A Novel Neutralizing Antibody Targeting Receptor Binding Domain of SARS-CoV-2


 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the current COVID-19 global pandemic. Vaccines and therapeutics are urgently needed for this highly transmissible virus. In this study, we screened human monoclonal antibodies (mAbs) targeting the receptor binding domain (RBD) of the SARS-CoV-2 spike protein from an antibody library constructed from peripheral blood mononuclear cells of a COVID-19 convalescent patient. A potent neutralizing antibody, termed CT-P59, was identified and found to be effective against various SARS-CoV-2 isolates including the D614G spike protein variant without antibody-dependent enhancement effect. Complex crystal structure of CT-P59 Fab/SARS-CoV-2 RBD showed that CT-P59 blocks interaction regions of SARS-CoV-2 RBD for its cellular receptor, angiotensin converting enzyme 2 (ACE2). The binding orientation of CT-P59 is notably different from the previously reported neutralizing mAbs targeting SARS-CoV-2 RBD suggesting that CT-P59 can be a novel binder to SARS-CoV-2 RBD. Therapeutic effects of CT-P59 were evaluated in three animal models (ferret, hamster, and rhesus monkey), and a substantial reduction in viral titre along with alleviation of clinical symptoms was observed. These findings suggest that the human monoclonal antibody, CT-P59, is a promising therapeutic candidate for treatment of COVID-19.


Structural basis of neutralization
To investigate the neutralizing mechanism of CT-P59, the crystal structure of the CT-P59 Fab/SARS-CoV-2 RBD complex was determined using X-ray crystallography at 2.7 Å resolution (Extended Data Table 2).
The complex structure shows that CT-P59 binds to the receptor binding motif (RBM) within SARS-CoV-2 RBD, which directly interacts with ACE2 (Fig. 2a). The association angle between CT-P59 and the RBD is different from that reported for other neutralizing antibodies in complex with the RBD (Fig. 2b) 14,15 .
These observations indicate that the epitope of CT-P59 are unique from those of other antibodies [14][15][16][17][18][19] (Fig. 2c). Further, the interactions of the RBD with the heavy and light chains of CT-P59 bury a solventaccessible surface area of 824.6 and 112.5 Å 2 , respectively. Consistently, most of the interaction between the two proteins is mediated by the heavy chain involving all three complementarity determining regions (CDRs). In total, 16 residues from the CT-P59 heavy chain interact with 19 residues of the RBD at a distance cut-off of 4.5 Å (Extended Data Table 3). Of note, the β-hairpin structure of the CDR H3 plays a crucial role in the strong association with the RBD, by forming eight hydrogen bonds as well as hydrophobic interactions involving several of aromatic residues in the middle of the ACE2 binding surface (Fig. 2d). The light chain shows marginal contact with the RBD involving parts of CDR L1 and L2 where only three residues interact with four residues of the RBD (Extended Data Table 3).
To further analyse the structural basis for blocking of the interaction between RBD and ACE2 by CT-P59, the complex structure of CT-P59-RBD was superimposed on the RBD-ACE2 structure (PDB 6LZG) 20 . CT-P59 binding does not alter the overall conformation of the RBD structure in which the pairwise root mean square deviation between the Cα atoms of the two RBD structures is 0.89 Å over 193 atoms. However, the β5-β6 loop region (residues 473-488) of the RBD shows a local conformational change, which might be induced by the interaction with CT-P59. The structural superposition reveals that the heavy chain of CT-P59 overlaps completely with ACE2 protein, while the light chain overlaps partially with the receptor (Extended Data Fig. 3a). In agreement with the superposition, there is substantial overlap between the CT-P59 and ACE2 binding surface areas on RBD (Extended Data Fig. 3b). Among the 21 residues of RBD that interact with ACE2, 12 are also involved in the interaction with CT-P59, when a distance cut-off of 4.5 Å is applied (Fig. 2c). These observations indicate that the binding of CT-P59 to RBD directly occludes the binding surface of ACE2.
In vivo e cacy in animal models To demonstrate in vivo antiviral e cacy of CT-P59 in terms of viral clearance and clinical symptoms, viral loads and lung pathology, we conducted virus challenge studies employing three animal models (ferrets, golden Syrian hamsters, and rhesus monkeys). In the ferret study, viruses were challenged via both intranasal and intratracheal routes, followed by intravenous treatment of CT-P59 and isotype control at 1 day post-infection (dpi). At 2 dpi, the infectious virus titre (TCID 50 ) in nasal wash was signi cantly decreased when compared to controls in time-and dose-dependent manners, and the infectious virus was not detected at 6 dpi in animals treated with 30 mg/kg of CT-P59. For rectal swabs, the viral RNA copies were signi cantly decreased from 4 dpi at both doses ( Fig. 3a and 3b). Further, the infectious virus titre was signi cantly attenuated in lung tissues with both doses at 3 dpi and not detectable with 30 mg/kg at 7 dpi (Extended Data Fig. 6a). The reduction of viral load in the upper and lower respiratory tract was consistent with an improvement in clinical symptoms and lung pathology (Supplementary Table 1 and Extended Data Fig. 7). To compare the therapeutic effect of CT-P59 with a World Health Organization (WHO) approved drug, Remdesivir was administered daily for 5 days (18 mg/kg) in ferrets following 1 day of SARS-CoV-2 infection. Remdesivir-treated ferrets showed attenuated virus titres and viral RNAs in respiratory tracts and rectal swabs respectively, compared with those of isotype control-treated animals, but the infectious virus was detected in the respiratory tract until 6 dpi (Fig. 3a, b) suggesting delayed clearance of SARS-CoV-2 in ferrets compared with the CT-P59-treated group.
In SARS-CoV-2-infected golden Syrian hamsters, the viral load reached peak levels on 3 dpi (8.3 log TCID 50 /g) in the lungs of vehicle-treated hamsters and slightly declined by 5 dpi (6.8 log TCID 50 /g, Extended Data Fig. 6b). In CT-P59-treated hamsters, there was signi cant attenuation of viral loads in lungs in the 15 mg/kg treated group, and all other groups (range, 30-90 mg/kg) showed no infectious virus in lung tissues 48 hours after CT-P59 treatment, suggesting complete inhibition of SARS-CoV-2 replication in lungs of golden Syrian hamster from 30 mg/kg dose (Extended Data Fig. 6b).
In the rhesus monkey study, no apparent clinical manifestations including fever, weight loss, and respiratory distress, were observed in both CT-P59-and vehicle control-treated animals. The viral load reached peak levels on 2 dpi (4.3 log TCID 50 /ml) in nasal swabs, and then gradually declined until 6 dpi in vehicle-treated control group (Fig. 3c). In contrast, CT-P59 treatment rapidly reduced virus titres and the infectious virus was not detected even at 2 dpi following the CT-P59 administration in both 45 and 90 mg/kg groups. Further no viral RNAs were detected in rectal swabs collected from CT-P59-treated animals from 4 dpi (Fig. 3d). All monkeys were euthanized at 6 dpi and individual lung lobes were collected to quantify the infectious virus titre. No infectious virus was detected in any of the lung lobes tested from any animals, including vehicle control-and CT-P59-treated groups (Extended Data Fig. 6c).
To further investigate possible adverse effect, we performed the in vitro ADE assay with authentic SARS-CoV-2. No increase in the viral infections was observed in Fc receptor-bearing cells (Extended Data Fig. 5).

Discussion
In this study, we demonstrated the potential therapeutic bene t of neutralizing antibody CT-P59 targeting RBD of SARS-CoV-2 in in vitro and in vivo studies. We found that CT-P59 binds to RBD of S protein, rendering complete steric hindrance interfering with the viral binding to ACE2 by BLI competition assay and X-ray crystallography. Importantly, CT-P59 signi cantly inhibited the viral replication of clinical isolates by in vitro PRNT. Unlike RBD-targeting antibodies with similar sensitivities to the viral neutralization on a D614G variant 21 , CT-P59 can effectively neutralize D614G variant; the underlying mechanism remains to be elucidated in terms of S protein stability and viral kinetics. SARS-CoV-2 RBD mutations might alter the binding a nity of the virus for ACE2 9,12,13 . For instance, V367F, W436R, and D364Y were reported to increase the binding a nity for ACE2, which might accelerate viral spread further perpetuating the pandemic. We found that CT-P59 binds to RBD mutant proteins and also interferes with ACE2 (Extended Data Table 1 and Fig. 1b). In addition, according to the X-ray crystallography data ( Fig. 2 and Extended Data Fig. 3), CT-P59 does not bind to the amino acid residues at position 367, 436, or 364 of the RBD. These results suggest that CT-P59 might be able to neutralize naturally occurring potential variants.
The complex structure of CT-P59 shows that CT-P59 inhibits SARS-CoV-2 RBD binding to its cellular receptor, ACE2, by blocking substantial areas of the ACE2 interaction regions. Among the previously reported neutralizing antibodies against SARS-CoV-2 RBD that speci cally block ACE2 binding, we compared the publicly available atomic coordinates with those of CT-P59 to evaluate association mode between antibodies and RBD (Extended Data Fig. 4a). We found that the majority of the ACE2 blocking antibodies-including CB6 16 , B38 17 , CV30 18 , CC 12.1 19 , CC 12.3 19 and REGN10933 14 -adopt a similar orientation when bound to RBD. Each of these antibodies belong to the immunoglobulin heavy chain variable region genes (IGHV) 3 germline that is the most frequently used IGHV gene among the known SARS-CoV-2 neutralizing antibodies 22 . The neutralizing antibody P2B-2F6 15 which is based on the IGHV4-38 gene, on the other hand, interacts with RBD at about a 90 degree angle from the previous group. Notably, CT-P59 (based on IGHV2-70) binds with an orientation in the middle of these mAb groups (Extended Data Fig. 4a) and shares portions of the epitope from each group (Fig. 2c). To our knowledge, CT-P59 is the rst SARS-CoV-2 RBD neutralizing antibody with an IGHV2 germline lineage that its high resolution structure reported. Cryo-electron microscopy has revealed that RBD of SARS-CoV-2 S protein trimer undergoes either "up" or "down" conformations and ACE2 can only bind to the "up" conformation 23,24 . Structural alignment of each group of neutralizing antibodies with the "down" form of SARS-CoV-2 S protein trimers showed that the IGHV3 antibody group heavily clashes with the adjacent RBD protomer, whereas P2B-2F6 can bind to the trimer without any collision with the adjacent molecules (Extended Data Fig. 4b). Although CT-P59 collides with the Asn343 glycosylated site on adjacent protomer, it has much fewer clashes compared with IGHV3 group antibodies. All the evaluated antibodies can interact with RBD on the "up" conformation without any steric hindrance. We therefore propose that CT-P59 may have more chance to block ACE2-RBD interaction in pre-fusion state than IGHV3 group antibodies if there is slight hinge-like movement in RBD region.
Because no animal models are available that accurately re ect clinical symptoms (e.g. lung damage) of patients with severe COVID-19 25-29 , ferrets, Syrian hamsters and rhesus monkeys have been used together for evaluation of SARS-COV-2 pathogenesis/transmission and to assess the e cacy of therapeutics and vaccines against COVID-19 30-33 . In vivo challenge studies using these models has demonstrated that CT-P59 is capable of quickly decreasing virus titres, particularly improving clinical symptoms and pathological changes in ferrets. Notably, when we compared the therapeutic e cacy of CT-P59 with Remdesivir, a drug for use in hospitalized patients with COVID-19, the CT-P59-treated ferrets showed more attenuated viral loads in upper respiratory tracts from 2 dpi. The early clearance of infectious virus suggests that CT-P59 might be an option for COVID-19 patients as combination therapy. Fig. 5), in line with no worsening of symptoms in CT-P59-treated animals as described above. Moreover, a recent animal study showed that ADE was not observed by vaccine targeting SARS-CoV-2 RBD 11 . Therefore, these observations suggest that CT-P59 can remarkably neutralize SARS-CoV-2 via binding to RBD and ameliorate pathological symptoms without ADE during clinical trials.  interval of 24 h were con rmed as negative for SARS-CoV-2 by PCR before blood sampling. Peripheral blood mononuclear cells (PBMCs) were isolated from the collected blood using Ficoll-Paque (GE Healthcare), and mRNA was extracted using the TRIzol reagent (Thermo Fisher). The isolated mRNA was immediately converted to cDNA using SuperScript TM III Reverse Transcriptase (Invitrogen).

Phage library construction and biopanning
Antibody variable regions (V L and V H ) were ampli ed by PCR with appropriate primers for phage display.
ScFvs were generated by linking V L and V H fragments and directly cloned into phagemid vector, pComb3xSS, for library construction. ER2738 cells (Lucigen) were transformed with the scFv library, then cultured in SB medium containing 50 μg/mL carbenicillin and VCSM13 helper phage (Stratagene) at 37℃ overnight. Next day, phages displaying scFv were harvested for biopanning to screen SARS-CoV-2 RBD-binding scFv displayed on phage. Brie y, SARS-CoV-2 RBD (Sino biological) was coated on magnetic beads (Invitrogen) and incubated with the phage library. Following incubation and washing, SARS-CoV-2 RBD-bound phages were eluted and used to infect fresh ER2738 cells. After several rounds of biopanning, scFv phages binding to SARS-CoV-2 RBD were identi ed by phage ELISA for further selection.
Preparation of scFv-Fc, full-length IgG and S proteins Each scFv identi ed by phage ELISA was cloned into the Fc fusion vector and transiently expressed in Chinese Hamster Ovary (CHO) cells. Next, for the expression of full-length IgG, synthesized DNAs of heavy chain and light chain for each mAb were inserted into MarEx vectors (Celltrion) by enzymatic digestion with NheI (NEB) / PmeI (NEB) and HpaI (NEB) / ClaI (NEB), respectively. CR3022 antibody was reconstituted with variable sequences for light and heavy chain according to the published sequence information (US2008/0014204A1). Thereafter, transient expression by co-transfection was performed in CHO cells. Each scFv-Fc and full-length IgG was puri ed with a nity chromatography on Protein A (GE Healthcare). For the production of SARS-CoV-2 RBD, DNA encoding the SARS-CoV-2 S protein RBD (YP_009724390.1: Arg319-Asn536) with a polyhistidine tag at the C-terminus was cloned into the MarEx vector and transiently expressed in CHO cells. SARS-CoV-2 RBD with polyhistidine tag was a nity puri ed using Ni-NTA Resin (Thermo Fisher). Recombinant proteins for RBD and its mutants (A435S, F342L, G476S, K458R, N354D, V367F, V483A, W436R), SARS-CoV S1, HCoV-HKU1 S1 and MERS-CoV RBD were commercial products (Sino Biological).

mAb neutralizing assays
To evaluate the neutralizing activity of monoclonal antibodies, plaque reduction neutralizing tests for SARS-CoV-2 were performed as described previously 37 . Brie y, 2-fold serially diluted mAbs ranging from 10 3 to 1 ng/ml and an equal volume of virus (40 pfu/well) were incubated at 37°C for 2 h. The antibodyvirus mixture was inoculated into a 24-well plate seeded with VeroE6 cells (1 × 10 5 cells/well) and incubated at 37°C for 1 h, followed by overlay of 1 ml of 0.5% agarose (Lonza). After 2 to 3 days of incubation, the cells were xed with 4% paraformaldehyde and visualized plaques with crystal violet. Two independent experiments were performed in duplicate for each mAb. The data were tted to a doseresponse inhibition model, and the half-maximal inhibitory concentration (IC 50 ) of each mAb was calculated using GraphPad Prism6 software.

Surface plasmon resonance for a nity
Binding a nity of CT-P59 to SARS-CoV-2 RBD was assayed using a Biacore T200 TM SPR instrument (Cytiva). SARS-CoV-2 RBD manufactured by Celltrion was covalently immobilized on the CM5 chip using an amine coupling reaction. Any unstable, SARS-CoV-2 RBD was removed by at least six washes of prerun solution before sample run. CT-P59 was serially diluted from 10 to 0.04 nM using HBS-EP buffer (pH 7.4), and then injected for 120 sec, followed by HBS-EP buffer (pH 7.4) for 120 sec to generate the binding and dissociation curves, respectively. After each cycle, the chip surface was treated with a brief pulse of 20 mM NaOH until the response unit (RU) signal returned to baseline, and then a new cycle was started. The dissociation constant was tted to a bivalent analyte model using Biacore evaluation software (Cytiva).

Biolayer interferometry (BLI)
Competitive binding to SARS-CoV-2 RBDs between CT-P59 and ACE2, binding speci city and binding a nity to SARS-CoV-2 RBDs were measured by biolayer interferometry (BLI) using the Octet QK e system (ForteBio). All samples were prepared with corresponding concentration by dilution in Kinetic Buffer (BMS). To determine the competitive characteristics between CT-P59 and ACE2, the immobilized wild type and mutant SARS-CoV-2 RBD proteins with a concentration of 50 nM were saturated with 267 nM of CT-P59 for 5 min, and then owed with CT-P59 (133.5 nM) in the presence or absence of ACE2 (133.5 nM) for 5 min. As a positive control, buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and owed ACE2 (133.5 nM). For the binding speci city assay, binding of CT-P59 to four virus S proteins (SARS-CoV-2 RBD, SARS-CoV S1, HCoV-HKU1 S1, MERS RBD) were measured. Each S protein (50 nM) was loaded onto Anti-Penta-HIS Biosensor, and then CT-P59 (267 nM) was owed for 5 min. Buffer and ACE2 were sequentially owed for a positive control. To evaluate binding a nity between CT-P59 and SARS-CoV-2 RBD wild type and mutants, CT-P59 (5 nM) was loaded onto Anti-Human IgG Fc Capture Biosensor for 7.5 min, and then each SARS-CoV-2 RBD was owed with concentration of 0 nM, 2.5 nM, 5 nM, 10 nM and 20 nM for 10 min and 15 min to generate association and dissociation curve, respectively.

Crystallization and structure determination
The CT-P59 Fab/SARS-CoV-2 RBD complex was prepared by mixing the puri ed SARS-CoV-2 RBD protein with CT-P59 Fab at a 1:1. Republic of Korea. The data set was processed using XDS program package 38 . The CT-P59 Fab/SARS-CoV-2 RBD complex structure was determined by molecular replacement using Phaser 39 . The SARS-CoV-2 RBD/CB6 complex structure (PDB code, 7C01) was used as a search model. Model building and re nement were performed using Coot 40 and the Phenix package 41 , respectively. The Ramachandran statistics of the nal structure are 96.81% in most favored region, 3.19% in allowed region and 0.00% in disallowed region. The X-ray diffraction and structure re nement statistics are summarized in Extended Data Table 2. All structure gures were generated with PyMol 42 .
After washing, the cells were probed with SARS-CoV-2 anti-nucleocapsid antibody (Sino Biologicals, 1:2,000) and bound antibody was detected by horseradish peroxidase (HRP)-conjugated anti-mouse antibody (Southern Biotech, 1:4,000). Tetramethylbenzidine (TMB) was added and incubated for 5 min, then stopped by H 2 SO 4 . Virus titres were assessed by optical density measured by spectrophotometer

Ferret study
Groups of 14-to 18-month-old female ferrets (6/group) which are seronegative for SARS-CoV-1 and SARS-CoV-2, were inoculated intranasally and intratracheally with 10 5.5 TCID 50 of NMC-nCoV02 (total 1 ml) under anaesthesia. Two doses, 3 and 30 mg/kg, of CT-P59 were administered intravenously 24 hr after virus inoculation in each group. Animals in the control group were given 30 mg/kg of human IgG isotype. Remdesivir (18 mg/kg) was administrated daily via oral gavage 24 h post-inoculation for 5 days.
Body weights and temperatures were measured and nasal washes, saliva and rectal swab specimens were collected every other day. Three ferrets per group were euthanized at 3 and 7 dpi, and the nasal turbinates and lungs were subjected to measure tissue viral titres and examine lung histopathology. Viral titres in nasal washes and tissues were determined by TCID 50 assay in Vero cells, while the viral loads in saliva and rectal swab specimens were assessed using quantitative real-time PCR (qRT-PCR).

Golden Syrian hamster study
Sixty male golden Syrian hamsters (n=12/group) were challenged with 6.4×10 4 PFU/80 μl of SARS-CoV-2 via the intranasal route. Vehicle and 15, 30, 60 and 90 mg/kg CT-P59 were administered via an intraperitoneal route 24 hr after virus inoculation. At 2, 3, and 5 dpi four animals from each group were sacri ced for quanti cation of viral load in the lungs.
Animals in the control group (n=3) were given an equal volume of vehicle. Viral load was measured by nasal, throat and rectal swabs daily until 6 dpi, and viral load in the lung was measured by necropsy at 6 dpi Virus titration and quantitation Virus titres in nasal washes and lungs (TCID 50 ) and in rectal swab (qRT-PCR) were determined. Brie y, viral titres for samples form the upper and lower airway were measured using Vero cells. All tissue samples were diluted 10-fold (w/v) with sterile phosphate buffered solution (PBS, pH 7.4) and homogenized using a tissue homogenizer (Precellys Homogenizer, Bertin Instruments, France). After centrifugation of all swabs and homogenized tissue samples at 3,000 g for 10 min, the supernatants were ltered through 0.2 μm pore size syringe lter (Millipore, USA) and directly inoculated into Vero cells.
The cells were incubated for 3 days at 37°C and then stained with crystal violet for cytopathic effect (CPE). The values of TCID 50 /ml were determined using a Reed and Muench method.
In the ferret study, viral RNA was extracted using RNeasy kit (Qiagen) and cDNAs were reverse transcribed with a SARS-CoV-2 speci c primer using QuantiTect Reverse Transcription (Qiagen). qRT-PCR reactions were performed using a SYBR Green Supermix (Bio-Rad). The viral RNA copy numbers were assessed by a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) with a S gene based -SARS-CoV-2-speci c primer and standard control 30 . In rhesus monkey study, total RNAs from the rectal swab sample were accessed by RT-qPCR. Viral RNA was extracted using a commercial viral RNA extraction kit (QIAamp Viral RNA Mini Kit, Qiagen). RT-qPCR was performed with a primer and probe set according to a previous report 43 . SARS-CoV-2 RNA standard samples were run in parallel for determination of virus copy number in all reactions.

Histology
Lung histology is evaluated as follows. Sections of the left caudal lung lobes were microscopically observed. Before collection, the lung lobes (with trachea intact) were insu ated with 10% neutral buffered formalin (NBF) and then submerged in 10% NBF for 2-3 days. Following xation, the desired sections of lungs were embedded in para n, sectioned (5 μm), placed on glass slides, and stained with hematoxylin and eosin (H&E).

Human samples
The human samples were obtained according to the procedures approved by Seoul National University Hospital and complied with all relevant ethical regulations regarding human research. The blood was taken from a convalescent COVID-19 patient after she/he signed the informed consent form.

Additional information
Correspondence and requests for materials should be addressed to S.Y.L and Y.K.C. Figure 1 | CT-P59 can effectively neutralize SARS-CoV-2 in vitro by blocking RBD-ACE2 binding. a, Serial 2-folddiluted CT-P59 were incubated with SARS-CoV-2 live viruses; wild type (blue) and D614G (red). The mixture was added to VeroE6 cells. After 2 to 3 days of incubation, the neutralization activity was evaluated by counting plaques. Two independent experiments were performed in duplicate. b, SARS-CoV-2 RBD immobilized on biosensor was saturated with CT-P59. Then, CT-P59 was owed over the biosensor surface in the presence or absence of ACE2 receptor. As a positive control, buffer was loaded onto SARS-CoV-2 RBD immobilized biosensor, and ACE2 was owed over the biosensor surface.

Figure 2
The structure of CT-P59 Fab in complex with SARS-CoV-2 RBD. a, The overall structure of the CT-P59 Fab/SARS-CoV-2 RBD complex. The RBD domain is green for the core subdomain and orange for RBM.
The heavy and light chains of CT-P59 are magenta and yellow, respectively. b, Superposition of the neutralizing antibodies in complex with RBD. RBD is shown as a surface model. CT-P59 is shown as a cartoon, and the other antibodies (CR3022: PDB 6XC3, PB2-2F6: PDB 7BWJ, REGN10933: PDB 6XDG) are shown as a ribbon model. The heavy and light chains of Fab are magenta and yellow, respectively. c, Assignment of the epitope residues for RBD-targeting neutralizing antibodies with a distance cut-off of 4.5 Å. RBD residues interacting with ACE2 are highlighted in red. d, The detailed interactions between the RBD and CDR loops of CT-P59. The interfaces between RBD and CDR H1/2 or H3 are shown in the top and bottom panels, respectively. The RBD domain is shown as a surface model with semi-transparent representation. The CDR loops and interacting residues on the interfaces are shown in ribbons and sticks, respectively. The residues are coloured as in a. Dashed lines indicate hydrogen bonds. Water molecules are shown as red spheres. | In vivo e cacy of CT-P59 in animal models. Female ferrets (n=6/group), and rhesus monkeys (three control; two 45 mg/kg; three 90 mg/kg) were challenged with 105.5 TCID50/ml and 106.4 TCID50 of SARS-CoV-2, respectively. Control (ferrets: 30 mg/kg of human IgG isotype and rhesus monkeys: vehicle) and CT-P59 (ferrets: 3, and 30 mg/kg, rhesus monkeys: 45, and 90 mg/kg) were administered intravenously after 24 hours of virus inoculation, respectively. To compare the e cacy of CT-P59, Remdesivir (18 mg/kg per ferret) was administered daily via oral gavage after 24 hours of virus inoculation in ferrets for 5 days. To detect the viral load, nasal wash/swab and rectal swabs specimens were collected at 2, 4, and 6 dpi. Virus titres (TCID50) were measured in nasal wash specimens from each group of (a) ferrets and, (c) rhesus monkeys. The number of viral RNA copies was measured in rectal swabs from each group of (b) ferrets and, (d) rhesus monkeys using qRT-PCR. Viral titres and RNA copy numbers are shown as means ± SEM from four animals and titres below the limit of detection are shown as 0.8 log10TCID50/ml or 0.3 log10 viral RNA copies/ml (dashed lines). * indicates P < 0.01, ** indicates P < 0.001, and *** indicates P < 0.0001 between the control and each group as determined by two-way ANOVA and subsequent Dunnett's test.

Supplementary Files
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