key: cord-0951682-ux8jqekp
authors: Baum, Alina; Copin, Richard; Ajithdoss, Dharani; Zhou, Anbo; Lanza, Kathryn; Negron, Nicole; Ni, Min; Wei, Yi; Atwal, Gurinder S.; Oyejide, Adelekan; Goez-Gazi, Yenny; Dutton, John; Clemmons, Elizabeth; Staples, Hilary M.; Bartley, Carmen; Klaffke, Benjamin; Alfson, Kendra; Gazi, Michal; Gonzales, Olga; Dick, Edward; Carrion, Ricardo; Pessaint, Laurent; Porto, Maciel; Cook, Anthony; Brown, Renita; Ali, Vaneesha; Greenhouse, Jack; Taylor, Tammy; Andersen, Hanne; Lewis, Mark G.; Stahl, Neil; Murphy, Andrew J.; Yancopoulos, George D.; Kyratsous, Christos A.
title: REGN-COV2 antibody cocktail prevents and treats SARS-CoV-2 infection in rhesus macaques and hamsters
date: 2020-08-03
journal: bioRxiv
DOI: 10.1101/2020.08.02.233320
sha: 59b1572f1ceb1ad883c60e6848d7d74d18b0f32a
doc_id: 951682
cord_uid: ux8jqekp

An urgent global quest for effective therapies to prevent and treat COVID-19 disease is ongoing. We previously described REGN-COV2, a cocktail of two potent neutralizing antibodies (REGN10987+REGN10933) targeting non-overlapping epitopes on the SARS-CoV-2 spike protein. In this report, we evaluate the in vivo efficacy of this antibody cocktail in both rhesus macaques and golden hamsters and demonstrate that REGN-COV-2 can greatly reduce virus load in lower and upper airway and decrease virus induced pathological sequalae when administered prophylactically or therapeutically. Our results provide evidence of the therapeutic potential of this antibody cocktail.

Fully human monoclonal antibodies are a promising class of therapeutics against SARS-CoV-2 39

infection (Cohen, 2020) . To date, multiple studies have described discovery and characterization 40 of potent neutralizing monoclonal antibodies targeting the spike glycoprotein of SARS-CoV-2 41 (Baum et al., 2020; Cao et al., 2020; Hansen et al., 2020; Ju et al., 2020; Pinto et may reflect remaining viral inoculum as well as newly replicating virus, subgenomic RNA 78 (sgRNA) should only result from newly replicating virus. For placebo-treated animals, the kinetics 79 of viral load measures was as previously reported, with peak in viral load on day 2 post-challenge, 80 although the majority of animals were still positive for viral RNA in nasal swabs on day 5; while 81 the kinetics of gRNA and sgRNA were similar, sgRNA levels were about a hundred-fold lower, 82 consistent with what others have reported (Chandrashekar et al., 2020; Mercado et al., 2020; Yu 83 et al., 2020; Zost et al., 2020) . For animals receiving REGN-COV2 prophylaxis we observed 84 markedly accelerated clearance of gRNA with almost complete ablation of sgRNA in the majority 85 of the animals, showing that REGN-COV2 can almost completely block establishment of virus 86 infection; this pattern was observed across all measurements in both nasopharyngeal swabs and 87 BAL compared to placebo animals, demonstrating that mAbs administered prophylactically can 88

greatly reduce viral load in both upper and lower airways ( Figure 1B ). 89 90

A second prophylaxis study (NHP Study #2) was designed to test whether REGN-COV2 could 91 protect against a 10-fold higher viral inoculum (1.05x10^6 PFU), and compared the 50mg/kg dose 92 of REGN-COV2 (25mg/kg of each antibody) with a much lower dose ( Figure 2A ). 93

Nasopharyngeal and oral swabs were collected and used to measure virus genomic and 94 subgenomic virus RNA. We observed that 50mg/kg of REGN-COV2 administered 3 days prior to 95 virus challenge was once again able to minimize virus replication even when animals were 96 challenged with this 10-fold higher viral challenge ( Figure 2B ), while the prophylactic effect was 97 greatly diminished with the 0.3mg/kg dose. Interestingly, in this study we observed increased 98 impact of mAb treatment on viral load in oral swabs versus nasopharyngeal swabs, potentially 99

indicating that mAb treatment may impact multiple physiological sources of virus replication 100

differentially. Additional studies in animal models and humans will be needed to assess whether 101 this is really the case. 102 103

Next, we assessed the impact of REGN-COV2 in the treatment setting by dosing animals 104 challenged with the higher 1x10^6 PFU of SARS-CoV-2 virus at 1-day post-infection with 105 25mg/kg or 150mg/kg of the antibody cocktail ( Figure 2A ). By day 1 post-challenge the animals 106 already reached peak viral load as measured by both genomic and subgenomic RNA, mimicking 107 a likely early treatment clinical scenario of COVID-19 disease, since it has been shown that most 108 SARS-CoV-2 infected individuals reach peak viral loads relatively early in the disease course and 109 often prior or just at start of symptom onset (He et al., 2020; Zou et al., 2020) . Compared to placebo 110 treated animals, REGN-COV2 treated animals displayed accelerated viral clearance in both 111 nasopharyngeal and oral swabs samples, including both genomic and subgenomic RNA samples 112 ( Figure 2C ), clearly demonstrating that the monoclonal antibody cocktail can impact virus load 113 even when administered post infection. Similar to the prophylaxis study, the decrease in viral load 114 appeared more dramatic in oral swabs versus nasopharyngeal swabs. Both treatment groups 115 displayed similar kinetics of virus clearance, suggesting that 25mg/kg and 150mg/kg demonstrate 116 similar efficacy in this study. The treated animals in the 150mg/kg group displayed approximately 117 10-fold higher titers on day 1, at the time of mAb administration, therefore potentially masking 118 enhanced effect of a higher drug dose. Similar impact of mAb treatment was observed on genomic 119 and subgenomic RNA for both NP and oral samples, indicating the mAb treatment is directly 120 limiting viral replication in these animals ( Figure 2C ). 121 122

The two antibody components of REGN-COV2 were selected to target non-overlapping sites on 123 the spike protein to prevent selection of escape mutants, which were readily detectable with single 124 mAb treatment (Baum et al., 2020) . To assess whether any signs of putative escape mutants are 125 observed in an in vivo setting with authentic SARS-CoV-2 virus, we performed RNAseq analysis 126 on all RNA samples obtained from all animals from the study. Analysis of the spike protein 127 sequence identified mutations in NHP samples that were not present in the inoculum virus ( Figure  128 S1) further indicating that the virus is actively replicating in these animals. However, we did not 129 observe any mutations that were unique to treated animals; all identified mutations were either 130 present in the inoculum or in both treated and placebo animals, indicating that they were likely 131 selected as part of virus replication in NHPs and were not selected by mAb treatment. 132 133

We next performed pathology analyses of lungs of infected animals. All four placebo monkeys 134

showed evidence of lung injury characterized in three monkeys by interstitial pneumonia ( Figure  135 2D), with minimal to mild infiltration of mononuclear cells (lymphocytes and macrophages) in the 136 septa, perivascular space, and/or pleura. In these three animals, the distribution of lesions was 137 multifocal and involved 2-3 of the 4 lung lobes. Accompanying these changes were alveolar 138 infiltration of lymphocytes, increased alveolar macrophages, and syncytial cells. Type II 139 pneumocyte hyperplasia was also observed in occasional alveoli. In the fourth placebo monkey, 140 lung injury was limited to type II pneumocyte hyperplasia, suggestive of a reparative process 141 secondary to type I pneumocyte injury. Overall, the histological lesions observed in the placebo 142 animals were consistent with an acute SARS-CoV-2 infection. In the prophylactic groups, 3 of 4 143 animals in the low dose (0.3mg/kg) and 1 of 4 animals in the high dose (50mg/kg) groups showed 144 evidence of interstitial pneumonia (Table S1 ) that was generally minimal and with fewer 145 histological features when compared to the placebo group. In the one affected high dose group 146 animal, only 1 of the 4 lung lobes had a minimal lesion. In the therapeutic treatment groups, 2 of 147 4 low dose (25mg/kg) and 2 of 4 high dose (150mg/kg) treated animals showed evidence of 148 interstitial pneumonia. In all affected low and high dose animals, only 1 of 4 lung lobes had lesions. 149

Finally, there was no test article related toxicities observed at any of the doses tested. In summary, 150 the incidence of interstitial pneumonia (number of animals as well as number of lung lobes 151 affected) and the severity were reduced in both prophylactic and therapeutic treatment modalities, 152 compared to placebo. The analyses demonstrated that prophylactic and therapeutic administration 153 of REGN-COV2 greatly reduced virus induced pathology in rhesus macaques and showed a clean 154 safety profile. 155 156

Unlike rhesus macaques which present with a mild clinical course of disease and transient virus 157

replication when infected with SARS-CoV-2, which may mimic mild human disease, the golden 158 hamster model is more severe, with animals demonstrating readily observable clinical disease, 159

including rapid weight loss accompanied by very high viral load in lungs, as well as severe lung 160

pathology. Thus, this model may more closely mimic more severe disease in humans, although 161 more extensive characterization of this model and severe human disease is needed to better 162 understand similarities and differences in pathology. To evaluate the ability of REGN-COV2 to 163

alter the disease course in this model, we designed a study which evaluated the prophylactic and 164 treatment efficacy of the antibodies ( Figure 3A ). Administration of 50, 5 or 0.5mg/kg of REGN-165 COV2 2 days before challenge with 2.3x10^4 PFU dose of SARS-CoV-2 virus resulted in dramatic 166 protection from weight loss at all doses. This protection was accompanied by greatly decreased 167 viral load in the lungs at the end of the study (day 7 post infection) ( Figure 3C ). Interestingly we 168 did observe high gRNA and sgRNA levels in the lungs of a few treated animals, however these 169 individual animals did not show decreased protection from weight loss than the animals with much 170 lower viral loads. It is possible that mAb treatment may provide additional therapeutic benefit in 171 this model not directly associated with viral load decrease. Alternatively, it is possible that the 172 increased detected viral RNA may not necessarily be associated with infectious virus. As viral 173

replication and lung pathology in the hamster model occur very rapidly, the treatment setting 174 represents a high bar for demonstrating therapeutic efficacy. We were able to observe therapeutic 175 benefit in animals treated with 50mg/kg and 5mg/kg doses of REGN-COV2 combination 1-day 176 post viral challenge ( Figure 3B ). Taken together the two hamster studies clearly demonstrate that 177 REGN-COV2 can alter the course of infection in the hamster model of SARS-COV-2 either when 178 administered prophylactically or therapeutically. 179 180 181

Discussion 182

In this study, we assessed the in vivo prophylactic and treatment efficacy of the REGN-COV2 183 mAb cocktail in two animal models, one of mild disease in rhesus macaques and one of severe 184 disease in golden hamsters. Our results demonstrated that the antibodies are efficacious in both 185 animal models, as measured by reduced viral load in the upper and lower airways, reduced virus 186 induced pathology in the rhesus macaque model, and by limited weight loss in the hamster model. 187 188

The ability of REGN-COV2 to almost completely block detection of subgenomic species of 189 SARS-COV-2 RNA matches or exceeds the effects recently shown in vaccine efficacy studies 190 using the same animal models (Corbett et al., 2020; Gao et al., 2020; Mercado et al., 2020; Patel 191 A. et al., 2020; van Doremalen et al., 2020) . Additionally, the observed accelerated reduction of 192 upper airway virus load in rhesus macaques treated with REGN-COV2 contrasts the lack of impact 193 on viral load in remdesivir treated animals, where reduced viral load could only be observed in 194

lower airways with no differences in nasal viral RNA levels (Williamson et al., 2020) . These 195 findings highlight the therapeutic potential of REGN-COV2 to both protect from and treat SARS-196 COV-2 disease. Additionally, the impact of REGN-COV2 prophylaxis on viral RNA levels in 197 nasopharyngeal and oral swabs may indicate the potential to not only prevent disease in the 198 exposed individual but also to limit transmission. 199 200

To our knowledge this is the first report demonstrating ability of any therapeutic to limit weight 201 loss in the treatment setting of SARS-CoV-2 infection in the hamster model, indicating potential 202 benefit of antibody treatment in the context of a severe infection. Further understanding of both 203 the hamster and the macaque model and how their disease course and pathology mimics the 204 breadth of human COVID-19 disease may help to gain more in depth understanding of how mAb 205 therapeutics may confer clinical benefit. 206 207

Importantly, in our studies we did not observe any signs of increased viral load and/or worsening 208 of pathology in presence of antibodies at either high or low doses in either animal model. Potential 209

for antibody mediated enhancement of disease (ADE) is a serious concern for antibody-based 210 therapeutics and vaccines. And although a recent report showed ability of some anti-spike mAbs 211

to mediate pseudovirus entry into FcγR expressing cell lines, these data do not address whether 212 similar behavior would be observed with authentic SARS-CoV-2 virus and primary immune cells 213 (Wang S. et al., 2020 The number of copies of RNA per mL was calculated by extrapolation from the standard Quantitative RT-PCR Assay for SARS-CoV-2 subgenomic RNA 289 SARS-CoV-2 E gene subgenomic mRNA (sgRNA or sgmRNA) was assessed by RT-PCR using 290 primers and probes as previously described (Chandrashekar et al., 2020 Animals were monitored at least twice daily and enrichment included commercial toys and food 317

supplements. Prior to all blood collections, animals were anesthetized using Telazol (Zoetis Inc., 318

Parsippany-Troy Hills, NJ, USA). At the end of the study, animals were euthanized with an 319 intravenous overdose of sodium pentobarbital. 320

Animal challenge 321

Twenty-four (24) rhesus macaques (13 female and 11 males) were used in this study, and randomly 322 assigned to one of six groups. Animals were obtained from the Southwest National Primate 323

Research Center (SNPRC) colony and were between 2.5 and 6 years of age and approximately 3 324

to 10 kg at the time of study enrollment. On Study Day 0, each NHP was exposed at ABSL-4 with 325 a targeted dose of 1.05 x 10 6 PFU of SARS-CoV-2 in a total volume of 500 µl (5.25 x 10 5 PFU in 326 250 µl via intranasal route and 5.25 x 10 5 PFU in 250 µl via intratracheal route kidney, and all 4 right lung lobes) were collected. Tissues were fixed by immersion in 10% neutral-388 buffered formalin for a minimum of fourteen days, then trimmed, routinely processed, and 389 embedded in paraffin. Sections of the paraffin-embedded tissues were cut at 5 µm thick, and 390 histology slides were deparaffinized, stained with hematoxylin and eosin (H&E), cover slipped, 391

and labeled. Slides were blindly evaluated by a board-certified veterinary pathologist. 392 393

Virus RNA Sequencing 394 10 ul of RNA combined with 25 ng Human Universal Reference RNA (Agilent) was purified by 395

PureBeads (Roche Sequencing). cDNA synthesis was performed using SuperScript™ IV First-396

Strand Synthesis System (Thermal Fisher) following vendor's protocol. Then one half of cDNA 397 (10 ul) was used to generate libraries using Swift Normalase™ Amplicon Panel (SNAP) SARS-398

CoV-2 Panel (Swift Biosciences) following vendor's protocol. Sequencing was run on NextSeq 399 (Illumina) by multiplexed paired-read run with 2X150 cycles. 400 401

RNAseq data analysis 402

RNAseq analysis was perform using Array Studio software package platform (Omicsoft). Quality 403 of paired-end RNA Illumina reads was assessed using the "raw data QC of RNA-Seq data suite". 404

Minimum and maximum read length, total nucleotide number, and GC% were calculated. Overall 405 quality report was generated summarizing the quality of all reads in each sample, along each base 406 pair. Swift amplicon bulk RNA-seq reads were aligned to the SARS-COV-2 reference genome 407

Wuhan-Hu-1 (MN908947) using Omicsoft Sequence Aligner (OSA) version 4. The alignments 408

were sorted by read name, and primers were clipped by the complementary Swiftbiosciences 409 primerclip software (v0.3.8) (https://github.com/swiftbiosciences/primerclip). Reads were 410 trimmed by quality score using default parameters (when aligner encountered nucleotide in the 411 read with a quality score of 2 or less, it trimmed the remainder of the read Figure 1 . Figure S1 . B Figure S1 . RNAseq analysis of viral RNA from NHP study #2. (A) Virus RNA was sequenced and RNAseq analysis was performed to identify amino acid changes relative to virus inoculum sequence. The graph shows the frequencies of all amino acid changes identified in the spike protein across all virus sequences. Each dot represents the frequency of the corresponding amino acid change in a specific virus sample. Samples are grouped based on treatment regiment: isotype control (Placebo), therapeutic antibodies administered prior (Prophylactic) or following (Treatment) viral challenge. (B) Detailed genomic information on all amino acid changes identified within the spike protein sequence across all samples. For each sample, the frequency of all mutations has been calculated. These frequencies are shown as percentage of the virus population with the amino acid change in the input virus or as range of frequency percentages (lowest to highest % ) in the virus populations isolated from the placebo, prophylactic and therapeutic groups. 2  1  0  3  2  0  2  3  1  0  0  0  0  1  1  0  1  0 Table S1 . Pathology analysis in rhesus macaque lungs (NHP Study #2). Pathology scores in individual animals treated with either REGN-COV-2 or placebo. Severity score of lesions: Minimal (1); Mild (2); Moderate (3); Severe (4) 

Potent neutralizing antibodies directed to multiple epitopes on SARS-476 CoV-2 spike

Single-shot Ad26 vaccine protects against SARS-479 CoV-2 in rhesus macaques

Respiratory disease 482 in rhesus macaques inoculated with SARS-CoV-2

Intradermal-delivered DNA vaccine provides anamnestic protection in a rhesus macaque 491 SARS-CoV-2 challenge model

Cross-neutralization of SARS-CoV-2 by a 494 human monoclonal SARS-CoV antibody

Convergent antibody responses

CoV-2 in convalescent individuals

Isolation of potent SARS-CoV-2 neutralizing antibodies and 500 protection from disease in a small animal model

Infection with novel coronavirus (SARS-CoV-2) causes pneumonia in 503 Rhesus macaques

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

CoV-2 in golden hamsters

ChAdOx1 511 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques

A human monoclonal antibody 514 blocking SARS-CoV-2 infection

An antibody-dependent enhancement (ADE) activity 519 eliminated neutralizing antibody with potent prophylactic and therapeutic efficacy against 520 SARS-CoV-2 in rhesus monkeys

Clinical benefit of 523 remdesivir in rhesus macaques infected with SARS-CoV-2

Virological assessment of hospitalized 526 patients with COVID-2019

DNA vaccine protection against SARS-529

CoV-2 in rhesus macaques

Rapid isolation and profiling of a diverse panel of 532 human monoclonal antibodies targeting the SARS-CoV-2 spike protein

SARS-CoV-2 Viral Load in Upper Respiratory Specimens of Infected Patients. 535

The following reagent was deposited by the Centers for Disease Control and