key: cord-0308688-7ts2hvdb
authors: Yadav, Pragya D; Mendiratta, Sanjeev Kumar; Mohandas, Sreelekshmy; Singh, Arun K; Abraham, Priya; Shete, Anita; Bandyopadhyay, Sanjay; Kumar, Sanjay; Parikh, Aashini; Kalita, Pankaj; Sharma, Vibhuti; Pandya, Hardik; Patel, Chirag G; Patel, Mihir; Soni, Swagat; Giri, Suresh; Jain, Mukul
title: ZRC3308 monoclonal antibody cocktail shows protective efficacy in Syrian hamsters against SARS-CoV-2 infection
date: 2021-09-17
journal: bioRxiv
DOI: 10.1101/2021.09.16.460724
sha: 7d5aa429d985b8526a2d0559585b91d9edfeaf78
doc_id: 308688
cord_uid: 7ts2hvdb

We have developed a monoclonal antibody (mAb) cocktail (ZRC-3308) comprising of ZRC3308-A7 and ZRC3308-B10 in the ratio 1:1 for COVID-19 treatment. The mAbs were designed to have reduced immune effector functions and increased circulation half-life. mAbs showed good binding affinities to non-competing epitopes on RBD of SARS-CoV-2 spike protein and were found neutralizing SARS-CoV-2 variants B.1, B.1.1.7, B.1.351, B.1.617.2 and B.1.617.2 AY.1 in vitro. The mAb cocktail demonstrated effective prophylactic and therapeutic activity against SARS-CoV-2 infection in Syrian hamsters. The antibody cocktail appears to be a promising candidate for the prophylactic use and for therapy in early COVID-19 cases which have not progressed to severe disease.

Since the first report of occurrence of SARS-COV-2 infection in China on December 30, 2019, 38 the virus has spread rapidly worldwide, accounting for more than 200 million cases and 4 million 39 deaths as on 9 th August 2021 1 . Even though, more than twenty vaccines have been granted 40 Emergency Use Authorization (EUA) in multiple countries, vaccination rate is skewed globally 41 due to limited accessibility to low-and middle-income countries 2,3 . Due to the inequitable access 42 of vaccines and emergence of new variants having immune escape property, the development of 43 herd immunity may take years 3 . Therefore, there remains an unfulfilled need for therapeutic 44 agents to restrict the morbidity and mortality. Convalescent plasma transfusion has been used as 45 therapeutic option in case of novel viral diseases like Ebola virus disease, SARS, MERS and 46 coronavirus disease 2019 (COVID-19) but the variability in the donor antibody titers and risk of 47 blood borne diseases remained as hurdles 4-7 . Monoclonal antibody (mAb) based therapy became 48 an alternative to overcome the limitations of convalescent plasma therapy. The favorable safety 49 profiles and comparatively lesser time for generation and approval made mAbs a feasible 50 therapeutic alternative in case of many emerging disease threats. 51 Monoclonal antibodies can serve as an adjunct to the prophylactic strategy for infections in high-risk groups like aged and immunocompromised people who have suboptimal 53 responses to vaccination 8 . The techniques of combinatorial display libraries, humanized mice 54 and B cell isolation methods have aided in rapid recovery of many antiviral mAbs 9 . Palivizumab 55 was the first mAb to be approved for treatment against Respiratory Syncitial Virus in 1998 10 . In 56 2020, a combination of 3 mAbs, atoltivimab, maftivimab, and odesivimab-ebgn were approved 57 by United States Food and Drug Administration (USFDA) for Zaire Ebola virus therapy 11,12 . 58 More than 200 research laboratories across world are working on developing highly potent 59 4 recombinant human mAbs against SARS-CoV-2 to provide unlimited supply of high-quality 60 product to patients. USFDA has granted EUA for few of the anti-SARS-CoV-2 mAbs targeting 61 epitopes on receptor binding domain (RBD) of the S protein like bamlanivimab plus etesevimab, 62 casirivimab plus imdevimab and sotrovimab for treatment of mild to moderate COVID-19 13-15 . 63 We have developed a cocktail (ZRC-3308) of two highly potent, neutralizing, humanized mAbs, 64 ZRC3308-A7 (CAS RN: 2640223-84-1) and ZRC3308-B10 (CAS RN: 2640224-48-0) that bind 65 with nanomolar affinities to non-competing epitopes on the RBD of the spike protein of SARS-66 CoV-2. Both of these mAbs were designed to have reduced immune effector functions and 67 increased circulation half-life. Here we describe the in vitro biological properties of the mAbs 68 and in vivo evaluation against SARS-CoV-2 infection in Syrian hamster model. 69 

Binding of ZRC3308 mAbs to RBD and Spike protein Trimer 71 ZRC-3308 cocktail showed similar binding profiles to RBD and S Trimer protein when 72 compared with its individual components, ZRC3308-A7 and ZRC3308-B10 ( Fig.1a-1f ). The 73 binding to both RBD and S trimer protein were with high affinities in the sub nM ranges for both 74 the individual mAbs and their cocktail (Table 1) . Pair-wise epitope binning experiment was 75 performed to determine whether each of the two mAbs of the cocktail would bind to RBD even 76 in the presence of the other one. The binding of each antibody to RBD was assessed after the 77 other mAb had been allowed to bind first. Binding of ZRC3308-B10 was observed on ZRC3308-78 A7 precaptured channels and vice versa indicating that both the antibodies bind to distinct 79 epitopes on RBD of spike protein as depicted in sensograms (Fig. 1g, 1h ). The binding of the mAbs to the RBD of SARS-CoV-2 S1 protein is expected to inhibit the 81 interaction of S1 protein to the ACE-2 receptor. The inhibition of RBD binding to ACE-2 in 82 presence of ZRC-3308 was measured in terms of 50 % inhibitory concentration (IC 50 ) by plotting 83 a 4 parameter fit curves of antibody concentration vs. % inhibition as shown in Fig.1i and 1j . 84 Both the mAbs were able to bind to the spike protein RBD and inhibit its binding to ACE2 85 receptor at sub nM IC 50 concentrations. 

For the study, hamsters (n=12/group) were treated with the ZRC3308 cocktail (50 mg/kg, 148 5mg/kg and 1 mg/kg ZRC3308 cocktail) 48 hours prior to the SARS-CoV-2 infection (Fig. 4a) . 149 No clinical signs post was observed following the mAb cocktail treatment and virus infection. In the 10 3.5 TCID50 virus dose group, only minimal bodyweight reduction was seen in the 50 250 mg/kg and 5 mg/kg mAb treated group (Fig 8a, 8b) . The 50 mg/kg group showed consistent viral 251 load reduction on 3 and 5DPI in nasal wash (p <.0.05), lungs (p <.0.05) and nasal turbinates (p 252 <.0.05) (Fig. 8c-e) . SgRNA levels in the lungs (p <.0.05) and nasal turbinates (p <.0.05) also 253 were significantly lower compared to placebo group (Fig.8f,8g) . On histopathological 254 examination, mild to moderate changes were seen in the 50 mg/kg dose group with a lower 255 histopathological score whereas the hamsters of 5 mg/kg group and placebo group showed 256 comparable disease severity (Fig 8i, Fig 9) . In the present study, we have characterized ZRC 3308 mAb cocktail for COVID 19 treatment, 353 which was found to be cross neutralizing and a promising candidate for the prophylactic use and 354 for therapy in early cases which have not progressed to severe disease. Initially ZRC3308-A7 and ZRC3308-B10 were run at saturating concentrations (at 50 nM) in 424 different channels immobilized with RBD. In the next run, ZRC3308-A7 and ZRC3308-B10 425 28 were individually run at 50 nM over both ZRC3308-A7and ZRC3308-B10 captured channels 426 and binding patterns were analyzed.

The inhibition potential of the mAbs was assessed using a competitive ELISA. Briefly, ACE-2 429 protein (Acro Biosystems, USA) was coated onto ELISA plates (Greiner, Germany) at 1 µg/mL 430 concentration in PBS and was incubated (for 12-72 hours) at 2-8°C in a humid chamber. mg/kg (2.5 mg/kg of ZRC3308-A7 + 2.5 mg/kg of ZRC3308-B10), 1.0 mg/kg (0.5 mg/kg of 497 ZRC3308-A7 + 0.5 mg/kg of ZRC3308-B10), and placebo consisting of 5 animals each except 498 for the placebo which consisted of only 3 animals. Cocktails of ZRC3308-A7 and ZRC3308-B10 499 were administered via intraperitoneal (I.P.) route and the total duration of the study was 7 days. Two separate ELISA methods were used to detect ZRC3308-A7 and ZRC3308-B10 antibodies. 506 In both the immunoassays, coating reagent used was SARS-CoV-2 S1 protein (Acro Biosystems, 507 USA 

A total of 60 female, Syrian hamsters, aged 7-10 weeks were used for the prophylactic study, 520 which were divided into 5 groups of 12 animals each. The groups were high (50mg/kg), medium 521 (5 mg/kg), low (1mg/kg) dose of mAb cocktail, an IgG1 isotype antibody (Trastuzumab @ 50 In the second study of therapeutic arm, 18 female hamsters aged 8-10 weeks were used and mAb 539 treatment was given 6 hours post infection with a virus challenge dose of 10 5.5 TCID50. The 50 540 mg/kg and 5 mg/kg mAb dose groups along with placebo group with 6 animals each were 541 included in the study. On day 3 and 5, 3 hamsters form each group were sacrificed to assess 542 collect organ and nasal wash samples. 543 In the third therapeutic study, a challenge virus dose of 10 3.5 TCID50 was given followed by study. Kruskal-Wallis test followed by Mann-Whitney test was used. P < 0.05 were considered 563 to be statistically significant. 564 

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