key: cord-0829430-ghs0zitc
authors: Bell, Benjamin N.; Powell, Abigail E.; Rodriguez, Carlos; Cochran, Jennifer R.; Kim, Peter S.
title: Neutralizing antibodies targeting the SARS-CoV-2 receptor binding domain isolated from a naïve human antibody library
date: 2021-01-08
journal: bioRxiv
DOI: 10.1101/2021.01.07.425806
sha: 849328fad11f25b4617cd88eb93a1c977c1763da
doc_id: 829430
cord_uid: ghs0zitc

Infection with SARS-CoV-2 elicits robust antibody responses in some patients, with a majority of the response directed at the receptor binding domain (RBD) of the spike surface glycoprotein. Remarkably, many patient-derived antibodies that potently inhibit viral infection harbor few to no mutations from the germline, suggesting that naïve antibody libraries are a viable means for discovery of novel SARS-CoV-2 neutralizing antibodies. Here, we used a yeast surface-display library of human naïve antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: one that blocks interaction with angiotensin-converting enzyme 2 (ACE2), the human receptor for SARS-CoV-2, and two that target other epitopes on the RBD. These three antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus with IC50 values as low as 60 ng/mL in vitro. Using a biolayer interferometry-based binding competition assay, we determined that these antibodies have distinct but overlapping epitopes with antibodies elicited during natural COVID-19 infection. Taken together, these analyses highlight how in vitro selection of naïve antibodies can mimic the humoral response in vivo, yielding neutralizing antibodies and various epitopes that can be effectively targeted on the SARS-CoV-2 RBD.

Over 84 million cases of Coronavirus Disease 2019 (COVID-19) and 1.8 million deaths worldwide in the past eleven months 1 have been caused by the novel human pathogen SARS-CoV-2 2,3 . Since the beginning of the COVID-19 pandemic, extensive work has been devoted to characterizing the antibody response to SARS-CoV-2 in response to both infection [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] and vaccination [14] [15] [16] [17] [18] [19] [20] . Analysis of antibody responses has revealed that patients elicit robust neutralizing antibodies targeting the SARS-CoV-2 spike 7, 8, 12, 21, 22 , a trimeric glycoprotein responsible for receptor recognition and host cell entry 2, 23, 24 . Specifically, the spike receptor binding domain (RBD), which binds the host cell receptor ACE2, has been shown to be an immunodominant region of the spike [6] [7] [8] [9] 12 .

Despite being only 25 kDa in size, the RBD elicits antibodies that fall into several subclasses targeting distinct, but overlapping, epitopes 7, 10 . Many of these subclasses contain neutralizing antibodies, demonstrating that multiple sites on the RBD are vulnerable to inhibition and raising the possibility that RBD-targeting antibodies neutralize viral entry via a variety of mechanisms of action.

Remarkably, many RBD antibodies described to date have high similarity to their germline antibody precursors, with low rates of somatic hypermutation [6] [7] [8] 10 . This observation is encouraging for RBD-and spike-based vaccine approaches, as it suggests that neutralizing antibodies can readily be elicited in healthy individuals. This germline similarity also highlights the potential use of naïve antibody libraries as a promising source of novel therapeutic antibodies and for comparison of clones selected in vitro to those elicited by natural COVID-19 infection or vaccination.

In order to identify RBD-targeting antibodies with high similarity to the germline, here we employed an in vitro approach based on a yeast surface-display library of human naïve antibodies expressed in a single-chain variable fragment (scFv) format. We subsequently isolated and characterized three novel neutralizing antibodies: one (ABP18) that blocks binding of RBD to ACE2, and two (NBP10, NBP11) that do not. These three antibodies neutralize SARS-CoV-2 spike-pseudotyped lentivirus in vitro with IC50 (halfmaximal inhibitory concentration) values of 0.06-13 μg/mL. When we performed binding competition experiments using biolayer interferometry (BLI), we found that ABP18, NBP10, and NBP11 have epitopes that are distinct but overlap with epitopes that are well-defined of antibodies elicited by natural COVID-19 infection. Taken together, these analyses demonstrate that in vitro selections of naïve antibodies can mimic the humoral response in vivo to identify neutralizing antibodies. In vitro selections therefore may constitute an important tool for both vaccine development and antibody discovery for SARS-CoV-2 therapeutics.

To isolate antibodies targeting the SARS-CoV-2 RBD, we panned a yeast surfacedisplay library developed by xCella Biosciences, xEmplar™ (Materials and Methods), that contains ~1 x 10 9 scFv sequences and has been used to identify antibodies against novel targets such as the immune checkpoint VISTA 25 . We performed a total of five rounds of selection that included both magnetic-and fluorescence-activated cell sorting to enrich for RBD-binding clones (Materials and Methods). In the final round, we also used biotinylated ACE2 labeled with Streptavidin-Alexa Fluor 647 to differentiate between scFvs that disrupted the ACE2/RBD interaction (ACE2-blocking population) and those that did not (non-blocking population; Fig. 1A ). We identified a single unique sequence from the ACE2-blocking population (scFv ABP18) and three unique sequences from the non-blocking population (scFvs NBP10, NBP11, and NBP18); only ABP18, NBP10, and NBP11 were confirmed to bind RBD (SI Fig. 1A) .

We cloned the variable heavy chain and variable light chain sequences into human IgG1 plasmids (Materials and Methods) to further characterize ABP18, NBP10, and NBP11. We used BLI to confirm binding to RBD and to determine whether these antibodies compete with ACE2, as expected based on the sorting scheme. RBD-coated biosensors bound ABP18, NBP10, and NBP11 in IgG form (Fig. 1B) , with low-nanomolar affinities calculated with a standard 1:1 binding model to simplify the bivalent interaction (Materials and Methods). To evaluate monovalent binding affinities, we processed the IgGs to Fab fragments using the endoprotease Lys-C (SI Fig. 1B) . ABP18, NBP10, and NBP11 Fabs bound RBD (Fig. 1C ) and exhibited substantially faster dissociation rates than the IgG forms (compare Fig. 1B,C) , suggesting that the slow off-rates for IgG/RBD binding were influenced by the avidity of the antibody binding site. Consistent with the sequence and structural diversity among coronavirus RBDs 26 , enzyme-linked immunosorbent assays indicated that ABP18, NBP10, and NBP11 minimally bind RBDs from SARS-CoV-1 and MERS-CoV (SI Fig. 1C ).

To confirm that our selection scheme differentiated between ACE2-blocking and non-blocking scFv clones, we employed BLI in two orientations to assess the ability of (B-C) Binding to SARS-CoV-2 RBD-coated BLI biosensors show substantial differences between (B) IgG antibodies (100, 33.3, and 11.1 nM) and (C) Fab antibody fragments (ABP18/NBP11: 500, 167, 56, 18 nM; NBP10: 720, 240, 80, 27 nM). Higher antibody concentrations are indicated with thicker lines. (D) Binding of 100 nM IgG antibodies to BLI biosensors coated with ACE2:RBD complex shows that NBP10 and NBP11 IgG retain binding while ABP18 shows no binding, consistent with ACE2-blocking activity. these antibodies to interact with RBD in the presence of ACE2 (SI Fig. 1D , E). When ACE2-coated biosensors were subsequently loaded with RBD, ABP18 showed no binding ( Fig. 1D) , while NBP10 and NBP11 IgG retained binding (Fig. 1D ). In the reverse binding orientation using RBD-coated biosensors loaded with ACE2, ABP18 IgG binding was reduced when biosensors were preloaded with ACE2 (SI Fig. 1E) ; however, NBP10 and NBP11 IgG binding to RBD was unaffected by ACE2 (SI Fig. 1E ), validating the ability of our selection scheme to distinguish between ACE2-blocking and non-blocking antibodies.

Not all RBD-targeting antibodies neutralize SARS-CoV-2 infection 10,27-29 ; further, antibodies isolated against the RBD alone may not effectively target the RBD in the context of the full-length spike trimer. We therefore tested the ability of ABP18, NBP10, and NBP11 IgG to neutralize SARS-CoV-2 pseudotyped lentivirus infection of ACE2expressing HeLa cells 19, 30 (Fig. 2A) . All three antibodies inhibited SARS-CoV-2 infection, with IC50 values ranging from 0.06 to 13 μg/mL (Fig. 2B ,C).

ABP18 IgG had an IC50 value of 0.41 nM (Fig. 2C ), similar to its apparent affinity of 1.8 nM (Fig. 2C) , while NBP10 and NBP11 IgG had IC50 values ~20-to 50-fold weaker than their apparent affinities (Fig. 2C) . This difference may arise from (i) distinct mechanisms of viral neutralization employed by ABP18 and NBP10/NBP11 or (ii) differential epitope accessibility in the context of full-length spike or up/down conformations of the RBD 10, 31 . Notably, all three antibodies have more potent IC50 values than their Fab binding affinities (Fig. 2C ), suggesting that antibody avidity plays an important role in viral neutralization.

Several classes of RBD antibodies have been described based on binding competition experiments and structure determination 6, 10, 13, 19, [27] [28] [29] 32, 33 . We therefore sought to map the epitopes of ABP18, NBP10, and NBP11 using a BLI-based binding competition assay. We expressed and purified (Materials and Methods) a panel of nine published RBD antibodies elicited by natural COVID-19 infection, four of which (CB6 33 , P2B-2F6 6 , EY6A 32 , CR3022 27 ) have high-resolution co-crystal structures available. Five antibodies were included from Brouwer et al. 28 , four of which (COVA1-12, COVA2-04, HeLa-ACE2

Antibody 28 . We included the fifth antibody from Brouwer et al. 28 , COVA2-05, because it represents a unique epitope class (compared to COVA1-12, COVA2-04, COVA2-07, and COVA2-15) and shares the same antibody heavy chain germline as NBP10 and NBP11 (SI Table 1 ). However, no structure is available for COVA2-05. This nine-antibody panel represents a range of RBD epitopes and antibody sequences, generated in vivo via patient responses to infection, by which to compare the antibodies that we isolated in vitro here.

To evaluate how ABP18, NBP10, and NBP11 compete with antibodies in this panel for binding to RBD, we used BLI to establish a binding competition matrix (Materials and Methods). RBD-coated biosensors were bound to saturation by an antibody (the "loaded" antibody). These loaded-antibody/RBD-coated biosensors were then bound to antibodies in solution (the "associating" antibody). The binding of an associating antibody to biosensors coated with RBD and loaded with a competing antibody was compared to biosensors only coated with RBD and is reported as a fraction of maximal binding ( NBP10 and NBP11 showed an identical pattern of binding competition (Fig. 3B ), suggesting that they share similar epitopes, consistent with their common heavy chain germline, IGHV5-51 (SI Table 1 ). NBP10 and NBP11 competed with COVA2-05, but not CR3022 or EY6A (Fig. 3B) . Interestingly, although EY6A and CR3022 have very similar epitopes 32 , COVA2-05 competed with CR3022, but not EY6A (Fig. 3B ). We therefore conclude that NBP10, NBP11, COVA2-05, CR3022, and EY6A share overlapping but distinct epitopes. One notable difference between the epitopes of CR3022 and EY6A is that the complementarity determining region loop 1 of the CR3022 light chain extends beyond the EY6A light chain footprint (Fig. 3A) , suggesting that this loop may be responsible for steric clashes between EY6A and NBP10/11. Combining our binding competition data and published structural data (Fig. 3C) , we determined putative footprints for NBP10, NBP11, and COVA2-05 (Fig. 3D ).

ABP18 demonstrated substantial competition with ACE2-blocking antibodies COVA1-12, COVA2-04, COVA2-07 (SI Fig. 2B ), and CB6 (Fig. 3B) , as expected.

However, ABP18 did not compete with COVA2-15 or P2B-2F6 (Fig. 3B) , which are other ACE2-competing antibodies. Based on the high-resolution X-ray crystal structure of P2B-2F6 6 and the low-resolution negative-stain electron-micrograph reconstruction of COVA2-15 28 , we determined an approximate antibody footprint for ABP18 (Fig. 3E) . Although the RBD makes up a small region of the spike trimer, these antibody competition results confirmed previous reports 7,10,29 that the RBD contains several unique epitopes targeted by monoclonal antibodies. Further, the varied footprints and competition patterns even within the set of ACE2-competing antibodies tested here indicates that there are likely varied mechanisms of viral neutralization mediated by ACE2-blocking monoclonal antibodies. 

Understanding the humoral responses to SARS-CoV-2 infection and vaccination is critical to efforts aimed at curtailing the COVID-19 pandemic and establishing effective correlates of protection. Accordingly, numerous patient-derived antibodies have been described, many of which target the RBD and have high similarity to germline antibody precursors. To exploit this similarity and to expand the repertoire of neutralizing antibodies, here we applied in vitro selections of a yeast surface-display library of human naïve antibodies to isolate and characterize three novel neutralizing antibodies that target the RBD: ABP18, NBP10, and NBP11 (Fig. 1) . These antibodies neutralized SARS-CoV-2 spike-pseudotyped lentivirus in vitro (Fig. 2) , targeting the ACE2-binding site (ABP18) and other RBD epitopes (NBP10 and NBP11) (Fig. 3) . BLI-based epitope mapping experiments revealed that the epitopes of these novel antibodies are distinct but overlapping with antibodies elicited by natural COVID-19 infection (Fig. 3B ). This work demonstrates that in vitro selections of naïve antibody repertoires can effectively mimic the antibody response in vivo in response to SARS-CoV-2 infection or vaccination, showcasing the value of this approach in rapidly isolating novel antibodies with therapeutic potential.

RBD-targeting antibodies bind numerous distinct, but overlapping, epitopes. We selected a panel of patient-derived RBD antibodies with well-defined epitopes and determined that none of them shared identical profiles with ABP18, NBP10, or NBP11 in binding competition experiments (Fig. 3B) . Each of our three novel antibodies demonstrated strong competition with at least one antibody in the binding panel, which in turn had strong competition with additional antibodies (Fig. 3B) , enabling us to delineate putative epitopes for ABP18, NBP10, and NBP11 (Fig. 3D,E) . High-resolution structural information is needed to more precisely determine these antibody binding sites and their binding angles. Binding angles can be a critical factor in viral neutralization 34 ; for example, antibodies such as CR3022 and EY6A complete strongly for binding the RBD, but only EY6A neutralizes SARS-CoV-2 entry 32 . Nevertheless, our results highlight how methods like BLI can quickly delineate classes of antibodies and provide low-resolution structural parameters for antibody:antigen interactions.

Antibodies with high similarity to the germline, and from diverse germline lineages, bind the RBD and can effectively neutralize SARS-CoV-2 7,10,29 . The antibodies described here also displayed high germline similarity, with low degrees of somatic hypermutation (SI Table 1 ). This observation is particularly promising for RBD-and spike-based vaccination strategies, as they may readily elicit neutralizing antibodies in patients presenting varied human antibody repertoires. There is well-documented enrichment of specific antibody germlines among antibodies elicited by SARS-CoV-2 infection, including IGHV1-24, IGVH1-69, IGHV3-30, IGHV3-53, and IGHV5-51 7, 8, 28, 35 . The antibodies that we isolated here have sequences derived from these major germlines elicited in COVID-19 patients (SI Table 1 ). ABP18 is an IGHV1-69 antibody, a common germline regularly elicited by influenza infection 36 and enriched among SARS-CoV-2 antibodies 28 . By contrast, NBP10 and NBP11 both utilize IGHV5-51, consistent with their shared epitope. NBP11 shares both heavy and light chain germlines (IGHV5-51 and IGKV1-5, respectively) with C110, a recently described neutralizing RBD antibody derived from a COVID-19 patient 29 . Interestingly, the epitope of P2B-2F6 overlaps with that of C110 10 , though P2B-2F6 does not compete with NBP11 (Fig. 3B) , suggesting that C110 and NBP11 target distinct epitopes. This result further illustrates the diversity of near-germline antibodies that can effectively target the RBD, and underscores that even antibodies with the same germline precursors can target distinct RBD epitopes.

Taken together, these analyses highlight how in vitro selections of naïve antibodies can mimic the humoral response in vivo to identify neutralizing antibodies against novel human pathogens like SARS-CoV-2. Additionally, these novel RBD-targeting antibodies demonstrate the numerous distinct epitopes that can be effectively targeted on the SARS-CoV-2 RBD, bolstering COVID-19 therapeutic and vaccine efforts. Plasmids were propagated overnight at 37 °C in Stellar cells (Takara) in 2xYT medium supplemented with 100 μg/mL carbenicillin (RBD and ACE2 plasmids) or 50 μg/mL kanamycin (IgG plasmids). Plasmids were isolated using a Machery Nagel maxi prep kit and filtered in a biosafety cabinet using a 0.22-µm filter prior to transfection.

IgGs, ACE2-huFc-Avi, and coronavirus RBDs were expressed and purified from Coronavirus RBDs (SARS-CoV-2, SARS1, and MERS) were purified using HisPur Ni-NTA affinity resin (Thermo). Supernatants were diluted 1:1 with 10 mM imidazole / 1X PBS and incubated with Ni-NTA resin for at least 1 h at 4 °C while stirring.

Resin/supernatant mixtures were then added to glass chromatography columns, washed with ~10 column volumes 10 mM imidazole / 1X PBS, and RBDs were eluted using 250 mM imidazole / 1X PBS. Elutions were concentrated using Amicon spin filters (MWCO 10 kDa; Millipore Sigma) and RBDs were subsequently loaded onto a GE Superdex S200 increase 10/300 GL column pre-equilibrated in 1X PBS on a GE ÄKTA Pure system. Protein-containing fractions were identified by A280 signal and/or SDS-PAGE, pooled, and stored at 4 °C or at -20 °C in 10% glycerol / 1X PBS until use.

Lys-C endopeptidase (Wako) was added at a final concentration of 4 μg/mL to 

Purified ACE2 expressed with a human IgG1 Fc domain and C-terminal Avi tag was site-specifically biotinylated using BirA-thioredoxin. BirA enzyme was expressed and purified as previously described 38 . Briefly, BirA was expressed recombinantly in Escherichia coli as a genetic fusion to thioredoxin and purified with HisPur Ni-NTA affinity resin (Thermo Fisher Scientific). BirA-thioredoxin was subsequently loaded onto a GE Superdex S200 increase 10/300 GL column pre-equilibrated in 1X PBS on a GE ÄKTA Pure system before being aliquoted and frozen with 10% glycerol.

To biotinylate purified ACE2, briefly, ACE2 was buffered with 50 mM bicine [pH For antibody binding competition assays, His-tagged SARS-CoV-2 RBD (300 nM) was loaded on His1K biosensors (Pall ForteBio) to a load threshold of 0.4-0.6 nm. After a baseline step in assay buffer, RBD-coated biosensors were dipped in a 500 nM solution of a particular "loaded" antibody for 4 min. "Loaded"-antibody/RBD-coated biosensors were then dipped in a 200 nM solution of a particular "associating" antibody for 3 min. The detected shift in nanometers of the "associating" antibody at 3 min in the absence of any "loaded" antibody was used as the maximal binding value. Values for the "loaded" antibody are reported as a fraction of this value. All BLI data were baseline-subtracted based on RBD-coated biosensors dipped into buffer for the "loading" and "associating" steps.

SARS-CoV-2 spike-pseudotyped lentivirus was produced as described 20 Neutralization assays were performed as described 19, 20 . HeLa cells overexpressing ACE2 19 were plated at 5,000 cells per well in clear-bottom white-walled 96-well plates 1 day prior to infection in D10 medium. Purified monoclonal antibodies were filtered using a 0.22-μm filter and then diluted using D10 medium. Virus was diluted in D10 medium and supplemented with polybrene (5 μg/mL final), added to inhibitor dilutions, and inhibitor/virus were incubated at 37 °C for 1 h. Medium was removed from HeLa/ACE2 cells and replaced with virus/inhibitor mixtures which were then incubated for signal from wells was determined using a BioTek plate reader.

Data from neutralization assays were processed using GraphPad Prism 8.4.1.

Relative luminescence units from virus-only wells and cells-only wells were averaged, and RLU values from inhibitor neutralization curves were normalized to these averages 

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