key: cord-0733022-z9vtw3r2
authors: Copin, Richard; Baum, Alina; Wloga, Elzbieta; Pascal, Kristen E.; Giordano, Stephanie; Fulton, Benjamin O.; Zhou, Anbo; Negron, Nicole; Lanza, Kathryn; Chan, Newton; Coppola, Angel; Chiu, Joyce; Ni, Min; Wei, Yi; Atwal, Gurinder S.; Hernandez, Annabel Romero; Saotome, Kei; Zhou, Yi; Franklin, Matthew C.; Hooper, Andrea T.; McCarthy, Shane; Hamon, Sara; Hamilton, Jennifer D.; Staples, Hilary M.; Alfson, Kendra; Carrion, Ricardo; Ali, Shazia; Norton, Thomas; Somersan-Karakaya, Selin; Sivapalasingam, Sumathi; Herman, Gary A.; Weinreich, David M.; Lipsich, Leah; Stahl, Neil; Murphy, Andrew J.; Yancopoulos, George D.; Kyratsous, Christos A.
title: The monoclonal antibody combination REGEN-COV protects against SARS-CoV-2 mutational escape in preclinical and human studies
date: 2021-06-05
journal: Cell
DOI: 10.1016/j.cell.2021.06.002
sha: c7a9ef0f2c6a9c6731c05589aef5965693b51a2e
doc_id: 733022
cord_uid: z9vtw3r2

Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. As rapidly emerging virus mutants are becoming the next major concern in the fight against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment-induced emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of virus variants in SARS-COV-2 isolates found in COVID-19 patients treated with the two-antibody combination REGEN-COV, as well as in preclinical in vitro studies using single, dual, or triple antibody combinations, and in hamster in vivo studies using REGEN-COV or single monoclonal antibody treatments. Our study demonstrates that the combination of non-competing antibodies in REGEN-COV provides protection against all current SARS-CoV-2 variants of concern/interest and also protects against emergence of new variants and their potential seeding into the population in a clinical setting.

The coronavirus disease 2019 global pandemic has motivated a widespread effort to understand adaptation of its etiologic agent, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Similar to all viruses, SARS-CoV-2 is constantly evolving, mutants are selected due to a fitness advantage, which can be associated with an ability to spread more efficiently in the human population or to overcome natural or vaccine-mediated immune responses. Ongoing viral evolution is a major concern for direct antivirals, especially for therapeutics targeting the main proteins recognized by humoral immunity, such as the viral glycoproteins. Such mutations can provide a fitness advantage in the context of drug treatment; therefore they can be rapidly selected during therapy, and may lead to treatment failure and/or spread of treatment resistant-associated variants. To date, multiple studies have investigated the development of viral resistance against neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 spike protein (Baum et al., 2020a; Liu et al., 2021b; Weisblum et al., 2020) . However, better understanding of virus evolution as the virus spreads in humans and the mechanisms driving resistance during treatment is necessary to ensure optimal efficacy of antivirals. REGEN-COV (previously known as REGN-COV2) is a cocktail of two fully-human noncompeting, neutralizing antibodies -casirivimab (REGN10933) and imdevimab (REGN10987) -that target the receptor binding domain (RBD) on the SARS-CoV-2 spike protein and thereby prevent viral entry into the host cell (Hansen et al., 2020) . The strategic decision to include a pair of highly potent non-competing antibodies that can simultaneously bind to distinct, non-overlapping epitopes of the RBD was made to safeguard against the rapid emergence of drug resistant variants and provide coverage against circulating viral variants (Baum et al., 2020a) . In vitro studies showed that combining two non-competing antibodies protected against rapid escape seen with its individual single-antibody components (Baum et al., 2020a) . Additionally, the benefit of this combination approach has been supported by subsequent studies showing that REGEN-COV retains neutralization potency against emerging SARS-CoV-2 variants potentially associated with increased infectivity (e.g. D614G and UK B.1.1.7 variants), J o u r n a l P r e -p r o o f as well as those associated with reduced vaccine-induced neutralization titers or vaccine efficacy (e.g. South Africa B.1.351), even when one of its components has partially reduced activity (Garcia-Beltran et al., 2021; Liu et al., 2021a; Madhi et al., 2021; Wang et al., 2021; Yurkovetskiy et al., 2020) . Concerns regarding single antibody approaches in the clinic are emerging, based on increasing prevalence of naturally circulating variants resistant to these single agents , and reports within clinical studies that single antibody treatment is associated with the emergence of resistant (FDA, 2021).

Although in vitro escape studies using recombinant viruses strongly support the rationale of using non-competing antibody combinations to avoid drug resistance, questions remain regarding the relevance of these findings to authentic SARS-CoV-2 virus, to in vivo infection models, and most importantly, to the clinical setting. In this work we first show that in vitro escape variants are rapidly selected against all individual clinical-stage antibodies targeting the RBD of the spike protein, independent of the targeted epitope or its degree of conservation, that such risk can be mitigated by utilizing combinations of two non-competing antibodies, and even further reduced by combining three non-competing antibodies. We extend these findings by showing that escape variants can also be efficiently selected in vivo upon treatment with individual antibodies, independent of dosage or treatment setting (prophylaxis or therapy), while the REGEN-COV combination fully protects against development of such resistance. Importantly, we confirm these findings in humans; we investigated the genetic diversity of the entire spike protein across 4,882 samples from 1,000 outpatients or hospitalized patients with confirmed COVID-19 from ongoing Phase 1-3 trials (Weinreich et al., 2021 . Analysis of baseline and post-treatment sequence diversity in placebo and REGEN-COV treated patients demonstrated that the non-competing antibody combination protects against selection of drug resistant variants.

We previously generated a large library of fully human monoclonal antibodies targeting the spike protein of SARS-CoV-2 and selected REGN10933 + REGN10987 as a pair of potently neutralizing molecules that can simultaneously bind to their target (Hansen et al., 2020) . We hypothesized that this non-competing antibody combination would retain its potency as the virus evolves in the human population and safeguard against selection of escape mutants following treatment (Baum et al., 2020a; Tegally et al., 2020) With the recent rise in rapidly emerging variants, we are able to test this hypothesis by evaluating the potency of the individual antibodies as well as the REGEN-COV combination against variants of concern/interest as defined by the Centers for Disease Davies et al., 2021; Nonaka et al., 2021; Tegally et al., 2020; West et al., 2021; Zhang et al., 2021) Importantly, even in instances where one of the antibodies is impacted, the combination retains full neutralization potency, a key property through which this combination of antibodies was originally selected, thus maintaining coverage against all assessed variants (Fig. 1A&B) .

We have previously shown that combination therapy with two anti-spike antibodies targeting non-overlapping epitopes of the SARS-CoV-2 spike protein minimizes the likelihood of rapid viral escape observed in vitro with monotherapy , as has also been demonstrated by others (Dong et al., 2021; Weisblum et al., 2020) .

Expanding on our previous work using VSV-SARS-CoV-2-spike recombinant virus to monitor resistance to mAb treatment, we now performed such escape studies with several RBD targeting mAbs approved under Emergency Use Authorization (EUA) or in late-stage clinical trials, and tested monotherapy as well as dual and triple combinations. We found that while only one to two passages led to complete virus resistance against all mAbs used as monotherapy, seven consecutive passages were needed to reach complete resistance to the REGEN-COV combination, requiring J o u r n a l P r e -p r o o f selection of multiple simultaneous mutations impacting each antibody (Fig.2A&B ).

Escape variants identified with COV2-2130, COV2-2196, and LY-COV555 antibodies were consistent with those previously described for these molecules (Table S1 ) (Dong et al., 2021; FDA, 2021) . Similar to the REGEN-COV combination, the COV2-2130 and COV2-2196 cocktail was also resistant to rapid escape and full escape was associated with acquiring multiple mutations within the same virus ( Fig.2A ) (Dong et al., 2021) . In contrast, the LY-CoV555/CB6 combination behaved similarly to monotherapy, where rapid escape driven by a single amino acid substitution was observed. Such rapid escape from combinations of competing antibodies has been demonstrated in our previous studies and is expected based on the overlapping nature of the LY-CoV555 and CB6 epitopes (Baum et al., 2020a; Jones et al., 2021; Shi et al., 2020; Starr et al., 2021) .

Importantly, rapid escape observed in the monotherapy setting was independent of the targeted epitope or its sequence conservation as escape variants were rapidly selected against an antibody targeting a broad coronavirus epitope, VIR-7831 (Pinto et al., 2020) ( Fig. 2A , Table S1 ). All VIR-7831 escape residues mapped to the published epitope of the antibody and the resultant escape virus maintained its ability to replicate, indicating that these mutations were not deleterious to the virus (data not shown). Furthermore, the identified escape variants can be found in publicly available genome sequences (GISAID), indicating that they are tolerated by the authentic SARS-CoV-2 virus and thus are likely to be selected under pressure. These experiments highlight the inherent risk of monotherapy against SARS-CoV-2 with any anti-spike mAb, regardless of the targeted epitope or its conservation.

We have previously described a triple antibody combination against Zaire ebolavirus (Pascal et al., 2018) and we similarly decided to assess potential advantages of combining three non-competing neutralizing mAbs targeting the RBD of SARS-CoV-2:REGN10933+REGN10987+REGN10985. As part of an effort to structurally characterize our SARS-CoV-2 neutralizing antibodies, we determined a cryo-EM structure of REGN10985 (plus another antibody, REGN10989) bound to RBD (Supp.

J o u r n a l P r e -p r o o f Fig. 4) . REGN10985 binds to a broad patch on the side of the RBD, directly below the region contacted by ACE2. Although there is very little overlap between the ACE2 and REGN10985 contact surfaces on the RBD, the REGN10985 Fab is angled so that it overlaps with the space that ACE2 would occupy, making this antibody a direct blocker of ACE2 binding like REGN10933 and REGN10987. By combining this cryoEM structure with our previously-determined structure of REGN10933+REGN10987+RBD (Hansen et al., 2020) we produced a model of the three-antibody complex showing that all three mAbs are able to bind simultaneously to the RBD of the spike protein in a nonoverlapping fashion ( Figure 2C ). We additionally demonstrated that the three-antibody combination has similar neutralization potency as REGEN-COV ( Figure 2B ). We found that addition of the third non-competing RBD mAb further increased protection against viral escape, with no loss of antiviral potency observed through eleven consecutive passages ( Figure 2A , Table S1 ). These results demonstrate that a three mAb neutralizing non-competing combination targeting the spike RBD provides a potential further advantage as a SARS-CoV-2 therapeutic.

To understand how these in vitro escape findings demonstrating that mAb combination treatments safeguard against development of drug resistance translate to a more physiological setting, we evaluated virus evolution in vivo following mAb administration as both monotherapy (REGN10987 or REGN10933) and as the REGEN-COV combination in the SARS-CoV-2 hamster challenge model. Animals were challenged with SARS-CoV-2 (USA-WA1/2020) and treated with either high or low dose (50mg/kg or 5mg/kg) of either REGN10987 or REGN10933 as monotherapy or with the REGEN-COV combination (50mg/kg or 5mg/kg) in both prophylactic and treatment settings ( Figure 3A ). These doses were previously shown to provide partial or complete efficacy in this animal model with REGEN-COV (Baum et al., 2020b) . Lungs from infected animals were collected on day 7 post-challenge, presumably after the virus has had an opportunity to go through multiple rounds of replication, to isolate viral RNA. RNAseq analysis was used to detect variants in the spike protein present even at low frequencies in the virus population (>5%). To identify putative treatment resistant variants, we focused our analysis on the spike RBD, where the REGEN-COV antibodies J o u r n a l P r e -p r o o f are known to bind, and where all previously identified resistant variants to either antibody are located. Analysis of the RBD sequence in saline treated animals did not reveal any variants, indicating that in the absence of treatment the virus is not under strong selection pressure in this model (Fig. 3B&C) . However, several variants in the RBD were identified in animals treated with either of the single antibodies as monotherapy (Fig. 3B&C) . Remarkably, our analysis demonstrated selection of resistance variants in almost half (18/40) of monotherapy treated animals versus none (0/20) of the animals treated with the REGEN-COV combination. Out of the 7 RBD variants identified in monotherapy treated animals, 6 have been previously described as resistance variants or mapped to the same amino acid position as other resistance variants to either REGN10933 or REGN10987 (Table S3 ). Four of these variants were previously identified in our in vitro escape studies, validating the relevance of using VSV-SARS-CoV-2-spike recombinant virus to identify SARS-CoV-2 escape mutations (Baum et al., 2020a) . Interestingly, variant selection occurred with monotherapy treatment in both the prophylactic and therapeutic settings, and in both low and high dose groups. Although rare unique variants outside of the RBD were also identified in a single animal treated with the REGEN-COV combination (Fig. 3B&C) , the low frequency of these variants and their location well outside the epitopes of the antibodies make it extremely unlikely that they were associated with the REGEN-COV treatment.

To assess the emergence of drug resistance in REGEN-COV treated COVID-19 patients, we characterized the genetic diversity of 1,336 and 3,546 SARS-CoV-2 RNA samples isolated from 272 hospitalized and 728 non-hospitalized individuals, respectively, at baseline and several time points post mAb cocktail administration (Table   S2 ). In both hospitalized and non-hospitalized study groups, patients were randomly assigned (1:1:1) to receive placebo, 2.4g of REGEN-COV, or 8.0g of REGEN-COV, and patient samples were collected at multiple time points from baseline (day 1) to study end point (day 29). RNA-seq analysis was performed to characterize the genetic diversity of the spike sequence from all swabs positive for SARS-CoV-2 RNA by qRT-PCR. Minor variants present at a frequency of at least 15% in the virus population were identified. In total, 244 and 514 amino acid changes were identified across all samples J o u r n a l P r e -p r o o f from hospitalized and non-hospitalized patients, respectively. For both studies, variant counts and frequencies in all three treatment groups were similar, indicating that REGEN-COV did not increase spike protein diversity relative to placebo (Table S2) .

To determine whether REGEN-COV treatment contributed to selection of resistant variants in the spike protein, we longitudinally compared the frequency of minor variants in each patient from all available RT-PCR positive samples. Among all patient samples from the hospitalized group, we identified a total of 89, 87 and 108 amino acid changes in the placebo, low-dose and high dose treated groups, respectively. These mutations were evenly spread across the entire spike protein sequence ( To date, we and others have identified antibody resistant variants through in vitro escape studies and have demonstrated that combinations of two non-competing antibodies provide protection against rapid viral escape (Baum et al., 2020a; Weisblum et al., 2020) . We hypothesized that a cocktail of three non-competing antibodies could further delay emergence of resistance. Although, neutralizing antibodies against the Nterminal domain (NTD) of spike have been described , this region is frequently deleted in emerging variants , therefore such molecules are not ideal as therapeutics. We thus screened for and identified a third, noncompeting, RBD-binding mAb and now show that a triple antibody combination containing this mAb is even less susceptible to escape than the double combination.

However, the question regarding the relevance of these in vitro escape studies remains, and it is important to examine viral dynamics under therapeutic antibody pressure in more relevant settings, including in in vivo efficacy models and in treated patients. Our J o u r n a l P r e -p r o o f study provides evidence that in vitro escape studies with recombinant virus are highly predictive of in vivo selection. Indeed, we are able to show that in animals and in humans, the relative risk of selecting for spike variants that are resistant to treatment is predicted by in vitro findings. Treatment with the single antibody components of the REGEN-COV combination results in rapid selection of escape variants, whereas the combination protects against viral escape in vitro, in vivo, and in COVID-19 patients.

Furthermore, by comparing escape mutations identified through in vitro assays utilizing recombinant VSV-based virus to those identified in SARS-CoV-2 infected hamsters, we show that many identical mutations are identified in both settings, further validating the in vitro findings.

Understanding pathogen population diversity and selection within the infected host has important implications for understanding virus evolution and resistance processes, and for proper identification of appropriate drug treatments. To date, the vast majority of SARS-CoV-2 sequencing data has come from global surveillance efforts and reporting of consensus sequences that include fixed mutations in isolates obtained from independent individuals. This is a large-scale report describing, within individual patients, the longitudinal evolution of the SARS-CoV-2 virus population. Moreover, this study evaluates the effects of antiviral antibody treatment on virus evolution in humans.

As predicted by our in vitro and in vivo preclinical studies, treatment with the REGEN-COV cocktail in the clinic did not seemingly select for resistant variants nor appreciably alter virus evolution. Our approach allowed us to observe minor variants and our data indicate the vast majority of infected individuals are already harboring spike protein variants at baseline, prior to use of any antiviral agent and before natural immunity is established, as half of the study participants had not yet mounted an antiviral immune response at baseline (Weinreich et al., 2020) . Indeed, from our clinical sequencing data we can observe multiple variants in amino acid residues identified in our in vitro escape studies expected to impact clinical antibody candidates, including VIR-7831 (P337, T345) and COV-2196 (G476) as well as our own individual antibodies. This clearly demonstrates that the SARS-CoV-2 circulating population is highly heterogeneous in Although the REGEN-COV combination maintains its neutralization potency against all variants of interest/concern to date, one of its components (REGN10933) demonstrates partially decreased potency against some variants. This highlights the potential risk that even non-competing combination therapies may not retain their full advantages in some geographical regions with high prevalence of select variants of concern. Thus, active surveillance of circulating viruses and development of next-generation antibody combinations where multiple components maintain coverage against emerging variants should be an important focus for the future of antiviral mAb therapeutics. This type of approach will require agility and may need to be analogous to the framework applied to seasonal updates of influenza vaccine components, where new anti-spike mAbs are substituted or added as necessary to maintain optimal efficacy against a rapidly mutating pathogen.

To ensure optimal and sustained efficacy, an antiviral therapeutic intervention should be prospectively designed to not only have coverage against major circulating variants, but also to prevent the selection of minor escape variants. And although the risk of viral resistance cannot be completely eliminated, it can be greatly reduced through selection of optimal combination therapies. This study demonstrates that carefully designed antibody combinations can achieve both of these critical objectives.

The current study focused on assessing likelihood of identifying REGEN-COV mediated 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Christos Kyratsous (christos.kyratsous@regeneron.com).

Regeneron materials described in this manuscript may be made available to qualified, academic, noncommercial researchers through a materials transfer agreement upon request at https://regeneron.envisionpharma.com/vt_regeneron/. For questions about how Regeneron shares materials, use the email address preclinical.collaborations@regeneron.com.

In vitro and in vivo preclinical sequencing data are available from the authors upon request. Protein Data Bank (PDB) identification number for the SARS-CoV-2 RBD complex with REGN10985 and REGN10989 Fabs is 7M42. The corresponding cryo-EM map is in the Electron Microscopy Data Bank (EMDB) with identification number EMD-23662.

The outpatient study is an ongoing adaptive, multicenter, randomized, double-blind, placebo-controlled, seamless phase 1/2/3 clinical trial in COVID-19 outpatients (clinicaltrials.gov: NCT04425629). Non-hospitalized patients > 18 years of age with a SARS-CoV2-positive test result <72 hours, and symptom onset < 10 days before J o u r n a l P r e -p r o o f randomization, were randomized 1:1:1 to receive placebo, 2.4g (1.2 g each of casirivimab and imdevimab) or 8.0 g (4.0 g each of casirivimab and imdevimab). For the present work, 3523 nasopharyngeal, 17 nasal and 6 saliva samples retrieved from a total of 728 patients were analyzed. Sex/gender of human donors was not associated with the samples.

The hospitalized study is an ongoing adaptive, multicenter, randomized, double-blind, placebo-controlled, seamless phase 1/2/3 clinical trial in hospitalized patients with Covid-19 (clinicaltrials.gov: NCT04426695) . Hospitalized patients > 18 years of age with a SARS-CoV2-positive test result <72 hours, and symptom onset < 7 days before randomization, were randomized 1:1:1 to receive placebo, 2.4g (1.2 g each of casirivimab and imdevimab) or 8.0 g (4.0 g each of casirivimab and imdevimab). For the present work, 968 nasopharyngeal, 249 nasal and 119 saliva samples retrieved from a total of 272 patients were analyzed. Sex/gender of human donors was not associated with the samples.

A total of 80 golden hamsters, male, approximately 9 weeks of age (130-150g) were used in the study. Animals were housed one to five per cage; caging complies with the cage requirements (type, floor space, height, etc.) of the Animal Welfare Act and the Guide for the Care and Use of Laboratory Animals. Animals were acclimated to ABSL-3 laboratory conditions prior to exposure. In the ABSL-3, prior to virus exposure, animals were observed at least once daily, and after virus exposure animals were observed at least twice daily for morbidity and mortality. On Day -2 relative to exposure, animals were sedated and received treatment via intraperitoneal injection. 

Non-replicative pseudoparticles were generated using a VSV genome encoding the firefly luciferase and GFP genes instead of the native viral glycoprotein (VSV-G).

Infectious particles complemented with VSV-G (VSV-∆G-Fluc-2A-GFP/VSV-G) were recovered and produced using standard techniques with minor modifications (Baum et al., 2020a; Hansen et al., 2020; Lawson et al., 1995) . The cells were incubated 1 hour at 37C with 5% CO2. Cells were washed three times with PBS to remove residual input virus and overlaid with DMEM with glutamine (Life Technologies) with 0.7% IgG-free BSA (Sigma), sodium pyruvate (Life Technologies), and Gentamicin (Life Technologies). After 24 hours at 37C with 5% CO2, the J o u r n a l P r e -p r o o f supernatant containing pseudoparticles was collected, centrifuged at 3000xg for 5 minutes to clarify, aliquoted, and frozen at -80C. Variants were cloned into the spike expression plasmid using site-directed mutagenesis and pseudoparticles were produced as described above.

Neutralization assays with VSV based pseudoparticles. hours prior to assay. Cells were allowed to reach approximately 85% confluence before use in assay. Antibodies were diluted in Infection Media containing DMEM with glutamine (Life Technologies), 0.7% Low IgG BSA (Sigma), 1X Sodium Pyruvate (Life Technologies), and 0.5% Gentamicin (Life Technologies) to 2X assay concentration and diluted 3-fold down in Infection media, for an 11-point dilution curve in the assay beginning at 3 ug/mL (20 nM). Antibody dilutions were mixed 1:1 with pseudoparticles for 30 minutes at room temperature prior to addition onto Vero cells. Cells were incubated at 37C, 5% CO2 for 24 hours. Supernatant was removed from cells prior to lysis with 100 uL Glo Lysis Buffer (Promega). 100 uL resuspended Bright Glo substrate (Promega) was then added and luminescence was read on a Spectramax i3x (Molecular Devices). Exported values were analyzed using GraphPad Prism (v8.4.1)

Escape studies were performed with recombinant VSV-SARS-CoV-2-Spike virus as previously described (Baum et al., 2020a) . Escape variants were selected by incubating VSV-SARS-CoV-2-Spike under antibody pressure ranging from 0.016ug/mL to 50ug/mL. After 30 minutes of incubation, the mixture was used to infect 2.5x10^5 Vero 

A total of 80 golden hamsters, male, approximately 9 weeks of age (130-150g) were used in the study. Animals were weighed prior to the start of the study. The animals were monitored twice daily for signs of COVID-19 disease (ruffled fur, hunched posture, responsiveness, nasal discharge, labored breathing, a.o.) during the study period. Body weights were measured once daily during the study period. Antibodies were dosed through intraperitoneal (IP) injection (0.200ml volume). Animals were challenged with 1x10^4 PFU of (USA-WA1/2020 (NR-52281; BEI Resources) by administration of 0.05ml total of viral inoculum, 0.025ml dropwise into each nostril (mock exposed animals received the same volume of sterile PBS). Prior to treatment and virus administration, animals were sedated using inhalational isoflurane. At study end (day 7 post-challenge), animals were euthanized via inhalational CO 2 and tissues samples were aseptically collected for RNAseq. The day 7 time-point was selected for RNA collection and analysis as the virus has had ample ability to replicate in the animal allowing for detection of selected variants. Tissue samples were inactivated using Trizol Reagent (Invitrogen, Carlsbad, CA, USA). All animal studies were conducted in compliance with all relevant local, state, and federal regulations and were approved by the Texas Biomed Institutional Animal Care and Use Committee (IACUC; protocol number 1739MA).

Outpatient clinical trial This is an ongoing adaptive, multicenter, randomized, double-blind, placebo-controlled, seamless phase 1/2/3 clinical trial in COVID-19 outpatients (clinicaltrials.gov: NCT04425629). Non-hospitalized patients > 18 years of age with a SARS-CoV2positive test result <72 hours, and symptom onset < 10 days before randomization, J o u r n a l P r e -p r o o f were randomized 1:1:1 to receive placebo, 2.4g (1.2 g each of casirivimab and imdevimab) or 8.0 g (4.0 g each of casirivimab and imdevimab). The primary virologic endpoint was the time weighted average daily change in viral load from baseline (Day 1) through Day 7. The key secondary clinical endpoint was the proportion of patients with 1 or more Covid-19 related medically-attended visit (MAV) through Day 29.

This is an ongoing adaptive, multicenter, randomized, double-blind, placebo-controlled, seamless phase 1/2/3 clinical trial in hospitalized patients with Covid-19 (clinicaltrials.gov: NCT04426695) . Hospitalized patients > 18 years of age with a SARS-CoV2-positive test result <72 hours, and symptom onset < 7 days before randomization, were randomized 1:1:1 to receive placebo, 2.4g (1.2 g each of casirivimab and imdevimab) or 8.0 g (4.0 g each of casirivimab and imdevimab). The primary clinical endpoint was the incidence of death or required mechanical ventilation through Day 29.

Viral RNA was extracted from hamster samples and from samples collected in the clinical trials (Viracor-Eurofins, MO, USA). RNAlater Stabilization Solution was aspirated from sample storage tubes, and 1-5mL Qiazol Lysis Reagent (Qiagen, #79306) was added. Tissues were homogenized in Qiazol using an Omni International Prep 96

Homogenizer. The resulting lysate was stored overnight at -20C. In preparation for extraction, lysates were thawed, vortexed, and set up on the deck of a Hamilton Robotics Microlab STAR. The STAR dispensed 150uL of chloroform (Sigma Aldrich, #C2432-500mL) per well to a 2 mL deep well plate (Life Technologies, #4388476), followed by the addition of either 375 or 750uL of lysate. Additional Qiazol was added to wells where only 375uL lysate was added to bring the total volume of lysate to 750uL.

Robotic 96-channel pipetting was used to mix the Qiazol and chloroform layers in the deep well plate, followed by a 10 minute incubation at room temperature. The deep well plate was transferred off the STAR to a High Res Biosolutions NanoCell modular robotic system, then centrifuged for 10 minutes at 18G. The plate was returned to the STAR, and using robotic 96-channel pipetting, 400 µL clear aqueous phase was aspirated from J o u r n a l P r e -p r o o f Qualitative and quantitative measurements of viral load were determined by quantitative RT-PCR,10 ul of RNA combined with 25 ng Human Universal Reference RNA (Agilent) was purified by PureBeads (Roche Sequencing). cDNA synthesis was performed using SuperScript™ IV First-Strand Synthesis System (Thermal Fisher) following vendor's protocol. Then one half of cDNA (10 ul) was used to generate libraries using Swift Normalase™ Amplicon Panel (SNAP) SARS-CoV-2 Panel (Swift Biosciences) following vendor's protocol. Sequencing was run on NextSeq (Illumina) by multiplexed pairedread run with 2X150 cycles. Overall quality report was generated summarizing the quality of all reads in each sample, along each base pair.

Swift amplicon bulk RNA-seq reads were aligned to the SARS-COV-2 reference genome Wuhan-Hu-1 (accession: MN908947) using Minimap2 (v2.17) (Li, 2018 calling. At each SNP, the frequency of viral mutations inferred from the sequencing reads were calculated from samtools (v1.9) pileup output (Li et al., 2009) . Variants were flagged if nucleotide positions were covered by at least 10 reads and frequency of mutated reads at these positions was at least 15%.

Variant calling from SARS-CoV-2 public genomes was performed as previously described . SARS-CoV-2 complete genome sequences were downloaded from GISAID Nucleotide database (https://www.gisaid.org) on February 4th, 2021.

Sequences were curated and genetic diversity of the spike-encoding gene was assessed across high quality genome sequences using custom pipelines. In brief, Blastn was used to align the Wuhan-Hu-1 spike nucleotide sequence (accession: MN908947) against each individual genome. Results were analyzed and presence of the gene was validated if alignment length was greater than 95% with an identity percentage greater than 70%. Homologous spike protein sequences were extracted, translated and aligned to identify amino-acid changes with respect to the reference.

The cryoEM structure of RBD-REGN10933-REGN10987 PDB (6XDG) and the cryoEM structure of RBD-REGN10989-REGN10985 ( Fig S4) were superimposed using the software Pymol with the command align for the RBD, where only REGN10933, REGN10987 and REGN10985 are shown.

J o u r n a l P r e -p r o o f Fab fragments of REGN10985 and REGN10989 antibodies were digested with FabALACTICA kit (Genovis) and purified by size-exclusion chromatography. 400 µg of the REGN10985 Fab and 400 µg of REGN10989 Fab were mixed with 200 µg of SARS-CoV-2 RBD and incubated on ice for ~30 min before being injected into a Superdex 200 increase gel filtration column equilibrated to 50 mM Tris pH 7.5, 150 mM NaCl. Peak fractions containing the REGN10985 Fab -REGN10989 Fab -RBD complex were collected and concentrated using a 10 kDa MWCO centrifugal filter (Amicon). For cryo-EM grid preparation, the protein sample was diluted to ~1.5 mg/mL and 0.15% PMAL-C8 amphipol was added. 3.5 µL of protein was deposited onto a freshly plasma cleaned UltrAufoil grid (1.2/1.3, 300 mesh). Excess solution was blotted away with filter paper for 4 s at blotting force 0, and plunge frozen into liquid ethane using a Vitrobot Mark IV (Thermo Fisher). The grid was loaded into a Titan Krios (Thermo Fisher) equipped with a K3 detector (Gatan) and 7,763 movies were collected using EPU (Thermo Fisher) at 105,000x magnification, corresponding to a pixel size of 0.85 Å. A dose rate of 15 electrons per pixel per second was used and each movie was 2 seconds, corresponding to a total dose of ~40 electrons per Å2.

All cryo-EM data processing was carried out using cryoSPARC v2.14.21 (Punjani et al., 2017) . The movies were aligned using patch motion correction and patch CTF estimation. An initial set of particles picked using blob picker were subjected to 2D classification to generate templates for template picking. ~5.5 million particles picked by template picking were subjected to multiple rounds of 2D classification to remove unbound Fabs and particles containing an incomplete complex, resulting in 607,309 'good' complex particles. Ab initio reconstruction with 6 classes generated a single class containing 146,054 particles that corresponded to the full REGN10985 Fab -REGN10989 Fab -RBD complex. Non-uniform refinement of the particles in this class followed by local refinement resulted in a 3.3 Å resolution (FSC=0.143) map that was used for model building. Into this map, we manually placed models of the RBD (taken from PDB code 6M17) and the two Fabs (taken from prior Regeneron antibody J o u r n a l P r e -p r o o f structures). These models were then manually rebuilt using Coot and real-space refined against the map using Phenix (Emsley et al., 2010; Liebschner et al., 2019) . 

Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies

REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters

SARS-CoV-2 Variant Classifications and Definitions

Increased mortality in community-tested cases of SARS-CoV-2 lineage B.1.1.7

Genetic and structural basis for recognition of SARS-CoV-2 spike protein by a two-antibody cocktail

Features and development of Coot

FACT SHEET FOR HEALTH CARE PROVIDERS EMERGENCY USE AUTHORIZATION (EUA) OF BAMLANIVIMAB

Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity

Mutational escape from the polyclonal antibody response to SARS-CoV-2 infection is largely shaped by a single class of antibodies

Studies in humanized mice and convalescent humans yield a SARS-CoV-2 antibody cocktail

Update on COVID-19 variants and impact on bamlanivimab distribution

Bamlanivimab/etesevimab

The neutralizing antibody, LY-CoV555, protects against SARS-CoV-2 infection in nonhuman primates

Recombinant vesicular stomatitis viruses from DNA

Minimap2: pairwise alignment for nucleotide sequences

The Sequence Alignment/Map format and SAMtools

Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike

Neutralizing Activity of BNT162b2-Elicited Serum

Identification of SARS-CoV-2 spike mutations that attenuate monoclonal and serum antibody neutralization

Efficacy of the

Covid-19 Vaccine against the B.1.351 Variant

The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data

Genomic Evidence of SARS-CoV-2 Reinfection Involving E484K Spike Mutation

Development of Clinical-Stage Human Monoclonal Antibodies That Treat Advanced Ebola Virus Disease in Nonhuman Primates

Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody

cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination

A human neutralizing antibody targets the receptor-binding site of SARS-CoV-2

Complete map of SARS-CoV-2 RBD mutations that escape the monoclonal antibody LY-CoV555 and its cocktail with LY-CoV016

Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa

Antibody Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7

REGEN-COV Antibody Cocktail Clinical Outcomes Study in Covid-19 Outpatients. BioRxiv

REGN-COV2, a Neutralizing Antibody Cocktail, in Outpatients with Covid-19

Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

SARS-CoV-2 lineage B.1.526 emerging in the New York region detected by software utility created to query the spike mutational landscape

Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant

Emergence of a Novel SARS-CoV-2 Variant in Southern California

The authors would like to thank Kristen Tramaglini, Yasmin Khan, Lisa Boersma and Sarah Whittleton for their continuous support.

A.B. and C.A.K. conceptualized the studies. E. W., K.E.P., S.G., and B.O.F. conducted