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Methods in
Molecular Biology 1045

Laurent Ducry Editor

Antibody-Drug
Conjugates
METHODS MOLECULAR BIOLOGY
TM
IN

Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:

http://www.springer.com/series/7651
.
Antibody-Drug Conjugates

Edited by

Laurent Ducry
Lonza Ltd, Visp, Switzerland
Editor
Laurent Ducry
Lonza Ltd
Visp, Switzerland

ISSN 1064-3745 ISSN 1940-6029 (electronic)

ISBN 978-1-62703-540-8 ISBN 978-1-62703-541-5 (eBook)
DOI 10.1007/978-1-62703-541-5
Springer New York Heidelberg Dordrecht London
Library of Congress Control Number: 2013943693

# Springer Science+Business Media, LLC 2013

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Preface

Antibody–drug conjugates (ADCs) represent a promising therapeutic approach for cancer

patients by combining the antigen-targeting specificity of monoclonal antibodies (mAbs)
with the cytotoxic potency of chemotherapeutic drugs. The FDA approval of Adcetris®
(brentuximab vedotin) in 2011 and Kadcyla® (trastuzumab emtansine or T-DM1) in 2013
has validated the idea of making “armed” antibodies, attracting a lot of attention into this
field. ADC technology has been an active area of research in recent years, resulting in a
number of ADCs in development for various tumor types. The number of immunoconju-
gates or ADCs undergoing clinical trial will thus further increase, possibly replacing some
of the existing naked monoclonal antibodies, and becoming the next generation of
anticancer biotherapeutics.
Although the ADC concept is quite simple, successfully designing and developing such
a “smart bomb” is a complex task. Despite a tremendous increase in our understanding in
recent years, a lot of work is necessary in order to identify a suitable target; properly design
the mAb, the linker, and the payload; as well as conjugate them in a reproducible and
scalable fashion.
The success of the current conjugation technologies has been achieved thanks to the
development of new methodologies. The aim of this book is to provide detailed protocols
for many of the key ADC techniques necessary for working in the field. Each method is
described by an author who has regularly used the technique in his or her laboratory. In
addition, several review chapters are included to summarize the current knowledge and
results in the ADC area. These should make this book useful to readers with no previous
ADC experience as well as those already working in the field. It is my hope that this
publication will further drive ADC development and thus help towards improving cancer
treatments of the future.

Visp, Switzerland Laurent Ducry

v
Contents

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

1 Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review. . . . . . . . . . . . . . 1

Ingrid Sassoon and Véronique Blanc
2 Antibody–Drug Conjugate Target Selection: Critical Factors . . . . . . . . . . . . . . . 29
Neil H. Bander
3 Selecting an Optimal Antibody for Antibody–Drug Conjugate Therapy:
Internalization and Intracellular Localization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Jay Harper, Shenlan Mao, Patrick Strout, and Adeela Kamal
4 Antibody–Drug Conjugate Payloads . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Jan Anderl, Heinz Faulstich, Torsten Hechler, and Michael Kulke
5 Linker Technologies for Antibody–Drug Conjugates . . . . . . . . . . . . . . . . . . . . . . 71
Birte Nolting
6 In Vivo Testing of Drug-Linker Stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Pierre-Yves Abecassis and Céline Amara
7 Pharmacokinetics and ADME Characterizations
of Antibody–Drug Conjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Kedan Lin, Jay Tibbitts, and Ben-Quan Shen
8 Safe Handling of Cytotoxic Compounds in a Biopharmaceutical
Environment. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Miriam I. Hensgen and Bernhard Stump
9 Micro- and Mid-Scale Maleimide-Based Conjugation of Cytotoxic
Drugs to Antibody Hinge Region Thiols for Tumor Targeting . . . . . . . . . . . . . 145
James E. Stefano, Michelle Busch, Lihui Hou, Anna Park,
and Diego A. Gianolio
10 Protocols for Lysine Conjugation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Marie-Priscille Brun and Laurence Gauzy-Lazo
11 Engineering THIOMABs for Site-Specific Conjugation
of Thiol-Reactive Linkers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Sunil Bhakta, Helga Raab, and Jagath R. Junutula
12 Enzymatic Antibody Modification by Bacterial Transglutaminase. . . . . . . . . . . . 205
Patrick Dennler, Roger Schibli, and Eliane Fischer
13 Formulation Development of Antibody–Drug Conjugates . . . . . . . . . . . . . . . . . 217
William J. Galush and Aditya A. Wakankar
14 Conjugation Process Development and Scale-Up . . . . . . . . . . . . . . . . . . . . . . . . . 235
Bernhard Stump and Jessica Steinmann
15 Methods for Conjugating Antibodies to Nanocarriers . . . . . . . . . . . . . . . . . . . . . 249
Anil Wagh and Benedict Law
16 Drug-to-Antibody Ratio (DAR) by UV/Vis Spectroscopy . . . . . . . . . . . . . . . . . 267
Yan Chen

vii
viii Contents

17 Drug-to-Antibody Ratio (DAR) and Drug Load Distribution

by Hydrophobic Interaction Chromatography and Reversed Phase
High-Performance Liquid Chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Jun Ouyang
18 Drug-to-Antibody Ratio (DAR) and Drug Load Distribution
by LC-ESI-MS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
Louisette Basa
19 Determination of Charge Heterogeneity and Level of Unconjugated
Antibody by Imaged cIEF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Joyce Lin and Alexandru C. Lazar
20 Risk-Based Scientific Approach for Determination
of Extractables/Leachables from Biomanufacturing
of Antibody–Drug Conjugates (ADCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Weibing Ding

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
Contributors

PIERRE-YVES ABECASSIS  DSAR (Disposition, Safety & Animal Research), Sanofi,

Vitry-sur-Seine, France
CÉLINE AMARA  DSAR (Disposition, Safety & Animal Research), Sanofi,
Vitry-sur-Seine, France
JAN ANDERL  Heidelberg Pharma GmbH, Ladenburg, Germany
NEIL H. BANDER  Weill-Cornell Medical College, Cornell University, New York,
NY, USA
LOUISETTE BASA  Protein Analytical Chemistry, Genentech Inc., South San Francisco,
CA, USA
SUNIL BHAKTA  Genentech Inc., South San Francisco, CA, USA
VÉRONIQUE BLANC  Sanofi Oncology, Vitry-sur-Seine, France
MARIE-PRISCILLE BRUN  Natural Product and Protein Chemistry, Sanofi,
Vitry-sur-Seine, France
MICHELLE BUSCH  Transitional Research, Genzyme, a Sanofi Company, Framingham,
MA, USA
YAN CHEN  Protein Analytical Chemistry, Genentech Inc., South San Francisco,
CA, USA
PATRICK DENNLER  Center for Radiopharmaceutical Sciences, Paul Scherrer Institute,
Villigen, Switzerland
WEIBING DING  SLS Global Technical Support, Pall Corporation, Port Washington,
NY, USA
HEINZ FAULSTICH  Heidelberg Pharma GmbH, Ladenburg, Germany
ELIANE FISCHER  Center for Radiopharmaceutical Sciences, Paul Scherrer Institute,
Villigen, Switzerland
WILLIAM J. GALUSH  Early Stage Pharmaceutical Development, Genentech Inc.,
South San Francisco, CA, USA
LAURENCE GAUZY-LAZO  Natural Product and Protein Chemistry, Sanofi,
Vitry-sur-Seine, France
DIEGO A. GIANOLIO  Drugs and Biomaterials R&D, Genzyme, a Sanofi Company,
Cambridge, MA, USA
JAY HARPER  Oncology Research, MedImmune Inc., Gaithersburg, MD, USA
TORSTEN HECHLER  Heidelberg Pharma GmbH, Ladenburg, Germany
MIRIAM I. HENSGEN  Bioconjugates R&D, Lonza Ltd, Visp, Switzerland
LIHUI HOU  Transitional Research, Genzyme, a Sanofi Company, Framingham,
MA, USA
JAGATH R. JUNUTULA  Genentech Inc., South San Francisco, CA, USA
ADEELA KAMAL  Oncology Research, MedImmune Inc., Gaithersburg, MD, USA
MICHAEL KULKE  Heidelberg Pharma GmbH, Ladenburg, Germany
BENEDICT LAW  Department of Pharmaceutical Sciences, College of Pharmacy,
Nursing and Allied Sciences, North Dakota State University, Fargo, ND, USA
ALEXANDRU C. LAZAR  Analytical & Pharmaceutical Sciences, ImmunoGen,
Inc., Waltham, MA, USA
ix
x Contributors

JOYCE LIN  Analytical & Pharmaceutical Sciences, ImmunoGen, Inc., Waltham,

MA, USA
KEDAN LIN  Early Development Pharmacokinetics and Pharmacodynamics,
Genentech Inc., South San Francisco, CA, USA
SHENLAN MAO  Oncology Research, MedImmune Inc., Gaithersburg, MD, USA
BIRTE NOLTING  Biotherapeutics Research and Development, Pfizer, Pearl River,
NY, USA
JUN OUYANG  Pharma Technical Regulatory, Genentech Inc., South San Francisco,
CA, USA
ANNA PARK  Transitional Research, Genzyme, a Sanofi Company, Framingham,
MA, USA
HELGA RAAB  Genentech Inc., South San Francisco, CA, USA
INGRID SASSOON  Sanofi Oncology, Vitry-sur-Seine, France
ROGER SCHIBLI  Center for Radiopharmaceutical Sciences, Paul Scherrer Institute,
Villigen, Switzerland
BEN-QUAN SHEN  Early Development Pharmacokinetics and Pharmacodynamics,
Genentech Inc., South San Francisco, CA, USA
JAMES E. STEFANO  Transitional Research, Genzyme, a Sanofi Company, Framingham,
MA, USA
JESSICA STEINMANN  Bioconjugates R&D, Lonza Ltd, Visp, Switzerland
PATRICK STROUT  Oncology Research, MedImmune Inc., Gaithersburg, MD, USA
BERNHARD STUMP  Bioconjugates R&D, Lonza Ltd, Visp, Switzerland
JAY TIBBITTS  Drug Metabolism and Pharmacokinetics, UCB Celltech Slough,
Berkshire, UK
ANIL WAGH  Department of Pharmaceutical Sciences, College of Pharmacy, Nursing
and Allied Sciences, North Dakota State University, Fargo, ND, USA
ADITYA A. WAKANKAR  Late Stage Pharmaceutical Development, Genentech Inc.,
South San Francisco, CA, USA
 Chapter 1

Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review

Ingrid Sassoon and Véronique Blanc

Abstract
Biological therapies play an increasing role in cancer treatment, although the number of naked antibodies
showing clinical efficacy as single agent remains limited. One way to enhance therapeutic potential of
antibodies is to conjugate them to small molecule drugs. This combination is expected to bring together
the benefits of highly potent drugs on the one hand and selective binders of specific tumor antigens on the
other hand. However, designing an ADC is more complex than a simple meccano game, requiring
thoughtful combination of antibody, linker, and drugs in the context of a target and a defined cancer
indication. Lessons learned from the first-generation antibody–drug conjugate (ADC) and improvement of
the technology guided the design of improved compounds which are now in clinical trials. Brentuximab
vedotin (Adcetris®), an anti-CD30 antibody conjugated to a potent microtubule inhibitor for the treat-
ment of Hodgkin’s lymphoma and anaplastic large cell lymphomas, is the only marketed ADC today.
A total of 27 ADC are currently undergoing clinical trials in both hematological malignancies and solid
tumor indications. Among them, T-DM1 (trastuzumab emtansine), an ADC comprised of trastuzumab
conjugated to DM1, via a non-cleavable linker, is showing very promising results in phase III for the
treatment of HER2-positive refractory/relapsed metastatic breast cancer. Other compounds, such as
CMC-544, SAR3419, CDX-011, PSMA-ADC, BT-062, and IMGN901 currently in clinical trials, target-
ing varied antigens and bearing different linker and drugs, contribute to the learning curve of ADC, as do
the discontinued ADC. Current challenges include improvement of the therapeutic index, linked to a
careful selection of the targets, a better understanding of ADC mechanism of action, the management and
understanding of ADC off-target toxicities, as well as the selection of appropriate clinical settings (patient
selection, dosing regimen) where these molecules can bring highest clinical benefit.

Key words Antibody–drug conjugate, Cancer, Cytotoxic, Linker, Antibody, Maytansine, Auristatin,
Calicheamicin, T-DM1, SGN-35, CMC-544

1 Introduction

Decades of intensive research in oncology have been devoted to

find drugs able to fight cancer and improve patient’s life. Nowa-
days, cancer biologics (antibodies, peptides, and proteins) play an
increasing role in the arsenal of therapeutic molecules, usually in
combination with radiotherapy or chemotherapy. Despite clear

Laurent Ducry (ed.), Antibody-Drug Conjugates, Methods in Molecular Biology, vol. 1045,
DOI 10.1007/978-1-62703-541-5_1, # Springer Science+Business Media, LLC 2013

1
2 Ingrid Sassoon and Véronique Blanc

advantages of antibodies compared to small molecules in terms

of (a) exquisite selectivity towards antigen-positive cells, leading
to decreased off-target toxicity and (b) long half-life, only 13 thera-
peutic antibodies are marketed today for the treatment of cancer
[1], highlighting the difficulty to identify targets whose modulation
will impact tumor growth as well as the difficulty to identify
antibodies with clinical efficacy as single agent. Arming antibodies
or antibody fragments with toxins, cytotoxic drugs, and radionu-
clides can be viewed as a means of enhancing tumor-cell
killing while sparing normal cells. Several of such armed molecules
are marketed, namely, denileukin diftitox (Ontak®), an engineered
protein combining interleukin-2 (which binds to IL2R) and
Diphteria toxin, for the treatment of persistent or recurrent cuta-
neous T cell lymphoma, ibritumomab tiuxetan (Zevalin®), and
131
I-tositumomab (Bexxar®), two murine anti-CD20 antibodies
conjugated to 90Y and 131I, respectively, for the treatment of
relapsed/refractory follicular lymphoma, as well as the antibody–
drug conjugate (ADC) brentuximab vedotin (Adcetris®), an anti-
CD30 antibody conjugated to a potent microtubule inhibitor
for the treatment of Hodgkin’s lymphoma and anaplastic large
cell lymphomas.
The concept of arming antibodies is not recent, as the use of
ADC in animal models was already described in the literature in the
1970s, and clinical trials with murine IgG-based ADC were con-
ducted in the 1980s, although with limited success. This is only in
2000 that the first ADC, gemtuzumab ozogamicin (Mylotarg®), an
anti-CD33 antibody conjugated to calicheamicin (a very potent
DNA binding drug), was approved in the USA for the treatment
of acute myelocytic leukemia (AML), based on clear evidence of
blast decrease in patient bone marrows [2, 3]. In 2010, the product
was withdrawn from the market by the developer, Pfizer, following
interim results from post-approval study (SWOG S0106), because
of serious concerns about product’s safety and failure to demon-
strate clinical benefit [4].
This review will focus on ADC which are undergoing clinical
trials (cf. Table 1). Lessons learned from first-generation ADC and
improvement of the technology, both described in the first section,
guided the design of improved compounds which are currently at
different stages of clinical development. Adcetris® and the most
advanced ADC in clinical trials will be described in a second section.
The third section covers explored areas of improvement based on a
thorough understanding of key parameters for ADC safety and
efficacy retrieved from preclinical and clinical trials. The growing
number of ADC in the clinic reflects the interest and confidence of
clinicians and pharmaceutical companies that this approach can
bring high benefit to cancer patients.
Table 1
ADC in clinical trials and launched

Drug names Company MAb mode Target Drug Linker Highest phasea Indications
Adcetris® Seattle Genetics/ Chimeric CD30 Auristatin vc Launched Hodgkin’s
Brentuximab Takeda (Millenium) (MMAE) 8.2011 lymphoma
vedotin ALCL
SGN-35

Inotuzumab UCB (Celltech) Humanized CD22 Calicheamicin Hydrazone Phase 3 ALL

ozogamicin Pfizer AcBut 04.2011 NHL (DLBCL)
CMC-544
T-DM1 Roche (Genentech) Humanized Her-2 Maytansine SMCC Phase 3 Her2+ breast
Trastuzumab ImmunoGen (DM1) 3.2009 Gastric
emtansine
Glembatumumab- Celldex (Curagen) Fully GPNMB Auristatin vc Phase 2 Breast
vedotin Amgen (Abgenix) human (osteoactivin) (MMAE) 4.2008 Melanoma
CDX-011
CR-011-vcMMAE
IMGN-901 ImmunoGen Humanized CD56 Maytansine SPP Phase 2 MCC, SCLC
Lorvotuzumab (DM1) 3.2012
mertansine SCLC
SAR3419 ImmunoGen/Sanofi Humanized CD19 Maytansine SPDB Phase 2 NHL, ALL
HuB4-DM4 (DM4) 9.2011
BT-062 Biotest Chimerized CD138 Maytansine SPDB Phase 1/2 MM
(Syndecan1) (DM4) 8.2010 Solid tumors
IMMU-110 Immunomedics Humanized CD74 Doxorubicin Hydrazone Phase 1/2 Multiple myeloma
Milatuzumab- 6.2010
doxorubicin
Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review

AGS-16M8F Astellas (Agensys) Fully ENPP3 Auristatin mc Phase 1 RCC

AGS-6MF human (MMAF) 8.2010
3

(continued)
 4

Table 1
(continued)

Drug names Company MAb mode Target Drug Linker Highest phasea Indications
AGS-22M6E Astellas (Agensys) Fully human Nectin-4 Auristatin vc Phase 1 Solid tumors
ASG-22ME (MMAE) 5.2011
AMG-172 Amgen nd nd nd nd Phase 1 Renal cancer
12.2011
Ingrid Sassoon and Véronique Blanc

AMG-595 Amgen Fully human EGFRvIII Maytansinoid Non-cleavable Phase 1 Glioma

3.2012
ASG-5ME Astellas (Agensys) Fully human SLC44A4 Auristatin vc Phase 1 Pancreas
AGS-5M2E (MMAE) 7.2010 Prostate
BAY 94-9343 Bayer Fully human mesothelin Maytansine SPDB Phase 1 Solid tumors
MorphoSys (DM4) 9.2011
DEDN-6526A Roche (Genentech) Humanized ET8R Auristatin vc Phase 1 Melanoma
Anti-ETBR-vc-E (endothelin B) (MMAE) 3.2012
IMGN 529 ImmunoGen Humanized CD37 Maytansine SMCC Phase 1 NHL
K7153A-SMCC-DM1 DM1 2.2012
IMMU-130 Immunomedics Humanized CEACAM5 SN-38 CL2 Phase 1 Breast, colorectal,
hMN14-SN38 8.2011 lung
MDX-1203 BMS (Medarex) Fully human CD70 MGBA vc Phase 1 B-NHL
7.2009 ccRCC
PSMA-ADC Progenics Fully human PSMA Auristatin vc Phase 1 Prostate
PSMA-ADC-1301 (MMAE) 9.2008
 RG-7450 Roche (Genentech) nd nd Auristatin nd Phase 1 Prostate (CRPC)
DSTP-3086S 3.2011
RG-7458 Roche (Genentech) nd MUC16 (CA125) Auristatin nd Phase 1 Ovary
4.2011
RG-7593 Roche (Genentech) Humanized CD22 Auristatin vc Phase 1 NHL
DCDT-2980S (MMAE) 10.2010
RG-7596 Roche (Genentech) nd CD79b Auristatin nd Phase 1 CLL, NHL
DCDS-4501A 3.2011
RG-7598 Roche (Genentech) nd nd Auristatin nd Phase 1 MM
9.2011
RG-7599 Roche (Genentech) nd MUC16 (CA125) Auristatin nd Phase 1 Ovary
7.2011 Lung (NSCLC)
RG-7600 Roche (Genentech) nd nd Auristatin nd Phase 1 Ovary
12.2011 Pancreas
SAR-566658 ImmunoGen Humanized Muc1 (CA6) Maytansine SPDB Phase 1 Solid tumors
huDS6-DM4 Sanofi (DM4) 9.2010
SGN-75 Seattle Genetics Humanized CD70 Auristatin vc Phase 1 NHL
h1F6-vcMMAF (MMAF) 11.2009 RCC
a
Current highest phase for cancer/first date/first indication
nd not disclosed
Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review
5
6 Ingrid Sassoon and Véronique Blanc

2 ADC Building Blocks

2.1 Definition An ADC can be defined as a prodrug. The antibody connected to

of an ADC the cytotoxic warhead (drug) via a linker serves as targeted delivery
system to the tumor expressing the antigen/target recognized by
the antibody. Ideally, in blood, after systemic administration, this
prodrug is nontoxic. Upon binding of the antibody to the targeted
tumor antigen and internalization of the complex into the cancer
cell, the drug is then released in its active form and in sufficient
quantity to kill the cell.
Designing an ideal ADC is more complex than a simple
meccano game. On top of the careful choice of a target/antigen
expressed in specific tumor indication, it requires finding the best
combination between the antibody, the linker, and the drug, which,
besides its own characteristics and constraints, are linked and
impact each other.

2.2 Target/Antigen The target/antigen is the starting point to build an ADC. It first
for ADC determines which tumor indication will be targeted by the ADC
and potentially impacts the choice of the conjugated drug.
In addition, the target will also drive the criteria which will be
defined for the selection of the targeted patient population within
the tumor indication.
Many targets have been evaluated for an ADC approach across
the years (for a review, see ref. 5), showing that a high variety of
targets, either single or multiple transmembrane domains proteins
or glycosylphosphatidylinositol (GPI)-anchored, can lead to ADC
internalization and subsequent tumor growth delay and regression
in preclinical mouse models.
The basis for the selection of the antigen is a high expression
level in tumor tissues and a restricted normal tissue distribution, in
order to limit on-target toxicity of the future ADC. However,
tumor-specific antigens with no expression in normal tissues are
rare, and most of the time, the antigen is expressed at the surface of
epithelial cells in a subset of normal tissues/organs. The type of
organ expressing the antigen (vital organs vs. reproductive organs,
for example), the cellular subtype and cell-cycle status (dividing
cells vs. differentiated quiescent cells), and the differential of
expression between normal antigen-positive cells and tumor cells
are to be considered for selection of the target.
It is important to notice that expression in normal organs may
not always mean subsequent toxicity in clinical trials. Several ADC
with normal tissue cross-reactivity have been well tolerated in
patients, causing minimal or manageable and reversible toxicities,
namely, cantuzumab mertansine/IMGN242 (targeting CanAg
antigen, a glycotope on Mucin-like protein [6, 7]), BT-062 (target-
ing CD138; see below), or CDX-011 (targeting gpNMB;
 Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review 7

Table 2
Discontinued ADC

Reasons for
Product name Target name Drug/linker discontinuation Year References
BAY79-4620 CAIX MMAE/vc Not disclosed 2011 Press release
IMGN388 Integrinαvβ3 DM4/SPDB Change in business strategy 2011 Press release
MEDI547 EphA2 MMAF/mc Safety issues: bleeding 2012 [122]
and coagulation events
Mylotarg CD33 Calicheamicin/ Failure to demonstrate 2010 [4]
hydrazone clinical benefit
BIIB015 Crypto1 DM4/SPDB Not disclosed 2010
IMGN242 CanAg DM4/SPDB Not disclosed 2009 Press release
AVE9633 CD33 DM4/SPDB Lack of clinical efficacy 2008 [12]
MLN2704 PSMA DM1/SPP Not disclosed 2006 [94, 123],
Press release
CMD-193 LeY Calicheamicin/ Not disclosed 2006 ClinicalTrials.
carbohydrate hydrazone gov
Bivatuzumab CD44v6 DM1/SPP Safety issues: fatal case of 2006 [9, 11]
mertansine toxic epidermal
necrolysis
SGN-15 LeY Doxorubicin/ Change in business strategy 2005 Press release
carbohydrate hydrazone
CMB-401 MUC1 Calicheamicin/ Lack of clinical efficacy 1999 [124, 125]
hydrazone

see below). Inversely it has clearly been demonstrated in the case of

bivatuzumab mertansine (targeting CD44v6), whose trial was pre-
maturely stopped in phase I (cf. Table 2), that the expression of the
CD44v6 target in skin keratinocytes [8] led to severe skin toxicity,
including a fatal case of toxic epidermal necrolysis [9–11].
While expression of the target should remain limited and at low
level in normal tissues, on the contrary, the level of expression
(antigen density) at the surface of cancer cells should be high and
combined to the ability of the antigen/antibody complex to inter-
nalize and be processed in the right subcellular compartments, in
order to release enough quantity of the active drug in the cytosol.
The use of tumor models mimicking the target expression pattern
and level found in patient biopsies is a very critical element to
translate preclinical data into clinical efficacy. AVE9633, an immu-
noconjugate targeting CD33 antigen, did not show clinical efficacy
in phase I [12] in part because of too limited antigen expression on
the malignant cell population, suggesting an insufficient delivery of
8 Ingrid Sassoon and Véronique Blanc

molecules in the cytoplasm to achieve cell death. In contrast,

preclinical models showed good response to AVE9633 [13] but
displayed a much higher CD33 antigen level than the one measured
in patient biopsies (unpublished internal data, sanofi, 2009).

2.3 Drugs Many conventional therapeutic agents have been conjugated to

and Linkers antibodies, but it soon became clear that they were not potent
enough, when conjugated, to achieve antitumor activity in the
clinic [14–16]. Efforts have then been turned towards natural
small cytotoxic molecules with higher potency but which have
been found too toxic as free drug in clinical trials. Currently, only
few highly potent natural cytotoxics, derivatives, or synthetic ana-
logues have been conjugated to antibodies and progressed to the
clinic. They fall into the following two classes: microtubule desta-
bilizing agents (auristatin derivatives, MMAE and MMAF and
maytansinoid derivatives, DM1 and DM4) and DNA minor groove
binders (calicheamicin and duocarmycin derivatives). Both classes
are extremely potent towards proliferating tumor cell lines [16].
IC50 of proliferation/viability of tumor cell lines are in the range of
10 10–10 12 M for DM1/DM4 maytansinoid derivatives [17, 18],
10 7–10 10 M for MMAF/MMAE auristatin derivatives [19],
around 10 10 M for N-acetyl-γ calicheamicin DMH [20], and
10 11–10 12 M for DC1 and CC-1065 duocarmycin precursors
[14, 21].
Importantly, the engineered linker connecting the cytotoxic
molecule to the antibody has been deeply studied as it is considered
to be an important parameter for preclinical, clinical efficacy and
safety of ADC: linkers must be stable enough in circulation since
release of the cytotoxic payload may be associated with undesired
and untargeted toxicities, but they must also be able to efficiently
release cytotoxics in their active form in the cytosol of the target cell
following internalization and trafficking in specific subcellular com-
partments [16, 22, 23]. Indeed, upon binding of the ADC to its
target, and subsequent internalization of the antigen/ADC com-
plex by receptor-mediated endocytosis, the ADC is trafficked in
acidifying endosomal and then in lysosomal vesicles, a compart-
ment rich in proteolytic enzymes. Due to the chemical environment
or to the metabolic properties of these intracellular compartments,
the ADC is activated/metabolized. This metabolization depends
on the type of linker connected to the drug:
– The acid labile hydrazone linkers are relatively stable at neutral
pH (pH 7.3–7.5, pH of the bloodstream) but undergo hydroly-
sis once the ADC is internalized into acidic endosomes (pH
5–6.5) and lysosomes (pH 4.5–5). They have been conjugated
to doxorubicin, calicheamicin, and auristatin. Their relative
stability depends on the antibody part attached, but they have
 Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review 9

been associated with high nonspecific release of the drug in

circulation in preclinical studies [24].
– The disulfide-based linkers have been combined with DM1 and
DM4 maytansinoids. The corresponding ADC is activated by
lysosomal degradation of the antibody part, resulting in meta-
bolites consisting of intact maytansinoid drug and linker
attached to lysines [23, 25]. Linkers are subsequently reduced
with more or less efficiency, depending on the level of steric
hindrance at carbon atoms adjacent to the disulfide linkage,
optimized linkers being the best compromise between high
ADC plasma stability and efficient metabolization/release of
the metabolites in tumor cells [26].
– The peptide-based linkers, already used for a number of years
with doxorubicin, mitomycin C, camptothecin, and talysomycin
[16], have been designed for the auristatin and the duocarmycin
derivatives. The type of linker which has been progressed to
clinical stage is composed of a valine–citrulline dipeptide selec-
tively hydrolized by cathepsin B and plasmin enzymes, a self
immolative spacer that spatially separates the drug from the
site of enzymatic cleavage, and the auristatin E microtubule
disruptive agent or duocarmycin prodrug derivative. In the
case of an auristatin E conjugate, the membrane-permeable
monomethyl auristatin E accounts for the only detectable
metabolite found in antigen-positive cells [27].
– Contrary to the above linker types, which are considered as
“cleavable,” thioether bond containing linkers are considered
as “non-cleavable,” and the corresponding ADC have been
clinically tested with DM1 and MMAF cytotoxics. In this case,
the degradation of the mAb component into the lysosomes
releases the drug still attached to the linker via a Lys or Cys
residue of the antibody. These charged entities are not able to
cross membranes with high efficiency, by contrast to metabolites
of maytansine and auristatin ADC conjugated to “cleavable”
linkers. In this case, the diffusion of metabolites induces killing
of surrounding cells, a property named “bystander effect”
[27–29].

2.4 Antibody All ADC currently in oncology clinical trials are canonical (i.e., full
Selection length) IgG molecules, mostly of the IgG1 isotype. They are either
chimeric, humanized, or fully human antibodies (cf. Table 1). The
generation of an immune response to these ADC has remained very
limited, highlighting the benefit of antibody engineering technol-
ogies over the last decades, as well as the fact that small molecule
cytotoxics, contrary to natural toxins, are not immunogenic.
Attention has also been focused on drug conjugation technol-
ogies on the selected antibodies. On top of the fact that drug
10 Ingrid Sassoon and Véronique Blanc

conjugation should not disturb antigen/antibody interaction, the

localization, the number, and the nature of the attachment between
linker and antibody have been shown to influence pharmacokinet-
ics, tumor exposure, and ADC plasma stability [30, 31]. So far, the
two conjugation technologies which progressed to clinical trials are
based on the following two principles: either conjugation through
Lysine side chain amines (with drugs such as DM1, DM4, or
calicheamicin) or conjugation through cysteine sulfhydryl groups
activated by reducing interchain disulfide bonds (with drugs such as
MMAE, MMAF, or duocarmycin) of the antibody. Both processes
give more or less heterogeneous mixtures of ADC with variable
drug load per antibody and variable sites of conjugation to the
protein. This heterogeneous mixture is defined by an average
drug–antibody ratio (DAR) and is challenging from a development
point of view, although robust analytical technologies and processes
are available to ensure constant quality control of the final
product [32].

3 Current Clinical Results of Antibody–Drug Conjugates

A total of 27 ADC are currently in clinical trials, 20 in phase I, 5 in

phase II, 2 in phase III, and 1 launched ADC (cf. Table 1). A total
of 12 ADC have been stopped and are listed in Table 2.

3.1 Brentuximab CD30, a type II transmembrane protein belonging to the TNF

Vedotin (Adcetris®) (tumor necrosis factor) superfamily, is abundantly and selectively
Clinical Overview expressed on the surface of Hodgkin’s lymphoma (HL), Reed–
Sternberg (RS) cells, anaplastic large cell lymphomas (ALCL), and
other lymphoid malignancies as well as on several nonlymphoid
malignancies [33]. RS cells and ALCL cells express high levels of
CD30, but the downstream signalling of CD30 may differ between
both diseases [34, 35]. In non-pathological conditions, CD30
expression is highly regulated and restricted to activated B and T
lymphocytes and NK cells, low expression being also noticed in
monocytes and eosinophils (for review, see refs. 34, 36), making it a
good candidate target for an ADC strategy.
HL is considered as one of the most curable cancers, with a
5-year survival rate of above 85 % although up to 20 % of patients
are refractory and advanced-stage patients often relapse [37].
In frontline systemic ALCL treatment, disease recurs in 40–65 %
of patients [38].
Clinical trials have been reported for unconjugated anti-CD30
antibodies [39]. Acceptable safety profile but modest antitumor
clinical activity precluded further development as naked but
supported exploration and development of a conjugated version:
SGN-35. SGN-35 (Adcetris®, brentuximab vedotin) is an ADC
comprised of a chimeric anti-CD30 antibody (cAC10) conjugated
 Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review 11

through interchain disulfide bonds to monomethyl auristatin

E (MMAE) via a valine–citrulline dipeptide cleavable linker, with
an average DAR of 4 [40].
Based on preclinical data showing good efficacy of SGN-35 at
low doses in lymphoma models [40], a phase I study was conducted
in 2006. Forty-five patients (42 HL, 3 ALCL) were enrolled, in a
dose escalation study ranging from 0.2 to 3.6 mg/kg with intrave-
nous (IV) administration once every 3 weeks (q3w) [41]. The
maximum tolerated dose (MTD) was found to be 1.8 mg/kg,
and drug-related dose-limiting toxicities (DLT) were febrile neu-
tropenia and hyperglycemia. At the MTD, objective clinical
responses were observed, with an objective response rate (ORR)
of 38 %, including 4 complete responses (CR) and 2 partial
responses (PR) out of 12 patients. In terms of pharmacokinetics
(PK), terminal half-life of the ADC and MMAE, at 1.8 mg/kg, was
estimated to be 4–6 and 3–4 days, respectively [41]. In a second
phase I study, enrolling 44 patients, a more frequent regimen was
investigated, at doses ranging from 0.4 to 1.4 mg/kg administered
weekly for 3 out of 4 weeks, for a total of four cycles. The MTD was
1.2 mg/kg and the ORR was 59 %, with 34 % CR. Most common
grade 3 adverse events (AE) were peripheral sensory neuropathy
(14 %), anemia (9 %), neutropenia (7 %), peripheral motor neurop-
athy (7 %), and hyperglycemia, diarrhea, and vomiting (5 % each).
Overall, 32 patients (73 %) experienced one or more events of
peripheral neuropathy. Compared to the q3w schedule, there was
a marked increase in neuropathy which led to the adoption of the
q3w schedule for further clinical studies [42].
In a phase II study, 102 heavily pretreated relapsed or refractory
HL patients were treated at the dose of 1.8 mg/kg in a q3w
schedule [43]. The ORR was 75 % including 34 % CR and 40 %
PR. The more severe AE were grade 3 neutropenia (14 %), periph-
eral sensory neuropathy (5 %), fatigue and hyperglycemia (3 %
each), grade 4 hematological toxicities (neutropenia 4 %; thrombo-
cytopenia 1 %), and pulmonary embolism and abdominal pain (1 %
each). In a second phase II trial, 58 patients with relapsed systemic
ALCL were treated with 1.8 mg/kg of with a q3w schedule [38].
The ORR was 86 % with 53 % achieving CR. Grade 3–4 AE were
similar to the previous studies.
Based on these outstanding data, SGN-35 has been granted
accelerated approval by the FDA in August 2011 for the treatment
of HL that had relapsed after autologous stem cell transplant
(ASCT) and for the management of relapsed ALCL, making it
the first approved drug over 30 years in HL. In July 2012, a positive
opinion was issued in the EU, recommending conditional market-
ing authorization for treatment of adults with relapsed or refractory
CD30-positive HL following ASCT or following at least two prior
therapies when ASCT or multi-agent chemotherapy is not a treat-
ment option as well as for the treatment of adults with relapsed
12 Ingrid Sassoon and Véronique Blanc

or refractory systemic ALCL. SGN-35 is currently evaluated in a

phase III randomized, double-blind, placebo-controlled study
(AETHERA) in HL patients following autologous stem cell trans-
plant [35]. Interim results show that 75 % of patients responded to
the drug, including 34 and 40 % achieving CR and PR, respectively
[44]. Future results of the AETHERA trial expected to be com-
pleted in June 2013 will form the basis for full FDA approval. Other
trials are ongoing, including another phase III trial evaluating
SGN-35 versus methotrexate or bexarotene in patients with
CD30-positive cutaneous T cell lymphomas [44].

3.2 Trastuzumab- ErbB2/neu/HER2 is a member of the ErbB receptor tyrosine

DM1 (T-DM1) Clinical kinase family which is involved in cell growth, survival, and differ-
Overview entiation [45]. Breast cancer accounts for 28 % of all new cases of
cancer in women, and 15–25 % of these new cases contain gene
amplification or overexpression of HER2 [46]. The humanized
anti-HER2 monoclonal antibody trastuzumab (Herceptin®; Gen-
entech), and the dual epidermal growth factor EGFR/HER2 tyro-
sine kinase inhibitor lapatinib (Tykerb®, GSK), in combination
with chemotherapy, prolongs survival of HER2-positive breast can-
cer patients in metastatic and adjuvant settings. However, a signifi-
cant portion of these patients relapse and finally die from
their cancer, highlighting the need for new therapeutic approaches
[47, 48].
T-DM1 (trastuzumab emtansine) is an ADC comprised of tras-
tuzumab conjugated through lysines to DM1, via a non-cleavable
thioether linker (N-succinimidyl 4-(N-maleimidomethyl) cyclohex-
ane-1-carboxylate, SMCC), with an average DAR of 3.5 [49].
Preclinical studies of T-DM1 suggested that the ADC retained
all activities of unconjugated trastuzumab, inhibition of PI3K/
AKT signalling, inhibition of HER2 shedding, and Fcγ receptor
engagement triggering ADCC [50]. Moreover, T-DM1 showed a
strong growth inhibitory effect on trastuzumab-resistant breast
cancer cell lines in vitro, as well as a significant inhibition of
tumor growth when administered in trastuzumab and lapatinib
resistant tumor-bearing mice [49, 51].
Four phase I/II studies evaluated T-DM1 as single agent for the
treatment of HER2-positive refractory/relapsed metastatic breast
cancer. In 2006, 24 patients were enrolled in a phase I dose escala-
tion study, with doses ranging from 0.3 to 4.8 mg/kg, in a q3w
schedule [52]. T-DM1 MTD was identified at 3.6 mg/kg without
cardiotoxicity or neuropathy. Transient grade 4 thrombocytopenia
was the most common adverse event and was defined as the DLT
[52]. Encouraging antitumor activity was observed: out of 15
patients enrolled in the 3.6 mg/kg group, four had a confirmed
objective partial response. One confirmed PR was also observed in
the 2.4 mg/kg group [52]. A phase I weekly dosing [53] reported
MTD at 2.4 mg/kg, with thrombocytopenia being also the DLT
 Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review 13

and showing the same range of activity. Different phase II studies

evaluated T-DM1 at 3.6 mg/kg, q3w (for review, see refs. 54, 56).
In one study [57] a median of seven doses was administered to 112
patients with HER2-positive metastatic breast cancer previously
treated with chemotherapy and progressed under trastuzumab ther-
apy. The ORR evaluated by independent review was 25.9 %, all PR.
Interestingly, in the group of tumors confirmed HER2-positive in a
retrospective central testing by immunohistochemistry (IHC) or
fluorescence in situ hybridization (FISH), the ORR was 33.8 %
versus 4.8 % for the group of tumors with normal HER2 expression.
The most common grade 3 or 4 AE were hypokalemia (8.9 %),
thrombocytopenia (8.0 %), and fatigue (4.5 %). PK parameters
showed a terminal half-life of T-DM1 of around 4 days, which was
found to be lower than the total trastuzumab half-life. No accumu-
lation of T-DM1 was reported [57]. In a second study, T-DM1 was
administered in 110 patients with metastatic breast cancer previ-
ously treated with an anthracycline, a taxane, and capecitabine, as
well as lapatinib and trastuzumab [58]. The ORR by independent
review was 34.5 % without CR and again rose to 41.3 % for patients
with tumors centrally confirmed for HER2-positivity (FISH and
IHC) compared to 20 % in the patient group displaying HER2-
normal expression levels. The most common grade 3 and 4 AE were
thrombocytopenia (9.1 %), fatigue (4.5 %), and cellulitis (3.6 %).
In the different studies, thrombocytopenia was one of the most
reported grade 3 or 4 abnormalities, but the decrease in platelets
was generally reversible and not associated with serious hemorrhage
[56–58]. Increased serum concentrations of hepatic enzymes was
observed [56]. T-DM1 exposure did not correlate with clinical
responses, grade 3 thrombocytopenia or grade 3 increase in hepatic
enzymes serum concentrations [59]. The comparison of pharmaco-
kinetics data from phase I and phase II studies, as single agent,
demonstrated a positive correlation between DM1 and T-DM1
exposure with neither accumulation of T-DM1 nor DM1 [59,
60]. At the MTD, T-DM1 showed a median terminal half-life of
4.5 days which is shorter than the one from total trastuzumab
(around 9 days) [59, 60]. The PK profile of T-DM1 was not affected
by circulating levels of HER2 or residual trastuzumab [59, 60]. On
a total of 286 patients, 4.5 % developed an antibody response to T-
DM1 but no impact on PK parameters, safety or efficacy profiles
were observed [59].
Interestingly a randomized phase II study was conducted to
compare T-DM1 versus trastuzumab plus docetaxel [55] in the
first-line treatment of HER-2-positive locally advanced or meta-
static breast cancer. A total of 137 patients, with no prior chemo-
therapy for metastatic disease, were randomized to T-DM1
(3.6 mg/kg, q3w) or trastuzumab (8 mg/kg first cycle, then
6 mg/kg) plus docetaxel (75 or 100 mg/m2). Assessment by
investigators showed equivalent ORR of 47.8 % with T-DM1 and
14 Ingrid Sassoon and Véronique Blanc

41.4 % with docetaxel plus trastuzumab [61] but with improved

therapeutic ratio in the case of T-DM1. Primary efficacy and update
on safety results were presented at ESMO 2011 [62] with a signifi-
cant improvement of progression-free survival (PFS) in the T-DM1
population (14.2 months vs. 9.2 months) and a confirmed favor-
able safety profile with grade 3 AE reported less frequently in the
T-DM1 arm (46.4 % vs. 89.4 %). The most frequent AE were also
different between the two arms, with increased level of liver
enzymes, fatigue and thrombocytopenia in the T-DM1 arm versus
alopecia, neutropenia, fatigue, and diarrhea in the trastuzumab/
docetaxel arm.
In addition, three phase III trials (EMILIA, MARIANNE and
TH3RESA) are ongoing [55]. EMILIA is a randomized trial
designed to evaluate the safety and efficacy of T-DM1 in compari-
son to lapatinib plus capecitabine in patients with HER-2 positive,
unresectable, locally advanced breast cancer or metastatic breast
cancer, following prior trastuzumab and taxane containing che-
motherapies. Recent publication of the first and second interim
analysis of the 991 patients enrolled indicates that T-DM1 signifi-
cantly improves PFS (9.6 months vs. 6.4 months) and overall
survival (OS) (30.9 months vs. 25.1 months) as compared to
lapatinib plus capecitabine treatment [63]. In addition, as previ-
ously shown in phase II, the safety profile of T-DM1 was different
and more favorable than lapatinib plus capecitabine, as shown by
the reduced incidence of grade 3 and 4 AE (40.8 % vs. 57.0 %).
Thrombocytopenia (12.9 %) and elevated AST (4.3 %) were the
most commonly reported AE for T-DM1, while diarrhea (20.7 %),
palmar–plantar erythrodysesthesia (16.4 %), vomiting (4.5 %), and
neutropenia (4.3 %) were the ones reported for lapatinib plus
capecitabine [63]. On the basis of this study, a Biologics License
Application (BLA) was filed in August 2012. MARIANNE is a
randomized trial of T-DM1 with or without pertuzumab compared
with trastuzumab plus taxane for first-line treatment of HER2-
positive, progressive, or recurrent locally advanced or metastatic
breast cancer. TH3RESA is a randomized trial to evaluate the
efficacy of T-DM1 compared with treatment of physician’s choice
in patients with HER2-positive metastatic breast cancer who have
received at least two prior regimens of HER2-directed therapy.

3.3 CMC-544 CD22 is a glycoprotein belonging to the sialic-acid-binding

(Inotuzumab immunoglobulin-like lectins (siglecs) expressed at the surface of
Ozogamicin) Clinical normal immature and mature B cells but neither on hematopoietic
Overview stem cells nor on memory B cells. Its function is still unclear, but
it is thought to be involved in cellular adhesion, B -cell homing,
and B-cell activation [64]. CD22 has been shown to be rapidly
internalized upon ligand binding, an attractive property supporting
CD22 as target for ADC [65]. CD22 is expressed in more
than 90 % of diffuse large B-cell non-Hodgkin lymphomas
 Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review 15

(DLBCL) and follicular lymphomas (FL) [66]. It is also expressed

in up to 100 % of mature B-cell acute lymphoblastic leukemia
(B-ALL) [67].
CMC-544 (Inotuzumab ozogamicin) is an ADC comprised
of a humanized anti-CD22 IgG4 monoclonal antibody (G544)
conjugated through a cleavable acid-labile hydrazone linker to
calicheamicin with an average DAR of 6 (73 μg calicheamicin/mg
of antibody) [4]. G544 binds to CD22 with subnanomolar affinity
and has no effector functions and no antitumor activity as naked
monoclonal antibody [4].
Based on encouraging preclinical data [68], two phase I single
agent studies were conducted in relapsed/refractory B-cell NHL.
The first phase I enrolled 36 patients in the dose escalation phase
with a q3w schedule and 43 patients in the expanded MTD cohort
[69]. In the dose escalation phase, DLT were grade 4 thrombocy-
topenia and grade 4 neutropenia, and the MTD was declared
1.8 mg/m2 (0.048 mg/kg) in a q4w schedule in order to allow
platelets recovery. Among the 49 patients treated at the MTD, the
common grade 3 or grade 4 AE were thrombocytopenia (63.3 %)
and neutropenia (34.7 %). At MTD, the ORR was 68 % for FL and
15 % for aggressive DLBCL with CR observed [69]. Drug disposi-
tion for CMC-544 and total calicheamicin was nonlinear with dose
or number of doses suggesting an accumulation of the drug, which
could be explained by the decrease in CD22 target after the first
dose [4]. After the first cycle, terminal half-life of CMC-544 at the
MTD was 17.1 h, increasing to 34.7 h at the second cycle [69]. The
second phase I dose escalation study was conducted in 13 Japanese
patients with relapsed/refractory FL. The MTD was confirmed at
1.8 mg/m2 q4w, with most common grade 3 and 4 AE being also
thrombocytopenia (54 %) and neutropenia (31 %). The ORR was
80 %, CR included [70]. PK parameters were similar to what was
observed previously.
Based on preclinical studies suggesting superior activities of
CMC-544 with rituximab [71], several phases I/II studies have
been initiated in recurrent/refractory FL or DLBCL [4, 66]. The
MTD was determined at 375 mg/m2 rituximab given on day 1 and
1.8 mg/m2 CMC-544 given on day 2 every 28 days for four cycles
[72, 73]. Pharmacokinetic and safety profile of CMC-544 were
shown to be equivalent to single-agent, dose-limiting toxicities
being again thrombocytopenia and neutropenia [66]. In one of
the study [72, 74], enrolling 110 patients treated at the combina-
tion MTD, the ORR of relapsed FL and DLBCL were 84 and 80 %,
respectively. Response to rituximab in prior treatment appeared to
be a very strong prognostic of response to the combination as when
rituximab-refractory patients were considered; the ORR was only
20 % [66, 72, 74]. A randomized open-label phase III trial is now
recruiting, comparing rituximab plus CMC-544 to rituximab plus
gemcitabine or bendamustine in relapsed/refractory aggressive
B-cell NHL [4].
16 Ingrid Sassoon and Véronique Blanc

CMC5-44 was also explored in refractory/relapsed acute

lymphoblastic leukemia (ALL) patients. The first published report
evaluating CMC-544 at 1.8 mg/m2 in a q3w schedule was
promising, as the ORR was 56 % [75]. A phase II trial has therefore
been undertaken in patients with refractory/relapsed ALL with the
same dosing schedule [76]. A total of 49 patients were treated, with
CD22 expressed in more than 50 % of blasts in all patients. The
ORR was 57 % with 18 % complete marrow response of short
duration and 39 % with no platelets or incomplete blood cell
count recovery. Thrombocytopenia was, like in NHL, a notable
adverse event, but based on the leukemia risk, treatment was not
delayed. Grade 3–4 fever was the most common AE (31 %). Further
clinical evaluation in ALL is ongoing with a weekly schedule [76].

3.4 Other ADC in Beside SGN-35, T-DM1, and CMC-544, 24 other ADC are
Early Clinical Trials currently being evaluated in phase I and II (cf. Table 1). The
more advanced ones, for which efficacy data are available, are
described below.
SAR3419: CD19 is a type I transmembrane glycoprotein of the
immunoglobulin (Ig) superfamily, expressed from the earliest
stages of pre-B-cell development until terminal B-cell differentia-
tion into plasma cells. CD19 expression covers all types of
B-lymphomas and non-T acute lymphoblastic leukemia, with mod-
erate to high homogeneous expression [77]. SAR3419 is com-
posed of a humanized IgG1 monoclonal anti-CD19 antibody,
huB4, conjugated via a cleavable disulfide linker to DM4 (huB4-
SPDB-DM4). In a first phase I study with refractory or relapsed
B-cell NHL (R/R NHL) [78], SAR3419 was evaluated in a q3w
schedule. The MTD was determined at 160 mg/m2 (4.3 mg/kg),
and the DLT was reversible severe blurred vision associated with
microcystic epithelial corneal changes. Tumor reduction from base-
line was observed in 74 % of patients bearing a variety of lymphoma
subtypes including DLBCL. The ORR was of 23.5 % at MTD [78].
A second dose escalation study was performed with a weekly sched-
ule, again in R/R NHL patients. The regimen consisting of
4 weekly doses of 55 mg/m2 followed by four additional doses
administered every 2 weeks showed a favorable safety profile and
was therefore retained for further clinical studies. In particular there
was no grade 3 or 4 ocular toxicity observed and hematotoxicity
incidence was low, allowing potential combination of SAR3419
with other agents used to treat NHL. In addition, no dose-limiting
cumulative side effects were observed in this cohort of 21 patients
[79]. In this heavily pretreated patient population, antitumor activ-
ity with around 30 % ORR in both aggressive (e.g., DLBCL) and
indolent (e.g., FL) subtypes of NHL was obtained. A phase II
program in patients with R/R DLBCL is underway testing
the drug as a single agent and also in combination with rituximab
(NCT01472887 and NCT01470456, respectively) in order
 Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review 17

to confirm the clinical benefit of SAR3419 in a more homogeneous

population. Based on encouraging preclinical data, the activity of
SAR3419 is also explored in adult patients with R/R ALL [80].
CDX-011 (glembatumumab vedotin): gpNMB (glycoprotein
nonmetastatic melanoma protein b/osteoactivin) is a type I trans-
membrane glycoprotein identified in melanoma cell lines and
shown to be expressed in several tumor indications including mela-
noma and breast [81, 82]. CDX-011 is an ADC comprising a fully
human IgG2 anti-gpNMB antibody conjugated to MMAE via the
cleavable protease-sensitive valine–citrulline linker [83]. A phase
I/II was undertaken in 117 unresectable, stage III/IV melanoma
patients treated with a q3w schedule, or with more frequent dosing
regimens, q2/3w and weekly. In the q3w dose escalation, DLT
were grade 3 rash and desquamation [83]. The MTD were 1.88,
1.5, and 1 mg/kg, respectively [84], with most common grade 3 or
4 AE being rash (20 %) and neutropenia (15 %) across the studies.
At 1.88 mg/kg q3w, the half-life of CDX-011 was around 28 h and
the half-life of the total antibody was 40 h [83, 85]. At MTD, the
ORR of the q3w, q2/3w, and qw were 15 % (5/34), 33 % (2/6),
and 29 % (2/7), respectively, and a clear correlation of skin rash
with outcome was observed [83, 84]. Another phase I/II was
completed in 42 locally advanced or metastatic breast cancers.
Among the 34 patients, without preselection of gpNMB expres-
sion, treated at 1.88 mg/kg q3w, ORR was 13 % [81, 83, 86].
In the subgroup of patients expressing gpNMB, the ORR reached
29 %. A phase II clinical study with breast cancer patients expressing
high gpNMB measured by IHC is ongoing [81]. It is interesting
to note that one of the most common treatment-related toxicities
with the melanoma and breast cancer studies were dermatologic
events (pruritus, rash, alopecia). The AE could be linked to the
expression of gpNMB in normal melanocytes [87].
PSMA-ADC (PSMA-vc-MMAE): PSMA (prostate-specific
membrane antigen) is a type II transmembrane glycoprotein dis-
playing carboxypeptidase activity and expressed mainly in normal
prostate epithelium [88, 89]. PSMA has been shown to be highly
expressed at the membrane of prostate cancer cells [90–92]
providing a rationale for the design of PSMA-ADC. The PSMA-
vc-MMAE ADC is a fully humanized IgG1 antibody, linked to
MMAE via the cleavable valine–citrulline linker [93]. It is the
second PSMA ADC to be evaluated in the clinic. The first one
(PSMA-SPP-DM1/MLN2704) was stopped in 2008 (Table 2).
Clinical data of MLN2704 showed low efficacy and limiting periph-
eral neuropathy [94]. A phase I, dose escalation trial with PSMA-
vc-MMAE, is being conducted in men with taxane-refractory
metastatic castration-resistant prostate cancer in a q3w schedule
for up to four cycles [95, 96]. As of today 26 patients have been
enrolled in a dose escalation study up to 2.0 mg/kg, and the MTD
has not been reached [95]. Evidence for antitumor activity, as
18 Ingrid Sassoon and Véronique Blanc

reflected by declines in PSA, circulating tumor cells and/or bone

pain, has been observed in 4 of 12 subjects treated at 1.6 or
1.8 mg/kg. Dose proportional increases in serum concentrations
of PSMA ADC have been seen with half-life of around 50 h [96].
From the last EORTC update, dose escalation has been completed
and 2.5 mg/kg has been identified as the MTD. DLT observed at
2.8 mg/kg were neutropenia and reversible liver function alter-
ation [97].
BT-062: CD138 (Syndecan-1) is a member of the family of
transmembrane heparan sulfate proteoglycans overexpressed in var-
ious solid tumors and hematological malignancies. In the normal
human hematopoietic compartment, CD138 expression is
restricted to plasma cells [98], and in malignant hematopoiesis,
CD138 is expressed on the majority of multiple myeloma (MM)
cells making it a good candidate antigen for this indication [99].
BT-062 is an antibody–drug conjugate, comprised of the anti-
CD138 chimeric IgG4 antibody conjugated to DM4 via a
cleavable disulfide linker. In a phase I trial enrolling a total of
32 MM patients, receiving 1 of 7 dose levels ranging from
0.27 to 5.4 mg/kg in a q3w schedule, the MTD was defined at
4.3 mg/kg, with mucositis and palmar–plantar erythrodysesthesia
syndrome being the DLTs [100]. Mucositis side effect could be
correlated with the target expression observed in stratified squa-
mous epithelium (mucosa) of the esophagus [99]. Of the 28
patients who were evaluated for response, 4 % achieved a PR.
A phase I/IIa study in MM has been initiated to further evaluate
the safety and efficacy of BT-062 using a more frequent dosing
regimen of three weekly doses [100]. Combination trials with
lenalidomide and dexamethasone are also ongoing.
IMGN901 (lorvotuzumab mertansine): CD56 antigen,
a neural cell adhesion molecule implicated in cell–cell adhesion,
neurite outgrowth, and other brain functions is overexpressed in
a variety of cancers including small-cell lung cancer (SCLC),
neuroblastoma and other neuroendocrine malignancies, multiple
myeloma, and ovarian cancers. The expression of CD56 on normal
tissues is restricted to NK cells and a subset of T lymphocytes [101].
IMGN901 is an anti-CD56 IgG1 antibody conjugated to DM1 via
a hindered disulfide cleavable SPP linker. It has been evaluated in
several phase I trials in patients with SCLC, MM, or other neuro-
endocrine tumors. A phase I dose escalation trial in 32 patients with
MM established the MTD at 112 mg/m2 (3 mg/kg) when the
ADC was administered weekly for 2 consecutive weeks every
3 weeks [102]. DLT was grade 3 fatigue in 2 out of 6 patients
treated at 140 mg/m2. One sustained PR was documented in a
patient treated at 140 mg/m2/week. In a small phase I trial
enrolling 6 patients with Merkel cell carcinoma, the MTD was
established at 75 mg/m2 (2 mg/kg) when the ADC was adminis-
tered daily for 3 consecutive days every 3 weeks [103]. In this trial,
 Antibody–Drug Conjugate (ADC) Clinical Pipeline: A Review 19

DLTs were grade 3 myalgia, headache, and back and shoulder pain.
Out of 6 patients, there was 1 CR and 1 PR. A similar schedule of
administration was used during another phase I on CD56-positive
solid tumors from different types [104]. The MTD was also estab-
lished at 75 mg/m2/day, and DLT were grade 3 headache, neu-
ropathy, fatigue, and myalgia, as previously reported. Half-life of
IMGN901 at MTD was 34 h. Evidence of activity was observed
with 1 CR and 1 PR in MCC and 1 unconfirmed PR in SCLC.
Combination trials were also initiated. Escalating doses of
IMGN901, given weekly for 3 weeks in a 4-week cycle, were
evaluated in combination with lenalidomide/dexamethasone at
their usual doses in patients with R/R CD56-expressing MM.
Among the 12 patients enrolled, all had previously been treated
with chemotherapy, with 42 % having received prior lenalidomide.
No DLT has been reported and no grade 4 toxicities have been
observed. One serious AE and 7 grade 3 toxicities related to com-
bination treatment have been observed in four patients. On 12
efficacy-evaluable patients, 2 had a very good PR (VGPR) [105],
and 4 had a PR. A phase I/II study to assess the safety and efficacy
of IMGN901 in combination with carboplatin/etoposide in
patients with advanced solid tumors including extensive stage
small-cell lung cancer is ongoing. The NORTH trial is the phase
II portion of this trial in which the ADC is administered for
3 consecutive days every 21 days at the dose of 60 mg/m2/day
(IMGN website, clinicaltrial.gov). Another phase I/II combination
study with panobinostat and carfilzomib is currently ongoing in
patients with R/R multiple myeloma [106].

4 Challenges and Perspectives

ADC have made tremendous progress over the last decades as

demonstrated by the outstanding clinical efficacy observed in
both hematological malignancies, with Adcetris® for the treat-
ment of Hodgkin’s lymphoma, and solid tumors, with T-DM1
for the treatment of metastatic breast cancer. The conjunction of
the evolution of monoclonal antibodies from murine to huma-
nized and human versions and the technological advances in the
conjugation of highly potent non-immunogenic small molecules
have been the pillars of these progresses. The increasing number
of ADC reaching the clinic, targeting different antigens, and
bearing different linkers and cytotoxics have contributed to the
learning curve and stepwise progress of ADC. Lessons learned
from the past experience of successful and stopped ADCs (see
Tables 1 and 2) highlight the major axes that shall guide the
development of future ADC.
Targets are at the heart of ADC development. Through their
expression in some normal organs/tissues, they can contribute to
Exploring the Variety of Random
Documents with Different Content
 SECT, i] PHYSICO-CHEMICAL SYSTEM 315 takes short cuts
in its development, and jumps from branch to branch of its
genealogical tree instead of climbing steadily upwards. Thus the little
West Indian frog, Hylodes, produces eggs which contain a larger
amount of yolk than those of the ordinary English frog. The young
Hylodes is consequently enabled to pass through the tadpole stage
before hatching, and to attain the form of the frog before leaving the
c:gg\ the tadpole stage is, in fact, only imperfectly recapitulated, the
formation of gills, for instance, being entirely omitted." The more
yolk, then, the longer the embryo can remain an embryo before
having to face the external world, and the more preparations it can
make for that event. It is probable that this question is intimately
bound up with the penetration of fresh-water surroundings by the
originally marine forms. "It has long been noticed", said Milnes-
Marshall, following the classical exposition of Sollas, " that marine
animals lay small eggs whereas their fresh-water allies lay eggs of
much larger size. The eggs of the salmon or trout are much larger
than those of the cod or the herring, and the crayfish, though only a
quarter the length of the lobster, lays eggs of actually larger size.
The larger size of the eggs of the fresh-water forms appears to be
dependent on the nature of the environment to which they are
exposed. Considering the geological instability of the land as
compared with the ocean, there can be no doubt that the fresh-
water fauna is, speaking generally, derived from the marine fauna,
and the great problem with regard to fresh-water life is to explain
why it is that so many groups of animals which flourish abundantly
in the sea should have failed to establish themselves in fresh water.
Sponges and Coelenterates abound in the sea, but their fresh- water
representatives are extremely few in number; Echinoderms are
exclusively marine ; there are no fresh-water Cephalopods, no
Ascidians, and of the smaller groups of Worms, Molluscs, and
Crustacea, there are many that do not occur in fresh water. Direct
experiment has shown that in many cases this distribution is not due
to the inability of the adult animals to live in fresh water, and the
real explanation appears to be that the early larval stages are unable
to establish themselves under such conditions. To establish itself in
fresh water permanently an animal must either be fixed, or else be
strong enough to withstand and make headway against the currents
of the streams or rivers it inhabits, for otherwise it will in the long
run be swept out to sea, and this condition applies to larval
 3i6 THE UNFERTILISED EGG AS A [pt. iii forms equally with
adults. The majority of marine invertebrates leave the egg as minute
ciliated larvae, which are quite incapable of holding their own in
currents of any strength. Hence it is only forms which have got rid of
the free-swimming ciliated larval stage, and which leave the egg as
organisms of considerable size and strength, that can establish
themselves as fresh-water animals. This is effected most readily by
the acquisition of yolk — hence the large size of the eggs of fresh-
water animals — and is often supplemented by special devices."
Here is an explanation for the well-known paucity of eggs in
freshwater plankton. In certain cases it is possible to induce an
embryo to skip the larval stage which it should normally pass
through. Thus Child could abolish the free-swimming larval stage in
the ascidian Corella willmeriana, simply by removing the eggs from
the parental atrial chamber {p¥L j'^.) to normal sea-water (/>H 8-
4). Giard had also noticed the discrepancy in egg-size between
closely related marine and fresh-water forms, and had classed it
among those cases where like adults have unlike larvae
("Poecilogony"). The classical instance is perhaps that of the shrimp
Palaemonetes varians, one variety of which {microgenitor) lives in
the sea near Wimereux and has eggs 0-5 mm. diam. (32 1 per
female) and another of which {macro genitor) lives in fresh water at
Naples and has eggs 1-5 mm. diam. (25 per female). Giard has
reviewed this subject in a very interesting paper. "Dans un groupe
determine", he said {(Euvres diverses, p. 18), "la condensation
embryogenique va en croissant des types marins aux types d'eau
douce ou terrestres." The correlated proposition, namely, that the
fresh-water forms generally lay fewer eggs than the marine ones, is
illustrated by the following instances collected by Carpenter: No. of
eggs laid per female per annum A Lamellibranchs ... Gastropods
Fishes Crustacea Marine form Ostrea edulis i ,800,000 Buccinum
undatum 12,000 Haddock 9,000,000 Lobster 5,000 Fresh-water form
Uniopictorum 220,000 Anodonta cygnea 18,000 Average of many
snails 100 Average of many limpets 6 Ovoviviparous pond-snails 15
Brook-trout 750 Crayfish 200 Another reason for the poverty of
fresh-water fauna was suggested by von Martens who pointed out
that the fresh-water climate, with its periods of desiccation and
freezing, was much more severe than
 SECT, i] PHYSICO-CHEMICAL SYSTEM 317 that of the sea.
But even these two causes together cannot fully account for the
phenomenon, for there are many cases of individual species which
they will not cover; thus the Cephalopods, which hatch out as
minute but very active copies of their parents, i.e. which pass their
larval stage within the egg, and which should therefore be immune
from the disadvantage described by Sollas, never penetrated into
fresh water. A third reason must be added to those of Sollas and of
von Martens. As will be shown in Sections 12 and 13 the marine
invertebrate embryo depends largely on the salts of the sea water
for its supply of ash, and therefore could not be expected to develop
in a medium very poor in inorganic matter. Colonisation of the fresh
water could not occur, then, until animals had begun to provide in
each egg sufficient ash to make one finished embryo. There seem to
be few data concerning the capacity of marine invertebrate eggs to
develop in fresh water, although the adult animals have been found
often enough to accustom themselves to a fresh-water environment
(see the instances given in Semper) . Many studies of the effect of
hypotonic solutions on marine embryos can, however, be called to
mind, and in all the cases the results are teratogenic. The fate of the
Cephalopods, it is interesting to note, is explained by this third
factor, for Ranzi has demonstrated the intake of the salts in the sea
water by the octopus egg. As for the general statement that animals
can afford their young a better chance of survival by providing them
with larger amounts of yolk and therefore a longer incubation-
period, there is a striking parallel here with the seeds of leguminous
plants which are packed with nourishment. In the Origin of Species
(6th ed. p. 56), Darwin wrote, "From the strong growth of young
plants produced from such seeds as peas and beans when sown in
the midst of long grass, it may be suspected that the chief use of the
nutriment in the seed is to favour the growth of the seedlings, whilst
struggling with other plants growing vigorously all round". It is
interesting that the birds show an adaptation exactly similar to the
poecilogony of the invertebrates and fishes. Tree-nesting birds are
usually nidicolous, but the defenceless state of the newly-hatched
squab has brought it about that ground-nesting birds are usually
nidifugous. As Table 30 shows, the composition of the eggs of all
animals other than those of the frog, the silkworm, and certain
fishes, is still, to
 3i8 THE UNFERTILISED EGG AS A [pt. m use a phrase of
William Harvey's, "hid in obscurity and deep night". It is as yet much
too early to try to draw any conclusions from the very fragmentary
figures which are all that we have at our disposal, and we may well
admit that one of the most urgent needs of chemical embryology is
a much wider extension of our knowledge of the static chemistry of
the egg. This is a quite indispensable preliminary to the investigation
of the metabolism of the embryo in the lesser known forms. The
attempt has already once been made to link up in some way the
chemistry of the egg with what is known of the type of embryonic
development which takes place in it. Wetzel in 1907 analysed the
eggs of a sea-urchin, a crab, a cephalopod, and an elasmobranch
fish. He pointed out that the eggs he studied were examples of
varying richness in yolk, of total and partial, equal and unequal,
superficial and discoidal cleavage, as well as chemical systems.
Taking the egg of Strongylocentrotus lividus as his first case, he
regarded it as typical of a class of alecithic eggs, of a total and equal
cleavage type, and he drew attention to the fact that it was rich in
water and in salts, but poor in fatty substances, in nitrogen, and in
phosphorus. Similarly, in the case of the mollusca, where there is no
very definite type of development, the egg of Sepia could not stand
as representative of any wider class than the cephalopods, but, as
far as it went, it showed that the cephalopod egg was rich in
nitrogen, poor in fat and inorganic substances, with a moderate
phosphorus and water-content. The decapod Crustacea, to which
Maia squinado belongs, have a purely superficial type of cleavage,
with no cell-multiplication in that part of the egg which holds the
yolk. Accordingly, the egg possessed a moderate fat and water-
content, a moderate ash, and much protein and phosphorus. The
mammalian ovum is still as unknown chemically as it was when
Wetzel was writing, and it may be found to have a constitution not
unlike the alecithic echinoderm eggs. For the eggs of birds (and of
reptiles, which only differ from them in having very little egg-white)
Wetzel found a low protein and water-content, a high proportion of
fat and ash, and a large amount of calcium and phosphorus. Here
cleavage would only take place at one isolated point on the surface
of the mass of food-material. In the amphibia, the richness of yolk,
while much more significant than in lower classes, does not reach
the level of birds and reptiles.
 SECT. I] PHYSICO-CHEMICAL SYSTEM 319 and this is duly
reflected in the chemical composition by the moderate water-
content, the high proportion of protein which is yet only double that
of the fat. The case of the dogfish is again different, for there the
egg is rich in yolk and the cleavage is meroblastic; thus the water is
rather low, the fat rather high, the nitrogen very high, and the ash
and phosphorus moderate. But these conclusions of Wetzel's,
interesting though they are, cannot really be assessed until a great
deal more comparative work has been done. They must rather be
taken to represent the kind of correlation we may hope for in the
future. However, one of Wetzel's generalisations may be accepted, if
with some reserve. He pointed out that the fat-content of eggs
showed great variations, rising from 12 per cent, of the dry weight
of the Sepia ^gg to 66 per cent, of the dry weight of the (yolk of
the) hen's tgg. Again, the nitrogen gave very variable results, rising
from 5-3 per cent, of the dry weight in the (yolk of the) hen's ^gg to
6-9 per cent, in the egg of the grass-snake, 1 2 per cent, in the egg
of the dogfish, and even in the case of the cod 14 per cent. On the
other hand, the phosphoruscontent varied only between the
(outside) limits of 2 • i per cent, for the sea-urchin tgg and 3-6 per
cent, for that of the grass-snake. Wetzel, therefore, suggested that a
distinction might be made, at any rate, roughly, between those
constituents of the egg which may serve as sources of energy for
the growing embryo, and those which in no circumstances do so.
Protein, fat, and carbohydrate would come in the former class;
phosphorus (for nucleoprotein) and cholesterol, for example, would
come in the latter class. The former would show great variations
among eggs of different species, the latter would not. He thus
supposed that one might be able to deduce, as it were, the
constitution of any given egg, if one knew what substances, and in
what proportions, were used by the embryo as combustible material
during its development, as well as the constitution of the newly born
or hatched organism. From this standpoint Wetzel distinguished four
types of substances in the unincubated egg : ( i ) material for the
embryo to burn during the course of its development, (2)
constituents of the finished protoplasm of the embryo, (3)
constituents of the finished embryo, but not for incorporation into
the protoplasm itself, but into the paraplasm (in Le Breton's
terminology), (4) the protoplasm of the original egg-cell. No aspect
of chemical embryology needs attention more
 320 THE UNFERTILISED EGG AS A [pt. iii urgently than
this, and the correlation of chemical constitution with developmental
type should offer a most attractive field for research. But it is not
only correlations of this type that lie hidden under the enigmatic
character of analytical figures. The water-content of the eggs may
have a powerful effect on the sex-ratio, for King found in 191 2 that
reducing the water-content of fertilised frog's eggs considerably
lowered the proportion of males, while increasing it by means of
treatment with dilute acid considerably raised the proportion. A
discussion of these facts in relation to genetics as a whole will be
found in the review of Huxley. It is probable that the effect which
delayed fertilisation has upon the sex-ratio is to be explained by
difference in water-content of the eggs. Hertwig was the first to
observe this delayed fertilisation phenomenon in some work which
he published in 1905, and since then it has many times been
observed not only for amphibia but also for trout (Kuschakevitsch;
Huxley; Mrsic) . Riddle has suggested that the mammalian egg may
be subject to such influences as it passes from ovary to uterus. He
quotes van der Stricht's histological work on the bat's egg during this
process, and points out that the swelling of the yolk-granules would
indicate an absorption of water. The exact degree of hydration of the
mammalian egg might thus conceivably have an effect on the
mammalian sex-ratio. Table 30 has several more important points
which have not, so far, been touched upon. It is interesting to follow
in the figures of Milroy the difference between the fish eggs which
float at the surface of the water during their development (pelagic
ova), and those which sink, or rather float, at lower and denser
levels (demersal ova) — the former have a water-content of about
90 per cent., the latter of about 70 per cent. A knowledge of the
chemical composition of fish eggs throws a great deal of light upon
their distribution in the sea, and so indirectly upon ecological
problems. Their fat-content, for example, has been treated from this
point of view by Polimanti, whose work will be discussed in the
section on the general metabolism of the embryo; and the
investigations of the specific gravity of fish eggs, which are
discussed in Section 5, have also an important bearing upon these
problems. Another point worth notice is the approximately constant
percentage of cholesterol in different eggs, nearly always about 500
mgm. per cent, of the wet weight, a proportion which, roughly
speaking, holds for the egg of the hen as well.
 SECT, i] PHYSICO-CHEMICAL SYSTEM 321 It would be as
well to emphasise the fact that no principle of selection has been
used in the preparation of Table 30, on the ground that results such
as those of Roffo & Correa on a Brazilian gastropod, and McCrudden
on fresh-water fishes, which seem obviously wrong, may not be so
at all. The estimation methods and analytical processes which are by
general consent judged most satisfactory at the present time cannot
be considered in any way final, and to have excluded certain results
on account of the technique employed in obtaining them would not
have been justifiable. Table 30 does not, therefore, absolve
investigators fi'om the duty of looking up the original papers in such
cases as touch them most closely, and forming an independent
judgment, according to the best opinion of the time, on the stress
which can be laid upon them. It is needless to say that I leave out of
account all doubtful figures in the generalisations made here. I -12.
Egg-shells and Egg-membranes Very little is known about the
relative proportions of yolk, white, and shell, in the eggs of the lower
animals, or rather, in most cases, egg-contents and shell or
surrounding membrane. Table 32 gives a few figures. The
discrepancy between the results of Ford & Thorpe, on the one hand,
and Wetzel, on the other, is very strange, especially as they both
used Scyllium canicula eggs, but it is probably due to insufficiency of
the statistical element. Ford & Thorpe's proportions are more likely
to be accurate. Much work, however, has been done on the
membranes and hard coverings which invest the unincubated eggs
of diflferent kinds of animals. For instance, the gelatinous substance
which surrounds the undeveloped amphibian egg was examined
chemically by Brande in 1 810, who noticed that it absorbed water
and was not precipitated by tannin or by strong acids. Later work
has shown that it consists almost entirely of mucoprotein and water.
Wetzel's figures for its weight are shown in Table 32. Giacosa
isolated mucin in a pure state from it in 1882, and the figures which
he obtained for its percentage composition are shown in Table 33.
He was able to show the presence of a reducing sugar on hydrolysis,
but he could isolate nothing else from the jelly, and therefore
concluded that it was pure mucin. The presence of glucosamine in
the mucoprotein was afterwards confirmed by Hammarsten, by
Schulz & Ditthorn and by Wolfenden, who confirmed Giacosa's
finding that
 322 THE UNFERTILISED EGG AS A [PT. Ill Table 32. In % of
total egg-weight Species Egg-membranes Whit( Herring Carp ... Cod
... Pike ... 2-4 3-7 4-4 4-1 — Dogfish Silkworm 5-4 26-9 8-87 (wet)
19-3 36-5 Trout ... Octopus 25-97 (dry) I3-57-20-29 86-0 — Yolk
Investigator and date — Konig & Grossfeld (191 3) 3? 33 75-3 Ford
& Thorpe (1920) 36-5 Wetzel (1907) — Tichomirov (1882) 14-0 3J
Kronfeld & Scheminzki (1926) Ranzi (1930) Tomita's figures. Marine
turtle ( Thalassochelys cortica) Weight Shell White Yolk Total m gm.
2-0 13-5 18-9 34-4 % 5-8 39-2 55-0 Wetzel's figures. I Frog {Rana
temporaria) Ovarial egg (no jelly)... Egg with unswoUen jelly Jelly
alone Swollen jelly ... Water content of ovarial egg jelly „ „ egg and
jelly Empty dry jelly Dry egg Dry egg + dry jelly ... Thus of dry
weight egg jelly Weight in mg. 1-897 4-674 2-777 8-97 0-62 0-90 1-
52 Melvin's figures. Shell-weights of insects Squash-bug {Anasa
tristis) Luna moth [Tropoeoa luna) Cecropia moth {Sarnia cecropia)
... Smartweed-borer {Pyrausta ainsleii) 52-5 78-65 67-48 59-28 40-
72 % of total weight of eggs 29-2 23-3 22-0 31-0 it was remarkably
resistant to putrefaction, and studied the effect of enzymes such as
pepsin upon it. The resistance of frog ovomucin to putrefaction was
for long a puzzle to biochemists, but it seems to be explained by the
unwillingness of most
 SECT, i] PHYSICO-CHEMICAL SYSTEM 323 bacteria to grow
on pure proteins, and as the jelly contains no enzymes of an
autolytic character no protein breakdown products are formed, and
consequently no bacterial growth takes place. This might be
considered a protection of the developing embryo from bacterial
attack. It is very probable, moreover, that the mucoprotein acts as a
source of nourishment for the young tadpoles immediately after
hatching, for they invariably attach themselves to it after they
emerge from the egg-membrane, and hang on to it by their oral
suckers (for histological details consult Nussbaum and Lebrun). On
the other hand, development will readily proceed in the absence of
the jelly, for as Hluchovski has shown it is disintegrated by exposure
to ultra-violet light and may thus be removed without harming the
eggs. The swelling which takes place in the gelatinous covering
when the eggs are shed into the water was studied as long ago as
1824 by Prevost & Dumas, who measured the size of the eggs at
intervals after they were laid. Their table is as follows : Hours after
laying Diameter of egg (mm.) 0 2-5 1-5 5-0 2-5 6-3 3-5 7-1 4-5 7-2
5-5 7-1 6-5 7-3 They observed that dyes would pass through the
jelly as soon as it had swollen, but not before. Similar work by
Wintrebert on Discoglossus pinctus gave the following figures : after
laying Diameter of egg (mm, o-oo 2-5 X 2-3 0-03 3-0 X 2-7 016 3-3
X 3-0 0-66 5-8 X 3-2 800 5-OX4-6 As regards the mineralogical and
morphological structure of the egg-shells of the lower animals, a
good deal is known, and for full detail the reviews of Prenant and of
Biedermann should be referred to. The majority of reptile egg-shells
have their calcium carbonate in the form of calcite, as Kelly;
Schmidtt, and Meigen have shown, but the two first-named
investigators discovered that the tggsheUs of chelonia were of
aragonite, and later Lacroix observed a similar phenomenon in the
case of certain saurians. The tgg 
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 326 THE UNFERTILISED EGG AS A [pt. m membranes of
snake's eggs which show all variations as to lime-content (see Table
9) are, as Kelly has shown, composed of amorphous and unstable
calcium carbonate. The eggs of gastropods, such as Helix,
Ampullaria, Bulimus, Amphidromus, etc., are, as Turpin {Helix
aspersa) and Rose {Helix pomatia) , besides the workers mentioned
above, have demonstrated, like those of birds in having their lime in
the form of calcite. For a general theory explaining these differences
see the paper of Prenant. The shells of eggs may also contain
calcium phosphate. In the hen and in birds generally there is very
little, but the globules seen in their egg-shells are believed to be
calcium phosphate, though no analysis has given a figure of more
than i per cent, of this salt. In other eggs, however, there may be
more; thus Gmelin found 7-3 per cent, in the egg-shells of a
tortoise, and Kelly noted its presence also in those of Bulimus and
Lophohelia, though she gives no analytical figures. It is interesting to
note that the mineralogical form of lime in the egg-shell may vary
during the development of the embryo; thus Kelly says that the shell
of many full-grown mollusca is conchite, while that of their
respective embryos and eggs is calcite. Kelly found that the organic
substance was a remarkably constant proportion of the shells of
mollusca, reptilia and birds (see Table 9). Some eggcoverings
contain almost no water at all (birds), others have more than the
egg-contents, as has been shown for the trout's egg by Kronfeld &
Scheminzki (membrane 75 per cent., egg 66 per cent.). By far the
commonest substance of which egg-membranes are composed is
keratin, though this protein seems to take many forms, and not to
have exactly the same properties in different situations. The earlier
workers were content to assert the presence of it on the basis
merely of solubility tests. Thus in 1874 Schenk studied the egg-shell
of Raia quadrimaculata, and decided that it was 95 per cent, keratin
after the application to it of the protein colour reactions and an
examination of its behaviour towards various solvents. The same
conclusion was arrived at by the same methods by Hussakov &
Welker for the egg-cases of Raia erinacea, and the Port Jackson
shark, Heterodontus philippi. The keratin of these egg-cases was
insoluble in all solvents except acid and alkali. They found that
sulphur was present, but no phosphorus, and they were unable to
find any reducing sugar after total hydrolysis. Irvine, using an optical
test for chitin, found
 SECT, i] PHYSICO-CHEMICAL SYSTEM 327 none in
elasmobranch egg-cases. Krukenberg in 1885 decided that the egg-
case of Scyllium stellare was of a keratinoid nature, because of its
percentage composition, in which he found a marked amount of
sulphur. He observed the interesting fact that the egg-cases of this
fish, while still in the uterus of the parent animal, would dissolve in
pepsin and trypsin, while after they were laid they would not
dissolve in solutions of either enzyme. He also isolated tyrosine and
leucine firom the keratin of the egg-cases of Scyllium stellare. He
made very similar researches on the egg-cases of Scyllium canicula
and Myliobatis aquila, finding that they possessed rather different
properties and seemed to be of different constitution; thus on
hydrolysis he recovered a great deal of leucine and hardly any
tyrosine from the keratin of Scyllium canicula, while from the keratin
of Myliobatis the yields were precisely reversed. The latter substance
was also considerably more resistant to digestion than the former,
and Krukenberg considered that the former was not a keratin at all.
He had already decided (wrongly, as it turned out) that the shell-
membrane of the hen's egg was mucin, not keratin, and now he
concluded that this also applied to the egg-case oi Scyllium stellare,
as well as to that ofLoligo vulgaris, of which he made a separate
examination. He thought it possible also that the jelly which
surrounds the egg in the ovo viviparous selachians might be a mucin
too, especially as, according to Schenk, it was not precipitated by
chromic acid, and he himself found that it was extremely resistant to
digestion by enzymes. This material has received no further chemical
investigation since the time of Krukenberg. Other workers who
identified the proteins of egg-membranes by the aid of colour tests
and solubility reactions were Leuckart, who showed, as far as
anything could be shown with such preliminary methods, that the
membranes of planarian eggs were of chitin, and Yoshida & Takano
and Jammes & Martin, who drew a similar conclusion about the
coats of the eggs of Ascaris lumbricoides, which they found were
readily soluble in gastric juice or in any acid.^ The case of the
parasitic nematodes is of special interest, for the chitinous
membrane does not arise until after the fertilisation of the egg,
being, therefore, in a sense, analogous to the fertilisation
membranes of echinoderms. Whether the chitin is formed as it is
required during these early stages, or whether it is already present
in the unfertilised egg-cell in some soluble form, is uncertain. Faure-
Fremiet in an ^ See also Campbell on the chitin of insect egg-
membranes.
 328 THE UNFERTILISED EGG AS A [pt. iii attempt to throw
light on this question, prepared pure samples of chitin from the
newly fertilised eggs ofAscaris megalocephala by boiling them with
strong potash, and identified the chitin chemically, isolating
glucosamine hydrochloride from it. Remembering that Weinland
showed that chitin is probably formed from glycogen during insect
metamorphosis, Faure-Fremiet estimated the glycogen in the Ascaris
eggs before and after fertilisation. Before fertilisation there was an
average amount of 20 gm. per cent, dry weight, but afterwards only
4-67, the extreme values being 5-91 and 3-23, so that no less than
17 per cent, of glycogen had disappeared. Estimations of chitin in
the egg-envelopes after fertilisation gave results of between 8-3 and
10-7 per cent, dry weight of glucosamine (calculated as glycogen)
with an average of 9-23. The total glucose, then, in the fertilised
eggs was 12-83 to 15-08, as against 20-0 in the unfertilised ones, a
loss of 7 to 9 per cent. All the glucose lost, therefore, could not have
transformed itself into chitin, but must have had some other
destination, perhaps butyric and valerianic acid if Weinland's view is
correct. The eggs o^ Ascaris have also an " ovospermatic
membrane", but for the discussion of the significance of this
reference should be made to the memoir of Faure-Fremiet, and
nothing is known about it chemically. Their third membrane, the
internal one, would seem to be composed to a large extent of
ascaristerol (see p. 352), for the histological evidence demonstrates
a collection of the ascaristerol globules at the periphery of the
cytoplasm. After fertilisation, FaureFremiet found the saponification
number of ascaristerol lowered from 199 to 145, from which he
concluded that its constitution had been slightly altered. Zavadovski
has also described the egg-shells of many nematodes. • Neumeister,
who found more than 5 per cent, of sulphur in the shells of the
reptiles, Calotes jubatus, Ptychozoon homalocephalus, and
Crocodilus biporcatus, concluded that they consisted of a true
keratin, and the reactions given by the egg-membrane protein of a
monotreme, Echidna aculeata, led him to the same conclusion in
that case also. Table 9 gives the figures which he obtained for the
calcium and other constituents of some of these egg-shells, as well
as the very similar investigations of Wicke & Brummerstadt on
Alligator sclerops. From these fragmentary results, it would seem
that the eggmembrane protein is here keratin, and a quantity of
calcium is secreted into the membrane by the animal, varying in
amount from
 SECT, i] PHYSICO-CHEMICAL SYSTEM 329 90 per cent, to
10 per cent,, according to the species. Again, the egg-membrane of
the Brazihan gastropod studied by RofFo & Correa is said, on the
basis of qualitative tests only, to be a true keratin, containing no
reducing sugar and associated with no other substances, save 2*45
per cent, of ash. It contained calcium the amount of which did not
vary during development. The transparent horny egg-membrane of
the selachian Mustelus ^ laevis, which disappears half-way through
the development of the ■ embryo, has also been investigated by
Krukenberg, who compared it with the egg-membrane of the grass-
snake, Tropidonotus natrix. The former resembled the shell-
membrane of the hen's egg rather than the true keratin of the
Myliobatis egg-case. The latter seemed to have some of the
properties of elastin and some of those of keratin ; from it he was
able to isolate a reducing carbohydrate as well as glycine, tyrosine
and leucine. Krukenberg was also one of the earliest workers to
make quantitative investigations on this subject. His figures for the
protein of the egg-shells of Murex trunculatus and the whelk
Buccinum undatum, which are given in Table 33, led him to make a
new class of such substances, the conchiolins. As no data exist for
the sulphur content of most of these proteins, it is impossible to say
whether they are keratins or not, and the whole subject needs re-
investigation. About five years later, Engel also investigated the egg-
membrane protein of Murex, and, obtaining 0-5 per cent, of sulphur
from it, concluded, its other properties taken into account, that it
was a keratin. Engel also agreed with Hilger, whose figures for the
egg-membrane of the snake, Coluber natrix (see Table 30),
suggested an elastin as its principal component. He had not been
able to find any sulphur in it. About the same time, Wetzel examined
the conchiolin in_the_egg-shells of, Mytilus edulis, and obtained from
it, after hydrolysis, leucine, tyrosine, glycine, Various hexone bases
and ammonia, but no phenylalanine. The first efforts at quantitative
discrimination between egg-membrane proteins were contented with
ascertaining the elementary composition ; thus von Fiirth analysed
the protein of Loligo vulgaris eggs in this way (39 per cent,
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