20
cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20
cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20
cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15
arylene, optionally substituted C2-C15 heteroarylene, O, S, NR.sup.i, P, carbonyl, thiocarbonyl,
sulfonyl, phosphate, phosphoryl, or imino,

(2405) wherein R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20
heteroalkyl, optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl,
optionally substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally
substituted C3-C20 cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted
C4-C20 cycloalkenyl, optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20
cycloalkynyl, optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or
optionally substituted C2-C15 heteroaryl.

(2406) 196. The conjugate of embodiment 194 or 195, wherein L or L′ is oxo substituted.

(2407) 197. The conjugate of any one of embodiments 113-196, wherein the backbone of L or L′
comprises no more than 250 atoms.

(2408) 198. The conjugate of any one of embodiments 113-197, wherein L or L′ is capable of forming
an amide, a carbamate, a sulfonyl, or a urea linkage.

(2409) 199. The conjugate of any one of embodiments 113-193, wherein L or L′ is a bond.

(2410) 200. The conjugate of any one of embodiments 113-193, wherein L or L′ is an atom.
(2411) 201. The conjugate of any one of embodiments 113-200, wherein each L is described by
formula (M-L-I):

J.sup.1-(Q.sup.1).sub.g-(T.sup.1).sub.h-(Q.sup.2).sub.i-(T.sup.2).sub.j-(Q.sup.3).sub.k-(T.sup.3).sub.l-
(Q.sup.4).sub.m-(T.sup.4).sub.n-(Q.sup.5).sub.o-J.sup.2

(2412) wherein J.sup.1 is a bond attached A.sub.1;

(2413) J.sup.2 is a bond attached to E or a functional group capable of reacting with a functional group
conjugated to E (e.g., maleimide and cysteine, amine and activated carboxylic acid, thiol and
maleimide, activated sulfonic acid and amine, isocyanate and amine, azide and alkyne, and alkene
and tetrazine);

(2414) each of Q.sup.1, Q.sup.2, Q.sup.3, Q.sup.4 and Q.sup.5 is, independently, optionally
substituted C1-C20 alkylene, optionally substituted C1-C20 heteroalkylene, optionally substituted C2-
C20 alkenylene, optionally substituted C2-C20 heteroalkenylene, optionally substituted C2-C20
alkynylene, optionally substituted C2-C20 heteroalkynylene, optionally substituted C3-C20
cycloalkylene, optionally substituted C3-C20 heterocycloalkylene, optionally substituted C4-C20
cycloalkenylene, optionally substituted C4-C20 heterocycloalkenylene, optionally substituted C8-C20
cycloalkynylene, optionally substituted C8-C20 heterocycloalkynylene, optionally substituted C5-C15
arylene, or optionally substituted C2-C15 heteroarylene;

(2415) each of T.sup.1, T.sup.2, T.sup.3, T.sup.4 is, independently, O, S, NR.sup.i, P, carbonyl,
thiocarbonyl, sulfonyl, phosphate, phosphoryl, or imino;

(2416) R.sup.i is H, optionally substituted C1-C20 alkyl, optionally substituted C1-C20 heteroalkyl,
optionally substituted C2-C20 alkenyl, optionally substituted C2-C20 heteroalkenyl, optionally
substituted C2-C20 alkynyl, optionally substituted C2-C20 heteroalkynyl, optionally substituted C3-C20
cycloalkyl, optionally substituted C3-C20 heterocycloalkyl, optionally substituted C4-C20 cycloalkenyl,
optionally substituted C4-C20 heterocycloalkenyl, optionally substituted C8-C20 cycloalkynyl,
optionally substituted C8-C20 heterocycloalkynyl, optionally substituted C5-C15 aryl, or optionally
substituted C2-C15 heteroaryl; and each of g, h, i, j, k, l, m, n, and o is, independently, 0 or 1.

(2417) 202. The conjugate of any one of embodiments 1-201, wherein R.sub.1 is —
NHC(═NH)NH.sub.2.

(2418) 203. The conjugate of any one of embodiments 1-202, wherein R.sub.2 is —F.

(2419) 204. The conjugate of any one of embodiments 1-203, wherein R.sub.3 is —F.

(2420) 205. The conjugate of any one of embodiments 1-204, wherein R.sub.4 is —CO.sub.2H.

(2421) 206. The conjugate of any one of embodiments 1-205, wherein R.sub.5 is —COCH.sub.3.

(2422) 207. The conjugate of any one of embodiment 1-206, wherein the squiggly line connected to E
indicates that the L of each A.sub.1-L or each A.sub.1-L-A.sub.2 is covalently attached to a nitrogen
atom of a solvent-exposed lysine of E.

(2423) 208. The conjugate of any one of embodiment 1-206, wherein the squiggly line connected to E
indicates that the L of each A.sub.1-L or each A.sub.1-L-A.sub.2 L is covalently attached to a sulfur
atom of a solvent-exposed cysteine of E.

(2424) 209. The conjugate of any one of embodiments 1-208, wherein each E is an Fc domain
monomer.

(2425) 210. The conjugate of embodiment 209, wherein n is 2, and each E dimerizes to form an Fc
domain.
(2426) 211. The conjugate of any one of embodiments 2-4, wherein n is 2, each E is an Fc domain
monomer, each E dimerizes to form an Fc domain, and the conjugate is described by formula (D-I-1):

(2427) ##STR01000## wherein J is an Fc domain; and

(2428) T is an integer from 1 to 20,

(2429) or a pharmaceutically acceptable salt thereof.

(2430) 212. The conjugate of embodiment 211, wherein the conjugate has the structure of

(2431) ##STR01001##

(2432) or a pharmaceutically acceptable salt thereof.

(2433) 213. The conjugate of embodiment any one of embodiments 113-115, wherein n is 2, each E is
an Fc domain monomer, each E dimerizes to form an Fc domain, and the conjugate is described by
formula (M-I-1)

(2434) ##STR01002##

(2435) wherein J is an Fc domain; and

(2436) T is an integer from 1 to 20,

(2437) or a pharmaceutically acceptable salt thereof.

(2438) 214. The conjugate of any one of embodiments 209-213, wherein each E comprises an amino
acid sequence at least 95% identical to the sequence of any one of SEQ ID NOs: 1-138.

(2439) 215. The conjugate of embodiment 214, wherein each E comprises and amino acid sequence
at least 95% identical to the sequence of any one of SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64,
SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 76, SEQ ID NO: 77,
SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 90,
SEQ ID NO: 91, SEQ ID NO: 94, SEQ ID NO: 95.

216. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 62.

217. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 63.

218. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 64.

219. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 67.

220. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 68.

221. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 72.

222. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 73.

223. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 76.
224. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 77.

225. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 80.

226. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 81.

227. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 82.

228. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 85.

229. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 86.

230. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 90.

231. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 91.

232. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 94.

233. The conjugate of embodiment 215, wherein each E comprises the amino acid sequence of SEQ
ID NO: 95.

234. The conjugate of any one of embodiments 209-233, wherein T is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

235. A population of conjugates of any one of embodiments 209-233, wherein the average value of T
is 1 to 10.

236. The population of conjugates of embodiment 235, wherein the average value of T is 1 to 5.

237. The population of conjugates of embodiment 235, wherein the average value of T is 3 to 7.

238. The population of conjugates of embodiment 236 or 237, wherein the average value of T is 3.5 to
5.5.

239. The population of conjugates of embodiment 236 or 237, wherein the average value of T is about
4.5.

240. The population of conjugates of embodiment 235, wherein the average value of T is 5 to 10.

241. A pharmaceutical composition comprising a conjugate of any of embodiments 1-234, or a

population of conjugates of embodiments 235-240, or a pharmaceutically acceptable salt thereof, and
a pharmaceutically acceptable excipient.

242. A method for the treatment of a subject having a viral infection or presumed to have a viral
infection, the method comprising administering to the subject an effective amount of a conjugate or
composition of any one of embodiments 1-241.

243. A method for the prophylactic treatment of a viral infection in a subject in need thereof, the
method comprising administering to the subject an effective amount of a conjugate or composition of
any one of embodiments 1-241.
244. The method of embodiment 242 or 243, wherein the viral infection is caused by influenza virus or
parainfluenza virus.

245. The method of any one of embodiments 242-244, wherein the viral infection is influenza virus A,
B, or C, or parainfluenza virus.

246. The method of any one of embodiments 242-245, wherein the subject is immunocompromised.

247. The method of any one of embodiments 242-246, wherein the subject has been diagnosed with
humoral immune deficiency, T cell deficiency, neutropenia, asplenia, or complement deficiency.

248. The method of any one of embodiments 242-247, wherein the subject is being treated or is about
to be treated with an immunosuppressive therapy.

249. The method of any one of embodiments 242-248, wherein said subject has been diagnosed with
a disease which causes immunosuppression.

250. The method of embodiment 249, wherein the disease is cancer or acquired immunodeficiency
syndrome.

251. The method of embodiment 250, wherein the cancer is leukemia, lymphoma, or multiple
myeloma.

252. The method of any one of embodiments 242-251, wherein the subject has undergone or is about
to undergo hematopoietic stem cell transplantation.

253. The method of any one of embodiments 242-252, wherein the subject has undergone or is about
to undergo an organ transplant.

254. The method of any one of embodiments 242-253, wherein the subject has or is at risk of
developing a secondary infection.

255. A method of preventing a secondary infection in a subject diagnosed with an influenza infection,
wherein the method includes administering to the subject the conjugate or composition of any one of
embodiments 1-241.

256. The method of embodiments 254 or 255, wherein the secondary infection is a respiratory
infection.

257. The method of any one of embodiments 254-256, wherein the secondary infection is associated
with pneumonia.

258. The method of any one of embodiments 254-257, wherein the secondary infection is a bacterial
infection, a viral infection, or a fungal infection.

259. The method of embodiment 258, wherein the secondary infection is a bacterial infection.

260. The method of embodiment 259, wherein the bacterial infection is a methicillin-resistant
Staphylococcus aureus (MRSA), Streptococcus pneumoniae, Pseudomonas aeruginosa, or
Haemophilus influenzae infection.

261. The method of embodiment 260, wherein the bacterial infection is MRSA.

262. The method of embodiment 260, wherein the bacterial infection is S. pneumoniae.

263. The method of any one of embodiments 242-262 wherein the conjugate of composition is
administered intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally,
intralesionally, intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally,
intravitreally, intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously,
subconjunctival, intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally,
locally, by inhalation, by injection, or by infusion.

264. The method of any one of embodiments 242-263, wherein the subject is treated with a second
therapeutic agent.

265. The method of embodiment 264, wherein the second therapeutic agent is an antiviral agent.

266. The method of embodiment 264, wherein the antiviral agent is selected from pimovidir,
oseltamivir, zanamivir, peramivir, laninamivir, amantadine, or rimantadine.

267. The method of embodiment 266, wherein the second therapeutic agent is an antiviral vaccine.

268. The method of embodiment 267, wherein the antiviral vaccine elicits an immune response in the
subject against influenza virus A, B, or C, or parainfluenza virus.

269. The method of embodiment 265, wherein the antiviral agent is baloxavir.

270. The method of embodiment 265, wherein the conjugate and baloxavir are administered
sequentially.

271. The method of embodiment 265, wherein the conjugate and baloxavir are administered
simultaneously.

272. The method of any one of embodiments 242-271, wherein conjugate is described by formula (D-
II-6).

273. The method of embodiment 272, wherein R.sub.7 is selected from C1-C20 alkyl, C3-C20
cycloalkyl, C3-C20 heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl.

274. The method of embodiment 272 or 273, wherein R.sub.7 is selected from methyl, ethyl, propyl, or
butyl.

275. The method of embodiments 272-274, wherein the conjugate is described by formula (D-II-7).

276. The method of any one of embodiments 242-275, wherein each E has a sequence at least 95%
identical to the sequence of any one of SEQ ID NOs: 63-68.

277. The method of any one of embodiments 242-275, wherein each E has a sequence at least 95%
identical to the sequence of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 72, or SEQ ID NO: 73.

278. The method of any one of embodiments 242-275, wherein each E has a sequence at least 95%
identical to the sequence of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 76, or SEQ ID NO: 77.

279. The method of any one of embodiments 242-275, wherein the conjugate is conjugate 45 or
conjugate 46.

280. A method for treating or preventing a viral infection in a subject, the method comprising
administering to the subject:

(2440) a) a conjugate or composition of any one of embodiments 1-241; and

(2441) b) a second therapeutic agent.

(2442) 281. The method of embodiment 280, wherein conjugate is administered to the subject after
the subject has a viral infection, is presumed to have a viral infection, or has been exposed to a virus.

(2443) 282. The method of embodiment 280, wherein the conjugate is administered to the subject
prophylactically.
(2444) 283. The method of any one of embodiments 278-282, wherein the second therapeutic agent is
administered to the subject after the subject has a viral infection, is presumed to have a viral infection,
or has been exposed to the virus.

(2445) 284. The method of any one of embodiments 278-283, wherein the second therapeutic agent is
administered to the subject prophylactically.

(2446) 285. The method of any one of embodiments 278-284, wherein the second therapeutic agent is
administered within 2 days of the conjugate.

(2447) 286. The method of any one of embodiments 278-285, wherein the second therapeutic agent is
an antiviral agent.

(2448) 287. The method of embodiment 286, wherein the antiviral agent is pimovidir, oseltamivir,
zanamivir, peramivir, laninamivir, amantadine, baloxavir marboxil, baloxavir acid, rimantadine, or a
pharmaceutically acceptable salt thereof.

(2449) 288. The method of embodiment 287, wherein the antiviral agent is baloxavir marboxil,
baloxavir acid, or a pharmaceutically acceptable salt thereof.

(2450) 289. The method of any one of embodiments 288, wherein the baloxavir marboxil, baloxavir
acid, or a pharmaceutically acceptable salt thereof, is administered in an amount between 20 mg and
90 mg.

(2451) 290. The method of embodiment 286, wherein the baloxavir marboxil, baloxavir acid, or a
pharmaceutically acceptable salt thereof, is administered orally.

(2452) 291. The method of embodiment 289 or 290, wherein the baloxavir marboxil, baloxavir acid, or
a pharmaceutically acceptable salt thereof, is administered as a single dose.

(2453) 292. The method of embodiment 289 or 290, wherein the baloxavir marboxil, baloxavir acid, or
a pharmaceutically acceptable salt thereof, is administered as more than one dose.

(2454) 293. The method of any one of embodiments 289-292, wherein the baloxavir marboxil,
baloxavir acid, or a pharmaceutically acceptable salt thereof, is administered in an amount between 20
mg and 40 mg.

(2455) 294. The method of any one of embodiments 289-292, wherein the baloxavir marboxil,
baloxavir acid, or a pharmaceutically acceptable salt thereof, is administered in an amount between 30
and 80 mg.

(2456) 295. The method of embodiment any one of embodiments 278-294, wherein the conjugate is
described by formula (D-II-6).

(2457) 296. The method of embodiment 295, wherein R.sub.7 is selected from C1-C20 alkyl, C3-C20
cycloalkyl, C3-C20 heterocycloalkyl; C5-C15 aryl, and C2-C15 heteroaryl.

(2458) 297. The method of embodiment 295 or 296, wherein R.sub.7 is selected from methyl, ethyl,
propyl, or butyl.

(2459) 298. The method of any one of embodiments 295-297, wherein the conjugate is described by
formula (D-11-7).

(2460) 299. The method of any one of embodiments 280-298, wherein each E has a sequence at least
95% identical to the sequence of any one of SEQ ID NOs: 63-68.

(2461) 300. The method of embodiment 299, wherein each E has a sequence at least 95% identical to
the sequence of SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 72, or SEQ ID NO: 73.
(2462) 301. The method of embodiment 300, wherein each E has a sequence at least 95% identical to
the sequence of SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 76, or SEQ ID NO: 77.

(2463) 302. The method of any one of embodiments 280-298, wherein the conjugate is conjugate 45
or conjugate 46.

(2464) 303. The method of any one of embodiments 280-302, wherein the conjugate is administered
intramuscularly, intravenously, intradermally, intraarterially, intraperitoneally, intralesionally,
intracranially, intraarticularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally,
intravaginally, intrarectally, topically, intratumorally, peritoneally, subcutaneously, subconjunctival,
intravesicularlly, mucosally, intrapericardially, intraumbilically, intraocularally, orally, locally, by
inhalation, by injection, or by infusion.

304. The method of embodiment 303, wherein the conjugate is administered intravenously.

305. The method of embodiment 303, wherein the conjugate is administered subcutaneously.

306. The method of embodiment 303, wherein the conjugate is administered intramuscularly.

307. The method of any one of embodiments 280-306, wherein the viral infection is caused by an
influenza virus or a parainfluenza virus.

308. The method of embodiment 307, wherein the virus is influenza virus A, B, or C, or parainfluenza
virus.

Other Embodiments

(2465) While the invention has been described in connection with specific embodiments thereof, it will
be understood that it is capable of further modifications and this application is intended to cover any
variations, uses, or adaptations of the invention following, in general, the principles of the invention
and including such departures from the invention that come within known or customary practice within
the art to which the invention pertains and may be applied to the essential features hereinbefore set
forth, and follows in the scope of the claims. All publications, patents, and patent applications
mentioned in the above specification are hereby incorporated by reference to the same extent as if
each individual publication, patent or patent application was specifically and individually indicated to be
incorporated by reference in its entirety.

(2466) Detailed descriptions of one or more preferred embodiments are provided herein. It is to be
understood, however, that the present invention may be embodied in various forms. Therefore,
specific details disclosed herein are not to be interpreted as limiting, but rather as a basis for the
claims and as a representative basis for teaching one skilled in the art to employ the present invention
in any appropriate manner.

Claims

1. A conjugate described by formula (D-II-6): ##STR01003## wherein each E comprises an Fc domain

monomer; n is 1 or 2; T is an integer from 1 to 20; L is a linker; R.sub.7 is selected from C1-C20 alkyl;
and the squiggly line indicates that L is covalently attached to E; or a pharmaceutically acceptable salt
thereof.

2. The conjugate of claim 1, wherein the squiggly line indicates that L is covalently attached to a
nitrogen atom of a solvent-exposed lysine of E.

3. The conjugate of claim 1, wherein the squiggly line indicates that L is covalently attached to a sulfur
atom of a solvent-exposed cysteine of E.

4. The conjugate of claim 1, wherein n is 2, and the two Es dimerize with each other to form an Fc
domain.
5. The conjugate of claim 1, wherein T is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

6. A population of conjugates of claim 1, wherein the average value of T is 1 to 10.

7. The conjugate of claim 1, wherein R.sub.7 is methyl, ethyl, propyl, or butyl.

8. The conjugate of claim 7, wherein the conjugate is described by formula (D-II-7): ##STR01004## or
a pharmaceutically acceptable salt thereof.

9. The conjugate of claim 8, wherein the conjugate is described by formula (D-II-8): ##STR01005##
wherein L′ is the remainder of L, and y.sub.1 and y.sub.2 are each independently an integer from 1-20,
or a pharmaceutically acceptable salt thereof.

10. The conjugate of claim 9, wherein the conjugate has the structure of: ##STR01006## or a
pharmaceutically acceptable salt thereof.

11. The conjugate of claim 10, wherein the conjugate has the structure of: ##STR01007## or a
pharmaceutically acceptable salt thereof.

12. The conjugate of claim 11, wherein n is 2, and the two Es dimerize with each other to form an Fc
domain.

13. The conjugate of claim 11, wherein T is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

14. A population of conjugates of claim 11, wherein the average value of T is 1 to 10.

15. The conjugate of claim 1, wherein each E comprises an amino acid sequence independently
selected from of any one of SEQ ID NOs: 1-138.

16. The conjugate of claim 1, wherein each E comprises an amino acid sequence independently
selected from any one of SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 76, and SEQ ID NO: 77.

17. The conjugate of claim 16, wherein each E comprises an amino acid sequence of SEQ ID NO: 72.

18. The conjugate of claim 16, wherein each E comprises an amino acid sequence of SEQ ID NO: 73.

19. The conjugate of claim 16, wherein each E comprises an amino acid sequence of SEQ ID NO: 76.

20. The conjugate of claim 16, wherein each E comprises an amino acid sequence of SEQ ID NO: 77.

21. A conjugate described by the structure: ##STR01008## wherein J is an Fc domain comprising two
Fc domain monomers, T is an integer from 1 to 20, L is a linker; R.sub.7 is selected from C1-C20 alkyl;
and the squiggly line indicates that L is covalently attached to J; or a pharmaceutically acceptable salt
thereof.

22. The conjugate of claim 21, wherein the squiggly line indicates that L is covalently attached to a
nitrogen atom of a solvent-exposed lysine of J.

23. The conjugate of claim 21, wherein the squiggly line indicates that L is covalently attached to a
sulfur atom of a solvent-exposed cysteine of J.

24. The conjugate of claim 21, wherein R.sub.7 is methyl, ethyl, propyl, or butyl.

25. The conjugate of claim 21, wherein T is an integer from 1 to 10.

26. The conjugate of claim 21, wherein the conjugate has the structure of: ##STR01009## or a
pharmaceutically acceptable salt thereof.
27. The conjugate of claim 26, wherein the conjugate has the structure of: ##STR01010## or a
pharmaceutically acceptable salt thereof.

28. The conjugate of claim 27, wherein each Fc domain monomer comprises an amino acid sequence
independently selected from any one of SEQ ID NOs: 1-138.

29. The conjugate of claim 28, wherein each Fc domain monomer comprises an amino acid sequence
independently selected from any one of SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 76, and SEQ ID
NO: 77.

30. The conjugate of claim 29, wherein each Fc domain monomer comprises an amino acid sequence
of SEQ ID NO: 72.

31. The conjugate of claim 29, wherein each Fc domain monomer comprises an amino acid sequence
of SEQ ID NO: 73.

32. The conjugate of claim 29, wherein each Fc domain monomer comprises an amino acid sequence
of SEQ ID NO: 76.

33. The conjugate of claim 29, wherein each Fc domain monomer comprises an amino acid sequence
of SEQ ID NO: 77.

34. The conjugate of 27, wherein T is an integer from 1 to 10.

35. A population of conjugates of claim 27, wherein the average value of T is 1 to 10.

Sequence Listing

CWU

SEQUENCE LISTING NEW RULES

1671252PRTArtificial SequenceSynthetic Construct 1Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu
Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys 20 25
30Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe 35 40 45Pro Pro Lys Ile Lys Asp
Val Leu Met Ile Ser Leu Ser Pro Ile Val 50 55 60Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp
Val Gln Ile65 70 75 80Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr 85 90 95His
Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro 100 105 110Ile Gln His Gln Asp Trp Met
Ser Gly Lys Glu Phe Lys Cys Lys Val 115 120 125Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile
Ser Lys Pro 130 135 140Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu145 150 155
160Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp 165 170 175Phe Met Pro Glu
Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr 180 185 190Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val
Leu Asp Ser Asp Gly Ser 195 200 205Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val
Glu 210 215 220Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His225 230 235 240His
Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 245 2502232PRTMus musculus 2Pro Arg Gly Pro Thr Ile
Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala1 5 10 15Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe
Pro Pro Lys Ile 20 25 30Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val 35 40 45Val
Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val 50 55 60Asn Asn Val Glu Val His Thr Ala
Gln Thr Gln Thr His Arg Glu Asp65 70 75 80Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln
His Gln 85 90 95Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp 100 105 110Leu
Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val 115 120 125Arg Ala Pro Gln Val Tyr Val Leu
Pro Pro Pro Glu Glu Glu Met Thr 130 135 140Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe
Met Pro Glu145 150 155 160Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr 165 170
175Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr 180 185 190Ser Lys Leu Arg
Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr 195 200 205Ser Cys Ser Val Val His Glu Gly Leu His
Asn His His Thr Thr Lys 210 215 220Ser Phe Ser Arg Thr Pro Gly Lys225 2303251PRTArtificial
SequenceSynthetic Construct 3Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu1 5
10 15Val Thr Asn Ser Met Val Arg Ser Asp Lys Thr His Thr Cys Pro Pro 20 25 30Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 35 40 45Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr 50 55 60Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn65 70
75 80Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 85 90 95Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 100 105 110Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser 115 120 125Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 130
135 140Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu145 150 155 160Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 165 170 175Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu 180 185 190Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe 195 200 205Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 210 215
220Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr225 230 235 240Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 245 2504231PRTArtificial SequenceSynthetic Construct 4Met Val Arg
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro1 5 10 15Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 20 25 30Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
35 40 45Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr65 70 75 80Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His Gln Asp 85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
100 105 110Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 130 135 140Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp145 150 155 160Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 165 170 175Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185
190Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220Leu Ser Leu Ser Pro Gly Lys225
2305258PRTArtificial SequenceSynthetic Construct 5Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu
Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys 20 25
30Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe 35 40 45Pro Pro Lys Ile Lys Asp
Val Leu Met Ile Ser Leu Ser Pro Ile Val 50 55 60Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp
Val Gln Ile65 70 75 80Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr 85 90 95His
Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro 100 105 110Ile Gln His Gln Asp Trp Met
Ser Gly Lys Glu Phe Lys Cys Lys Val 115 120 125Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile
Ser Lys Pro 130 135 140Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu145 150 155
160Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp 165 170 175Phe Met Pro Glu
Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr 180 185 190Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val
Leu Asp Ser Asp Gly Ser 195 200 205Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val
Glu 210 215 220Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His225 230 235 240His
Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys His His His His 245 250 255His His6238PRTArtificial
SequenceSynthetic Construct 6Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala1 5
10 15Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile 20 25 30Lys Asp Val Leu Met
Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val 35 40 45Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
Ser Trp Phe Val 50 55 60Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp65 70 75
80Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln 85 90 95Asp Trp Met Ser Gly Lys
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp 100 105 110Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro
Lys Gly Ser Val 115 120 125Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr 130 135
140Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu145 150 155 160Asp Ile Tyr Val
Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr 165 170 175Lys Asn Thr Glu Pro Val Leu Asp Ser
Asp Gly Ser Tyr Phe Met Tyr 180 185 190Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn
Ser Tyr 195 200 205Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys 210 215 220Ser
Phe Ser Arg Thr Pro Gly Lys His His His His His His225 230 2357257PRTArtificial SequenceSynthetic
Construct 7Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn
Ser Met Val Arg Ser Asp Lys Thr His Thr Cys Pro Pro 20 25 30Cys Pro Ala Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro 35 40 45Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr 50 55 60Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn65 70 75 80Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 85 90 95Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val 100 105 110Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
115 120 125Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 130 135 140Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu145 150 155 160Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe 165 170 175Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu 180 185 190Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 195 200
205Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 210 215 220Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr225 230 235 240Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Lys His His His His His 245 250 255His8237PRTArtificial SequenceSynthetic Construct 8Met Val
Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro1 5 10 15Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys 20 25 30Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 35 40 45Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr65 70 75 80Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp 85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu 100 105 110Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 130 135 140Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp145 150 155 160Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys 165 170 175Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180
185 190Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205Cys Ser Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220Leu Ser Leu Ser Pro Gly Lys His His
His His His His225 230 2359262PRTArtificial SequenceSynthetic Construct 9Met Tyr Arg Met Gln Leu
Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser Met Val Arg Ser Asp Lys Thr His
Thr Cys Pro Pro 20 25 30Cys Pro Pro Cys Lys Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 35 40
45Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 50 55 60Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His Glu Asp Pro65 70 75 80Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala 85 90 95Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 100 105
110Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 115 120 125Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 130 135 140Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu145 150 155 160Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys 165 170 175Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 180 185 190Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 195 200 205Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser 210 215 220Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala225 230 235 240Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
245 250 255His His His His His His 26010242PRTArtificial SequenceSynthetic Construct 10Met Val
Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Pro Cys1 5 10 15Lys Cys Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe 20 25 30Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val 35 40 45Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 50 55 60Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro65 70 75 80Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr 85 90 95Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
100 105 110Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 115 120 125Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 130 135 140Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly145 150 155 160Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro 165 170 175Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 180 185
190Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 195 200 205Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn His 210 215 220Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro Gly Lys His His His His225 230 235 240His His11236PRTArtificial SequenceSynthetic Construct
11Met Val Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Pro Cys1 5 10 15Lys Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 20 25 30Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val 35 40 45Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 50 55
60Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro65 70 75 80Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 85 90 95Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val 100 105 110Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 115 120
125Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 130 135 140Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly145 150 155 160Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 165 170 175Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser 180 185 190Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 195 200 205Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 210 215 220Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys225 230 23512258PRTArtificial SequenceSynthetic Construct 12Met Tyr Arg
Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser Pro Arg Gly Pro Thr Ile
Lys Pro Cys Pro Pro Cys 20 25 30Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe
35 40 45Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val 50 55 60Thr Cys Val Val Val
Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile65 70 75 80Ser Trp Phe Val Asn Asn Val Glu Val His Thr
Ala Gln Thr Gln Thr 85 90 95His Arg Glu Asp Tyr Ala Ser Thr Leu Arg Val Val Ser Ala Leu Pro 100 105
110Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val 115 120 125Asn Asn Lys Asp
Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro 130 135 140Lys Gly Ser Val Arg Ala Pro Gln Val Tyr
Val Leu Pro Pro Pro Glu145 150 155 160Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr
Asp 165 170 175Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr 180 185 190Glu
Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser 195 200 205Tyr Phe Met Tyr Ser Lys
Leu Arg Val Glu Lys Lys Asn Trp Val Glu 210 215 220Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly
Leu His Asn His225 230 235 240His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys His His His His 245
250 255His His13238PRTArtificial SequenceSynthetic Construct 13Pro Arg Gly Pro Thr Ile Lys Pro
Cys Pro Pro Cys Lys Cys Pro Ala1 5 10 15Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro
Lys Ile 20 25 30Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val 35 40 45Val Asp Val
Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val 50 55 60Asn Asn Val Glu Val His Thr Ala Gln Thr
Gln Thr His Arg Glu Asp65 70 75 80Tyr Ala Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln 85
90 95Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp 100 105 110Leu Pro Ala Pro
Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val 115 120 125Arg Ala Pro Gln Val Tyr Val Leu Pro Pro
Pro Glu Glu Glu Met Thr 130 135 140Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro
Glu145 150 155 160Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr 165 170 175Lys
Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr 180 185 190Ser Lys Leu Arg Val Glu
Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr 195 200 205Ser Cys Ser Val Val His Glu Gly Leu His Asn His
His Thr Thr Lys 210 215 220Ser Phe Ser Arg Thr Pro Gly Lys His His His His His His225 230
23514257PRTArtificial SequenceSynthetic Construct 14Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala
Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser Met Val Arg Ser Asp Lys Thr His Thr Cys Pro Pro 20 25
30Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 35 40 45Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 50 55 60Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn65 70 75 80Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 85 90
95Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 100 105 110Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 115 120 125Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys 130 135 140Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu145
150 155 160Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 165 170 175Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 180 185 190Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe 195 200 205Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly 210 215 220Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr225 230
235 240Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys His His His His His 245 250
255His15237PRTArtificial SequenceSynthetic Construct 15Met Val Arg Ser Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro1 5 10 15Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
20 25 30Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 35 40 45Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr65 70 75 80Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 85 90
95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105 110Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys 130 135 140Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp145 150 155 160Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 165 170 175Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205Cys Ser Val Met His Glu Ala Leu His Asn His
Tyr Thr Gln Lys Ser 210 215 220Leu Ser Leu Ser Pro Gly Lys His His His His His His225 230
23516252PRTArtificial SequenceSynthetic Construct 16Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe
Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Ile Ser Ala Met Val Arg Ser Asp Lys Thr His Thr Cys Pro 20 25
30Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55 60Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe65 70 75 80Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90
95Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 115 120 125Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala 130 135 140Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg145
150 155 160Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 165 170 175Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 180 185 190Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser 195 200 205Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln 210 215 220Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His225 230
235 240Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 25017266PRTArtificial
SequenceSynthetic Construct 17Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5
10 15Tyr Ser Ile Ser Ala Met Val Arg Ser Asp Lys Thr His Thr Cys Pro 20 25 30Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 50 55 60Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe65 70 75
80Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90 95Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val 115 120 125Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135
140Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg145 150 155 160Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 165 170 175Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 180 185 190Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser 195 200 205Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 210 215 220Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His225 230 235 240Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser 245 250 255Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
260 26518248PRTArtificial SequenceSynthetic Construct 18Ile Ser Ala Met Val Arg Ser Asp Lys Thr
His Thr Cys Pro Pro Cys1 5 10 15Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
20 25 30Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu65 70 75 80Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90
95His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu 130 135 140Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr145 150 155 160Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 210 215 220Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Glu Gln225
230 235 240Lys Leu Ile Ser Glu Glu Asp Leu 24519252PRTArtificial SequenceSynthetic Construct
19Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Ile Ser Ala Met
Val Arg Ser Asp Lys Thr His Thr Cys Pro 20 25 30Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe 35 40 45Pro Pro Lys Pro Ser Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55
60Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe65 70 75 80Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90 95Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr 100 105 110Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 115
120 125Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135 140Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg145 150 155 160Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly 165 170 175Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro 180 185 190Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 195 200 205Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 210 215 220Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His225 230 235 240Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 245 25020234PRTArtificial SequenceSynthetic Construct 20Ile Ser Ala Met Val Arg Ser Asp Lys
Thr His Thr Cys Pro Pro Cys1 5 10 15Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro 20 25 30Lys Pro Ser Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu65 70 75 80Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90
95His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu 130 135 140Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr145 150 155 160Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 210 215 220Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys225 23021266PRTArtificial
SequenceSynthetic Construct 21Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5
10 15Tyr Ser Ile Ser Ala Met Val Arg Ser Asp Lys Thr His Thr Cys Pro 20 25 30Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45Pro Pro Lys Pro Ser Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val 50 55 60Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe65 70 75
80Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90 95Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val 115 120 125Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135
140Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg145 150 155 160Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 165 170 175Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro 180 185 190Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser 195 200 205Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 210 215 220Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His225 230 235 240Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser 245 250 255Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
260 26522248PRTArtificial SequenceSynthetic Construct 22Ile Ser Ala Met Val Arg Ser Asp Lys Thr
His Thr Cys Pro Pro Cys1 5 10 15Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
20 25 30Lys Pro Ser Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu65 70 75 80Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90
95His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu 130 135 140Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr145 150 155 160Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175Asn
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180 185 190Leu Tyr Ser Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 210 215 220Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Glu Gln225
230 235 240Lys Leu Ile Ser Glu Glu Asp Leu 24523266PRTArtificial SequenceSynthetic Construct
23Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Ile Ser Ala Met
Val Arg Ser Asp Lys Thr His Thr Cys Pro 20 25 30Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
Val Phe Leu Phe 35 40 45Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55
60Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe65 70 75 80Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90 95Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val
Ser Val Leu Thr 100 105 110Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 115
120 125Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135 140Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg145 150 155 160Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly 165 170 175Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro 180 185 190Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 195 200 205Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 210 215 220Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu His Asn His225 230 235 240Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Gly Gly Gly Gly Ser 245 250 255Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 260 26524248PRTArtificial
SequenceSynthetic Construct 24Ile Ser Ala Met Val Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys1 5
10 15Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 20 25 30Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 35 40 45Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp 50 55 60Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu65 70 75
80Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 85 90 95His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 100 105 110Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly 115 120 125Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 130 135
140Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr145 150 155 160Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 165 170 175Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe 180 185 190Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 195 200 205Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215
220Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Glu Gln225 230 235 240Lys Leu Ile Ser
Glu Glu Asp Leu 24525266PRTArtificial SequenceSynthetic Construct 25Met Lys Trp Val Thr Phe Ile
Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Ile Ser Ala Met Val Arg Ser Asp Lys Thr His
Thr Cys Pro 20 25 30Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55 60Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe65 70 75 80Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro 85 90 95Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110Val Leu
Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 115 120 125Ser Asn Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135 140Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg145 150 155 160Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 165
170 175Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 180 185 190Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 195 200 205Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln 210 215 220Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn Ala225 230 235 240Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser 245 250
255Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 260 26526248PRTArtificial SequenceSynthetic Construct
26Ile Ser Ala Met Val Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys1 5 10 15Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 20 25 30Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys 35 40 45Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu65 70 75 80Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 85 90 95Ala Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn 100 105 110Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 130 135 140Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr145 150 155 160Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 165 170 175Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180
185 190Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn Ala Tyr Thr 210 215 220Gln Lys Ser Leu Ser Leu Ser Pro
Gly Gly Gly Gly Gly Ser Glu Gln225 230 235 240Lys Leu Ile Ser Glu Glu Asp Leu
24527266PRTArtificial SequenceSynthetic Construct 27Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe
Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Ile Ser Ala Met Val Arg Ser Asp Lys Thr His Thr Cys Pro 20 25
30Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55 60Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe65 70 75 80Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90
95Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 115 120 125Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala 130 135 140Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg145
150 155 160Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 165 170 175Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 180 185 190Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser 195 200 205Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln 210 215 220Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His225 230
235 240Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser 245 250 255Glu Gln Phe
Leu Ile Ser Glu Glu Asp Leu 260 26528248PRTArtificial SequenceSynthetic Construct 28Ile Ser Ala
Met Val Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys1 5 10 15Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 20 25 30Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys 35 40 45Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu65 70 75 80Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 85 90 95His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 100 105 110Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 130 135 140Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr145 150 155 160Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 165 170 175Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180
185 190Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220Gln Lys Ser Leu Ser Leu Ser Pro
Gly Gly Gly Gly Gly Ser Glu Gln225 230 235 240Phe Leu Ile Ser Glu Glu Asp Leu
24529266PRTArtificial SequenceSynthetic Construct 29Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe
Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Ile Ser Ala Met Val Arg Ser Asp Lys Thr His Thr Cys Pro 20 25
30Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55 60Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val Lys Phe65 70 75 80Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90
95Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 115 120 125Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala 130 135 140Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg145
150 155 160Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 165 170 175Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 180 185 190Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser 195 200 205Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln 210 215 220Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His225 230
235 240Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser 245 250 255Glu Gln Phe
Leu Ile Ser Glu Glu Asp Leu 260 26530248PRTArtificial SequenceSynthetic Construct 30Ile Ser Ala
Met Val Arg Ser Asp Lys Thr His Thr Cys Pro Pro Cys1 5 10 15Pro Ala Pro Glu Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro 20 25 30Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys 35 40 45Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 50 55 60Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu65 70 75 80Glu Gln Tyr Ala Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu 85 90 95His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn 100 105 110Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 115 120 125Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 130 135 140Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr145 150 155 160Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn 165 170 175Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 180
185 190Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 195 200 205Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 210 215 220Gln Lys Ser Leu Ser Leu Ser Pro
Gly Gly Gly Gly Gly Ser Glu Gln225 230 235 240Phe Leu Ile Ser Glu Glu Asp Leu
24531259PRTArtificial SequenceSynthetic Construct 31Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe
Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 20 25
30Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val65 70 75 80Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 85 90 95Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala 115 120 125Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 130 135 140Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln145 150
155 160Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu 195 200 205Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 210 215 220Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser225 230 235
240Leu Ser Pro Gly Gly Gly Gly Gly Ser Glu Gln Phe Leu Ile Ser Glu 245 250 255Glu Asp
Leu32245PRTArtificial SequenceSynthetic Construct 32Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe
Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 20 25
30Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val65 70 75 80Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 85 90 95Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala 115 120 125Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 130 135 140Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln145 150
155 160Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu 195 200 205Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser 210 215 220Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser225 230 235
240Leu Ser Pro Gly Lys 24533231PRTArtificial SequenceSynthetic Construct 33Met Val Arg Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro1 5 10 15Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys 20 25 30Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val 35 40
45Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 50 55 60Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr65 70 75 80Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp 85 90 95Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 100 105
110Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 115 120 125Glu Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys 130 135 140Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp145 150 155 160Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
165 170 175Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 180 185 190Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 195 200 205Cys Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser 210 215 220Leu Ser Leu Ser Pro Gly Lys225
23034250PRTArtificial SequenceSynthetic Construct 34Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe
Leu Phe Ser Ser Ala1 5 10 15Tyr Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 20 25
30Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 35 40 45Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 50 55 60Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp65 70 75 80Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 85 90
95Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 100 105 110His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 115 120 125Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly 130 135 140Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu145
150 155 160Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 165 170 175Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 180 185 190Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 195 200 205Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn 210 215 220Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr225 230
235 240Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 25035245PRTArtificial SequenceSynthetic
Construct 35Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 20 25 30Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr 35 40 45Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50
55 60Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val65 70 75 80Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 85 90 95Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu 100 105 110Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 115
120 125Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln145 150 155 160Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr 180 185 190Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 195 200
205Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser225 230 235 240Leu Ser Pro Gly Lys
24536226PRTArtificial SequenceSynthetic Construct 36Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly1 5 10 15Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile 20 25
30Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 35 40 45Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His 50 55 60Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg65 70 75 80Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 85 90 95Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 100 105 110Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 115 120 125Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 130 135 140Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp145 150
155 160Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 165 170 175Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 180 185 190Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser Cys Ser Val Met His 195 200 205Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Pro 210 215 220Gly Lys22537226PRTArtificial SequenceSynthetic Construct 37Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly1 5 10 15Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 20 25 30Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
35 40 45Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 50 55 60Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg65 70 75 80Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys 85 90 95Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
100 105 110Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 115 120 125Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 130 135 140Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp145 150 155 160Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val 165 170 175Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 180 185
190Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His 195 200 205Glu Ala Leu His Ser
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 210 215 220Gly Lys22538259PRTArtificial
SequenceSynthetic Construct 38Met Lys Trp Val Thr Phe Ile Ser Leu Leu Phe Leu Phe Ser Ser Ala1 5
10 15Tyr Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 20 25 30Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val
Val Val Asp Val 50 55 60Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val65 70 75
80Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 85 90 95Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 115 120 125Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135
140Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln145 150 155 160Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr 180 185 190Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu 195 200 205Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 210 215 220Val
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser225 230 235 240Leu Ser Pro Gly Gly Gly
Gly Gly Ser Glu Gln Lys Leu Ile Ser Glu 245 250 255Glu Asp Leu39226PRTArtificial
SequenceSynthetic Constructmisc_feature(32)..(32)Xaa is Met or Tyrmisc_feature(34)..(34)Xaa is Ser
or Thrmisc_feature(36)..(36)Xaa is Thr or Glumisc_feature(136)..(136)Xaa is Asp or
Glumisc_feature(138)..(138)Xaa is Leu or Metmisc_feature(208)..(208)Xaa is Met or
Leumisc_feature(214)..(214)Xaa is Asn or Sermisc_feature(265)..(265)Xaa is Asn or Ser 39Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Xaa 20 25 30Ile Xaa Arg Xaa Pro Glu Val Thr Cys Val Val Val Asp Val
Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr
Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Xaa 195 200 205His Glu
Ala Leu His Xaa His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly22540226PRTArtificial
SequenceSynthetic Constructmisc_feature(136)..(136)Xaa is Asp or Glumisc_feature(138)..(138)Xaa
is Leu or Met 40Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys Asn Gln Val Ser 130 135 140Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro
Gly22541226PRTArtificial SequenceSynthetic Constructmisc_feature(136)..(136)Xaa is Asp or
Glumisc_feature(138)..(138)Xaa is Leu or Met 41Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25
30Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys
Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150
155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 210 215 220Pro Gly22542226PRTArtificial SequenceSynthetic Construct 42Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Tyr 20 25 30Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly22543226PRTArtificial
SequenceSynthetic Construct 43Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5
10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr 20 25 30Ile Thr Arg Glu Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75
80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130
135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Met 195 200 205His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220Pro Gly22544226PRTArtificial SequenceSynthetic Constructmisc_feature(136)..(136)Xaa is Asp or
Glumisc_feature(138)..(138)Xaa is Leu or Met 44Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25
30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys
Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150
155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Leu 195 200 205His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser 210 215 220Pro Gly22545226PRTArtificial SequenceSynthetic Construct 45Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5 10 15Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75 80Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185
190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 195 200 205His Glu Ala Leu
His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220Pro Gly22546226PRTArtificial
SequenceSynthetic Construct 46Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly1 5
10 15Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
Gly Val Glu Val 50 55 60His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr65 70 75
80Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val 115 120 125Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser 130
135 140Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu145 150 155 160Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys
Ser Val Leu 195 200 205His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215
220Pro Gly22547265PRTArtificial SequenceSynthetic Constructmisc_feature(71)..(71)Xaa is Met or
Tyrmisc_feature(73)..(73)Xaa is Ser or Thrmisc_feature(75)..(75)Xaa is Thr or Glumisc_feature(175)..
(175)Xaa is Asp or Glumisc_feature(177)..(177)Xaa is Leu or Metmisc_feature(247)..(247)Xaa is Met
or Leumisc_feature(253)..(253)Xaa is Asn or Ser 47Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala
Thr Ala Thr Gly1 5 10 15Val His Ser Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 20 25
30Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 35 40 45Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 50 55 60Lys Pro Lys Asp Thr Leu Xaa Ile Xaa Arg Xaa Pro
Glu Val Thr Cys65 70 75 80Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 85 90
95Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 100 105 110Glu Gln Tyr Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 115 120 125His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 130 135 140Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly145
150 155 160Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu 165 170 175Xaa Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 180 185 190Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn 195 200 205Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe 210 215 220Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn225 230
235 240Val Phe Ser Cys Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr 245 250 255Gln Lys Ser
Leu Ser Leu Ser Pro Gly 260 26548266PRTArtificial SequenceSynthetic Construct 48Met Gly Trp Ser
Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 20 25 30Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 35 40
45Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 50 55 60Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys65 70 75 80Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp 85 90 95Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 100 105
110Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 115 120 125His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 130 135 140Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly145 150 155 160Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
165 170 175Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 180 185 190Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 195 200 205Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 210 215 220Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn225 230 235 240Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 245
250 255Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 260 26549266PRTArtificial SequenceSynthetic
Construct 49Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 20 25 30Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys 35 40 45Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 50
55 60Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys65 70 75 80Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 85 90 95Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 100 105 110Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 115 120
125His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 130 135 140Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly145 150 155 160Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 165 170 175Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 180 185 190Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 195 200 205Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 210 215 220Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn225 230 235 240Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 245 250 255Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 260 26550266PRTArtificial
SequenceSynthetic Construct 50Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5
10 15Val His Ser Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 20 25 30Lys Val Glu Pro Lys
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 35 40 45Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro 50 55 60Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys65 70
75 80Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 85 90 95Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 100 105 110Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 115 120 125His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
130 135 140Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly145 150 155 160Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 165 170 175Leu Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr 180 185 190Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 195 200 205Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 210 215
220Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn225 230 235 240Val Phe Ser
Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr 245 250 255Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 260 26551266PRTArtificial SequenceSynthetic Construct 51Met Gly Trp Ser Cys Ile Ile Leu Phe
Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
20 25 30Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 35 40 45Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 50 55 60Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys65 70 75 80Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
85 90 95Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 100 105 110Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 115 120 125His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn 130 135 140Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly145 150 155 160Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 165 170 175Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 180 185 190Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 195 200 205Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe 210 215 220Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn225
230 235 240Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr 245 250 255Gln Lys Ser
Leu Ser Leu Ser Pro Gly Lys 260 26552266PRTArtificial SequenceSynthetic Construct 52Met Gly Trp
Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys 20 25 30Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys 35
40 45Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 50 55 60Lys Pro Lys Asp Thr
Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys65 70 75 80Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp 85 90 95Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 100 105
110Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 115 120 125His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 130 135 140Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly145 150 155 160Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
165 170 175Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 180 185 190Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 195 200 205Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe 210 215 220Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn225 230 235 240Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 245
250 255Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 260 26553266PRTArtificial SequenceSynthetic
Construct 53Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 20 25 30Lys Val Glu Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys 35 40 45Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 50
55 60Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys65 70 75 80Val Val Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 85 90 95Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu 100 105 110Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 115 120
125His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 130 135 140Lys Ala Leu Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly145 150 155 160Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Glu Glu 165 170 175Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 180 185 190Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 195 200 205Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 210 215 220Leu Tyr Ser Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn225 230 235 240Val Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr 245 250 255Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 260 26554267PRTArtificial
SequenceSynthetic Construct 54Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5
10 15Val His Ser Ile Ser Ala Met Val Arg Ser Asp Lys Thr His Thr Cys 20 25 30Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 35 40 45Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu 50 55 60Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys65 70 75
80Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 85 90 95Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu 100 105 110Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys 115 120 125Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 130 135
140Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser145 150 155 160Arg Glu Glu Met
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 165 170 175Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln 180 185 190Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly 195 200 205Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 210 215 220Gln
Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser225 230 235 240His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly 245 250 255Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu
260 26555267PRTArtificial SequenceSynthetic Construct 55Met Gly Trp Ser Cys Ile Ile Leu Phe Leu
Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ile Ser Ala Met Val Arg Ser Asp Lys Thr His Thr Cys 20 25
30Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 35 40 45Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 50 55 60Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys65 70 75 80Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 85 90
95Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 100 105 110Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 115 120 125Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys 130 135 140Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser145
150 155 160Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 165 170 175Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 180 185 190Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly 195 200 205Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln 210 215 220Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser225 230
235 240His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly 245 250 255Ser Glu Gln Lys
Leu Ile Ser Glu Glu Asp Leu 260 26556267PRTArtificial SequenceSynthetic Construct 56Met Gly Trp
Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ile Ser Ala Met Val Arg Ser
Asp Lys Thr His Thr Cys 20 25 30Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
35 40 45Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu 50 55 60Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys65 70 75 80Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr Lys 85 90 95Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 100 105
110Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 115 120 125Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 130 135 140Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser145 150 155 160Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys 165 170 175Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 180 185 190Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 195 200 205Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln 210 215 220Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn225 230 235 240His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly 245
250 255Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 260 26557267PRTArtificial SequenceSynthetic
Construct 57Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5 10 15Val His Ser Ile
Ser Ala Met Val Arg Ser Asp Lys Thr His Thr Cys 20 25 30Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu 35 40 45Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu 50
55 60Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys65 70 75 80Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 85 90 95Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val
Val Ser Val Leu 100 105 110Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 115
120 125Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 130 135 140Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser145 150 155 160Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu Val Lys 165 170 175Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln 180 185 190Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 195 200 205Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 210 215 220Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn225 230 235 240His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
Gly Gly Gly Gly Gly 245 250 255Ser Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 260
26558275PRTArtificial SequenceSynthetic Construct 58Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val
Ala Thr Ala Thr Gly1 5 10 15Val His Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 20 25
30Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 35 40 45Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr 50 55 60Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn65 70 75 80Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 85 90
95Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 100 105 110Leu His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser 115 120 125Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys Ala Lys 130 135 140Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp145
150 155 160Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 165 170 175Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu 180 185 190Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe 195 200 205Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly 210 215 220Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr225 230
235 240Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gln 245 250 255Arg Asn Pro
Arg Leu Arg Leu Ile Arg Arg His Pro Thr Leu Arg Ile 260 265 270Pro Pro Ile 27559275PRTArtificial
SequenceSynthetic Construct 59Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly1 5
10 15Val His Ser Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro 20 25 30Cys Pro Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro 35 40 45Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr 50 55 60Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn65 70
75 80Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg 85 90 95Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val 100 105 110Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser 115 120 125Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys 130
135 140Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp145 150 155 160Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe 165 170 175Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu 180 185 190Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe 195 200 205Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 210 215
220Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr225 230 235 240Thr Gln Lys Ser
Leu Ser Leu Ser Pro Gly Gly Gly Gly Gly Ser Gln 245 250 255Arg Asn Pro Arg Leu Arg Leu Ile Arg
Arg His Pro Thr Leu Arg Ile 260 265 270Pro Pro Ile 27560247PRTArtificial SequenceSynthetic
Constructmisc_feature(20)..(20)Glx is Cys or Sermisc_feature(52)..(52)Xaa is Met or
Tyrmisc_feature(54)..(54)Xaa is Ser or Thrmisc_feature(56)..(56)Xaa is Thr or Glumisc_feature(156)..
(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is Leu or Metmisc_feature(228)..(228)Xaa is Met
or Leumisc_feature(234)..(234)Xaa is Asn or Ser 60Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu1 5 10 15Pro Lys Ser Glx Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25
30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Xaa Ile
Xaa Arg Xaa Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90
95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr
Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser 210 215 220Cys Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys
Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 24561247PRTArtificial SequenceSynthetic
Constructmisc_feature(52)..(52)Xaa is Met or Tyrmisc_feature(54)..(54)Xaa is Ser or
Thrmisc_feature(56)..(56)Xaa is Ser or Thrmisc_feature(156)..(156)Xaa is Asp or
Glumisc_feature(158)..(158)Xaa is Leu or Metmisc_feature(228)..(228)Xaa is Met or
Leumisc_feature(234)..(234)Xaa is Asn or Ser 61Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Xaa Ile Xaa Arg
Xaa Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150 155
160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser 210 215 220Cys Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Lys 24562247PRTArtificial SequenceSynthetic
Constructmisc_feature(156)..(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is Leu or Met 62Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Xaa Glu Xaa Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185
190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 24563247PRTArtificial
SequenceSynthetic Construct 63Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5
10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70
75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130
135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys145 150 155 160Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215
220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser
Pro Gly Lys 24564247PRTArtificial SequenceSynthetic Construct 64Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser225
230 235 240Leu Ser Leu Ser Pro Gly Lys 24565247PRTArtificial SequenceSynthetic Construct 65Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185
190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Leu His Glu Ala Leu His
Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 24566247PRTArtificial
SequenceSynthetic Construct 66Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5
10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70
75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130
135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys145 150 155 160Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215
220Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser
Pro Gly Lys 24567247PRTArtificial SequenceSynthetic Construct 67Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr
Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser225
230 235 240Leu Ser Leu Ser Pro Gly Lys 24568247PRTArtificial SequenceSynthetic Construct 68Asn
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185
190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 24569246PRTArtificial
SequenceSynthetic Constructmisc_feature(19)..(19)Glx is Cys or Sermisc_feature(52)..(52)Xaa is Met
or Tyrmisc_feature(54)..(54)Xaa is Ser or Thrmisc_feature(56)..(56)Xaa is Thr or
Glumisc_feature(156)..(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is Leu or
Metmisc_feature(228)..(228)Xaa is Met or Leumisc_feature(234)..(234)Xaa is Asn or Ser 69Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Glx Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Xaa Ile Xaa Arg Xaa Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180
185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Xaa His Glu Ala Leu
His Xaa His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly 24570246PRTArtificial
SequenceSynthetic Constructmisc_feature(52)..(52)Xaa is Met or Tyrmisc_feature(54)..(54)Xaa is Ser
or Thrmisc_feature(56)..(56)Xaa is Thr or Glumisc_feature(156)..(156)Xaa is Asp or
Glumisc_feature(158)..(158)Xaa is Leu or Metmisc_feature(228)..(228)Xaa is Met or
Leumisc_feature(234)..(234)Xaa is Asn or Ser 70Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Xaa Ile Xaa Arg
Xaa Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150 155
160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser 210 215 220Cys Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly 24571246PRTArtificial SequenceSynthetic Constructmisc_feature(156)..
(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is is Leu or Met 71Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu
Xaa Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165
170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly 24572246PRTArtificial SequenceSynthetic Construct
72Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
Gln Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120
125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly
24573246PRTArtificial SequenceSynthetic Construct 73Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25
30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys145 150
155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly 24574246PRTArtificial SequenceSynthetic Construct 74Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105
110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr
Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly 24575246PRTArtificial SequenceSynthetic
Construct 75Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Leu His Gln Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200
205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Leu
His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly
24576246PRTArtificial SequenceSynthetic Construct 76Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25
30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Tyr Ile Thr
Arg Glu Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys145 150
155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly 24577246PRTArtificial SequenceSynthetic Construct 77Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
35 40 45Asp Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 100 105
110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly 24578246PRTArtificial SequenceSynthetic
Constructmisc_feature(19)..(19)Glx is Cys or Sermisc_feature(51)..(51)Xaa is Met or
Tyrmisc_feature(53)..(53)Xaa is Ser or Thrmisc_feature(55)..(55)Xaa is Thr or Glumisc_feature(155)..
(155)Xaa is Asp or Glumisc_feature(157)..(157)Xaa is Leu or Metmisc_feature(227)..(227)Xaa is Met
or Leumisc_feature(233)..(233)Xaa is Asn or Ser 78Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro1 5 10 15Lys Ser Glx Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25
30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Xaa Ile Xaa
Arg Xaa Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90
95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys
Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys 210 215 220Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys Ser
Leu225 230 235 240Ser Leu Ser Pro Gly Lys 24579246PRTArtificial SequenceSynthetic
Constructmisc_feature(51)..(51)Xaa is Met or Tyrmisc_feature(53)..(53)Xaa is Ser or
Thrmisc_feature(55)..(55)Xaa is Thr or Glumisc_feature(155)..(155)Xaa is Asp or
Glumisc_feature(157)..(157)Xaa is Leu or Metmisc_feature(227)..(227)Xaa is Met or
Leumisc_feature(233)..(233)Xaa is Asn or Ser 79Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Xaa Ile Xaa Arg Xaa
Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys Asn145 150 155
160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys 210 215 220Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys Ser Leu225 230 235
240Ser Leu Ser Pro Gly Lys 24580246PRTArtificial SequenceSynthetic Constructmisc_feature(155)..
(155)Xaa is Asp or Glumisc_feature(157)..(157)Xaa is Leu or Met 80Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr
Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170
175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu225 230 235 240Ser Leu Ser Pro Gly Lys 24581246PRTArtificial SequenceSynthetic Construct
81Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120
125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly Lys
24582246PRTArtificial SequenceSynthetic Construct 82Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25
30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn145 150
155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu225 230 235
240Ser Leu Ser Pro Gly Lys 24583246PRTArtificial SequenceSynthetic Construct 83Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105
110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln
Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly Lys 24584246PRTArtificial SequenceSynthetic
Construct 84Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200
205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Leu His
Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly Lys
24585246PRTArtificial SequenceSynthetic Construct 85Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25
30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Tyr Ile Thr Arg
Glu Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn145 150
155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu225 230 235
240Ser Leu Ser Pro Gly Lys 24586246PRTArtificial SequenceSynthetic Construct 86Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
35 40 45Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105
110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu
Glu Met Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly Lys 24587245PRTArtificial SequenceSynthetic
Constructmisc_feature(19)..(19)Glx is Cys or Sermisc_feature(51)..(51)Xaa is Met or
Tyrmisc_feature(53)..(53)Xaa is Ser or Thrmisc_feature(55)..(55)Xaa is Thr or Glumisc_feature(155)..
(155)Xaa is Asp or Glumisc_feature(157)..(157)Xaa is Leu or Metmisc_feature(227)..(227)Xaa is Met
or Leumisc_feature(233)..(233)Xaa is Asn or Ser 87Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro1 5 10 15Lys Ser Glx Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25
30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Xaa Ile Xaa
Arg Xaa Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90
95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys
Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys 210 215 220Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys Ser
Leu225 230 235 240Ser Leu Ser Pro Gly 24588245PRTArtificial SequenceSynthetic
Constructmisc_feature(51)..(51)Xaa is Met or Tyrmisc_feature(53)..(53)Xaa is Ser or
Thrmisc_feature(55)..(55)Xaa is Thr or Glumisc_feature(155)..(155)Xaa is Asp or
Glumisc_feature(157)..(157)Xaa is Leu or Metmisc_feature(227)..(227)Xaa is Met or
Leumisc_feature(233)..(233)Xaa is Asn or Ser 88Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Xaa Ile Xaa Arg Xaa
Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys Asn145 150 155
160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys 210 215 220Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys Ser Leu225 230 235
240Ser Leu Ser Pro Gly 24589245PRTArtificial SequenceSynthetic Constructmisc_feature(155)..
(155)Xaa is Asp or Glumisc_feature(157)..(157)Xaa is Leu or Met 89Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr
Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170
175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu225 230 235 240Ser Leu Ser Pro Gly 24590245PRTArtificial SequenceSynthetic Construct 90Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180
185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly 24591245PRTArtificial
SequenceSynthetic Construct 91Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5
10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70
75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130
135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn145 150 155 160Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215
220Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro
Gly 24592245PRTArtificial SequenceSynthetic Construct 92Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20
25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90
95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys 210 215 220Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225
230 235 240Ser Leu Ser Pro Gly 24593245PRTArtificial SequenceSynthetic Construct 93Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100
105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu Met Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Leu His Glu Ala Leu His Ser His Tyr Thr
Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly 24594245PRTArtificial SequenceSynthetic
Construct 94Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Tyr Ile Thr Arg Glu Pro Glu Val Thr Cys Val Val Val Asp
50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200
205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly
24595245PRTArtificial SequenceSynthetic Construct 95Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25
30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Tyr Ile Thr Arg
Glu Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn145 150
155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu225 230 235
240Ser Leu Ser Pro Gly 24596247PRTArtificial SequenceSynthetic Constructmisc_feature(1)..(1)Xaa
is Asn or absentmisc_feature(20)..(20)Glx is Cys or Sermisc_feature(52)..(52)Xaa is Met or
Tyrmisc_feature(54)..(54)Xaa is Ser or Thrmisc_feature(56)..(56)Xaa is Thr or Glumisc_feature(97)..
(97)Xaa is Asn or Alamisc_feature(109)..(109)Xaa is Leu or Aspmisc_feature(111)..(111)Xaa is Gln or
Hismisc_feature(156)..(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is Leu or
Metmisc_feature(228)..(228)Xaa is Met or Leumisc_feature(234)..(234)Xaa is Asn or
Sermisc_feature(247)..(247)Xaa is Lys or absent 96Xaa Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu1 5 10 15Pro Lys Ser Glx Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25
30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Xaa Ile
Xaa Arg Xaa Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90
95Xaa Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Xaa His Xaa Asp 100 105 110Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr
Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser 210 215 220Cys Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys
Ser225 230 235 240Leu Ser Leu Ser Pro Gly Xaa 24597247PRTArtificial SequenceSynthetic
Constructmisc_feature(1)..(1)Xaa is Asn or absentmisc_feature(97)..(97)Xaa is Asn or
Alamisc_feature(109)..(109)Xaa i sLeu or Aspmisc_feature(111)..(111)Xaa is Gln or
Hismisc_feature(156)..(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is Leu or
Metmisc_feature(234)..(234)Xaa is Asn or Sermisc_feature(247)..(247)Xaa is Lys or absent 97Xaa Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 85 90 95Xaa Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Xaa His Xaa
Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180
185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu
His Xaa His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Xaa 24598247PRTArtificial
SequenceSynthetic Constructmisc_feature(1)..(1)Xaa is Asn or absentmisc_feature(97)..(97)Xaa is
Asn or Alamisc_feature(156)..(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is Leu or
Metmisc_feature(247)..(247)Xaa is Lys or absent 98Xaa Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25
30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Xaa
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150
155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Xaa 24599247PRTArtificial SequenceSynthetic
Constructmisc_feature(97)..(97)Xaa is Asn or Alamisc_feature(156)..(156)Xaa is Asp or
Glumisc_feature(158)..(158)Xaa is Leu or Met 99Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Xaa Ser
Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150 155
160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu
Ser Leu Ser Pro Gly Lys 245100247PRTArtificial SequenceSynthetic Constructmisc_feature(156)..
(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is Leu or Met 100Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu
Xaa Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165
170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys
Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 245101247PRTArtificial SequenceSynthetic
Construct 101Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200
205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met
His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys
245102247PRTArtificial SequenceSynthetic Construct 102Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20
25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90
95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys145
150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Lys 245103246PRTArtificial SequenceSynthetic Construct 103Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp Trp
100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185
190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Ser
His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly Lys 245104246PRTArtificial
SequenceSynthetic Construct 104Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5
10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70
75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135
140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn145 150 155 160Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser
Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly Lys
245105247PRTArtificial SequenceSynthetic Construct 105Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20
25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90
95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys145
150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Lys 245106247PRTArtificial SequenceSynthetic Construct 106Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp
100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185
190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His
Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 245107245PRTArtificial
SequenceSynthetic Construct 107Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5
10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70
75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135
140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn145 150 155 160Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser
Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly
245108245PRTArtificial SequenceSynthetic Construct 108Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20
25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90
95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn145
150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230
235 240Ser Leu Ser Pro Gly 245109247PRTArtificial SequenceSynthetic Constructmisc_feature(156)..
(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is Leu or Met 109Asn Val Asn His Lys Pro Ser
Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
Gln Tyr 85 90 95Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu
Xaa Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165
170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys
Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 245110247PRTArtificial SequenceSynthetic
Construct 110Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr
Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln
Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200
205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met
His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys
245111247PRTArtificial SequenceSynthetic Construct 111Asn Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25
30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Ala
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys145 150
155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Lys 245112246PRTArtificial SequenceSynthetic Construct 112Val Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr
Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Ala 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp Trp
100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185
190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Ser
His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly Lys 245113246PRTArtificial
SequenceSynthetic Construct 113Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5
10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70
75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 85 90 95Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135
140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn145 150 155 160Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser
Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly Lys
245114247PRTArtificial SequenceSynthetic Construct 114Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20
25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90
95Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys145
150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Lys 245115247PRTArtificial SequenceSynthetic Construct 115Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Ser Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 85 90 95Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp
100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185
190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His
Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 245116245PRTArtificial
SequenceSynthetic Construct 116Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5
10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70
75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 85 90 95Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135
140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn145 150 155 160Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser
Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly
245117245PRTArtificial SequenceSynthetic Construct 117Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20
25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 85 90
95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn145
150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230
235 240Ser Leu Ser Pro Gly 245118247PRTArtificial SequenceSynthetic Constructmisc_feature(1)..
(1)Xaa is Asn or absentmisc_feature(97)..(97)Xaa is Asn or Alamisc_feature(109)..(109)Xaa is Leu or
Aspmisc_feature(111)..(111)Xaa is Gln or Hismisc_feature(156)..(156)Xaa is Asp or
Glumisc_feature(158)..(158)Xaa is Leu or Metmisc_feature(234)..(234)Xaa is Asn or
Sermisc_feature(247)..(247)Xaa is Lys or absent 118Xaa Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu1 5 10 15Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25
30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Xaa
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Xaa His Xaa Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150
155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Xaa His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Xaa 245119247PRTArtificial SequenceSynthetic
Constructmisc_feature(1)..(1)Xaa is Asn or absentmisc_feature(97)..(97)Xaa is Asn or
Alamisc_feature(156)..(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is Leu or
Metmisc_feature(247)..(247)Xaa is Lys or absent 119Xaa Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu1 5 10 15Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25
30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Xaa
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150
155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Xaa 245120247PRTArtificial SequenceSynthetic
Constructmisc_feature(97)..(97)Xaa is Asn or Alamisc_feature(156)..(156)Xaa is Asp or
Glumisc_feature(158)..(158)Xaa is Leu or Met 120Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu1 5 10 15Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25
30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Xaa
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150
155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Lys 245121247PRTArtificial SequenceSynthetic
Constructmisc_feature(156)..(156)Xaa is Asp or Glumisc_feature(158)..(158)Xaa is Leu or Met
121Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Cys Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55
60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115
120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200
205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met
His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys
245122247PRTArtificial SequenceSynthetic Construct 122Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20
25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90
95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys145
150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Lys 245123247PRTArtificial SequenceSynthetic Construct 123Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp
100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185
190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His
Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 245124246PRTArtificial
SequenceSynthetic Construct 124Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5
10 15Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70
75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135
140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn145 150 155 160Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser
Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly Lys
245125246PRTArtificial SequenceSynthetic Construct 125Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90
95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn145
150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230
235 240Ser Leu Ser Pro Gly Lys 245126247PRTArtificial SequenceSynthetic Construct 126Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp
100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185
190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His
Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 245127247PRTArtificial
SequenceSynthetic Construct 127Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5
10 15Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70
75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Asn Ser Thr Tyr Arg
Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130
135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys145 150 155 160Asn Gln Val
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215
220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser
Pro Gly Lys 245128245PRTArtificial SequenceSynthetic Construct 128Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170
175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser
Leu225 230 235 240Ser Leu Ser Pro Gly 245129245PRTArtificial SequenceSynthetic Construct
129Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55
60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp
His His Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115
120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200
205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His
Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly
245130247PRTArtificial SequenceSynthetic Constructmisc_feature(156)..(156)Xaa is Asp or
Glumisc_feature(158)..(158)Xaa is Leu or Met 130Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys Lys Val Glu1 5 10 15Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25
30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Ala
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Xaa Glu Xaa Thr Lys145 150
155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235
240Leu Ser Leu Ser Pro Gly Lys 245131247PRTArtificial SequenceSynthetic Construct 131Asn Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 85 90 95Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp
100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp Glu Leu Thr Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185
190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His
Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly Lys 245132247PRTArtificial
SequenceSynthetic Construct 132Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5
10 15Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70
75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Ala Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135
140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys145 150 155 160Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys
Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly
Lys 245133246PRTArtificial SequenceSynthetic Construct 133Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala
85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225
230 235 240Ser Leu Ser Pro Gly Lys 245134246PRTArtificial SequenceSynthetic Construct 134Val
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Cys Asp Lys Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Ala 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp
Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Glu Glu Met Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180
185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His
Ser His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly Lys 245135247PRTArtificial
SequenceSynthetic Construct 135Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu1 5
10 15Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 20 25 30Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp65 70
75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 85 90 95Ala Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 130 135
140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys145 150 155 160Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 210 215 220Cys
Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225 230 235 240Leu Ser Leu Ser Pro Gly
Lys 245136247PRTArtificial SequenceSynthetic Construct 136Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu1 5 10 15Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro 20 25 30Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 35 40 45Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 50 55 60Asp Val Ser His Glu Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp65 70 75 80Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
85 90 95Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp 100 105 110Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 115 120 125Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys
Ala Lys Gly Gln Pro Arg 130 135 140Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
Lys145 150 155 160Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 165 170 175Ile
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 180 185 190Thr Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 195 200 205Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn Val Phe Ser 210 215 220Cys Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser225
230 235 240Leu Ser Leu Ser Pro Gly Lys 245137245PRTArtificial SequenceSynthetic Construct
137Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Cys Asp Lys
Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55
60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 85 90 95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp
His His Asp Trp 100 105 110Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115
120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn145 150 155 160Gln Val Ser Leu Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 195 200
205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 210 215 220Ser Val Met His
Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230 235 240Ser Leu Ser Pro Gly
245138245PRTArtificial SequenceSynthetic Construct 138Val Asn His Lys Pro Ser Asn Thr Lys Val
Asp Lys Lys Val Glu Pro1 5 10 15Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
20 25 30Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 35 40 45Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 50 55 60Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly65 70 75 80Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 85 90
95Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Asp His His Asp Trp 100 105 110Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 115 120 125Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 130 135 140Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn145
150 155 160Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 165 170 175Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 180 185 190Thr Pro Pro Val Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys 195 200 205Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 210 215 220Ser Val Met His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu225 230
235 240Ser Leu Ser Pro Gly 245139585PRTHomo sapiens 139Asp Ala His Lys Ser Glu Val Ala His
Arg Phe Lys Asp Leu Gly Glu1 5 10 15Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu
Gln 20 25 30Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu 35 40 45Phe Ala Lys
Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60Ser Leu His Thr Leu Phe Gly Asp Lys
Leu Cys Thr Val Ala Thr Leu65 70 75 80Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu
Pro 85 90 95Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110Pro Arg
Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125Asp Asn Glu Glu Thr Phe Leu
Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe
Ala Lys Arg145 150 155 160Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170
175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185 190Ser Ala Lys Gln
Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195 200 205Arg Ala Phe Lys Ala Trp Ala Val Ala
Arg Leu Ser Gln Arg Phe Pro 210 215 220Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu
Thr Lys225 230 235 240Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp 245 250
255Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser 260 265 270Ser Lys Leu Lys Glu
Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His 275 280 285Cys Ile Ala Glu Val Glu Asn Asp Glu Met
Pro Ala Asp Leu Pro Ser 290 295 300Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr
Ala305 310 315 320Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330
335Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350Tyr Glu Thr Thr Leu
Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys
Pro Leu Val Glu Glu Pro 370 375 380Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly
Glu385 390 395 400Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln
Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425 430Val Gly Ser Lys Cys Cys Lys
His Pro Glu Ala Lys Arg Met Pro Cys 435 440 445Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu
Cys Val Leu His 450 455 460Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser465
470 475 480Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr 485 490 495Tyr Val
Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp 500 505 510Ile Cys Thr Leu Ser Glu Lys
Glu Arg Gln Ile Lys Lys Gln Thr Ala 515 520 525Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys
Glu Gln Leu 530 535 540Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys545 550
555 560Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575Ala Ala Ser
Gln Ala Ala Leu Gly Leu 580 585140591PRTHomo sapiens 140Arg Gly Val Phe Arg Arg Asp Ala His
Lys Ser Glu Val Ala His Arg1 5 10 15Phe Lys Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile
Ala 20 25 30Phe Ala Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu 35 40 45Val Asn Glu
Val Thr Glu Phe Ala Lys Thr Cys Val Ala Asp Glu Ser 50 55 60Ala Glu Asn Cys Asp Lys Ser Leu His
Thr Leu Phe Gly Asp Lys Leu65 70 75 80Cys Thr Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp
Cys 85 90 95Cys Ala Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys 100 105 110Asp Asp
Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val 115 120 125Met Cys Thr Ala Phe His Asp
Asn Glu Glu Thr Phe Leu Lys Lys Tyr 130 135 140Leu Tyr Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala
Pro Glu Leu145 150 155 160Leu Phe Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln 165
170 175Ala Ala Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg 180 185 190Asp Glu Gly
Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser 195 200 205Leu Gln Lys Phe Gly Glu Arg Ala
Phe Lys Ala Trp Ala Val Ala Arg 210 215 220Leu Ser Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser
Lys Leu225 230 235 240Val Thr Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu 245 250
255Leu Glu Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu 260 265 270Asn Gln Asp Ser Ile
Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro 275 280 285Leu Leu Glu Lys Ser His Cys Ile Ala Glu
Val Glu Asn Asp Glu Met 290 295 300Pro Ala Asp Leu Pro Ser Leu Ala Ala Asp Phe Val Glu Ser Lys
Asp305 310 315 320Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe 325 330
335Leu Tyr Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu 340 345 350Leu Arg Leu Ala Lys
Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala 355 360 365Ala Ala Asp Pro His Glu Cys Tyr Ala Lys Val
Phe Asp Glu Phe Lys 370 375 380Pro Leu Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu
Leu385 390 395 400Phe Glu Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg 405 410
415Tyr Thr Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val 420 425 430Ser Arg Asn Leu Gly
Lys Val Gly Ser Lys Cys Cys Lys His Pro Glu 435 440 445Ala Lys Arg Met Pro Cys Ala Glu Asp Tyr
Leu Ser Val Val Leu Asn 450 455 460Gln Leu Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val
Thr465 470 475 480Lys Cys Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala 485 490
495Leu Glu Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr 500 505 510Phe Thr Phe His
Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln 515 520 525Ile Lys Lys Gln Thr Ala Leu Val Glu Leu
Val Lys His Lys Pro Lys 530 535 540Ala Thr Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala
Phe545 550 555 560Val Glu Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu 565 570
575Glu Gly Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu 580 585 590141590PRTMus
musculus 141Arg Gly Val Phe Arg Arg Glu Ala His Lys Ser Glu Ile Ala His Arg1 5 10 15Tyr Asn Asp
Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu Ile Ala 20 25 30Phe Ser Gln Tyr Leu Gln Lys Cys Ser
Tyr Asp Glu His Ala Lys Leu 35 40 45Val Gln Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp Glu
Ser 50 55 60Ala Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu65 70 75 80Cys Ala
Ile Pro Asn Leu Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys 85 90 95Cys Thr Lys Gln Glu Pro Glu Arg
Asn Glu Cys Phe Leu Gln His Lys 100 105 110Asp Asp Asn Pro Ser Leu Pro Pro Phe Glu Arg Pro Glu
Ala Glu Ala 115 120 125Met Cys Thr Ser Phe Lys Glu Asn Pro Thr Thr Phe Met Gly His Tyr 130 135
140Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu145 150 155 160Leu Tyr Tyr Ala
Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys Cys Ala 165 170 175Glu Ala Asp Lys Glu Ser Cys Leu Thr
Pro Lys Leu Asp Gly Val Lys 180 185 190Glu Lys Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys
Ser Ser 195 200 205Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg 210 215
220Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr Lys Leu225 230 235 240Ala Thr Asp
Leu Thr Lys Val Asn Lys Glu Cys Cys His Gly Asp Leu 245 250 255Leu Glu Cys Ala Asp Asp Arg Ala
Glu Leu Ala Lys Tyr Met Cys Glu 260 265 270Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Thr Cys Cys
Asp Lys Pro 275 280 285Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val Glu His Asp Thr Met 290 295
300Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp Phe Val Glu Asp Gln Glu305 310 315 320Val Cys Lys Asn
Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Thr Phe 325 330 335Leu Tyr Glu Tyr Ser Arg Arg His Pro
Asp Tyr Ser Val Ser Leu Leu 340 345 350Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys
Cys Ala 355 360 365Glu Ala Asn Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln 370 375
380Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys Asp Leu385 390 395 400Tyr Glu Lys
Leu Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu Val Arg 405 410 415Tyr Thr Gln Lys Ala Pro Gln Val Ser
Thr Pro Thr Leu Val Glu Ala 420 425 430Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu
Pro Glu 435 440 445Asp Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn 450 455
460Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His Val Thr465 470 475 480Lys Cys Cys Ser
Gly Ser Leu Val Glu Arg Arg Pro Cys Phe Ser Ala 485 490 495Leu Thr Val Asp Glu Thr Tyr Val Pro Lys
Glu Phe Lys Ala Glu Thr 500 505 510Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu Lys Glu Lys
Gln 515 520 525Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys Pro Lys 530 535 540Ala Thr
Ala Glu Gln Leu Lys Thr Val Met Asp Asp Phe Ala Gln Phe545 550 555 560Leu Asp Thr Cys Cys Lys
Ala Ala Asp Lys Asp Thr Cys Phe Ser Thr 565 570 575Glu Gly Pro Asn Leu Val Thr Arg Cys Lys Asp
Ala Leu Ala 580 585 590142330PRTHomo sapiens 142Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25
30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu Leu
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145
150 155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu225 230 235
240Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly
Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330143326PRTHomo sapiens 143Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100
105 110Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140Val Ser His Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly145 150 155 160Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
Asn 165 170 175Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205Ala Pro Ile Glu Lys Thr Ile
Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met
Thr Lys Asn225 230 235 240Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250
255Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270Thr Pro Pro Met
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys 290 295 300Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu305 310 315 320Ser Leu Ser Pro Gly Lys 325144377PRTHomo sapiens 144Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35
40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys Val Asp Lys 85 90 95Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100
105 110Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125Cys Pro Glu
Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140Pro Glu Pro Lys Ser Cys Asp Thr
Pro Pro Pro Cys Pro Arg Cys Pro145 150 155 160Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro Lys 165 170 175Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180
185 190Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220Gln Tyr Asn Ser Thr Phe Arg Val Val Ser
Val Leu Thr Val Leu His225 230 235 240Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys 245 250 255Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro
Glu Asn Asn305 310 315 320Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325
330 335Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350Phe Ser Cys Ser
Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365Lys Ser Leu Ser Leu Ser Pro Gly Lys
370 375145327PRTHomo sapiens 145Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
Arg1 5 10 15Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr65
70 75 80Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110Glu Phe Leu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys 115 120 125Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val 130 135 140Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp145 150 155 160Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 180 185 190Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
Asn Lys Gly Leu 195 200 205Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215
220Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys225 230 235 240Asn Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys 260 265 270Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser 275 280 285Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300Cys
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser305 310 315 320Leu Ser Leu Ser Leu Gly
Lys 3251466PRTArtificial SequenceSynthetic Construct 146His His His His His His1
514720PRTArtificial SequenceSynthetic Construct 147Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala
Leu Ser Leu Ala Leu1 5 10 15Val Thr Asn Ser 2014822PRTArtificial SequenceSynthetic
Constructmisc_feature(1)..(2)Xaa can be any naturally occurring amino acidmisc_feature(4)..(8)Xaa
can be any naturally occurring amino acidmisc_feature(10)..(11)Xaa can be any naturally occurring
amino acidmisc_feature(14)..(14)Xaa can be any naturally occurring amino acidmisc_feature(18)..
(20)Xaa can be any naturally occurring amino acid 148Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Cys Xaa
Xaa Phe Cys Xaa Asp Trp1 5 10 15Pro Xaa Xaa Xaa Ser Cys 201499PRTArtificial SequenceSynthetic
Constructmisc_feature(4)..(6)Xaa can be any naturally occurring amino acid 149Val Cys Tyr Xaa Xaa
Xaa Ile Cys Phe1 51507PRTArtificial SequenceSynthetic Constructmisc_feature(3)..(3)Xaa can be any
naturally occurring amino acidmisc_feature(6)..(6)Xaa can be any naturally occurring amino acid
150Cys Tyr Xaa Pro Gly Xaa Cys1 515111PRTArtificial SequenceSynthetic Constructmisc_feature(2)..
(2)Xaa can be any naturally occurring amino acidmisc_feature(6)..(6)Xaa can be any naturally
occurring amino acid 151Asp Xaa Cys Leu Pro Xaa Trp Gly Cys Leu Trp1 5 1015212PRTArtificial
SequenceSynthetic Constructmisc_feature(4)..(5)Xaa can be any naturally occurring amino
acidmisc_feature(7)..(7)Xaa can be any naturally occurring amino acidmisc_feature(9)..(9)Xaa can be
any naturally occurring amino acid 152Trp Cys Asp Xaa Xaa Leu Xaa Ala Xaa Asp Leu Cys1 5
1015310PRTArtificial SequenceSynthetic Constructmisc_feature(4)..(4)Xaa can be any naturally
occurring amino acid 153Asp Leu Val Xaa Leu Gly Leu Glu Cys Trp1 5 1015411PRTArtificial
SequenceSynthetic Construct 154Asp Leu Cys Leu Arg Asp Trp Gly Cys Leu Trp1 5
1015511PRTArtificial SequenceSynthetic Construct 155Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1
5 1015615PRTArtificial SequenceSynthetic Construct 156Met Glu Asp Ile Cys Leu Pro Arg Trp Gly
Cys Leu Trp Gly Asp1 5 10 1515720PRTArtificial SequenceSynthetic Construct 157Gln Arg Leu Met
Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Glu Asp Asp Glu 2015820PRTArtificial
SequenceSynthetic Construct 158Gln Gly Leu Ile Gly Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5
10 15Gly Arg Ser Val 2015921PRTArtificial SequenceSynthetic Construct 159Gln Gly Leu Ile Gly Asp
Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp1 5 10 15Gly Arg Ser Val Lys 2016015PRTArtificial
SequenceSynthetic Construct 160Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp1 5
10 1516118PRTArtificial SequenceSynthetic Construct 161Arg Leu Met Glu Asp Ile Cys Leu Pro Arg
Trp Gly Cys Leu Trp Glu1 5 10 15Asp Asp16216PRTArtificial SequenceSynthetic Construct 162Met
Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp Asp1 5 10 1516315PRTArtificial
SequenceSynthetic Construct 163Met Glu Asp Ile Cys Leu Pro Arg Trp Gly Cys Leu Trp Glu Asp1 5
10 1516418PRTArtificial SequenceSynthetic Construct 164Arg Leu Met Glu Asp Ile Cys Leu Ala Arg
Trp Gly Cys Leu Trp Glu1 5 10 15Asp Asp16520PRTArtificial SequenceSynthetic Construct 165Glu
Val Arg Ser Phe Cys Thr Arg Trp Pro Ala Glu Lys Ser Cys Lys1 5 10 15Pro Leu Arg Gly
2016620PRTArtificial SequenceSynthetic Construct 166Arg Ala Pro Glu Ser Phe Val Cys Tyr Trp Glu
Thr Ile Cys Phe Glu1 5 10 15Arg Ser Glu Gln 2016711PRTArtificial SequenceSynthetic Construct
167Glu Met Cys Tyr Phe Pro Gly Ile Cys Trp Met1 5 10

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