key: cord-0993980-xik7pmj7
authors: Westendorf, Kathryn; Žentelis, Stefanie; Wang, Lingshu; Foster, Denisa; Vaillancourt, Peter; Wiggin, Matthew; Lovett, Erica; van der Lee, Robin; Hendle, Jörg; Pustilnik, Anna; Sauder, J. Michael; Kraft, Lucas; Hwang, Yuri; Siegel, Robert W.; Chen, Jinbiao; Heinz, Beverly A.; Higgs, Richard E.; Kallewaard, Nicole L.; Jepson, Kevin; Goya, Rodrigo; Smith, Maia A.; Collins, David W.; Pellacani, Davide; Xiang, Ping; de Puyraimond, Valentine; Ricicova, Marketa; Devorkin, Lindsay; Pritchard, Caitlin; O’Neill, Aoise; Dalal, Kush; Panwar, Pankaj; Dhupar, Harveer; Garces, Fabian A.; Cohen, Courtney A.; Dye, John M.; Huie, Kathleen E.; Badger, Catherine V.; Kobasa, Darwyn; Audet, Jonathan; Freitas, Joshua J.; Hassanali, Saleema; Hughes, Ina; Munoz, Luis; Palma, Holly C.; Ramamurthy, Bharathi; Cross, Robert W.; Geisbert, Thomas W.; Menacherry, Vineet; Lokugamage, Kumari; Borisevich, Viktoriya; Lanz, Iliana; Anderson, Lisa; Sipahimalani, Payal; Corbett, Kizzmekia S.; Yang, Eun Sung; Zhang, Yi; Shi, Wei; Zhou, Tongqing; Choe, Misook; Misasi, John; Kwong, Peter D.; Sullivan, Nancy J.; Graham, Barney S.; Fernandez, Tara L.; Hansen, Carl L.; Falconer, Ester; Mascola, John R.; Jones, Bryan E.; Barnhart, Bryan C.
title: LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants
date: 2022-04-25
journal: Cell Rep
DOI: 10.1016/j.celrep.2022.110812
sha: 912ce38187da4370aef2f94417b0d3384739e7f7
doc_id: 993980
cord_uid: xik7pmj7

SARS-CoV-2 neutralizing monoclonal antibodies (mAbs) can reduce the risk of hospitalization from COVID-19 when administered early. However, SARS-CoV-2 variants of concern (VOC) have negatively impacted the therapeutic use of some authorized mAbs. Using a high throughput B-cell screening pipeline, we isolate LY-CoV1404 (bebtelovimab), a highly potent SARS-CoV-2 spike glycoprotein receptor binding domain (RBD)-specific antibody. LY-CoV1404 potently neutralizes authentic SARS-CoV-2, B.1.1.7, B.1.351 and B.1.617.2. In pseudovirus neutralization studies, LY-CoV1404 potently neutralizes variants including B.1.1.7, B.1.351, B.1.617.2, B.1.427/B.1.429, P.1, B.1.526, B.1.1.529, and the BA.2 subvariant. Structural analysis reveals that the contact residues of the LY-CoV1404 epitope are highly conserved, except for N439 and N501. Notably, the binding and neutralizing activity of LY-CoV1404 is unaffected by the most common mutations at these positions (N439K and N501Y). The broad and potent neutralization activity and the relatively conserved epitope suggest that LY-CoV1404 has the potential to be an effective therapeutic to treat all known variants.

Variants of SARS-CoV-2 continue to alter the trajectory of the COVID-19 pandemic, which at the time of writing has infected over 416 million people world-wide and is responsible for more than 5.8 million deaths (https://covid19.who.int/ accessed 17 February 2022). As predicted, SARS-CoV-2 has continued to evolve as the pandemic has progressed (Mercatelli and Giorgi, 2020; Pachetti et al., 2020) . Selective pressures and viral adaptation during prolonged, suboptimally-treated infections are thought to have generated numerous variants (McCormick et al., 2021) , some significantly diminishing the effectiveness of COVID-19 clinical countermeasures (Altmann et al., 2021; Plante et al., 2020) . Variants of concern (VOC) represent a closely monitored subset of the many detected SARS-CoV-2 variants, due to their potential for increased transmissibility, and ability to evade immunity produced by infection or vaccines while reducing the efficacies of antibody-based treatments (Hoffmann et al., 2021; Kuzmina et al., 2021; McCormick et al., 2021; Thomson et al., 2021; Wang et al., 2021b) . The impact of VOC continues to increase (World Health Organization, 2021) , with emerging variants threatening to slow the pace and success of global vaccination efforts and limit the effectiveness of existing COVID-19 treatments Munnink et al., 2021; Plante et al., 2020; Tegally et al., 2021) .

Many studies have demonstrated the clinical safety and efficacy of antibody-based COVID-19 therapies and their potential for easing the strain on economies and healthcare infrastructures during the pandemic Jiang et al., 2020; Jones et al., 2020) . Key target populations for such monoclonal antibody treatments include immunocompromised individuals, patients that are particularly susceptible due to comorbidities (cardiovascular disease, diabetes, chronic respiratory conditions), and those aged over 65 (Jiang et al., 2020) . To date, several antibody therapies targeting the spike (S) protein of SARS-CoV-2 have been successfully used in the fight against the virus Gottlieb et al., 2021) . One example is Eli Lilly J o u r n a l P r e -p r o o f 4 and Companies' bamlanivimab (LY-CoV555), which was the first anti-SARS-CoV-2 monoclonal antibody tested clinically in June 2020, three months after initiating discovery efforts (Jones et al., 2021 ). Lilly's combination of bamlanivimab with another antibody, etesevimab, received emergency authorization in several countries. Clinical testing has shown that antibodies from Regeneron, GSK/Vir, AstraZeneca, and others are also safe and effective (AstraZeneca, 2020; GlaxoSmithKline, 2021; Regeneron, 2021; Tada, et al., 2021) . As variants continue to emerge and spread, antibodies can provide highly effective therapeutic options to vulnerable populations against COVID-19. However, for antibody therapies to be successful, they must retain potent neutralizing breadth against emerging SARS-CoV-2 lineages carrying mutations. A high neutralization potency may also provide the opportunity to explore lower clinical doses (Andreano et al., 2021) and alternate routes of administration. With the rise of Omicron and BA.2 VOC many of the authorized antibodies may no longer be an option with a strong negative impact on their neutralization capacity (Iketani et al., 2022) .

Multiple reports have consistently shown that mutations in SARS-CoV-2 can substantially reduce the binding affinity and neutralization of antibodies (Rees-Spear et al., 2021; Yao et al., 2021) . Variants such as B.1.1.529, BA. 2, B.1.1.7, B.1.351, P.1, B.1.526, B.1.427, and B.1.429 affect the in vitro binding of antibodies being tested clinically or on those already authorized for emergency use to varying degrees (Kuzmina et al., 2021; Liu et al., 2021; Rees-Spear et al., 2021; Iketani et al., 2022) . Mutations at amino acid positions 417, 439, 452, 484, and 501 were found to have the greatest consequences on the functionality of both antibodies and vaccines (Thomson et al., 2021) . Therefore, antibodies that retain binding and potent neutralization activity in the presence of these mutations could be extremely valuable tools to rapidly respond to these variants.

J o u r n a l P r e -p r o o f 5 In this report, we describe LY-CoV1404 (also known as bebtelovimab), a fully human IgG1 monoclonal SARS-CoV-2 antibody targeting the receptor binding domain (RBD), identified in our ongoing pandemic-response efforts. As of February 2022, LY-CoV1404 binds and potently neutralizes all currently known VOC of SARS-CoV-2 including B.1.1.529 and BA.2 (the Omicron variant and sub-variant). LY-CoV1404 binds to an epitope that is largely distinct from the mutations identified to be widely circulating within the newly emerged variants, including those mutations that reduce the effectiveness of vaccines. Importantly, the LY-CoV1404 interaction with the S protein is driven by amino acids that are rarely mutated in the global GISAID EpiCoV database, indicating LY-CoV1404 has the potential to be a long-term, solution for reducing COVID-19-related illness and death. Additionally, bebtelovimab received Emergency Use Authorization from the US FDA on February 11, 2022 (https://www.fda.gov/news-events/pressannouncements/coronavirus-covid-19-update-fda-authorizes-new-monoclonal-antibodytreatment-covid-19-retains).

Variants of SARS-CoV-2 continue to be identified globally ( Figure 1A ) and can abrogate the binding of monoclonal antibody therapies or reduce vaccine effectiveness (Hoffmann et al., 2021; Wang et al., 2021b) . To identify potent neutralizing antibodies to SARS-CoV-2 variants, a high-throughput screening approach (Jones et al., 2021) was conducted on peripheral blood mononuclear cells (PBMC) that were isolated from a COVID-19 convalescent donor. The sample was obtained approximately 60 days after the onset of symptoms.

Three different screening strategies were employed to identify SARS-CoV-2-binding antibodies from the PBMCs in this donor sample. We used a soluble antigen assay with fluorescently J o u r n a l P r e -p r o o f 6 labeled prefusion-stabilized full-length trimeric SARS-CoV-2 S protein as well as the SARS-CoV-2 RBD subunit, a live-cell-based assay using mammalian cells that transiently expressed the SARS-CoV-2 S protein, and a multiplexed bead assay using S proteins from multiple coronaviruses ( Figure 1B) . A total of 740,000 cells were screened in this discovery effort and machine learning (ML)-based analyses were used to automatically select and rank 1,692 antibody "hits" (0.22% frequency). Of these, 1,062 single antibody-secreting cells were selected for recovery. Libraries of antibody genes from recovered single B cells were generated and sequenced using next-generation sequencing, and a custom bioinformatics pipeline with MLbased sequence curation was used to identify paired-chain antibody sequences, with 290 unique high-confidence paired heavy and light chain sequences identified ( Figure 1B) . The antibody sequences corresponded to 263 clonal families and used a diverse set of 41 VH genes. The mean sequence identity to germline was 96.8% from this 60-days post-symptomonset blood draw in this donor.

High-throughput SPR experiments on a selected subset of 69 recombinantly-expressed antibodies (including benchmark controls) were performed to assess S protein epitope coverage. These studies included epitope binning, isolated subdomain binding, and binding competition with ACE2 (Figures 1C/S1). We employed 21 benchmark antibodies including those known to bind to S protein subunits S1, N-terminal domain (NTD), RBD, S2 subunit, and several internally identified antibodies with known binding domains. The antibodies (including benchmarks) were clustered into 46 bins using Carterra epitope analysis software based on the competition heatmap ( Figure 1C ) and were delineated into bins among known subunits of the S protein. Several of the antibodies discovered are cross-blocked with those that are currently in clinical testing or authorized for use in COVID-19 patients including bamlanivimab. We therefore focused analyses on antibodies outside of these established epitopes and identified LY-CoV1404, an ACE2-blocking antibody, which is found in a unique bin shared with one other J o u r n a l P r e -p r o o f 7 antibody. LY-CoV1404 was observed to compete directly for S protein RBD binding with the S309 (Pinto et al., 2020) and REGN10987 (Hansen et al., 2020) antibodies, suggesting a binding epitope somewhat distinct from other ACE2-blocking antibodies such as bamlanivimab or etesevimab.

Characterization of LY-CoV1404 binding kinetics revealed that the Fab fragment of the antibody bound to the S protein of D614G with high affinity, characterized by binding constant (KD) values ranging between 790 pM and 4nM, depending on the assay ( Figure 1D , Table 1 ). We further analyzed the binding affinity of LY-CoV1404 as a human IgG1 antibody and determined the KD to be between 75 pM and 220 pM (depending on the assay design) for LY-CoV1404 binding to the S protein ( Figure 1E , Table 1 ). There was no loss of binding to the S protein carrying the D614G mutation (Asp 614 →Gly).

We explored the ability of LY-CoV1404 to bind variant S proteins from the more widely circulating VOC lineages of SARS-CoV-2. LY-CoV1404 retained binding to key VOC including B.1.351 and B.1.1.7. Critically, when compared to wild type virus, LY-CoV1404 bound to these variants with no loss in affinity ( Figure 1E ). These data suggest that LY-CoV1404 binds to a region that is not significantly affected by the prevalent VOC mutations. We further tested the binding of LY-CoV1404 to a wider range of S mutants and VOC using an assay system with S protein expressed on the surface of mammalian cells. In these assays, we compared binding of LY-CoV1404 to isotype control antibodies. Consistent with the affinity data, LY-CoV1404 maintained binding to all variants and mutants tested ( Figure 1F) . These data indicate that SARS-CoV-2 S protein variants identified and reported to be of significant concern in this pandemic are still potently bound by LY-CoV1404.

J o u r n a l P r e -p r o o f 8 The in vitro viral neutralization activity LY-CoV1404 was assessed against an authentic viral strain (SARS-CoV-2/MT020880.1) using an immunofluorescence assay (IFA) readout. In these studies, LY-CoV1404 potently neutralized the authentic virus with half maximal inhibitory concentration (IC50) values ranging from 9 ng/mL to 22.1 ng/mL. Bamlanivimab (LY-CoV555), another potent anti-RBD binding antibody, consistently had a 2-3-fold higher (less potent) IC50 compared to LY-CoV1404 (Figure 2A) .

We also tested the VIR-S309 antibody that was recently described to have a unique binding epitope, outside of many of the mutated positions in current VOC (Pinto et al., 2020) . Vir's authorized antibody, Vir-7831, is cross-reactive to SARS-CoV-1 S protein (Cathcart et al., 2021) . Comparing separate assay runs, we observed VIR-S309 had an IC50 of 118 ng/mL (highly consistent with a recent report on the clinically active derivative VIR-7831 (Cathcart et al., 2021) ) compared to bamlanivimab with a range of 19.2 to 29.7 ng/mL IC50 in multiple independent assays ( Figure 2B ). Amongst other antibodies tested, LY-CoV1404 exhibited the most potent neutralization of authentic virus in this assay ( Figure 2C ). We also tested authentic virus neutralization in a plaque reduction neutralization test (PRNT) against a Canadian strain of SARS-CoV-2 (hCoV-19/Canada/ON_ON-VIDO-01-2/2020) with multiple antibodies (Figure 2C ), and against other natural SARS-CoV-2 isolates tested with LY-CoV1404 alone and in combination with other antibodies ( the amino acid sequence of the spike protein, and 15 of those mutations located in the RBD, has raised concerns for the activity of a number of clinically tested therapeutic monoclonal antibodies. The ability of these antibodies to neutralize B.1.1.529 was compared in pseudotyped neutralization assays. LY-CoV1404 was the only antibody that retained full potency against the Omicron variant in these assays; separately, Vir-7831 (sotrovimab) has been shown to exhibit a minor loss in neutralization potency (2.7-fold reduction) against this same variant (Cathcart et al., 2021) . (Table 3C , 3D). These assays were performed using two different systems with highly comparable results. In addition, we tested the subvariant of B.1.1.529 known as BA.2 in pseudovirus neutralization (Table 3B) . LY-CoV1404 retained activity against this variant as well.

The data indicate that LY-CoV1404 retains its potency against VOCs, including those dominant or rapidly spreading worldwide (such as B.1.617.2, B.1.1.529, and BA.2).

To fully characterize the binding epitope of LY-CoV1404 we determined the X-ray crystal structure of LY-CoV1404 Fab bound to S protein RBD and CryoEM structure (EMD-26560) of intact S protein bound to LY-CoV1404 (Figure 3) . The Fab of LY-CoV1404 binds to a region overlapping the ACE2-interacting site of the S protein that is accessible in both the "up" and "down" conformers of the RBD on the S protein ( Figure S2 ). While this property would suggest that LY-CoV1404 is a Class 2 binder (Barnes et al., 2020) , the structural location of the epitope is closer to the canonical Class 3 binder, VIR-S309 (Wang et al., 2021b) . Interestingly, the binding epitope of LY-CoV1404 is very similar to previously described antibodies imdevimab J o u r n a l P r e -p r o o f 10 (REGN10987) (Hansen et al., 2020) and Fab 2-7 (Cerutti et al., 2021; . This similarity is apparent from the structural superposition of the Fabs as well as which RBD residues interact with the antibodies (Figure S3) . Furthermore, though independently discovered, LY-CoV1404 and Fab 2-7 (Cerutti et al., 2021) share 92% amino acid sequence identity in the variable regions of both their heavy and light chains, and they both engage the RBD similarly through their heavy and light chains. By contrast, REGN10987 is more sequence divergent and nearly all of its interactions with the RBD are through its heavy chain. LY-CoV1404 has a contact surface area on the RBD of 584 Å 2 , compared to 496 Å 2 for Fab 2-7, and only 343 Å 2 for REGN10987 (Table S2) .

To determine the frequency of mutations of the amino acid residues of the S protein that are close contacts with LY-CoV1404 as determined by structural analysis, we used the publicly deposited mutations observed at these sites in the GISAID EpiCoV database (Table 3E) . There were very few changes to these identified amino acids, with the only two residues below the >99% unchanged threshold: N439 and N501 (reported as having 99.371% and 78.189% conservation at these positions respectively), demonstrating that the LY-CoV1404 epitope has remained relatively unchanged during the pandemic. We identified the most prevalent mutations at epitope residues in the GISAID EpiCoV database and characterized their impact on the functional activity of LY-CoV1404. Interestingly, the observed N439K and N501Y mutations had no impact on LY-CoV1404 binding or neutralization ( Table 4A ). The lack of effect by the N493K mutation is interesting in the context of the loss of antibody binding previously observed for REGN10987 (Thomson et al., 2021) demonstrating that beyond the epitope, the specific interactions are critical to determining whether resistant mutations will arise for any particular antibody. A loss of binding, ACE2 competition and neutralization activity was observed with mutations at positions 444 and 445 (which could arise by a single nucleotide change) ( Table   4A ). Importantly however, these changes are extremely rare in the general population as J o u r n a l P r e -p r o o f 11 reported in the GISAID database ( Table 3E ). The ability of LY-CoV1404 to maintain neutralization potency despite a variety of mutations that have been shown to nullify activity of several other potent neutralizing antibodies (Rappazzo et al., 2021) indicates that this antibody may be uniquely suited to combat the current VOC. Critically, potent activity against all of these variants suggests that LY-CoV1404 binds to an epitope that has acquired few mutations.

With the exception of N439 and N501, the residues with which LY-CoV1404 interacts are not prevalent in current VOC (e.g. 417, 439, 452, 484) . Importantly, while the LY-CoV1404 binding epitope includes residues both N439 and N501, LY-CoV1404 still binds the N501Y mutant Figure   S2 ). However, unlike imdevimab (Thomson et al., 2021) , LY-CoV1404 retains full functional neutralization against pseudovirus with the N439K mutant (Table 4A), indicating that these residues do not play critical roles in LY-CoV1404 interaction with S protein. In addition, the mutations present in B.1.1.529 that reside within the binding epitope for LY-CoV1404, specifically G446S, N440K, Q498R, and N501Y, do not impact the neutralization activity of LY-CoV1404. Importantly, potent activity against all the tested variants suggests LY-CoV1404 binds uniquely to an epitope that has acquired few mutations and is not sensitive to the mutations that have arisen to date.

We explored the potential for resistant mutations to arise by yeast-display of the S protein RBD.

A library was constructed in which all amino acid residues were sampled at positions 331 to 362 and 403 to 515 of the spike glycoprotein containing the epitope of LY-Cov1404. Cells containing J o u r n a l P r e -p r o o f 12 RBD substitutions that were still able to bind soluble ACE2 after antibody treatment were isolated and sequenced to determine the location and identity of the change. Further functional characterizations of isolates were limited to those containing a change of a single amino acid residue which can be generated by a single nucleotide change based on the codon used in the Wuhan isolate sequence.

Consistent with the structure of the RBD complex, selection using LY-CoV1404 identified potential susceptibility to some, but not all, substitutions at residues K444, V445, G446, and P499. None of the observed G446 changes identified from the selection could arise from a single nucleotide substitution but nevertheless suggested a potentially liable residue with LY-CoV1404. The presence of the G446V variant in the GISAID database, as well as the ability of this variant to be resistant to imdevimab treatment prompted further analysis. The ability of LY-CoV1404 to inhibit ACE2 binding to RBD substitutions that have been identified in common circulating viral variants and/or have been shown to impart resistance to the authorized antibodies bamlanivimab, etesevimab, casirivimab, and imdevimab was also analyzed ( Table   4B) .

We further characterized LY-CoV1404 activity in the presence of mutations known to weaken the effectiveness of other antibodies in clinical testing. Of the mutations tested, only K444Q, V445A, and G446V impacted LY-CoV1404 binding (Table 4A) . Interestingly, while the K444Q and V445A mutations also resulted in reduced ACE2 competition and neutralization activity, the G446V mutation only appeared to significantly impact the monomeric binding affinity, along with modest changes in ACE2 competition and neutralization. These residues are very rarely mutated according to GISAID reports (Tables 3E, 4A), thus suggesting a low risk of mutations emerging at these residues and restriction in functional activity of LY-CoV1404. We examined the frequency of all variants observed at residues 444 and 445 within the GISAID-EpiCov database (Shu and McCauley, 2017) , as of December 21, 2021; see Materials and Methods).

J o u r n a l P r e -p r o o f 13 These two sites were rarely mutated, with 0.036% at residue 444 and 0.014% at residue 445 of samples collected contain mutations. It is not known whether all variants at these locations would confer resistance. Of the specific mutations tested (Table 4A) , we find that only 0.0057% of samples contained the known resistance V445A, while no occurrences of K444Q were observed. This suggests LY-CoV1404 may remain effective even as new strains emerge. These data also indicate that LY-CoV1404 is not only likely to be highly effective against current variants, but is less likely to be impacted by future mutations given the low level of changes observed to date in its binding epitope (McCormick et al., 2021) .

We report the discovery of LY-CoV1404, a highly potent SARS-CoV-2 S protein RBD-binding antibody that maintains binding and neutralizing activity across currently known and reported authentic virus indicates that the antibody binds to an epitope on the S protein that is accessible and bound with high affinity by LY-CoV1404. In addition, LY-CoV1404 blocks interaction between ACE2 and the S protein, providing a strong, well-documented mechanism for the potent neutralizing activity. Multiple antibodies that rely upon this receptor-blocking mechanism have recently been developed as treatments for COVID-19 and clearly demonstrate that antibody-mediated viral neutralization is a safe and highly efficacious COVID-19 treatment strategy Eli Lilly and Company, 2020; Gottlieb et al., 2021; Regeneron, 2021; Tada, et al., 2021) . However, the emergence of SARS-CoV-2 variants has diminished the effectiveness of several of these therapeutic antibodies Munnink et al., 2021; Plante et al., 2020; Tegally et al., 2021) . The rapid spread of a new VOC (B1.1.529, Omicron) J o u r n a l P r e -p r o o f 14 has diminished the function of all the authorized and clinically tested antibodies. Notably, this is true even for two antibodies that have demonstrated cross-reactivity to SARS and were believed to be less susceptible to viral escape; ADG20 was found to be more than 300-fold less potent against B1.1.529, while the potency of S309 was reduced by approximately 10-fold. This underscores the unpredictability of SARS-CoV-2 spike mutation. Importantly, LY-CoV-1404 is the only clinically tested antibody that retains full functional potency in pseudotype virus neutralization assays against this mutant. Our data suggest that LY-CoV-1404 is more than 50fold more potent against omicron than any of the other clinical stage antibodies we have tested.

In addition, either Omicron or the BA.2 subvariant of Omicron have been reported to severely reduce or eliminate neutralization activity against all clinically tested antibodies (Iketani et al., 2022) . LY-CoV1404 retains full potency against both variants (Table 3B ) in pseudovirus assays.

The unique binding epitope of LY-CoV1404, together with the low frequency of mutations observed within this epitope, indicate that this antibody could provide an effective therapeutic option against current VOCs and emerging variants as a complementary approach to vaccinations and other COVID-19 therapies.

With more than 404 million infections world-wide it is not surprising that SARS-CoV-2 variants with a potential selective advantage have arisen. These resulting variants have acquired either increased affinity for ACE2 binding (Altmann et al., 2021) , increased transmissibility and infection severity (Gu et al., 2020) , or increased immune evasion (Altmann et al., 2021) , and threaten to reduce the effectiveness of currently available vaccinations (Mansbach et al., 2020; Yurkovetskiy et al., 2020) . The D614G mutation, reported early in the course of the pandemic, has been determined to stabilize the S protein, leading to more efficient ACE2 interactions and infections (Santos and Passos, 2021) . The rapid spread of the B.1.1.7 variant, which contains the key mutation N501Y, has been correlated with increased ACE2 binding affinity (Baric, 2020; Horby et al., 2021; Plante et al., 2020) . Certain recurring mutations within the S protein have J o u r n a l P r e -p r o o f 15 also been reported in variants from different geographical regions indicating that these common mutations may confer an advantage to the virus (Kuzmina et al., 2021; Liu et al., 2021; Rees-Spear et al., 2021) . Reports of particular key mutations such as E484K are present in several variants emerging independently in several geographic locations (South Africa B.1.351, Brazil P.1, New York B.1.526, and recently some UK B.1.1.7 strains) (Xie et al., 2021) . Investigations have indicated that vaccine-induced and natural immune responses may not be as effective against variants carrying the E484K mutation (Plante et al., 2020) . In addition, several other specific mutations within the S protein have been demonstrated to reduce binding and effectiveness of antibody therapies (Eli Lilly and Company, 2020; Regeneron, 2021) . With the proven clinical effectiveness of antibody therapies at preventing hospitalization, reducing severity of disease symptoms and death (Darby and Hiscox, 2021) , the importance of access to effective antibody treatments targeting conserved neutralizing epitopes for response to viral variants must be recognized.

It remains unknown whether vaccines will significantly alter the mutation profile of the virus (Choi et al., 2020; Kemp et al., 2021) . If, as vaccination continues world-wide, the virus responds to this selection pressure with particular mutations to avoid the effects of vaccination, it will be important to maintain alternative therapies to help overcome these variants. It has been hypothesized that certain variants may have arisen in immunocompromised individuals, which provided the virus with an extended timeframe for continued viral replication and the accumulation of many mutations, seeding these novel VOC (Aschwanden, 2021) . A potent neutralizing monoclonal antibody, such as LY-CoV1404, would be a potential therapeutic option for these immunocompromised patients, with rapid neutralization of the virus providing both protection for the patient, and less opportunity for viral mutation and evolution. Finally, immunocompromised individuals are less likely to respond well to vaccines, further highlighting the utility of monoclonal antibody therapy for protecting this population. Monoclonal antibodies J o u r n a l P r e -p r o o f with potent neutralizing effects could provide a safe and robust medical countermeasure against variants or a necessary, complementary alternative form of protection for those individuals who cannot, or do not receive vaccines. For the reasons listed above, it has been proposed that achieving "herd immunity" is unlikely in the near term for SARS-CoV-2 (Cathcart et al., 2021; Pinto et al., 2020) , further emphasizing the need for medical alternatives to combat viral resistance.

Recently, an antibody that binds to an epitope of the SARS-CoV-2 S protein that is distinct from current VOCs has been described. This antibody, VIR-7831(GSK, 2021), binds to and neutralizes current variants of concern, including Delta (B.1.617.2) and Omicron (B.1.1.529), and has recently been reported to have clinical efficacy at a 500mg dose (Cathcart et al., 2021) .

is several-fold more potent in viral neutralization assays (Cathcart et al., 2021) (Figure 2B ).

Though it remains to be tested, this increased potency has the potential to enable lower doses and subcutaneous administration for either treatment or prophylaxis. Interestingly, VIR-7831 was derived from the antibody S309, which was identified from a SARS-CoV-1 convalescent patient (Cathcart et al., 2021) . This antibody is cross-reactive to SARS and binds to one of the few epitopes shared between the viruses, which includes an N-linked glycan. By comparison, LY-CoV1404 was discovered from a SARS-CoV-2 convalescent patient and selected based on its cross-reactivity to SARS-CoV-2 variants, thereby allowing testing and identification of much more potent neutralizing epitopes. A more recently identified antibody from a COVID-19recovered patient, S2X259, was described with broad cross-reactivity to variants (including B.1.1.7, B.1.351, B.1.429, and P.1) and several zoonotic strains (Tortorici et al., 2021 ). This antibody, while able to bind widely to coronaviruses, had neutralizing capacity against SARS-CoV-2 in authentic virus-neutralization assays that was substantially less potent than LY-CoV1404. Finally, an engineered antibody with broad cross-reactivity against coronaviruses and J o u r n a l P r e -p r o o f 17 potent neutralization was recently described (Rappazzo et al., 2021) , which was optimized for binding following identification from a SARS convalescent patient.

Mutations within the S protein and in particular in the RBD are inevitable, particularly as SARS-CoV-2 has been present in the human population for only 18 months (Jones et al., 2020) . Since the pandemic began, we have continued to screen patient samples and have identified thousands of human antibodies with associated functional data. We analyzed this antibody database in response to the increase in VOC and identified many potential solutions based on these variants. LY-CoV1404 is one example of this screening and analysis. To fully prepare for the inevitability of additional mutants and reduction in treatment effectiveness, we propose 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Bryan E. Jones (jones_bryan_edward@lilly.com). 

All data associated with this study are available in the main text or the supplementary materials figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using this material.

This paper does not report original code.

Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Vero-E6 cells were maintained in plaque assay media (complete MEM media with 1% BGS + 1% low melting point agarose).

Authentic SARS-CoV-2 virus (MT020880.1) was obtained through the Biodefense and Emerging

Infections Research Resources Repository.

Recombinant antibodies and other proteins were produced as previously described (Jones et al., 2020) . The antigen binding fragment (Fab) portion of LY-CoV1404 was generated by proteolytic digestion using immobilized papain (ThermoFisher Scientific), followed by removal of un-cleaved protein using standard chromatography techniques. The reference S protein sequence was from Beads or cells were flowed onto microfluidic screening devices and incubated with single antibody-secreting cells, and mAb binding to cognate antigens was detected via a fluorescently labeled anti-human IgG secondary antibody, or soluble antigen labeled with fluorophore. Positive hits were identified using machine vision and recovered using automated robotics-based protocols.

Single-cell polymerase chain reaction (PCR) and custom molecular biology protocols generated NGS sequencing libraries (MiSeq, Illumina) using automated workstations (Bravo, Agilent).

Sequencing data were analyzed using a custom bioinformatics pipeline to yield paired heavy and light chain sequences for each recovered antibody-secreting cell (Jones et al., 2020) . Each sequence was annotated with the closest germline (V(D)J) genes, degree of somatic hypermutation, and potential sequence liabilities. Antibodies were considered members of the same clonal family if they shared the same inferred heavy and light V and J genes and had the same CDR3 length. The variable (V(D)J) region of each antibody chain was PCR amplified and inserted into expression plasmids using a custom, automated high-throughput cloning pipeline.

Plasmids were verified by Sanger sequencing to confirm the original sequence previously identified by NGS. Antibodies were recombinantly produced by transient transfection in either human-embryonic kidney (HEK293) or CHO cells.

All epitope binning and ACE-2 blocking experiments were performed on a Carterra® LSA™ instrument equipped with an HC-30M chip type (Carterra-bio), using a 384-ligand array format as previously described (Shu and McCauley, 2017) . For epitope binning experiments, antibodies coupled to the chip surface were exposed to various antibody:antigen complexes. Samples were prepared by mixing each antibody in 10-fold molar excess with antigen (1:1 freshly prepared mix of 400 nM antibody and 40 nM antigen, both diluted in 1X HBSTE + 0.05% BSA running buffer).

Each antigen-antibody premix was injected sequentially over the chip surface for 5 minutes (association phase to ligand printed onto chip previously), followed by a running buffer injection for 15 minutes (dissociation phase). Two regeneration cycles of 30 seconds were performed between each premix sample by injecting 10 mM glycine pH 2.0 onto the chip surface. An antigen-J o u r n a l P r e -p r o o f 28 only injection (20 nM concentration in the running buffer) was performed every 8 cycles to assess maximum binding to S protein and in-order to accurately determine the binning relationship.

The data were analyzed using the Carterra Epitope analysis software for heat map and competition network generation. Analyte binding signals were normalized to the antigen-only binding signal, such that the antigen-only signal average is equivalent to one RU (response unit).

A threshold window ranging from 0.5 RU to 0.9 RU was used to classify analytes into 3 categories:

blockers (binding signal under the lower limit threshold), sandwichers (binding signal over the higher limit threshold) and ambiguous (binding signal between limit thresholds). Antibodies with low coupling to the chip, poor regeneration or with absence of self-blocking were excluded from the binning analysis. Like-behaved antibodies were automatically clustered to form a heat map and competition plot.

To test the antibodies' ability to block ACE2, antibodies coupled to the HC-30M chip as described above were exposed to SARS-CoV-2 S protein:ACE2 complex. A freshly prepared 1:1 mix of 40 nM of SARS-CoV-2 S protein and 400 nM of untagged ACE2, both diluted in HBSTE + 0.05% BSA, were tested for binding to the immobilized mAbs on the prepared HC-30M chips, with association for 5 minutes and dissociation for 5 minutes. A SARS-CoV-2 S protein injection at 20 nM was included to assess for maximum binding, as well as a ACE2 injection at 200 nM to assess for non-specific binding. Regeneration was performed in 20 mM glycine pH 2.0 with 1M NaCl for 30 seconds twice. To investigate binding of LY-CoV1404 mAb to SARS-CoV-2 S protein, D614G S protein variant and RBD protein, a HC30M chip was used, and the array preparation was performed as described above, coupling antibody diluted to 3 µg/mL in 10 mM acetate, pH 4.0, for 10 minutes, and deactivation for 7 minutes in 1 M ethanolamine, pH 8.5.

To measure binding kinetics and affinity, the mAb-coupled HC30M chip surface was exposed to injections of the proteins, with an association period of 5 minutes and dissociation period of 15 used, and the array preparation was performed as described above, coupling Fc-fused RBD proteins diluted 5 µg/mL and 10 µg/mL in 10 mM acetate, pH 4.0, for 10 minutes, and deactivation for 7 minutes in 1 M ethanolamine, pH 8.5.

To measure binding kinetics and affinity, the RBD-coupled HC30M chip surface was exposed to The data were analyzed using the Biacore Insight Evaluation Software. The curves were referenced and blank subtracted and then fit to a 1:1 Langmuir binding model to generate apparent association (ka) and dissociation (kd) kinetic rate constants and binding affinity constants (KD).

Unique antibody sequences were confirmed to bind the screening target (SARS-CoV-2 full length Luciferase activity was measured on a PerkinElmer EnVision 2104 multilabel reader.

Neutralization assays were carried out as described (Nie et al., 2020) . Virus preparation volumes were normalized to equivalent signal output (RLU, relative light units) as determined by luciferase activity following infection with serially diluted virus. Eleven-point, 2-fold titrations of LY-CoV1404

were performed in 96-well plates in duplicate and pre-incubated with a fixed amount of pseudovirus for 20 minutes at 37°C. Following pre-incubation, the virus-antibody complexes were added to 20,000 VeroE6 cells/well in white, opaque, tissue culture-treated 96 well plates, and incubated 16 to 20 hours at 37°C. Control wells included virus only (no antibody, quadruplicate) and cells only (duplicate). Following infection, cells were lysed, and luciferase activity was measured.

SARS-CoV-2 spike pseudotyped lentiviruses that harbor a luciferase reporter gene were produced and neutralization assay was performed as described previously Naldini et al., 1996) . Pseudovirus was produced by co-transfection of 293T cells with plasmids encoding the lentiviral packaging and luciferase reporter, a human transmembrane protease 529 containing A67V, H69-, V70-, T95I, G142D, V143-, Y144-, Y145-, N211-,L212I,   ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A,   Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F amino acid changed compared to WA-1) or S variant genes (Wang et al., 2021a) using Lipofectamine 3000 transfection reagent (ThermoFisher, CA).

Forty-eight hours after transfection, supernatants were harvested, filtered and frozen. For neutralization assay serial dilutions (2 dilutions at 10 and 1 µg/ml for the initial screen assay or 8 dilutions for the full curve at 10-0.0006 µg/ml) of monoclonal antibodies were mixed with titrated pseudovirus, incubated for 45 minutes at 37 °C and added to pre-seeded 293T-ACE2 cells for RBD variants of interest expressed on the surface of yeast. The in vitro inhibition assay used 1 nM soluble hACE2 incubated with increasing concentrations of LY-CoV1404, ranging from 0 to 3.75 ug/mL with each yeast RBD variant of interest. Soluble hACE2 binding was performed at room temperature for 20 minutes followed by transfer of reactions to ice for another 10 minutes to quench dissociation. Fluorescence was measured after addition of streptavidin-phycoerythrin for 20 minutes on ice followed by multiple washes with cold buffer. Mean fluorescence intensity (MFI) was normalized for each concentration response curve (CRC) using the maximum MFI at no mAb inhibitor (MAX) and the background MFI (Bgnd). Percent inhibition of S protein binding to ACE2 was defined as follows:

where x was the MFI at the tested concentration of mAb. CRCs were fit with a four-parameter logistic function. All four parameters were estimated from the fitting. Absolute IC50 (50% absolute inhibition) values were reported. Standard error and 95% confidence intervals for IC50 estimates are reported and, in some cases, a fixed top (Top = 100) was used to stabilize the standard error estimate. IC50 ratios between mutant S protein and wild type (WT) were reported. For mutant S proteins that exhibited no mAb inhibition, fold-change was reported at 1x of the highest concentration of mAb tested. For any single experimental batch, inhibition data were obtained in duplicate or triplicate. WT RBD was analyzed with every experiment and the consensus estimate as of the date indicated is reported. If select mutants of interest were analyzed in more than one experimental batch, then the geometric mean is reported.

Work with authentic SARS-CoV-2 at USAMRIID was completed in BSL-3 laboratories in accordance with federal and institutional biosafety standards and regulations. Vero-76 cells were inoculated with SARS-CoV-2 (GenBank MT020880.1) at a MOI = 0.01 and incubated at 37°C with 5% CO2 and 80% humidity. At 50 h post-infection, cells were frozen at -80°C for 1 h, allowed to thaw at room temperature, and supernatants were collected and clarified by centrifugation at ~2,500 x g for 10 min. Clarified supernatant was aliquoted and stored at -80°C. Sequencing data from this virus stock indicated a single mutation in the S glycoprotein (H655Y) relative to isolates (in the case of the rWA1, rWA1 E484K, and rWA1 E484Q viral isolates) were constructed using reverse genetics as previously described (Xie et al., 2021b) . Virus stocks were grown by inoculating cultured Vero E6 cells, followed by incubation at 37ºC until cytopathic effects (CPE)

were evident (typically 48 to 72 hours). Expansion was limited to 1 to 2 passages in cell culture to retain integrity of the original viral sequence. The virus stock was quantified by standard plaque assay, and aliquots were stored at -80ºC. A freshly thawed aliquot was used for each neutralization experiment. following staining with secondary detection antibody (goat α-rabbit) conjugated to AlexaFluor 488.

Infected cells were enumerated using the Operetta high content imaging instrument and data analysis was performed using the Harmony software (Perkin Elmer).

Vero-E6 cells were seeded in a 24-well plate 48 hours before the assay (Figure 2 

A 10 mg/mL solution of LY-CoV1404 Fab (with CrystalKappa mutations (Lieu et al., 2020b) indexed and integrated using autoPROC (Vonrhein et al., 2011) /XDS (Kabsch, 2010) and merged and scaled in AIMLESS (Evans and Murshudov, 2013) from the CCP4 suite (Winn et al., 2011) .

Non-isomorphous data readily yielded initial structures by molecular replacement using the Fab portion of crystal structures from the proprietary Eli Lilly structure database and the SARS-CoV-2 S protein RBD from the public domain structure with the access code 6yla (Huo et al., 2020) .

The initial structure coordinates were further refined using Buster (Smart et al., 2012) CONTACT (Winn et al., 2011) and custom shell/Perl scripts.

A stabilized version of the SARS-CoV-2 S protein was employed for all studies (Hsieh 2020 (Scheres 2016) .

To estimate the incidence of SARS-CoV-2 variants with potential resistance to LY-CoV1404, we analyzed data from GISAID EpiCoV database (Shu and McCauley, 2017) , focusing on recent data, i.e. genomes collected in a 3 month window from Dec 16 replicative data were presented as mean ± SD. Value of p < 0.05 was considered to be statistically significant and represented as an asterisk ( * ). Value of p < 0.01 was supposed to be more statistically significant and described as double asterisks ( * * ). Value of p < 0.001 was considered the most statistically significant and represented as triple asterisks ( * * * ). Value of p < 0.0001 was supposed to be extremely statistically substantial and described as quadruple asterisks ( * * * * ).

For comparison between two treatments, a Student's t-test was used. For comparison between each group with the mean of every other group within a dataset containing more than two groups, one-way ANOVA with Tukey's multiple comparisons test was used. J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f Abbreviations: D = a spartate; del = deletion; E = glutamate; 0 = glyene; H = histidine; I = isoleuene; 1050 ,IC90 = concentration inhibiting maximal activity by 50% and 90%, respectively; K = lysine; L = leucine; N = asparagine; NA = not applicable; P = proline; Q = glutamine; R = arginine; S = serine; T= threonine; Y = tyrosine.

a Absolute IC estimates presented are a result of a meta-analysis of multiple replicate experiments. *Viral constructs for the lentivural platform include D614G ** Viral constructs for the VSV platform include D614G

J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f J o u r n a l P r e -p r o o f Abbreviations -= not tested; ACE2 = angiotensin-converting enzyme 2; CI = confidence interval; del= deletion; GISAID = Global Initiative on Sharing All Influenza Data; IC50 = concentration inhibiting maximal activity by 50%; mAb = monoclonal antibody; nc = no change (difference was less than ≤5-fold); NA = not applicable; SARS-CoV-2 = severe acute respiratory syndrome coronavirus 2.

a IC50 values presented are geometric mean if multiple experiments were performed and a weighted geometric mean for the Wuhan strain (n=10) 95% CI values were included for variants tested only once. IC50 for ACE2-binding inhibition of Wuhan sequence is presented as the consensus estimate as of 26 February 2021 achieved across repeat experiments. b Fold shifts are calculated comparing to the in-experiment Wuhan control. The geometric mean of the fold changes is provided if multiple experiments were performed. For ACE2-binding inhibition, no inhibition represents <50% ACE2-binding inhibition with up to 3.75 μg/mL mAb. c SARS-CoV-2 S GenBank MN908947.3. show that LY-CoV1404 is a potent SARS-CoV-2-binding antibody that neutralizes all known variants of concern and whose epitope is rarely mutated.

• LY-CoV1404 potently neutralizes SARS-CoV-2, Omicron, BA.2 Omicron, and Delta variants • No loss of potency against currently circulating variants • Binding epitope on RBD of SARS-CoV-2 is rarely mutated based on current GISAID data • Breadth of neutralizing activity and potency supports clinical development Table S1 . Crystallographic statistics Table S2 . Summary of atomic interactions at the RBD epitope J o u r n a l P r e -p r o o f

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Space group P2 (1)2(1)