SCIENCE ADVANCES | RESEARCH ARTICLE

CORONAVIRUS Copyright © 2020

The Authors, some
Antibody-like proteins that capture and rights reserved;
exclusive licensee
neutralize SARS-CoV-2 American Association
for the Advancement
of Science. No claim to
T. Kondo1, Y. Iwatani2,3, K. Matsuoka2, T. Fujino1, S. Umemoto1, Y. Yokomaku2, K. Ishizaki1, original U.S. Government
S. Kito1, T. Sezaki1, G. Hayashi1,4, H. Murakami1,5* Works. Distributed
under a Creative
To combat severe acute respiratory syndrome–related coronavirus 2 (SARS-CoV-2) and any unknown emerging Commons Attribution
pathogens in the future, the development of a rapid and effective method to generate high-affinity antibodies or NonCommercial
antibody-like proteins is of critical importance. We here report high-speed in vitro selection of multiple high-­ License 4.0 (CC BY-NC).
affinity antibody-like proteins against various targets including the SARS-CoV-2 spike protein. The sequences of
monobodies against the SARS-CoV-2 spike protein were successfully procured within only 4 days. Furthermore,
the obtained monobody efficiently captured SARS-CoV-2 particles from the nasal swab samples of patients and
exhibited a high neutralizing activity against SARS-CoV-2 infection (half-maximal inhibitory concentration,
0.5 nanomolar). High-speed in vitro selection of antibody-like proteins is a promising method for rapid development
of a detection method for, and of a neutralizing protein against, a virus responsible for an ongoing, and possibly
a future, pandemic.

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INTRODUCTION mation step using phage DNA, the efficiency of which limited the
The coronavirus disease 2019 (COVID-19) pandemic, caused by library size practically from 109 to 1011, is the main disadvantage of
the severe acute respiratory syndrome–related coronavirus 2 phage display. To generate highly diverse protein libraries, typically
(SARS-CoV-2), has negatively and deeply affected our lives and 1012 to 1013, a cell-free translation system has been used for mRNA
societies. To control the COVID-19 pandemic in addition to any display (18–20). However, this display method requires multistep
emerging, heretofore unknown pathogens in the future, it is of manipulations to synthesize protein/mRNA/puromycin linker (PuL)
paramount importance to rapidly generate multiple high-affinity complexes, the transcription of the mRNA, the attachment of the
antibodies or antibody-like proteins (ALPs) against virus proteins, PuL to the mRNA, and the translation of library proteins. These
thus developing both a detection method for the virus (1) and a multistep manipulations render mRNA display a time-consuming
neutralizing protein against it (2). method, with more than weeks typically being necessary to com-
Identification of a cross-reactive antibody among those developed plete six to seven rounds of the procedure (2 to 3 days per round).
against SARS-CoV is the fastest way to obtain a therapeutically useful We previously modified the mRNA display method and devel-
antibody (3–7). In principle, however, this approach would yield oped a high-speed in vitro selection method, i.e., transcription-­
lower-affinity antibodies with cross-reactivity to the original virus. translation coupled with association of PuL (TRAP) display, for the
The isolation of monoclonal antibodies from virus-infected patients identification of macrocyclic peptides (fig. S1A) (21). In the TRAP
is another possible approach (8), but the collection of B cell samples display, macrocyclic peptide/mRNA/PuL complexes are automati-
and the identification and large-scale production of effective anti- cally synthesized via the simple addition of the peptide template
bodies represent a long-term research and development project. DNA, thus skipping two time-consuming steps: transcription of
In vitro selection is advantageous in terms of the isolation speed mRNA and ligation of PuL. We reported the completion of six
of ALPs because time-consuming animal immunization is not rounds of selection in approximately 14 hours using the TRAP dis-
necessary to generate ALPs. This method also allows the use of play and obtained macrocyclic peptides with nanomolar affinity to
immunoglobulin protein scaffolds, including single-chain vari- their target protein. We also performed selection against the vascu-
able fragments (9, 10), single-domain antibodies (11), and non-­ lar endothelial growth factor receptor 2 (VEGFR2) and obtained a
immunoglobulin proteins. Non-immunoglobulin proteins such macrocyclic peptide with VEGF-induced angiogenesis inhibitory
as a lipocalin (12), a domain of fibronectin (13), the Z domain of activity (22).
protein A (14), the ankyrin repeat motif (15), and the SH3 domain Here, we report the development of an improved TRAP display
of Fyn (16) allow a greater chance to obtain high-affinity ALPs. to facilitate the selection of ALPs (Fig. 1A and fig. S1A). Subse-
Phage display is the method that is used most commonly for quently, we performed selection trials against the human EGFR1
in vitro selection (17). ALP libraries were expressed in Escherichia coli (HER1) and HER2 using a nanobody (the camelid single-domain
and displayed on the surface protein of phage. The E. coli transfor- antibody) (11, 23, 24) and a monobody (the 10th type III domain of
human fibronectin) (13) as backbone proteins. We further used this
1
method to rapidly obtain monobodies against the S1 subunit
Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya
University, Nagoya, Japan. 2Department of Infectious Diseases and Immunology,
of the SARS-CoV-2 spike protein. The selected high-affinity
Clinical Research Center, National Hospital Organization Nagoya Medical Center, monobodies were used to capture SARS-CoV-2 particles from
Nagoya, Japan. 3Division of Basic Medicine, Graduate School of Medicine, Nagoya the nasal swab samples of patients. We also tried to increase the
University, Nagoya, Japan. 4Japan Science and Technology Agency (JST), PRESTO, affinity of a monobody by dimerization. The monomeric and
Saitama, Japan. 5Institute of Nano-Life-Systems, Institutes of Innovation for Future
Society, Nagoya University, Nagoya, Japan. dimeric monobodies were used as a neutralizing protein against
*Corresponding author. Email: [email protected] SARS-CoV-2 infection.

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Fig. 1. Selection of monobodies against the SARS-CoV-2 spike protein using the TRAP display. (A) Schematic representation of the TRAP display. Monobody/mRNA
complexes were synthesized simply by adding the template DNA to the TRAP system. After reverse transcription (RT), selection, and PCR, the amplified DNAs were added
to the TRAP system to reproduce a monobody library for the subsequent round of selection. (B) Structure of the monobody backbone (10th type III domain of fibronectin,
1TTG). The BC and FG loops (labeled in blue) were randomized with the indicated number of residues. (C) Progress of the TRAP display selection. After each round of
selection, the recovered cDNA was quantified by real-time PCR. The recovery of cDNA was calculated by dividing the amount of recovered cDNA by the theoretical
amount of mRNA/PuL (1 M). At the fourth round, the selection pressure was increased by decreasing the target concentration from 20 to 2 nM. After the sixth-round
selection, the recovered DNA (*) was sequenced. The sequences of the monobodies are shown in Table 1. Stv, streptavidin; Bio, biotin; NTD, N-terminal domain; Pu, puromycin.

RESULTS We then used the 2′-OMe–modified PuL (mPuL) in the TRAP

Development of the improved TRAP display display. As expected, formation efficiency of the mRNA/mPuL com-
for ALP selection plex increased from 11% (dPuL) to 60% (mPuL), and display effi-
We started our study by measuring the display efficiencies of the ciency increased from 3% (dPuL) to 22% (mPuL) (fig. S1C). We also
nanobody and monobody on the mRNA because they determine analyzed the display efficiency of the anti-triclocarban nanobody
the diversity of ALP pools and the selection efficiency against a (23, 24) on the mRNA. Formation efficiency of the mRNA/mPuL
target protein. The monobody DNA template and the DNA-PuL complex was increased, from 9% (dPuL) to 45% (mPuL); display
(dPuL; fig. S1B) were added to the TRAP system, followed by efficiency of the nanobody on the mRNA also increased, from 2%
determination of the display efficiency of the monobody on the (dPuL) to 15% (mPuL) (fig. S1C).
mRNA. In contrast with our expectations, less than 3% of the dPuL The TRAP display was very useful for simplifying the selection
formed a monobody/mRNA/dPuL complex; moreover, most of the procedure, but it was not ideal for first-round selection because the
dPuL (87%) did not form an mRNA/dPuL complex (fig. S1C). As diversity of the library was limited by the amount of the DNA tem-
an mRNA sufficient to capture the dPuL (1 M) was produced plate added in the translation mixture (see fig. S2C for the optimi-
within 10 min in the reaction mixture (fig. S2A), an unexpected reaction zation of DNA concentration). To maximize the diversity of libraries,
occurred that prohibited the formation of the mRNA/dPuL complex. we usually stock an mRNA pool and add it to the translation
We found that the DNA portion of the dPuL complementary to reaction after the formation of the mRNA/PuL complex in the first-
the 21 mers of the 3′-end mRNA sequence (fig. S1B) was tran- round selection. Therefore, we also analyzed the display efficiency
scribed to RNA in a promoter-independent manner (25) and formed of the nanobody/monobody on the mRNA in the first-round
an unwanted DNA/RNA duplex, thus inhibiting formation of the format. We found that the display efficiencies were similar to that
mRNA/dPuL complex (fig. S2B). To prevent promoter-independent of the TRAP display format for both the monobody (19%) and
transcription, we added a 2′–methoxy (OMe) modification to the nanobody (15%) (fig. S1D).
ribose because of its reported prevention of T7 promoter–dependent Next, we conducted a selection trial using the monobody and
transcription (26). The 2′-OMe modification prevented promoter-­ nanobody libraries against EGFR1 and HER2 (fig. S3). High-affinity
independent transcription even after an incubation of 30 min binders (KD = 1.3 and 3.5 nM for EGFR1 and KD = 4.4 and 13 nM
(fig. S2B). for HER2) were obtained from the monobody library, whereas

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Table 1. Monobodies obtained by the TRAP display selection against the SARS-CoV-2 spike protein S1 subunit. The table contains the following
information: sequences of the BC and FG loops of the monobodies, the kinetic parameters determined by BLI, the inhibitory activity of the S1 subunit and ACE2
interaction, SARS-CoV-2 S1 subunit specificity, and clones with binding competition. Mutations were identified in the body sequence of clone 6 (S55W), clone
11 (L62M), and clone 12 (D67H). A Ser-to-Thr mutation in the BC loop of clone 11 was introduced during cloning. These mutations were reversed in the clones
6b, 11b, and 12b. Clones TD4, TD6b, and TD12b are tandem dimers of the corresponding clones. N.D., not detected.
kon (1/Ms) koff (1/s) × CoV-2 S1/ACE2 Binding
Clones BC loop FG loop KD (nM)
× 105 10−4 specificity inhibition competition
1 GVYDELGH SLWGYYTMWD 2.47 3.10 7.66 − − 11, 12, 18
4 PSSRYEHYQF WTGDVPWYWLVN 1.94 3.48 6.76 + + 9
6 GGDYVGYY TYNGPWIYGYEEI 0.76 1.47 1.11 + + 10
9 VYNVYPGT GSGVKYVVYRRS 17.8 1.83 32.6 + + 4
10 GHQDYGVS YYMGPDVYGRSEY 2.02 1.24 2.51 + + 6
11 TYGSSYGL ELWGYLTSWD 3.23 2.87 9.25 − − 1, 12, 18
12 EIYYEIGD RLWGYYTQWD 1.37 2.89 3.97 − − 1, 11, 18
16 PRSFDDSQ GYYQFVVYRYGG 24.8 1.22 30.3 N.D. − N.D.
18 MYGVVHGV SLWGYETYWD 0.65 2.19 1.42 − − 1, 11, 12

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6b GGDYVGYY TYNGPWIYGYEEI 0.42 2.34 0.99
11b SYGSSYGL ELWGYLTSWD 2.53 3.56 9.01
12b EIYYEIGD RLWGYYTQWD 0.62 3.44 2.13
TD4 PSSRYEHYQF WTGDVPWYWLVN <0.001 5.45 <0.001
TD6b GGDYVGYY TYNGPWIYGYEEI <0.001 2.48 <0.001
TD12b EIYYEIGD RLWGYYTQWD <0.001 6.11 <0.001
WT PAVT GRGDSPASSK N.D. N.D. N.D. N.D. N.D. N.D.
6b* GGDYVGYY TYNGPWIYGYEEI 1.18 0.95 1.13
TD4* PSSRYEHYQF WTGDVPWYWLVN 3.13 1.66 5.21
TD6b* GGDYVGYY TYNGPWIYGYEEI 2.92 0.54 1.59
TD12b* EIYYEIGD RLWGYYTQWD 1.96 0.82 1.62

*Monobody was immobilized on sensor chip.

mid-affinity ones were obtained from the nanobody library (KD = 47 maximize the diversity of the monobody library (1 M mRNA in a
and 32 nM for EGFR1 and KD = 11 and 9.9 nM for HER2). 500-l scale; monobody display efficiency on the mRNA of about
10%; 3 × 1013 calculated molecules; fig. S4). We used mixed targets
High-speed selection of monobodies against the in the first round to optimize the effort of the large-scale preparation
SARS-CoV-2 spike protein S1 subunit of the cell-free translation system. From the second round, we used
According to the results presented above, we focused on the mono- the TRAP display to boost the speed of selection and simultaneously
body for the subsequent library construction. We introduced 8 or conducted selection procedures against each target. We observed an
10 random residues at the BC loop (Pro 25-Val29) and 10 or 12 enrichment of binder monobodies against both targets in the third
random residues at the FG loop (Gly77-Lys86) of the monobody round of selection (Fig. 1C). To obtain higher-affinity monobodies,
(Fig. 1B), according to previous reports (13, 20, 27, 28). We used a we performed an additional three rounds of selection using a 10 times
Tyr-, Ser-, Gly-, and Trp-rich codon mix to construct the random lower target concentration (2 nM). After the six rounds of selection,
residues (27, 28). The preparation of the mRNA library took 6 months the recovered complementary DNAs (cDNAs) of monobodies
after we ordered the oligonucleotides due to the oligonucleotide were sequenced. Notably, because of the high-speed in vitro selec-
synthesis using trinucleotide phosphoramidites (29) and the large- tion feature of the TRAP display (Fig. 1A), we successfully completed
scale polymerase chain reaction (PCR) and transcription proce- the selection within 3 days and obtained the monobody sequences on
dures that were applied to maintain the diversity of the monobody the 4th day.
library. However, once the pool of mRNA was prepared, it could be
used for multiple in vitro selection studies against various targets RBD-specific monobodies with low- to sub-nanomolar
due to the large amount of mRNA produced. KD values
With the pool mRNA in our hands, we received the two bioti- As the same clones were enriched through the selection against
nylated targets, the SARS-CoV-2 spike protein S1 subunit (S1-biotin) both the S1 subunit and the RBD, we selected the nine most en-
and the receptor binding domain (RBD-HRP-biotin), and started riched clones in the pool (Table 1) for further study. To confirm the
the TRAP display selection (Fig. 1A). In the first round of the selec- binding activity, we immobilized the purified monobodies (table S1
tion, an mRNA template, rather than a DNA one, was used to and fig. S5) on a microwell plate and added either the S1 subunit

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(S1-HRP) or the (RBD-HRP) conjugated with streptavidin–horseradish nM; clone 10, KD = 2.02 nM; clone 11, KD = 3.23 nM; clone 12,
peroxidase (Stv-HRP). The results of the enzyme-linked immuno- KD = 1.37 nM; clone 18, KD = 0.65 nM). Since mutations were found
sorbent assay (ELISA) showed that the all monobodies were able to in clone 6 (S55W), clone 11 (L62M and S to T in the BC loop), and
bind to both targets (Fig. 2A). The wild-type (WT) monobody (the clone 12 (D67H), we also determined the kinetic parameters of the
control protein with the original loop sequences) did not bind to back mutants (6b, 11b, and 12b). The affinities were similar to those
either of the targets. These results clearly indicated that the selected observed for the parental ones, suggesting that these mutations do
loop sequences were responsible for the binding to the RBD of the not contribute to the binding affinity. It was interesting to obtain
S1 subunit. high-affinity monobodies because the in vitro selection of ALPs
To study the stability of monobodies, C-terminally biotinylated sometimes required additional affinity maturation after the initial
monobodies prepared by sortase A reaction (monobody-biotin; selection to obtain high-affinity monobodies. We believe that this
fig. S5) were incubated overnight at various temperatures, and the finding is attributable to either the high antigenicity of the S1
remaining binding activity was measured by ELISA using an S1 subunit or the high diversity/quality of the library prepared using
subunit–immobilized microplate and Stv-HRP. We found that all trinucleotide phosphoramidite synthesis (27–29).
monobodies were stable at 37°C and that five of them retained half
of the binding activity after incubation at 50°C overnight (fig. S6). Monobodies bound to the SARS-CoV-2 spike protein S1
We further assessed the kinetic parameters of all nine monobodies subunit but not to that of SARS-CoV
using biolayer interferometry (BLI). S1-biotin was immobilized on Next, we studied the specificity of the monobodies for the SARS-
the streptavidin-immobilized sensor, and various concentrations of CoV-2 spike protein S1 subunit because this parameter is important
monobodies were added to determine the kinetic parameters based for the development of a future diagnostic assay for this virus. Since

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on global fitting (Fig. 2B and Table 1). The seven clones exhibited a the SARS-CoV-2 spike protein sequence exhibits greater similarity
sub-nanomolar to nanomolar-level affinity against the S1 subunit with that of SARS-CoV than it does with other human coronaviruses
(clone 1, KD = 2.47 nM; clone 4, KD = 1.94 nM; clone 6, KD = 0.76 (30), we decided to test the specificity of the monobodies against the

Fig. 2. Binding activity of each clone against the SARS-CoV-2 spike protein S1 subunit and the RBD. (A) Determination of the binding domain. A schematic repre-
sentation of the ELISA is provided on the left. S1-HRP or RBD-HRP (10 nM) was added to the monobody-immobilized microplate. Error bars indicate SD of each experiment
(in triplicate). (B) Determination of kinetic parameters by BLI. A schematic representation of the BLI is provided at the top. S1-biotin was immobilized on a streptavidin-­
sensor chip, and monobodies (2.5 to 40 nM) were used in the kinetic analysis. Some mutations were identified in clones 6, 11, and 12. These mutations were reversed in
the clones 6b, 11b, and 12b. The data are depicted in blue, and the fit data to a 1:1 binding model are shown in black. The determined kinetic parameters of the mono-
bodies are provided in Table 1. WT, 10th type III domain of fibronectin; RLU, relative luminescence units; Nus, Nus-Tag fused at the C terminus of the monobody.

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S1 subunit of SARS-CoV-2 and SARS-CoV. The pull-down experi- on a microplate, and the premixed solution of ACE2-HRP and each
ment using monobody-immobilized beads showed four monobodies monobody was added. The results of ELISA showed that monobody
(clones 4, 6, 9, and 10) bound to the SARS-CoV-2 S1 subunit but clones 4, 6, 9, and 10 at a concentration of 100 nM inhibited the
not to the SARS-CoV S1 subunit (Fig. 3A), suggesting that these binding of the S1 subunit to ACE2 (Fig. 3B); moreover, the inhibi-
monobodies are specific for the SARS-CoV-2 S1 subunit. In turn, tion was retained for three of the monobodies (clones 4, 6, and 10)
four monobodies (clones 1, 11, 12, and 18) bound to both targets, at the low concentration of 10 nM (Fig. 3C). These monobodies also
and clone 16 did not bind to either of the S1 subunits due to a low exhibited specificity toward the SARS-CoV-2 S1 subunit (Fig. 3A),
binding affinity (Table 1). Clone 16 pulled down a spike protein probably because the ACE2-binding subdomain of the RBD contains
trimer (Fig. 3A), probably due to a multivalent effect of the target. more substitutions than other regions of the spike protein (5, 6).

Monobodies inhibited the interaction between Application of monobodies in sandwich ELISA

angiotensin-converting enzyme 2 and the S1 subunit The nine monobodies studied above exhibited binding activity
To determine whether the binding site of the monobodies over- toward the S1 subunit. This observation led us to develop a sandwich
lapped with that of the angiotensin-converting enzyme 2 (ACE2), ELISA for the detection of the S1 subunit because it requires a pair
we also tested the inhibition of the S1 subunit/ACE2 interaction of antibodies that recognize distinct epitopes of the S1 subunit. To
(31) afforded by the monobodies. The S1 subunit was immobilized explore for these monobody pairs, we tested all combinations of the

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Fig. 3. Characterization of monobodies and the application for the sandwich ELISA. (A) A schematic representation of the pull-down assay is provided at the top. The
SARS-CoV S1 subunit (100 nM), SARS-CoV-2 S1 subunit (100 nM), or SARS-CoV-2 S protein trimer (100 nM) were pulled down by monobody-immobilized beads. The
supernatant and the heat elution from the beads were loaded onto SDS–polyacrylamide gel electrophoresis (PAGE), followed by staining with SYPRO Ruby. (B) A schematic
representation of the ELISA is provided on the left. ACE2-HRP (1 nM) was mixed with each monobody (100 nM) and was added to an S1-immobilized microplate. (C) ACE2-
HRP (1 nM) was mixed with each monobody (10 nM clones 4, 6, 9, or 10) and was added to an S1-immobilized microplate (before). Alternatively, a monobody was added
to an ACE2-HRP–incubated S1-immobilized microplate (after). (D) A schematic representation of the sandwich ELISA is provided on the left. The S1 subunit (10 nM) was
added to the monobody-immobilized microplate. The S1 subunit bound with the capturing monobody was identified by detecting monobody-HRP (10 nM). All possible
combinations were tested. (E) Titration curves of the SARS-CoV-2 S1 subunit, SARS-CoV-2 spike protein trimer, and SARS-CoV S1 subunit. After incubation of the analyte
with the detecting monobody-HRP (clone 12, 1 nM), the solution was added to the capturing monobody (clone 10)–immobilized microplate. Error bars indicate SD of
each experiment (in triplicate). S1, SARS-CoV or SARS-CoV-2 spike protein S1 subunit or SARS-CoV-2 spike protein trimer; N.D., not determined; MK, protein maker (kDa).

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cloned monobodies in a sandwich ELISA format and found that SARS-CoV-2 in a 100-l solution, corresponding to 1 fM spike
the monobodies recognized three different epitopes (Fig. 3D and protein), its combination with other detection methods, such as
Table 1). Monobody clones 1, 11, 12, and 18 shared an epitope that digital ELISA (32) or loop-mediated isothermal amplification (33),
was not located at the ACE2-binding surface (Fig. 3, B and D). The may help to overcome the problem of the detection limit in a future
FG loop sequences of these monobodies were similar (xLWGYxTxWD; study.
with x representing a nonconserved residue), suggesting that they
are the main antigen-binding loop. Monobody clones 6 and 10 Capture of SARS-CoV-2 virions using monobodies
shared another epitope that was likely located in the ACE2-binding from a cultivated sample
subdomain (Fig. 3, B and D). These clones exhibited a conserved Next, we studied the binding of monobodies to SARS-CoV-2. Each
sequence in the FG loop (xYxGPxxYGxxEx), which will be investi- monobody-biotin was incubated with SARS-CoV-2 particles in
gated in a future study. Monobody clones 4 and 9 shared another phosphate-buffered saline (PBS), and the viral particles bound to
epitope that was also likely located in the ACE2-binding surface the monobodies were collected on the magnetic beads. A reverse
(Fig. 3, B and D). No conserved sequence was detected in the BC transcription quantitative PCR (RT-qPCR) assay of the SARS-
loop or the FG loop of these clones. Therefore, we identified 16 CoV-2 RNA extract in the supernatants and the bead eluents
pairs that can be used in further sandwich ELISA. Among them, we showed that a large proportion of the SARS-CoV-2 particles were
tested clone 10 as a capturing monobody and clone 12 as a detecting detected in the bead eluents for all monobodies, except for the WT
monobody because the pair gave one of the highest signal-to-noise monobody (Fig. 4A), proving the binding activity of all selected
ratios in the sandwich ELISA experiment (Fig. 3D). The results of monobodies toward SARS-CoV-2 particles.
sandwich ELISA showed that a combination of monobodies could On the basis of the experiment described above, we realized that

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specifically detect the SARS-CoV-2 spike protein trimer and the S1 this pull-down method would also be useful for enhancing the
subunit (Fig. 3E). Although the sensitivity of this system was not detection limit of SARS-CoV-2. To test this idea, we suspended
sufficient to detect the target in actual samples (103 particles of SARS-CoV-2 particles in a PBS solution at various concentrations

Fig. 4. Monobody binding to SARS-CoV-2 from culture and patient samples. (A) Monobody binding activities toward SARS-CoV-2 particles. SARS-CoV-2 particles
bound with monobody-biotin were pulled down by M280-streptavidin beads. The RNA genomes in the supernatant and on the beads were quantified by RT-qPCR after
RNA extraction. (B) The detection limits of the traditional RT-qPCR method and the pull-down RT-qPCR method. Various concentrations of SARS-CoV-2 (0.1 to 10,000
particles/l) were assayed. SARS-CoV-2 particles were collected from 700 l of solution by pull down using monobody clone 4, and viral RNA was quantified by RT-qPCR
after RNA extraction. Alternatively, the original solution (14 l) was subjected to RT-qPCR. (C) Pull down of SARS-CoV-2 particles from nasal swab samples (35 l) of pa-
tients. The number of RNA genomes in the supernatant and on the beads was quantified by RT-qPCR after RNA extraction. (D) Pull-down direct RT-qPCR using nasal swab
samples of patients. SARS-CoV-2 particles were collected from 400 l of nasal swab samples by pull down and were directly added to an RT-qPCR reaction mixture.
Alternatively, an original nasal swab solution (2.5 l) was added to an RT–droplet digital PCR (ddPCR) reaction mixture. Error bars indicate SD of RT-qPCR results (in triplicate).
N.D., not detected.

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(0.1 to 10,000 particles/l) and analyzed the number of particles by detected only by the pull-down direct RT-qPCR but not by tradi-
pull-down RT-qPCR using monobody clone 4 or by a traditional tional direct RT-qPCR, suggesting the increased sensitivity of the
RT-qPCR. The result showed a wide linear dynamic range of this method pull-down direct RT-qPCR assay.
(Fig. 4B). The detection limit was increased from 1 to 0.1 particles/l.
Binding activity of monobody tandem dimers
Monobody capture of SARS-CoV-2 virions from nasal swab A dimeric form of a protein binder usually showed higher affinity
samples of patients than the monomeric one (7, 16); therefore, we next synthesized the
We then performed a pull down of SARS-CoV-2 particles from tandem dimers of a monobody. We chose clones 4, 6b, and 12b to
nasal swab samples of patients (Fig. 4C). Notably, the actual patient synthesize the corresponding tandem dimers (TD4, TD6b, and
samples (sometimes in the form of highly viscous liquids) were very TD12b) because these clones showed the highest affinity in each
different from cultured samples, primarily because they contain group defined by the binding competition assay (Fig. 3D). As ex-
many unknown components. We tested 16 samples and found that pected, the affinities of these tandem dimers were markedly im-
the monobody captured SARS-CoV-2 even in these nasal swab proved (Fig. 5A and Table 1): The KD value displayed less than 1 pM
samples. To our knowledge, this is the first example of an antibody due to the immeasurable koff value (10−7/s).
or an ALP to capture SARS-CoV-2 from patient nasal swab samples. To understand why the dimerization of monobodies markedly
For samples 8, 15, and 16, the capture rates were notably lower than improved the affinity, we first immobilized the decreased amounts of
the remaining cases. We reasoned that this might be because the S1 protein on the sensor chip and analyzed monobody dissociation.
monobody binding site was hidden by neutralizing antibodies de- The results showed that the monobody dimers still dissociated very
veloped in the patients. slowly (fig. S7A). Next, when the monobodies were immobilized on

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Last, we performed a pull-down direct RT-qPCR for nasal swab the sensor chip and the S1 protein was used as an analyte, we
samples (400 l) of patients (Fig. 4D). We tested four samples this observed a similar KD value for clone 6b (Fig. 2 versus fig. S7B and
way and found that the monobody captured SARS-CoV-2 even in Table 1) and no improvement on the KD values for the tandem
higher-volume nasal swab samples. The number of RNA genomes dimers (Fig. 2 versus Fig. 5B and Table 1). These results suggest that
was increased 25 to 77 times after the pull-down technique was the tandem connection of monobodies does not improve the affinity
used. Moreover, RNA genome in the nasal swab sample 4 was but bringing up the bridging effect, i.e., an avidity effect.

Fig. 5. The affinity of monobody tandem dimers and the neutralization of SARS-CoV-2 infection. (A) Determination of kinetic parameters of tandem dimers by
BLI. S1-biotin was immobilized on a streptavidin-sensor chip, and the tandem dimers (2.5 to 40 nM) were used in the kinetic analysis. (B) The biotinylated tandem dimers
of monobody were immobilized on a streptavidin-sensor chip (*), and the S1 subunit (2.5 to 40 nM) was used in the kinetic analysis. TD4, TD6b, and TD12b are tandem dimers
of the corresponding clones 4, 6b, and 12b connected with a (GGGGS)3 linker. The data are depicted in blue, and the fit data to a 1:1 binding model are shown in black.
The determined kinetic parameters of the monobodies are provided in Table 1. (C) Monobody-mediated neutralization of SARS-CoV-2 infection in VeroE6/TMPRSS2 cells.
The x-axis value indicates the final concentration of the indicated monobody (6b, TD6b, or WT) for each assay well. The experiment was performed with triplicate samples. The
copy number of each RNA in the supernatant is plotted.

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SCIENCE ADVANCES | RESEARCH ARTICLE

Monobodies exhibited a high neutralizing activity against RT-qPCR and positive control RNA for N2 were purchased from
SARS-CoV-2 infection Applied Biosystems Inc. (NY, USA). The synthetic DNAs were
The monobody clone 6b showed high affinity against the S1 subunit acquired from GenScript (NJ, USA). The sequences of the primers
of the SARS-CoV-2 spike protein and inhibited the interaction and synthetic DNAs are listed in data file S1. SARS-CoV-2 protein
between ACE2 and the S1 subunit (Fig. 3C). These findings led active trimer (SPN-C52H8), human ACE2 protein (AC2-H5257),
us to analyze the neutralizing activity of monobody clones 6b and biotinylated 2019-nCoV S1 subunit–Avitag (S1N-C82E8), biotinylat-
TD6b against SARS-CoV-2 infection. We added fourfold serially ed 2019-nCoV S protein RBD-Avitag (SPD-C82E9), SARS-CoV-2
diluted monobody solutions to VeroE6 cells expressing the trans- (COVID-19) S1 subunit (S1N-C52H4), and SARS S1 subunit (S1N-
membrane serine protease (TMPRSS2), VeroE6/TMPRSS2 cells, (34) S52H5) were all purchased from ACROBiosystems (DE, USA).
in 96-well culture plates, followed by infection with SARS-CoV-2 EGFR/Fc chimera and ErbB2/Fc chimera were obtained from R&D
[105 TCID50 (median tissue culture infectious dose)/ml] for 1 hour Systems (MN, USA). Creatine kinase, creatine phosphate, and E. coli
at 37°C. After 36 hours of incubation with fresh medium, SARS-CoV-2 transfer RNAs were purchased from Roche Diagnostics (Japan).
mRNA amounts in the supernatant were measured by one-step RT-­ The restriction enzymes were obtained from New England Biolabs
qPCR. The monobody clone 6b showed a very low half-maximal (MA, USA).
inhibitory concentration (IC50, 0.5 nM; Fig. 5C), even in the mono-
meric form. Its neutralizing activity was increased in the dimeric Preparation of monobody mRNA libraries for selection
form (IC50, 0.4 nM), although the data obtained for 390 pM TD6b against SARS-CoV-2 targets
fluctuated, probably due to the high sensitivity of VeroE6/TMPRSS2 To prepare an A-fragment DNA of the monobody library, FN3F0.
cells. These IC50 values were comparable to those of the high-affinity F83 (1 M), FN3F1-2.F29(P) (1 M), and the FN3FF1coR8.F73

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human neutralizing antibodies reported recently (35–37), indicating the (0.5 M) or FN3FF1coR10.F79 (0.5 M) were ligated by T4 DNA ligase
usefulness of the TRAP display for the selection of highly active ALPs. (75 l in total, 75 pmol for each oligonucleotide) with an assistance
of Fn3an1.R20(3NH2) (2 M) and Fn3an2-1.R20(3NH2) (2 M).
As codons for the randomized residues, we used a codon mix with
DISCUSSION the following ratios: 20% Tyr, 10% Ser, 15% Gly, 10% Trp, and 3%
Here, we developed a high-speed in vitro selection method to each of all the other amino acids except for Cys, which is similar to
obtain ALPs against various targets. We demonstrated a constant the original cocktail (30% Tyr, 15% Ser, 10% Gly, 5% Phe, 5% Trp,
development of high-affinity binders (KD values in the low- or and 2.5% each of all the other amino acids except for Cys) (27, 28).
sub-nanomolar range) against EGFR1, HER2, and the SARS-CoV-2 After the ligation, the entire mixture was added to the reaction mix-
spike protein. Furthermore, our selected monobodies recognized a ture [10 mM tris-HCl (pH 8.4), 100 mM KCl, 0.1% (v/v) Triton
recombinant spike protein and SARS-CoV-2 particles, demonstrat- X-100, 2% (v/v) dimethyl sulfoxide, 2 mM MgSO4, 0.2 mM each
ing the applicability for a sandwich ELISA for a rapid antigen test. deoxynucleotide triphosphate (dNTP), 0.375 M T7SD8M2.F44,
Using the monobody before performing pull-down RT-qPCR also 0.375 M FN3BsaI.R40, and 2 nM Pfu-S DNA polymerase] and the
increased the detection limit of SARS-CoV-2. The monobody amplified by PCR (15 ml in total, seven cycles of PCR). B-fragment
captured SARS-CoV-2 in nasal swab samples of patients with DNA was prepared by the same procedure using FN3FF2co.F72(p),
COVID-19. Actual patient samples are very different from cultured FN3F3coR10.F70(p), FN3F3coR12.F76(p), and Fn3an3.R20(3NH2)
samples because they consist of inhomogeneous liquid and contain for ligation and FN3BsaI.F33 and FN3Pri2.R44 for amplification.
many unknown components, including various proteases, mucins, The amplified A-fragment DNA and B-fragment DNA were
and neutralization antibodies. We believe that the monobodies pro- purified by phenol/chloroform extraction and isopropanol precipi-
cured in our study will soon be useful to develop effective diagnostic tation. One end of each DNA product was digested with Bsa I (New
tools and that these tools will contribute to the worldwide effort to England Biolabs, MA, USA), as per the manufacturer’s protocol,
overcome the COVID-19 pandemic. The monobody also had a very low and the DNA products were then purified by phenol/chloroform
IC50 value against SARS-CoV-2 infection, which was comparable to extraction and isopropanol precipitation. The products were ligated
those of the antibodies that were reported recently as being highly to each other (1 M, 200 l) to synthesize full-length DNA products,
active for neutralizing SARS-CoV-2 (35–37). Because the backbone and they were amplified using T7SD8M2.F44, G5S-4Gan21-3.R42,
of the monobody was the 10th type III domain of human fibronectin, and Pfu-S DNA polymerase (60 ml in total, four cycles of PCR). The
it might be useful as a neutralizing ALP against SARS-CoV-2 infection. products were purified through phenol/chloroform extraction and
We received the SARS-CoV-2 spike protein S1 subunit on 7 April 2020 isopropanol precipitation. The DNA template was transcribed by
as a target of the TRAP display selection and, using the methods in vitro run-off transcription, and the mRNA was purified by iso-
described herein, obtained high-affinity monobody sequences on propanol precipitation, followed by polyacrylamide gel electrophoresis
10 April 2020. The high-speed feature of the improved TRAP (PAGE) purification. The mRNA/hexachloro-fluorescein (HEX)–mPuL
display is, therefore, useful for rapid response to the subspecies of was prepared by a similar procedure described above. The resulting
SARS-CoV-2 (38, 39) in addition to potential new viruses causing complex was directly used in the first round of selection.
future pandemics.
In vitro selection of monobodies against SARS-CoV-2 spike
protein S1 subunit and RBD of the S1 subunit by
MATERIALS AND METHODS the improved TRAP display
Materials For first-round selection, 1 M mRNA/puromycin-OMe linker was
The oligonucleotides were purchased from either FASMAC Co. Ltd. added to a reconstituted translation system, and the reaction mixture
(Japan) or Nippon Bio Service (Japan). The primer/probe set for (500 l) was incubated at 37°C for 30 min. After the reaction, 41.7 l

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SCIENCE ADVANCES | RESEARCH ARTICLE

of 200 mM EDTA (pH 8.0) was added to the translation mixture. A step in the binding assay was as follows: equilibration for 150 s,
reverse transcription buffer [41.1 l of 0.78 M tris-HCl (pH 8.4), association for 900 s, and dissociation for 900 s (EGF and HER2);
1.16 M KCl, 0.37 M MgCl2, and 0.08 M dithiothreitol (DTT)], 5 mM equilibration for 150 s, association for 600 s, and dissociation for
dNTPs (66.7 l), 100 M FN3S.R29 (10 l), and 28.7 M in-house 600 s (S1 subunit). Buffer D′ [50 mM Hepes-KOH (pH 7.5), 300 mM
moloney murine leukemia virus reverse transcriptase (HMLV) NaCl, 0.1% (v/v) Tween 20, and 1% (w/v) PEG-6000] was used
(27.5 l) were added to the translation mixture, and the resulting when the S1 subunit was an analyte.
solution was incubated at 42°C for 15 min. The buffer was ex-
changed to HBST buffer [50 mM Hepes-KOH (pH 7.5), 300 mM Binding assay of monobodies against SARS-CoV-2 S1 or S1
NaCl, 0.05% (v/v) Tween 20] using Zeba Spin Desalting Columns. RBD proteins
To remove the bead binders, the resulting solution was mixed The ELISA microplate with 96 wells was coated with 100 l of 100 nM
with Dynabeads M-280/M-270 streptavidin (1:1) (Thermo Fisher Sci- monobody in HBS2 buffer [25 mM Hepes-K (pH 7.5) and 150 mM
entific) at 25°C for 10 min. The supernatant was mixed with 4.8 l of NaCl] overnight. The microplate was washed once with HBST2
7.3 M biotinylated 2019-nCoV S1 subunit–Avitag and 2.1 l of buffer [HBS2 with 0.05% (v/v) Tween 20], blocked with 1% (w/v)
16.5 M biotinylated 2019-nCoV S protein RBD-Avitag and then bovine serum albumin (BSA) in HBS2 buffer at room temperature
incubated at 25°C for 5 min. After recovering the target proteins with for 1 hour, and then washed once with HBST2 buffer. S1-HRP or
Dynabeads M-270 streptavidin, the resulting beads were washed with RBD-HRP was prepared by mixing S1-biotin or RBD-biotin with
HBST buffer thrice, and PCR premix (1 ml) was added. The beads the same amount of Stv-HRP in HBST2BP buffer [HBST2 with
were heated at 95°C for 5 min, and the amount of the eluted cDNAs 0.1% (w/v) BSA and 0.1% (w/v) PEG-6000] and was added to the
was quantified by SYBR green–based qPCR using T7SD8M2.F44 and microplate with a final concentration of 10 nM. The microplate was

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FN3Lip.R20 as primers. The eluted cDNAs were PCR-amplified incubated at room temperature for 1 hour. The microplate was
using T7SD8M2.F44, G5S-4Gan21-3.R42, and Pfu-S DNA polymerase washed five times with HBST2, and 200 l of SuperSignal ELISA
and was purified by phenol/chloroform extraction and isopropanol Pico was added to each well. The chemiluminescence was measured
precipitation. The posttranslation and post–reverse transcription immediately using the SpectraMax M5 plate reader (Molecular Devices).
reaction mixtures were analyzed using the same procedure as
described above. Pull down of S proteins with monobody-immobilized beads
For the second-round selection, the resulting DNA (about 3 to Biotinylated monobody (1 M, 3.3 l) in HBST2BP buffer was
10 nM in final concentration) was added to the TRAP system, and mixed with Dynabeads M-280 streptavidin (10 mg/ml, 6.6 l,
the reaction mixture (40 l) was incubated at 37°C for 30 min. After prewashed by HBST buffer) at room temperature for 5 min. After
the reaction, 8 l of 100 mM EDTA (pH 8.0) was added to the trans- removing the supernatant, the beads were washed twice with HBST
lation mixture. Reverse transcription mixture [24 l of 150 mM tris- buffer, and 2.5 l of 100 nM each analyte (SARS-CoV-2 spike
HCl (pH 8.4), 225 mM KCl, 75 mM MgCl2, 16 mM DTT, 1.5 mM protein S1 subunit, SARS-CoV spike protein S1 subunit, and SARS-
dNTPs, 7.5 M primer, and 3.4 M HMLV] were added to the CoV spike protein trimer) in buffer D was added to a one-third
translation mixture, and the resulting solution was incubated at fraction of the beads. After mixing at room temperature for 30 min,
42°C for 15 min. The buffer was exchanged to HBST buffer using the supernatant was collected. The beads were washed twice with
Zeba Spin Desalting Columns. To remove the bead binders, the re- HBST buffer. The supernatant and heat elution from the beads were
sulting solution was mixed thrice with M270/M280 magnetic beads loaded on SDS-PAGE gel and was stained using SYPRO Ruby.
(1:1) at 25°C for 20 min. Each target (20 nM final concentration)
was added to the half portion of the supernatant. After removing Inhibition of ACE2/CoV-2 S1 subunit interaction by
the supernatant, the beads were washed with HBST buffer thrice, monobodies
and the PCR premix was added to the beads. Quantitation of cDNA ELISA was performed using a microplate with 96 wells, coated with
and amplification and purification of DNA were performed by the 100 l of 2 nM SARS-CoV-2 spike protein S1 subunit in HBS2 buffer
same procedure as described for the first-round selection. overnight. The microplate was washed once with HBST2 buffer,
For the third- to sixth-round selection, the procedure was per- blocked with 1% (w/v) BSA in HBS2 buffer at room temperature for
formed similarly as that of the second round, except for the volume 1 hour, and then washed once with HBST2 buffer. Biotinylated
of the reaction mixtures (5 l), the target concentration (2 nM in the ACE2 complexed with same amount of Stv-HRP in HBST2BP buffer
fourth to sixth rounds), and bead washing conditions (an extensive was added to a final concentration of 1 nM together with 100 or 10 nM
wash in the sixth-round selection against S1 subunit). After the monobody. Alternatively, a monobody (10 nM in total) was added to
sixth-round selection, the sequences of the recovered DNAs were the ACE2-HRP–preincubated S1-immobilized microplate. The micro-
analyzed using an Ion Torrent instrument (Thermo Fisher Scientific). plate was incubated for 1 hour at 37°C. The plate was then washed
five times with HBST2 buffer. After the addition of SuperSignal ELISA
Affinity measurement of selected nanobodies Pico (200 l) to each well, the chemiluminescence was recorded im-
and monobodies mediately using a SpectraMax M5 plate reader.
The affinity measurement was performed against the EGFR/Fc
chimera or the ErbB2/Fc chimera immobilized on an anti-human Sandwich ELISA using monobodies
immunoglobulin G Fc capture biosensor (ForteBio), biotinylated The sandwich ELISA was performed using a microplate with 384
2019-nCoV S1 subunit–Avitag, or biotinylated monobodies immo- wells, which was coated with 25 l of 100 nM of the capturing
bilized on a streptavidin biosensor (ForteBio), using Octet system monobodies (nine clones and WT) in HBS2 buffer overnight. The
(ForteBio, CA, USA), as described in the manufacturer’s instruc- microplate was washed once with HBST2 buffer, blocked with 1%
tions. The binding assay was performed at 30°C in the buffer D. Each (w/v) BSA in HBS2 buffer at room temperature for 1 hour, and then

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SCIENCE ADVANCES | RESEARCH ARTICLE

washed once with HBST2 buffer. SARS-CoV-2 S1 subunit (10 nM) clone 4 in PBS [10 mM phosphate (pH 7.4), 2.7 mM KCl, and 137 mM
in HBST2BP buffer was added. The microplate was incubated at NaCl] containing 0.05% (v/v) Tween 20 at room temperature for
room temperature for 1 hour and was subsequently washed once 10 min. The solution was mixed with Dynabeads M-280 streptavidin
with HBST2 buffer. Biotinylated detecting monobody (nine clones (10 mg/ml, 8 l, prewashed by PBS buffer) at 25°C for 10 min. The
and WT) complexed with same amount of Stv-HRP in HBST2BP beads were washed with HBST three times and were suspended in
buffer was added to a final concentration of 10 nM, and the plate HBST. Viral RNA from the bead suspension or the original solu-
was incubated at room temperature for 30 min. The microplate was tion (14 l) was extracted using the QIAamp Viral RNA Mini Kit.
washed five times with HBST2 buffer, and 50 l of SuperSignal For RT-qPCR, the One-Step PrimeScript III RT-qPCR Mix with the
ELISA femto (Thermo Fisher Scientific) was added to each well. N2 primer/probe set was used. The target copy numbers were deter-
The chemiluminescence was immediately recorded using the mined using the Thermal Cycler Dice Real Time System III (Takara
SpectraMax M5 plate reader. Bio), as aforementioned.
For the other sandwich ELISA, a microplate with 96 wells was
coated with 100 l of 100 nM monobody clone 10 in HBS2 buffer Pull down of SARS-CoV-2 in nasal swab specimens
overnight. The microplate was washed once with HBST2, blocked from patients
with 1% (w/v) BSA in HBS2 buffer at room temperature for 1 hour, For the pull-down assay using nasal swab specimens from patients,
and washed once with HBST2 buffer. One of the analytes, the SARS- we used residual nasal samples after diagnostic tests. The study was
CoV-2 S1 subunit, the spike protein trimer, or the SARS-CoV S1 approved by the ethical committee at the Nagoya Medical Center
subunit, was incubated with the detecting monobody-HRP (clone (registration no. 2019-087) and conducted according to the tenets
12, 1 nM) on ice for 1 hour. The solution was then added to the of the Declaration of Helsinki. Written informed consent for the

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capturing monobody–immobilized microplate and was incubated use of the residual samples was obtained from all participants. Thirty-­
at room temperature for 1 hour. The microplate was washed five five microliters of each nasal sample was mixed with 2 nM monobody
times with HBST2 buffer, and 200 l of SuperSignal ELISA femto clone 4 to a total volume of 100 l in HBST buffer and incubated at
was added to each well. The chemiluminescence was immediately 25°C for 10 min. The mixture was then incubated for another 10 min
recorded using the SpectraMax M5 plate reader. with Dynabeads M-280 streptavidin (2.5 mg/ml, 40 l, prewashed
by HBST buffer). The RNA copies in both the supernatant and the
Binding assay of monobodies toward SARS-CoV-2 particles bead fraction were measured using RT-qPCR, as described above.
The SARS-CoV-2 virus was isolated from VeroE6 cells (American
Type Culture Collection) from a nasal swab sample of a patient in Pull-down direct RT-qPCR assay of SARS-CoV-2 in nasal swab
the Nagoya Medical Center, Japan. Virus-infected cells were grown specimens from patients
in Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) supple- Each residual sample of nasal swab from four patients was used for
mented with 10% fetal bovine serum and penicillin (100 U/ml) and this assay. Briefly, 0.5% Tween 20 (45 l) and monobody clone 4
streptomycin (100 g/ml) (Thermo Fisher Scientific). Four days (5 l of 200 nM) were added into 400 l of the swab sample in
after infection, the supernatant was harvested. The supernatant was PBS. The mixture was incubated at 25°C for 10 min, followed by
clarified by centrifugation and filtration through a 0.45-m pore- addition of Dynabeads M-280 streptavidin (2.5 mg/ml, 10 l, pre-
size syringe filter (Merck Millipore) and used for the virion-binding washed by HBST buffer). After 10 min of incubation, the beads
assay. The virus particle amounts were measured using droplet dig- were washed with 1 ml of HBST three times and resuspended with
ital PCR (ddPCR) using the One-Step RT-ddPCR Advanced Kit for 2.5 l of HBST containing carrier RNA. The bead suspension or the
Probes (Bio-Rad) with a primer/probe set for N2 (data file S1) (40). original swab solution (2.5 l) was mixed with an RT-qPCR reac-
SARS-CoV-2 particles (5 × 104 particles/140 l) and 10 nM mono- tion mixture (47.5 l; PrimeDirect Probe RT-qPCR Mix, Takara
body in HBST buffer were incubated at 25°C for 10 min. This was Bio) with a primer/probe set (data file S1). The RNA copy numbers
followed by the addition of Dynabeads M-280 streptavidin (10 mg/ml, were determined using the Thermal Cycler Dice Real Time System
10 l, prewashed by PBS buffer), which were incubated with the solu- III (Takara Bio), as aforementioned. The detection level of positive
tion for 10 min. After the incubation, the supernatant was collected control RNA using the PrimeDirect Probe RT-qPCR Mix was ≳25
for further analysis. The beads were washed three times with HBST copies per well.
and were suspended with 140 l of HBST buffer. Viral RNA was ex-
tracted from both the supernatant and the bead suspension using the Virus neutralization assay
QIAamp Viral RNA Mini Kit (QIAGEN), as described in the manu- SARS-CoV-2 neutralization assay was performed using VeroE6/
facturer’s protocol. The 5 l of the 60 l of RNA-­containing solution TMPRSS2 cells (34) that were obtained from the JCRB Cell Bank,
was added to an RT-qPCR reaction mixture (One-Step PrimeScript III Ibaraki, Japan. The cells (5 × 103 cells per well, 50 l) were seeded in
RT-qPCR Mix, Takara Bio) using a primer/probe set for N2 (40). 96-well culture plates and incubated at 37°C for 18 hours before in-
The target copy numbers were determined using the Thermal Cycler fection. Monobodies were fourfold serially diluted (from 400 nM to
Dice Real Time System III (Takara Bio), which constantly showed 24.4 pM), and 20 l of the diluted monobodies were added into each
about three times higher number of RNA genomes than that of above culture well. Cells were infected with 10 l of SARS-CoV-2 (105
RT-ddPCR. TCID50/ml) for 1 hour at 37°C. The supernatant was removed, and
80 l of Dulbecco’s modified Eagle’s medium (Sigma-Aldrich) was
Pull-down RT-qPCR for study of the detection limit of supplemented with 10% fetal bovine serum and penicillin (100 U/ml)
SARS-CoV-2 particles and streptomycin (100 g/ml) (Thermo Fisher Scientific). After
SARS-CoV-2 virus particles (0.1 to 10,000 particles/l based on viral incubation at 37°C supplied with 5% CO2 for 36 hours, the culture
RNA coy number, 700 l) were incubated with 2 nM monobody supernatants were harvested. The SARS-CoV-2 RNA amounts in

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the supernatants were measured by RT-qPCR using the SARS- 21. T. Ishizawa, T. Kawakami, P. C. Reid, H. Murakami, TRAP display: A high-speed selection
method for the generation of functional polypeptides. J. Am. Chem. Soc. 135, 5433–5440
CoV-2 Direct Detection RT-qPCR Kit (Takara Bio). The IC 50
(2013).
values were determined using the GraphPad Prism version 6.0. 22. T. Kawakami, T. Ishizawa, T. Fujino, P. C. Reid, H. Suga, H. Murakami, In vitro selection
of multiple libraries created by genetic code reprogramming to discover macrocyclic
SUPPLEMENTARY MATERIALS peptides that antagonize VEGFR2 activity in living cells. ACS Chem. Biol. 8, 1205–1214
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ (2013).
content/full/sciadv.abd3916/DC1 23. S. Tabares-da Rosa, L. A. Wogulis, M. D. Wogulis, G. González-Sapienza, D. K. Wilson,
Structure and specificity of several triclocarban-binding single domain camelid antibody
View/request a protocol for this paper from Bio-protocol.
fragments. J. Mol. Recognit. 32, e2755 (2018).
24. S. Tabares-da Rosa, M. Rossotti, C. Carleiza, F. Carrión, O. Pritsch, K. C. Ahn, J. A. Last,
REFERENCES AND NOTES B. D. Hammock, G. González-Sapienza, Competitive selection from single domain
1. L. J. Carter, L. V. Garner, J. W. Smoot, Y. Li, Q. Zhou, C. J. Saveson, J. M. Sasso, A. C. Gregg, antibody libraries allows isolation of high-affinity antihapten antibodies that are not
D. J. Soares, T. R. Beskid, S. R. Jervey, C. Liu, Assay techniques and test development favored in the llama immune response. Anal. Chem. 83, 7213–7220 (2011).
for COVID-19 diagnosis. ACS Cent. Sci. 6, 591–605 (2020). 25. G. Krupp, Unusual promoter-independent transcription reactions with bacteriophage
2. S. Jiang, C. Hillyer, L. Du, Neutralizing antibodies against SARS-CoV-2 and other human RNA polymerases. Nucleic Acids Res. 17, 3023–3036 (1989).
coronaviruses. Trends Immunol. 41, 355–359 (2020). 26. R. Padilla, R. Sousa, A Y639F/H784A T7 RNA polymerase double mutant displays superior
3. X. Tian, C. Li, A. Huang, S. Xia, S. Lu, Z. Shi, L. Lu, S. Jiang, Z. Yang, Y. Wu, T. Ying, Potent properties for synthesizing RNAs with non-canonical NTPs. Nucleic Acids Res. 30, e138
binding of 2019 novel coronavirus spike protein by a SARS coronavirus-specific human (2002).
monoclonal antibody. Emerg. Microbes. Infect. 9, 382–385 (2020). 27. A. Koide, J. Wojcik, R. N. Gilbreth, R. J. Hoey, S. Koide, Teaching an old scaffold new tricks:
4. M. Yuan, N. C. Wu, X. Zhu, C.-C. D. Lee, R. T. Y. So, H. Lv, C. K. P. Mok, I. A. Wilson, A highly Monobodies constructed using alternative surfaces of the FN3 scaffold. J. Mol. Biol. 415,
conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS- 393–405 (2012).
CoV. Science 368, 630–633 (2020). 28. J. Wojcik, O. Hantschel, F. Grebien, I. Kaupe, K. L. Bennett, J. Barkinge, R. B. Jones, A. Koide,

Downloaded from http://advances.sciencemag.org/ on June 16, 2021

5. C. Wang, W. Li, D. Drabek, N. M. A. Okba, R. V. Haperen, A. D. M. E. Osterhaus, G. Superti-Furga, S. Koide, A potent and highly specific FN3 monobody inhibitor
F. J. M. V. Kuppeveld, B. L. Haagmans, F. Grosveld, B.-J. Bosch, A human monoclonal of the Abl SH2 domain. Nat. Struct. Mol. Biol. 17, 519–527 (2010).
antibody blocking SARS-CoV-2 infection. Nat. Commun. 11, 2251 (2020). 29. B. Virnekäs, L. Ge, A. Plückthun, K. C. Schneider, G. Wellnhofer, S. E. Moroney,
6. D. Wrapp, N. Wang, K. S. Corbett, J. A. Goldsmith, C.-L. Hsieh, O. Abiona, B. S. Graham, Trinucleotide phosphoramidites: Ideal reagents for the synthesis of mixed
J. S. McLellan, Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. oligonucleotides for random mutagenesis. Nucleic Acids Res. 22, 5600–5607 (1994).
Science 367, 1260–1263 (2020). 30. P. Zhou, X. L. Yang, X. G. Wang, B. Hu, L. Zhang, W. Zhang, H. R. Si, Y. Zhu, B. Li,
7. D. Wrapp, D. D. Vlieger, K. S. Corbett, G. M. Torres, N. Wang, W. V. Breedam, K. Roose, C. L. Huang, H. D. Chen, J. Chen, Y. Luo, H. Guo, R. D. Jiang, M. Q. Liu, Y. Chen, X. R. Shen,
L. V. Schie; VIB-CMB COVID-19 Response Team, M. Hoffmann, S. Pöhlmann, B. S. Graham, X. Wang, X. S. Zheng, K. Zhao, Q. J. Chen, F. Deng, L. L. Liu, B. Yan, F. X. Zhan, Y. Y. Wang,
N. Callewaert, B. Schepens, X. Saelens, J. S. McLellan, Structural basis for potent G. F. Xiao, Z. L. Shi, A pneumonia outbreak associated with a new coronavirus of probable
neutralization of betacoronaviruses by single-domain camelid antibodies. Cell 181, bat origin. Nature 579, 270–273 (2020).
1436–1441 (2020). 31. J. Shang, G. Ye, K. Shi, Y. Wan, C. Luo, H. Aihara, Q. Geng, A. Auerbach, F. Li, Structural
8. X. Chen, R. Li, Z. Pan, C. Qian, Y. Yang, R. You, J. Zhao, P. Liu, L. Gao, Z. Li, Q. Huang, L. Xu, basis of receptor recognition by SARS-CoV-2. Nature 581, 221–224 (2020).
J. Tang, Q. Tian, W. Yao, L. Hu, X. Yan, X. Zhou, Y. Wu, K. Deng, Z. Zhang, Z. Qian, Y. Chen, 32. D. M. Rissin, C. W. Kan, T. G. Campbell, S. C. Howes, D. R. Fournier, L. Song, T. Piech,
L. Ye, Human monoclonal antibodies block the binding of SARS-CoV-2 spike protein P. P. Patel, L. Chang, A. J. Rivnak, E. P. Ferrell, J. D. Randall, G. K. Provuncher, D. R. Walt,
to angiotensin converting enzyme 2 receptor. Cell. Mol. Immunol. 17, 647–649 (2020). D. C. Duffy, Single-molecule enzyme-linked immunosorbent assay detects serum
9. R. E. Bird, K. D. Hardman, J. W. Jacobson, S. Johnson, B. M. Kaufman, S. M. Lee, T. Lee, proteins at subfemtomolar concentrations. Nat. Biotechnol. 28, 595–599 (2010).
S. H. Pope, G. S. Riordan, M. Whitlow, Single-chain antigen-binding proteins. Science 242, 33. T. Notomi, H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, T. Hase,
423–426 (1988). Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28, 63e–663e (2000).
10. J. S. Huston, D. Levinson, M. Mudgett-Hunter, M. S. Tai, J. Novotný, M. N. Margolies, 34. S. Matsuyama, N. Nao, K. Shirato, M. Kawase, S. Saito, I. Takayama, N. Nagata, T. Sekizuka,
R. J. Ridge, R. E. Bruccoleri, E. Haber, R. Crea, H. Oppermann, Protein engineering H. Katoh, F. Kato, M. Sakata, M. Tahara, S. Kutsuna, N. Ohmagari, M. Kuroda, T. Suzuki,
of antibody binding sites: Recovery of specific activity in an anti-digoxin single-chain Fv T. Kageyama, M. Takeda, Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells.
analogue produced in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 85, 5879–5883 (1988). Proc. Natl. Acad. Sci. U.S.A. 117, 7001–7003 (2020).
11. C. Hamers-Casterman, T. Atarhouch, S. Muyldermans, G. Robinson, C. Hammers, 35. R. Shi, C. Shan, X. Duan, Z. Chen, P. Liu, J. Song, T. Song, X. Bi, C. Han, L. Wu, G. Gao, X. Hu,
E. B. Songa, N. Bendahman, R. Hammers, Naturally occurring antibodies devoid of light Y. Zhang, Z. Tong, W. Huang, W. J. Liu, G. Wu, B. Zhang, L. Wang, J. Qi, H. Feng, F. S. Wang,
chains. Nature 363, 446–448 (1993). Q. Wang, G. F. Gao, Z. Yuan, J. Yan, A human neutralizing antibody targets the receptor
12. G. Beste, F. S. Schmidt, T. Stibora, A. Skerra, Small antibody-like proteins with prescribed binding site of SARS-CoV-2. Nature 584, 120–124 (2020).
ligand specificities derived from the lipocalin fold. Proc. Natl. Acad. Sci. U.S.A. 96, 36. B. Ju, Q. Zhang, J. Ge, R. Wang, J. Sun, X. Ge, J. Yu, S. Shan, B. Zhou, S. Song, X. Tang, J. Yu,
1898–1903 (1999). J. Lan, J. Yuan, H. Wang, J. Zhao, S. Zhang, Y. Wang, X. Shi, L. Liu, J. Zhao, X. Wang,
13. A. Koide, C. W. Bailey, X. Huang, S. Koide, The fibronectin type III domain as a scaffold Z. Zhang, L. Zhang, Human neutralizing antibodies elicited by SARS-CoV-2 infection.
for novel binding proteins. J. Mol. Biol. 284, 1141–1151 (1998). Nature 584, 115–119 (2020).
14. K. Nord, E. Gunneriusson, J. Ringdahl, S. Stahl, M. Uhlen, P. A. Nygren, Binding proteins 37. Y. Cao, B. Su, X. Guo, W. Sun, Y. Deng, L. Bao, Q. Zhu, X. Zhang, Y. Zheng, C. Geng, X. Chai,
selected from combinatorial libraries of an -helical bacterial receptor domain. R. He, X. Li, Q. Lv, H. Zhu, W. Deng, Y. Xu, Y. Wang, L. Qiao, Y. Tan, L. Song, G. Wang, X. Du,
Nat. Biotechnol. 15, 772–777 (1997). N. Gao, J. Liu, J. Xiao, X. D. Su, Z. Du, Y. Feng, C. Qin, C. Qin, R. Jin, X. S. Xie, Potent
15. H. K. Binz, M. T. Stumpp, P. Forrer, P. Amstutz, A. Plückthun, Designing repeat proteins: neutralizing antibodies against SARS-CoV-2 identified by high-throughput single-cell
Well-expressed, soluble and stable proteins from combinatorial libraries of consensus sequencing of convalescent patients' B cells. Cell 182, 73–84.e16 (2020).
ankyrin repeat proteins. J. Mol. Biol. 332, 489–503 (2003). 38. P. Forster, L. Forster, C. Renfrew, M. Forster, Phylogenetic network analysis of SARS-CoV-2
16. D. Grabulovski, M. Kaspar, D. Neri, A novel, non-immunogenic Fyn SH3-derived binding genomes. Proc. Natl. Acad. Sci. U.S.A. 117, 9241–9243 (2020).
protein with tumor vascular targeting properties. J. Biol. Chem. 282, 3196–3204 39. J. Ou, Z. Zhou, R. Dai, J. Zhang, W. Lan, S. Zhao, J. Wu, D. Seto, L. Cui, G. Zhang, Q. Zhang,
(2007). Emergence of RBD mutations in circulating SARS-CoV-2 strains enhancing the structural
17. G. P. Smith, Filamentous fusion phage: Novel expression vectors that display cloned stability and human ACE2 receptor affinity of the spike protein. bioRxiv, doi.
antigens on the virion surface. Science 228, 1315–1317 (1985). org/10.1101/2020.03.15.991844 (2020).
18. N. Nemoto, E. Miyamoto-Sato, Y. Husimi, H. Yanagawa, In vitro virus: Bonding of mRNA 40. K. Shirato, N. Nao, H. Katano, I. Takayama, S. Saito, F. Kato, H. Katoh, M. Sakata, Y. Nakatsu,
bearing puromycin at the 3'-terminal end to the C-terminal end of its encoded protein Y. Mori, T. Kageyama, S. Matsuyama, M. Takeda, Development of genetic diagnostic
on the ribosome in vitro. FEBS Lett. 414, 405–408 (1997). methods for novel coronavirus 2019 (nCoV-2019) in Japan. Jpn. J. Infect. Dis. 73, 304–307
19. R. W. Roberts, J. W. Szostak, RNA-peptide fusions for the in vitro selection of peptides (2020).
and proteins. Proc. Natl. Acad. Sci. U.S.A. 94, 12297–12302 (1997). 41. Y. Shimizu, A. Inoue, Y. Tomari, T. Suzuki, T. Yokogawa, K. Nishikawa, T. Ueda, Cell-free
20. C. A. Olson, H. I. Liao, R. Sun, R. W. Roberts, mRNA display selection of a high-affinity, translation reconstituted with purified components. Nat. Biotechnol. 19, 751–755 (2001).
modification-specific phospho-IkappaBalpha-binding fibronectin. ACS Chem. Biol. 3, 42. P. C. Reid, Y. Goto, T. Katoh, H. Suga, Charging of tRNAs using ribozymes and selection
480–485 (2008). of cyclic peptides containing thioethers. Methods Mol. Biol. 805, 335–348 (2012).

Kondo et al., Sci. Adv. 2020; 6 : eabd3916 14 October 2020 11 of 12

SCIENCE ADVANCES | RESEARCH ARTICLE

43. H. Ohashi, Y. Shimizu, B. W. Ying, T. Ueda, Efficient protein selection based on ribosome T.F.) from the Japan Society for the Promotion of Science and a donation from H.M.
display system with purified components. Biochem. Biophys. Res. Commun. 352, 270–276 Author contributions: T.K., T.F., S.U., K.I., S.K., and T.S. carried out the nanobody/monobody
(2007). selection experiment. Y.I. designed the virus detection study, and K.M. conducted the
44. Y. Wang, D. E. Prosen, L. Mei, J. C. Sullivan, M. Finney, P. B. Vander Horn, A novel strategy experiment. Y.Y. collected the virus samples. H.M. wrote the manuscript with support from
to engineer DNA polymerases for enhanced processivity and improved performance T.K., T.F., Y.I., and G.H. H.M. supervised the project. Competing interests: T.K., Y.I., T.F., S.U.,
in vitro. Nucleic Acids Res. 32, 1197–1207 (2004). G.H., and H.M. are inventors on the provisional patent application (U.S. application no.
45. N. Taniguchi, H. Murakami, Multiple site-directed and saturation mutagenesis by 63/026,802, filed 19 May 2020) submitted by the Tokai National Higher Education and
the patch cloning method. Methods Mol. Biol. 1498, 339–347 (2017). Research System and National Hospital Organization Nagoya Medical Center that covers
46. N. Taniguchi, S. Nakayama, T. Kawakami, H. Murakami, Patch cloning method for multiple monobody sequences against the SARS-CoV-2 spike protein. All other authors declare that
site-directed and saturation mutagenesis. BMC Biotechnol. 13, 91 (2013). they have no competing interests. Data and materials availability: All data needed to
47. C. N. Pace, F. Vajdos, L. Fee, G. Grimsley, T. Gray, How to measure and predict the molar evaluate the conclusions in the paper are present in the paper and/or the Supplementary
absorption coefficient of a protein. Protein Sci. 4, 2411–2423 (1995). Materials. The monobody genes can be provided by H.M., as well as pending scientific review
48. P. G. Blommel, K. J. Becker, P. Duvnjak, B. G. Fox, Enhanced bacterial protein expression and a completed material transfer agreement. Requests for the genes should be submitted to
during auto-induction obtained by alteration of lac repressor dosage and medium H.M. Additional data related to this paper may be requested from the authors.
composition. Biotechnol. Prog. 23, 585–598 (2007).
49. I. Chen, B. M. Dorr, D. R. Liu, A general strategy for the evolution of bond-forming Submitted 19 June 2020
enzymes using yeast display. Proc. Natl. Acad. Sci. U.S.A. 108, 11399–11404 (2011). Accepted 28 August 2020
Published First Release 18 September 2020
Acknowledgments: We thank Y. Baba and Y. Hasegawa for initial arrangement of the Published 14 October 2020
collaboration between the Murakami group and the Iwatani group for this research. Funding: 10.1126/sciadv.abd3916
This work was supported by the Grant-in-Aid for Scientific Research (A) (General) (grant
number 15H02006 to H.M.), Grant-in-Aid for Scientific Research (B) (General) (grant number Citation: T. Kondo, Y. Iwatani, K. Matsuoka, T. Fujino, S. Umemoto, Y. Yokomaku, K. Ishizaki,
19H03482 to Y.I.), Grant-in-Aid for Scientific Research on Innovative Areas (grant number S. Kito, T. Sezaki, G. Hayashi, H. Murakami, Antibody-like proteins that capture and neutralize

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20H04704 to G.H.), and Grant-in-Aid for Early-Career Scientists (grant number 18K14332 to SARS-CoV-2. Sci. Adv. 6, eabd3916 (2020).

Kondo et al., Sci. Adv. 2020; 6 : eabd3916 14 October 2020 12 of 12

Antibody-like proteins that capture and neutralize SARS-CoV-2
T. Kondo, Y. Iwatani, K. Matsuoka, T. Fujino, S. Umemoto, Y. Yokomaku, K. Ishizaki, S. Kito, T. Sezaki, G. Hayashi and H.
Murakami

Sci Adv 6 (42), eabd3916.

DOI: 10.1126/sciadv.abd3916originally published online September 18, 2020

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