key: cord-0316452-zr8lavaa
authors: Duty, J. Andrew; Kraus, Thomas; Zhou, Heyue; Zhang, Yanliang; Shaabani, Namir; Yildiz, Soner; Du, Na; Singh, Alok; Miorin, Lisa; Li, Donghui; Stegman, Karen; Ophir, Sabrina; Cao, Xia; Atanasoff, Kristina; Lim, Reyna; Kowdle, Shreyas; Carreño, Juan Manuel; Rivero-Nava, Laura; Raskin, Ariel; Moreno, Elena; Johnson, Sachi; Rathnasinghe, Raveen; Pai, Chin I; Kehrer, Thomas; Cabral, Elizabeth Paz; Jangra, Sonia; Healy, Laura; Singh, Gagandeep; Warang, Prajakta; Simon, Viviana; Sordillo, Mia Emilia; van Bakel, Harm; Liu, Yonghong; Sun, Weina; Kerwin, Lisa; Palese, Peter; Teijaro, John; Schotsaert, Michael; Krammer, Florian; Bresson, Damien; García-Sastre, Adolfo; Fu, Yanwen; Lee, Benhur; Powers, Colin; Moran, Thomas; Ji, Henry; Tortorella, Domenico; Allen, Robert
title: Discovery of a SARS-CoV-2 Broadly-Acting Neutralizing Antibody with Activity against Omicron and Omicron + R346K Variants
date: 2022-01-20
journal: bioRxiv
DOI: 10.1101/2022.01.19.476998
sha: 872c7b609dbc49f4c5dc31703ed3de21fedea4c5
doc_id: 316452
cord_uid: zr8lavaa

The continual emergence of SARS-CoV-2 variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant, has rendered ineffective a number of previously EUA approved SARS-CoV-2 neutralizing antibody therapies. Furthermore, even those approved antibodies with neutralizing activity against Omicron are reportedly ineffective against the subset of Omicron variants that contain a R346K substitution, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. Following a campaign of antibody discovery based on the vaccination of Harbour H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of Spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against the Omicron and Omicron + R346K variants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for use in human clinical trials.

The severe acute respiratory disease syndrome coronavirus 2 (SARS-CoV-2) pandemic has 60 continued to significantly impact the health and lives of people around the globe 1 . To date, 61 public health agencies have sought to combat infections leading to COVID-19 by relying on 62 quarantine, social distancing, vaccination, and antiviral countermeasure strategies 2,3 . Despite 63 these efforts, the continued spread of SARS-CoV-2 has led to the emergence of several variants 64 of concern (VOCs) that have risen in prevalence worldwide 2-7 . 65 Each VOC encodes multiple changes in the amino acid sequence of the SARS-CoV-2 spike that 66 can impact the neutralizing properties of manufactured SARS-CoV-2 neutralizing antibodies 67 (nAbs) as well as nAbs elicited following vaccination or during the course of natural infection. 68 Specifically, the Omicron VOC (B.1.1.529) live virus, when profiled in vitro using Vero cells 69 expressing human ACE2 and human TMPRSS2 for susceptibility to nAbs currently authorized or 70 approved for clinical use (AFCU nAbs), has been shown to be resistant to the neutralizing 71 activities of REGN10987 (imdevimab), REGN10933 (casirivimab), LY-CoV555 (bamlanivimab), LY-72 CoV016 (etesevimab), and CT-P59 (regdanvimab), at nAb concentrations ≤ 10 µg/mL (IC50), and 73 remained susceptible to nAbs COV2-2130 (cilgavimab) and COV2-2196 (tixagevimab) tested as 74 single nAb therapies or in combination (IC50 of 43, 126, and 181 ng/mL, respectively) 8-13 . In live 75 virus neutralization assays utilizing Vero cells overexpressing human TMPRSS2, S309 76 (sotrovimab) registered an IC50 of 373 ng/mL, consistent with previously published activity in 77 Omicron pseudovirus assays for this antibody. 78 A subset (approximately 23% of Omicron sequences in GISAID recorded on outbreak.info) of 79 Omicron viruses encode an additional mutation in the SARS-CoV-2 spike at position R346K in 80 the receptor binding domain (RBD) of the protein 14-16 . The R346K mutation was previously 81 identified among the defining mutations of the SARS-CoV-2 Mu VOC 7 . Using Omicron + R346K 82 pseudoviruses, neutralization potency was reported as substantially reduced for all tested AFCU 83 nAbs, including COV2-2130, COV2-2196, and S309 5,17-20 . Current antibodies in development, 84 including bebtelovimab and BRII-198 (romlusevimab), maintain activity in Omicron pseudotype 85 neutralization assays 12,21 . BRII-198 displays substantially reduced neutralizing activity in assays 86 using Omicron + R346K pseudoviruses while testing of bebtelovimab against the Omicron + 87 R346K variant has not yet been reported 12,21 . As such, there is a continued need for discovery 88 and development of nAbs that can provide potent immune protection against COVID-19 caused 89 by pandemic VOCs presently infecting the global population. 90 In the early COVID-19 disease setting, intravenous (IV) administration of nAbs is an effective 91 means of lessening progression and overall severity of disease 18 Figure 1B) . We observed that the serum from Mouse 1, 3, and 4 demonstrated a concentration 112 dependent and specific binding to 293ExpiF cells expressing SARS-CoV-2 spike. Given that 113 Mouse 3 and Mouse 4 had the highest titer humoral response against SARS-CoV-2 spike, the 114 spleens from these animals were used to generate hybridoma clones 26 . The hybridoma clones 115 (1,824 clones from Mouse 3 and 1,440 clones from Mouse 4) were screened for binding to 116 293ExpiF cells expressing SARS-CoV-2 spike by flow cytometry and RBD-spike (Wuhan) ( Figure   117 1). A representative heat map for Mouse 4 fusion was generated to summarize the mean 118 fluorescence intensity (MFI) for each hybridoma clone ( Figure 1C ). In parallel, hybridoma clones 119 were subjected to an RBD ELISA validating the clones that bound to SARS-CoV-2 spike. We 120 identified 188 clones with a >5-fold MFI over untransfected cells and classified these as 121 candidate SARS-CoV-2 binding antibodies. The supernatants from these clones were then 122 evaluated in a high-throughput neutralization assay using the replication competent VsV 123 reporter virus that utilizes SARS-CoV-2 spike (VsV CoV2-spike ) as its envelope protein and 124 expressing GFP as readout for infection 27 (Figure 1C the Mouse 3 fusion, 340 clones were found to bind to SARS-CoV-2 spike and 90 clones were 131 found to have neutralization activity against VsV CoV2-spike . The selected neutralizing clones with 132 IC50 values <125pM were then examined for IgG isotypes and expanded for further analysis. 133 Sequencing of the heavy chain from each hybridoma clone revealed diverse CDR3 lengths 134 ranging from 10-20 aa in length ( Figure 1D ). Clones that were identical copies of each other 135 were consolidated to a single candidate. 136 To identify the most effective human anti-SARS-CoV-2 spike neutralizing antibodies, we 137 performed a VsV CoV2-spike neutralization assay ( Figure 1D) To determine the effects of variant specific spike S1 domain mutations within and outside the 155 RBD region of S1 on antibody binding, the affinity of STI-9167 and EUA-approved SARS-CoV-2 156 nAbs sotrovimab, cilgavimab, and tixagevimab were determined for monomeric WA-1 spike S1 157 subunit binding as well as VOC-derived S1 domains using surface plasmon resonance (SPR). Of 158 note, the kD of STI-9167 was measured as 6.20 nM for the WA-1 isolate, 4.45 nM for the Delta 159 variant, and 22.6 nM for the Omicron variant. Binding kinetics for the Omicron variant were 160 compared between STI-9167, sotrovimab, cilgavimab, and tixagevimab. (Figure 2A , Table 1A 161 and Supplemental Figure 4) . STI-9167 and cilgavimab had a similar association rate and 162 sotrovimab was approximately 5-fold slower. The dissociation rate was slowest with sotrovimab 163 by a factor of approximately 10-fold as compared to STI-9167, and STI-9167 dissociated at an 164 approximately 2-fold slower rate than cilgavimab. Tixagevimab binding to Omicron S1 domain 165 monomer was insufficient to allow for quantitation. 166 In an effort to assess nAb binding to spike proteins in a native conformation, STI-9167 was Figure 5 ). Of note, half-maximal binding of STI-9167 to the Omicron + R346K 176 spike (EC50=0.023 µg/mL) was equivalent to that measured for Omicron, suggesting that the 177 epitope recognized by STI-9167 is preserved in the context of Omicron + R346K as compared to 178 epitopes engaged by cilgavimab, which displayed reductions in Omicron + R346K spike binding 179 of over 60-fold as compared to EC50 values in assays targeting Omicron spike. Based on the 180 spike S1 and full-length spike protein binding data, STI-9167 was further profiled to determine 181 the potency of virus neutralization and the breadth of neutralizing protection this antibody 182 provided against SARS-CoV-2 variants of concern in vitro. 183 Virus pseudotypes were used to determine the neutralization potency (IC50) of STI-9167 against 184 an index virus generated with a spike protein that carries a single D614G (VSV D614G-spike ) 185 mutation as compared to the WA-1 spike protein 35 . To approximate conditions found in the 186 setting of human SARS-CoV-2 infection, pseudovirus assays were carried out using HEK293 cells 187 which overexpressed human ACE2 and TMPRSS2 proteins. The average IC50 value for STI-9167 188 in assays using the VSV D614G-spike pseudovirus was 3.6 ng/mL ( Table 1C) . The STI-9167 189 neutralization potency for VSV Delta-spike (IC50=5.4 ng/mL), VSV Omicron-spike (IC50=14.8 ng/mL), and 190 VSV Omicron+R346K-spike (IC50=23.9 ng/mL) pseudotypes was maintained to within 7-fold of that 191 measured in assays with the VSV D614G-spike pseudotype ( Figure 2C) . Furthermore, STI-9167 192 neutralization potency was maintained to the same degree against the full catalog of VOC-193 based pseudovirus tested, including Alpha, Beta, Gamma, Delta Plus, Epsilon, Zeta, Iota, Kappa, 194 Lambda, and Mu VOCs (Supplemental Data Figure 1 ). The concentration of nAbs required to 195 achieve half-maximal and eighty-percent-maximal levels of neutralization potency for VOC 196 pseudotypes as well as for the VSV D614G-spike pseudotype are detailed in Table 1C . The pool of antibodies we identified following vaccination of mice with an SARS-CoV-2 RBD 295 protein based on the Wuhan spike protein sequence includes a candidate with potent 296 neutralizing activity against many SARS-CoV-2 variants of concern that have emerged in the Omicron: A67V, del69-70, T95I, G142D, del143-145, N211D, del212, G339D,   506   S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, for Spike S1 binding domain from the following isolates and VOCs: USA/WA-1/2020(WA-1) 666 isolate, Delta, and Omicron. The antibody affinities were measured using SPR on a BIAcore T200 Table 1 Supplemental Figure 1 

We thank R. Albrecht for 631 support with the BSL-3 facility and procedures at the Icahn School of Medicine at Mount Sinai

received funding from Swiss National Foundation (SNF) Postdoc Mobility fellowship 633 (P400PB_199292). M.S. laboratory is supported by NIH grant R01DK130425

S. laboratory has received research support from Pfizer, Senhwa Biosciences

Atea Pharma and Merck, 638 outside of the reported work

Applied Biological Laboratories, Pharmamar, Paratus

CureLab Veterinary and Pfizer, outside of the reported work. A.G.-S. is 642 inventor on patents and patent applications on the use of antivirals and vaccines for the 643 treatment and prevention of virus infections and cancer, owned by the Icahn School of 644 Medicine at Mount Sinai

005 mg/kg () at 24 hours post-administration as compared to 685 samples collected from untreated mice. Values represent mean ± SEM (n=3-4 animals no 686 treatment group, n=5 in treatment groups)

****, P < 0.0001. Pharmacokinetics: Concentration of STI-9167 (C)

in lungs and isolated serum collected from female CD-1 mice administered STI-9167 intranasally 689 (IN) at a dose of 5 mg/kg. Samples from treated mice were collected at the indicated timepoint 690 post-administration; antibodies concentrations were quantified by ELISA and compared to 691 samples collected from untreated mice. Values represent mean ± SD (n=3-6 animals no 692 treatment group

Efficacy of Intranasal (IN) delivery of STI-9167 Neutralizing Antibody in the K18-696 hACE2 murine model of COVID-19

A) K18-hACE2 transgenic mice were infected with 10,000 PFU of WA-1, Delta or Omicron SARS-698

CoV-2 treated with indicated concentration of isotype control antibody (Isotype) or STI-9167

intravenously 1 h post infection. (B) Body weight change of mice was measured daily (n = 5)

SARS-CoV-2 viral titers were measured in lung day 4 post infection (n = 5). (D)

000 PFU SARS-CoV-2 WA1, Delta, or Omicron strains and 702 treated with indicated concentration of Isotype or STI-9167 intranasally 12 h post infection

Body weight change of mice was measured daily (n = 5). (F) SARS-CoV-2 viral titers were 704 measured in lung day 4 post infection (n = 5) n.s. not significant, P* < 0.05, P** < 0.01, P*** < 705 0.001 or P****<0.0001. Unpaired t-test (C and F)

Supplemental Figure 1. Binding and neutralization of candidate antibody to VoCs

WA-1) isolate, Alpha, Beta, and Gamma. The antibody affinities were 719 measured using SPR on a BIAcore T200 instrument using a 1:1 binding model. Graphs are 720 representative of triplicate data and table data presented as mean ± SD. (B) Spike protein derived 721 from Alpha, Beta, Gamma, Delta Plus, and Lambda SARS-CoV-2 isolates were independently 722 expressed on the surface of HEK 293 cells. Serially-diluted STI-9167 was assayed for Spike protein 723 binding by flow cytometry. To quantify antibody binding, mean fluorescent intensity was 724 measured for each dilution tested and the EC50 value was calculated for each nAb. (C) Spike-725 pseudotyped VSV neutralization

The curves represent the average 727 of three independent experiments, with error bars representing one standard deviation. IC50 728 values for each pseudotype/antibody combination are indicated on the right. (D) PRNT assay 729 using STI-9167 with indicated SARS-COV-2 variants were performed as described in the methods

Supplemental Figure 2. Efficacy of Intranasal (IN) delivery of STI-9167 Neutralizing Antibody in the 732 K18-hACE2 murine model of COVID-19 VoCs

A) A schematic of experimental model, K18-hACE2 transgenic mice were infected with 10000

PFU of indicated variants of SARS-CoV-2 treated with indicated concentration of AB

intravenously 1 hour post infection. (B) Body weight change of mice was measured daily

SARS-CoV-2 viral titers were measured in lung day 5 post infection (n = 5). P****<0

Unpaired t test (C)

Supplemental Figure 3. Plaque Reduction Neutralization Fluorescent staining on Vero-ACE2

PRNT assay using STI-9167 and various neutralizing antibodies with SARS-COV-2 WA-1 or 741

Omicron were performed as described in the methods on Vero-ACE2-expressing cells and 742 visualized

SPR binding affinity graphs of STI-9167, Cilgavimab, Tixagevimab, and Sotrovimab

Omicron spike 748 protein was expressed on HEK 293 cells and binding of selected neutralizing antibodies was

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 627 This work was partly supported by CRIPT (Center for Research on Influenza Pathogenesis and   628 Transmission), an NIAID funded Center of Excellence for Influenza research and Response 629