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A SARS-CoV-2 antibody broadly neutralizes SARS-related coronaviruses and variants by coordinated recognition of a virus-vulnerable site

Potent neutralizing SARS-CoV-2 antibodies often target the spike protein receptor-binding site (RBS), but the variability of RBS epitopes hampers broad neutralization of multiple sarbecoviruses and drifted viruses. Here, using humanized mice, we identified an RBS antibody with a germline VH gene that potently neutralized SARS-related coronaviruses, including SARS-CoV and SARS-CoV-2 variants. X-ray crystallography revealed coordinated recognition by the heavy chain of non-RBS conserved sites and the light chain of RBS with a binding angle mimicking the angiotensin-converting enzyme 2 (ACE2) receptor. The minimum footprints in the hypervariable region of RBS contributed to the breadth of neutralization, which was enhanced by immunoglobulin G3 (IgG3) class switching. The coordinated binding resulted in broad neutralization of SARS-CoV and emerging SARS-CoV-2 variants of concern. Low-dose therapeutic antibody treatment in hamsters reduced the virus titers and morbidity during SARS-CoV-2 challenge. The structural basis for broad neutralizing activity may inform the design of a broad spectrum of therapeutics and vaccines.


In brief
Antigenic variability in SARS-related coronavirus decreases the likelihood of cross-neutralization by monoclonal antibodies. Onodera et al. identify a broad neutralizing SARS-CoV-2 antibody in humanized mice that targets a vulnerable site of SARS-CoV and SARS-CoV-2 variants. Structural analysis reveals that the broad neutralization is coordinated by the heavy and light chains and is enhanced by IgG3 class switching.

INTRODUCTION
The coronavirus disease 2019 (COVID-19) pandemic, triggered by the novel virus SARS-CoV-2 (CoV2), has caused more than 198 million infections and more than 4.2 million deaths world-wide as of August 3, 2021 (https://www.who.int/). Whereas host immune responses are considered to play both beneficial and detrimental roles for disease progression and outcome in the acute period, neutralizing antibodies that are maintained in the convalescent period protect against re-infection in animal models (Baum et al., 2020a;McMahan et al., 2021). Indeed, convalescent plasma and monoclonal antibody therapeutics received emergency use authorization by the U.S. Food and Drug Administration (Gottlieb et al., 2021;Weinreich et al., 2021), with more antibody seeds under preclinical and clinical development.
Major epitopes for CoV2-neutralizing antibodies reside in the receptor-binding domain (RBD) of spike (S) protein (Andreano et al., 2021;Piccoli et al., 2020). RBD epitopes are further classified into four classes based on the structure of antigen-antibody complex Yuan et al., 2021). Class 1 and class 2 epitopes overlap angiotensin-converting enzyme 2 (ACE2) binding sites (receptor-binding site [RBS]), thereby constituting the major target for highly potent neutralizing antibodies . However, low conservation of the RBS sequence between CoV2 and other coronaviruses (CoVs) decreases the likelihood of cross-neutralization of multiple CoVs by RBS antibodies. Supporting this, only 1.2% (6 clones) of CoV2-neutralizing antibodies (482 clones) deposited in the coronavirus antibody database (CoV-AbDab) cross-neutralize SARS-CoV (CoV1) among sarbecoviruses (Raybould et al., 2021). CoV1 cross-neutralizing activity was also absent among highly potent CoV2-neutralizing antibodies that were selected from 453 neutralizing antibodies (Andreano et al., 2021). In addition, many RBS antibodies are vulnerable to CoV2 escape mutations under monotherapeutic usage (Baum et al., 2020b;Li et al., 2020;Weisblum et al., 2020) because of the antigenic drift of the RBS epitopes (Piccoli et al., 2020;Starr et al., 2021). Thus, similar to antibodies to other mutating RNA viruses, potent neutralizing activity and breadth to antigenically divergent viruses are incompatible characters in most CoV2-neutralizing antibodies.
Neutralizing RBS antibodies are frequently encoded by structurally related V H genes and share similar binding modes and footprints (Robbiani et al., 2020;Yuan et al., 2020b). Such convergent antibody responses in many individuals could increase the risk of generating antibody escape mutations because of limited diversity of humoral immune responses in the population. Indeed, CoV2 variants of concern (VOCs) in multiple lineages have emerged in several countries, including the United Kingdom (B.1.1.7; Alpha) (Volz et al., 2021), South Africa (B.1.351; Beta) (Tegally et al., 2021), and Brazil (P.1; Gamma) (Faria et al., 2021). All of these VOCs possess the N501Y mutation in RBD, probably contributing to the enhanced transmissibility by increased ACE2 binding (Andreano et al., 2020). Beta and Gamma VOCs share two additional RBD mutations in K417 and E484 that contribute to the antigenic drift and escape from therapeutic monoclonal antibodies and human serum antibodies from convalescent individuals or vaccinees (Garcia-Beltran et al., 2021;Supasa et al., 2021;Wang et al., 2021b;Wibmer et al., 2021;Zhou et al., 2021). Furthermore, circulation of SARSrelated viruses in animal reservoirs pose continuing concerns as the next pandemic potential (Hu et al., 2017). At least two SARSrelated CoVs have the ability to infect primary human airway cells (Menachery et al., 2015(Menachery et al., , 2016. Therefore, isolation of broad neutralizing antibodies against SARS-related CoVs in animal reservoirs and drifted CoV2 in humans advances the development of vaccines and therapeutics against CoVs. In the present study, we have identified a highly potent CoV2neutralizing antibody, named , with the breadth to CoV1 and resistance to CoV2 escape mutations. Structures of the antibody bound to monomeric RBD revealed the distinct binding mode of this RBS antibody, by which the antibody mimics the binding angle of ACE2. Furthermore, the sites of vulnerability in CoV2 and other SARS-related CoVs were unveiled by crystallography as the NT-193 target. CoV1 cross-neutralizing activity of this antibody depended highly on the avidity effect of an immunoglobulin (Ig) G subclass, and robust cross-neutralizing activity was achieved by IgG3 switching. Given the V gene usage of near germline, this distinct class of antibody is a promising target for broad neutralizing vaccines.
RBS epitopes can be subdivided into either class 1 and class 2 ( Barnes et al., 2020) or RBS-A, RBS-B, and RBS-C (Yuan et al., 2021). In addition, there are non-RBS epitopes within RBD, (A) Schematic of the experimental workflow for isolating RBD monoclonal antibodies from TC-mAb mice. CoV2 spike (S) binding monoclonal antibodies were established by a single-cell culture approach from immune mice that express human antibodies.
(B) Binding of monoclonal antibodies in culture supernatant to the indicated S and RBD proteins was evaluated by ELISA. CoV2-neutralizing activity (CoV2 NT) in culture supernatant was assessed using a pseudovirus system. Two clones (#108, NT-108; #193, NT-193) exhibited neutralizing activity. The representative data from two independent experiments are shown. (C) V H /V k gene usage and somatic hypermutations in  (D) Amounts of CoV2 RBD bound to hACE2-coated plates were assessed after preincubation with serially diluted NT-108/NT-193. The antibody-mediated inhibition on hACE2-RBD binding is plotted as the percentage.
(E) After masking RBD-bound plates with competitive antibodies of the indicated class, NT-108 and NT-193 was loaded to assess the binding to unmasked RBD. (F and G) Neutralizing activity against CoV2 was assessed using pseudovirus (F) and authentic virus (G).
Values represent mean ± SD (F and G). The representative data from three independent experiments are shown (D-G). See also Figure S1. denoted as either class 3 and class 4 or S309-proteoglycan and CR3022-cryptic sites. Antibodies against the class 4 epitope are generally less potent than those against other classes of epitopes (Yuan et al., 2020a); therefore, we depriotized the antibodies against this epitope class from our initial screening. Recombinant human IgG1/kappa antibodies were expressed based on the NT-108 and NT-193 V H /V L sequences. We chose REGN10933 (isolate from a humanized mouse) and REGN10987 (isolate from convalescent individuals) as the benchmark of clinical-stage antibodies (Baum et al., 2020b), because REGN10933 exhibits the most potent neutralization activity among clinical-stage antibodies in vitro (Rappazzo et al., 2021) and the antibody cocktail is effective for therapeutic usage (Baum et al., 2020a;Weinreich et al., 2021). In addition, a S309 antibody, another clinical-stage antibody against the class 3 epitope, was expressed as a representative of CoV1/CoV2 cross-neutralizing antibody . Both NT-108 and NT-193 IgG1 inhibited RBD binding to human ACE2 (Figure 1D), suggesting their epitopes overlap RBS. We performed competitive ELISA in which the RBD class 1-3 epitopes were masked by a representative antibody of each class ( Figure 1E). Strong competition by a class 2 antibody (C002) confirmed that both NT-108 and NT-193 are RBS antibodies against the class 2 epitope. Additional competition by a class 1 antibody (REGN10933) suggested that NT-193 recognizes an epitope encompassing class 1 and 2. Moreover, NT-108 and NT-193 likely recognize partially overlapping epitopes, because they compete with each other. Both antibodies were highly potent neutralizers of CoV2-pseudotyped and CoV2-authentic viruses, with a halfmaximal inhibitory concentration (IC 50 ) as potent as that for the REGN antibodies (IC 50 < 100 ng/mL) ( Figures 1F and 1G). Thus, we identified highly neutralizing RBS antibodies with unconventional V H gene usage.
NT-193-neutralizing activity depends on the IgG subclass Only 24 ± 9 S trimers are present on the surfaces of individual CoV2 virions . Such a low density of RBS epitopes may interfere with bivalent binding by antibodies required for inter-RBS crosslinking, as previously observed in HIV-1 (Galimidi et al., 2015). For HIV-1, elevated neutralization was achieved by Env binding antibodies in the context of the IgG3 subclass, which bears a long and flexible hinge region (Richardson et al., 2019;Scharf et al., 2001). To assess the possible contribution of the IgG3-mediated avidity effect on CoV2 neutralization, we first compared the RBD binding affinities of NT-108 and NT-193 in the context of IgG1, IgG3, and Fab by surface plasmon resonance (SPR) analysis. Irrespective of the IgG subclasses, NT-193 bound to both CoV2 and CoV1 RBD at high affinity (apparent affinities of approximately 10 À10$À11 M) ( Figure 2A). However, the RBD binding affinities of NT-193 Fab dropped to less than 1/100-fold of NT-193 IgG antibodies (Figure 2A). The reduction of NT-108 affinity was modest (2-to 4fold decrease) in the same condition, underscoring the large contribution of the avidity effect on the NT-193 IgG affinities. Therefore, the possible impacts of avidity effects on the breadth and neutralizing potency were assessed using  The avidity difference between the IgG1 and the IgG3 subclasses could be underestimated by extremely high-affinity bind-ing under SPR analysis. Moreover, to mimic the low density of RBS epitopes on CoV2 virions, ELISA plates were coated at a non-saturating concentration and then NT-193 of the IgG1 and IgG3 subclasses were added to the plates to assess their binding avidity ( Figure 2B). Subclass-dependent differences were not observed in the binding curve under the saturating concentration of RBD, but increased amounts of IgG3 antibodies bound to a non-saturating concentration of RBD compared with the IgG1 subclass. Furthermore, the IgG3-dependent enhancement correlated with a modest but noticeable increase in neutralizing activity against CoV2-pseudotyped and CoV2-authentic viruses ( Figures 2C and 2D). The IgG3-dependent enhancement was further assessed using the D614G variant, which is considered to express S trimers more abundantly than the Wuhan strain by reduced shedding of the S1 . The variant also expresses RBD with one-open conformation at elevated frequency (Weissman et al., 2021). The usage of this variant exaggerated the IgG3-dependent enhancement ( Figure 2D), suggesting that the phenomenon depends on the density of S trimers and/or the frequencies of open conformation. The enhancement was also noticeable from REGN10933, whose epitope is masked in closed RBD conformation ( Figure 2D).
A distinct feature of NT-193 is its cross-reactivity with CoV1 ( Figures 1B and 2A). Although the cross-reactivity was not extended to other CoVs (MERS-CoV and four seasonal CoVs) ( Figure S2), the breadth to CoV1 in sarbecoviruses is a meaningful and important character, given the circulation of zoonotic CoV1-related viruses, including WIV-1, which has the ability to infect human airway cells (Hu et al., 2017;Menachery et al., 2015Menachery et al., , 2016. Therefore, the neutralizing activity of NT-193 against CoV1 and WIV-1 was examined in both IgG1 and IgG3 subclasses ( Figures 2E and 2F). First, we observed that NT-193 IgG3 potently neutralized these CoV1-related viruses at low IC 50 (<100 ng/mL). The potency and breadth of neutralization to the CoV1-related viruses and CoV2 are noteworthy, because the neutralizing activity against CoV1, CoV2, or both is generally weaker (IC 50 > 100 ng/mL) in the previously isolated, crossneutralizing antibodies (Brouwer et al., 2020;Hansen et al., 2020;Zost et al., 2020). Furthermore, potent cross-neutralizing activity was achieved by non-aggregated forms of the IgG3 subclass ( Figure 2E). No IgG3-dependent enhancement of the neutralization activity in a class 3 RBD antibody (S309) suggests that the phenomenon is not common in RBD antibodies but depends on the epitopes. Indeed, the class 3 epitope can be recognized by the S309 antibody independently of open and closed conformation , and the binding character is distinct from that of REGN10933 (class 1) with IgG3-dependent enhancement activity ( Figure 2D). Given IgG3-dependent enhancement in NT-193 activity, this antibody may recognize the epitope that is exposed under open RBD conformation.

Structure of the NT-193 Fab-RBD complex reveals a distinct binding mode
To gain the structural information on potent cross-neutralization activity by  and enhancement by the IgG3 subclass, we performed X-ray crystallographic analysis. Crystals of the complex of the RBD protein with NT-193 Fab were successfully obtained. The structure of the complex was determined at a resolution of 2.8 Å (Table S1). NT-193 recognizes the top areas of RBD, which widely overlap the binding site of ACE2 ( Figure 3A), but not those of S309 (class 3) and CR3022 (class 4) antibodies ( Figure S3A). Class 1/RBS-A and class 2/RBS-B antibodies also recognize the top areas of RBD Yuan et al., 2021); however, the recognition mode of NT-193 is clearly distinct from the benchmark antibodies B38 (class 1) and COVA2-39 (class 2) ( Figures 3B and S3B). The NT-193 antibody uses not only complementarity determining region (CDR)-1 and CDR-3 of the light chain (hereafter designated as CDR-Ls) but also the framework region, the DE loop, for recognition of the ACE2 binding site (Figures 3C and 3D; Table S2). Somatic hypermutations substituted six amino acids in the light chain, among which positions 31 and 66 with Ser to Gly mutations were located at the interface with RBD. These Ser to Gly substitutions are un-  likely to cause steric hindrance or unsuitable electrostatic complementarity (Figures S4A and S4B), suggesting little, if any, contribution of somatic hypermutations on NT-193 binding. However, the protruding CDR-3 of the heavy chain (hereafter designated as CDR-Hs), together with only small portions of CDR-H1 and CDR-H2 of the heavy chain, are responsible for binding to the CoV1/ CoV2 conserved area next to the ACE2 binding site ( Figures 3E-3G, S4C, and S5). This site is included in the cryptic epitope that faces inward to the closed prefusion trimer, presumably restricting NT-193 access to RBD of a closed/ down configuration. Given the predominance ($55%) of one-open RBD trimers , this raises the possibility that a longer hinge in the IgG3 subclass may enhance inter-S-trimer cross-linking by the avidity effect for achieving higher neutralizing activity. Furthermore, these structural features result in an atypical binding mode that permits broad coverage of ACE2 binding sites and potent cross-reactivity by avidity effect with minimum footprints (Figures 3F, 3G, and S5).
Looking in more detail at the binding interfaces of the CDR regions, F94 of CDR-L3 forms a p-p interaction with Y449 of RBD ( Figure 4A). Furthermore, Y505 of RBD is surrounded by a hydrophobic patch that includes W97 and L95 of CDR-L3, as well as Y33 of CDR-H1 ( Figure 4B). In contrast, Y33 of CDR-L1 makes a hydrophobic interaction with L455 and polar interactions with K417 and Y453 ( Figure 4C). The main chain of the (legend continued on next page) ll OPEN ACCESS Article DE loop of light chain forms hydrogen bonds with RBD (N487) and is sandwiched between Y489 and F486 ( Figure 4D). Numerous hydrophobic interactions were observed in the CDR-H3 heavy chain, including those formed by three continuous tyrosines, namely, Y103, Y104, and Y105 ( Figures 4E  and 4F), which recognize the CoV1/2 conserved region. Y104 protrudes deeply to establish p-cation interactions with K417 and R403 of RBD and hydrogen bonds with E406 and Q409 ( Figure 4F). However, N58 and S56 of CDR-H2 recognize the main-chain N atoms of G502 and G504 of the RBD loop (Figure 4G). In addition, N52 of CDR-H2 and Y33 of CDR-H1 make hydrogen bonds with D405 ( Figure 4G). Finally, the CoV2 RBD epitopes recognized by the NT-193 light chain encompass most of the ACE2 binding site (the 595 Å 2 surface area is buried), whereas the CDR-H3 of its heavy chain further mediates NT-193 anchoring into the CoV-1/2 conserved area, presumably relevant for the formation of the closed prefusion trimer, with the 498 Å 2 surface area buried by heavy chain (Figure S4C). This binding mode is in stark contrast to the representative class 1 antibodies B38 and REGN10933 that mainly rely on heavy chain to cover ACE2 binding sites (Figures 3F and S5). Overall, the previously mentioned binding residues of RBD for interaction with NT-193 are relatively conserved and functionally maintained within clade 1 sarbecoviruses that include clade 1a (CoV1 related) and clade 1b (CoV2 related) ( Figure 4H). With coordinated binding by heavy and light chains, NT-193 is capable of targeting these amino acid residues that are conserved among clade 1 sarbecoviruses, resulting in broad sarbecovirus-neutralizing activity ( Figure 3G).
NT-193 is resistant to escape mutations of SARS-CoV-2 Potential resistance to neutralization escape was evaluated by escape mutation screening in vitro. In this assay, the viruses were passaged up to three times in the VeroE6/TMPRSS2 cell line in the presence of serially diluted IgG1 antibodies (Figure 5A). After passaging three times, we observed cytopathic effects (CPEs) by the viruses that escaped from neutralization by NT-193 IgG1, but not by the NT-108/NT-193 cocktail that was set as the control for suppressing the emergence of escape variants ( Figure 5A). The RBD mutations required for NT-193 escape were determined by viral RNA sequencing from CPE + wells ( Figure 5B). More than 90% of the sequenced viruses carried a single replacement mutation at position 504 within RBD in two separate experiments. The outcome of escape mutation screening largely depends on initial viral stock and culture condition; therefore, we repeated the experiments using a different viral stock and culture condition and examined whether the same escape mutations are reproducibly isolated ( Figure S6A) (Kobayashi et al., 2008). We confirmed that the same G504V mutation emerged as a single RBD mutation using  IgG3 under a different screening condition. Whereas the amino acids next to G504 (N501, G502, and Y505) are directly interacting with ACE2 ( Figure 3F), G504 is not in the ACE binding site and is located at the outer ledge of the NT-193 heavy-chain footprint on the CoV1/CoV2 conserved site ( Figures 3D, 4G, and 5C), likely enabling escape from heavy-chain recognition. The possible impact of the G504V mutation on viral fitness was further assessed using G504V mutant clones, which were confirmed to remain resistant to NT-193 after cloning (Figure S6B). G504V mutants poorly replicated in VeroE6 cell lines ( Figure 5D), and the defect can be at least partly explained by the reduced ACE2 affinity of G504V RBD relative to that of parental RBD ( Figure 5E). Thus, the NT-193-escaping G504V mutation imposed a large cost on viral fitness, thereby losing the competitive advantage over circulating CoV2. This idea is supported by the rare frequency (<0.001%) of G504V mutation among CoV2 sequences in the GISAID database (as of August 3, 2021). CoV2 VOC have recently emerged with potential alternation in pathogenicity, transmissibility, and antigenicity. We thus evaluated the cross-neutralizing activity of NT-193 against these naturally emerged VOC strains (3 from Alpha and 2 from Gamma) ( Figure 5F). The Gamma variants carry the E484K mutation, causing a 10-to 1,000-fold reduction in the neutralization activities of RBS antibodies, including REGN10933 and LY-CoV555, upon monotherapeutic usage (Wang et al., 2021b). In addition, the Gamma variants bear the K417 mutation that permits escape from REGN10933 and LY-COV016 (Baum et al., 2020b;Shi et al., 2020;Starr et al., 2021). Despite these mutations, NT-193 IgG1 neutralized both VOCs even at the lower IC 50 required for neutralizing the original Wuhan strain, whereas REGN10933 reduced neutralizing activity to the Gamma variants below the detection limit (IC 50 > 1 mg/mL) ( Figure 5F). Increases in VOC-neutralizing activities have been previously observed in other antibodies, but the mechanisms remain to be addressed (Hoffmann et al., 2021;Dejnirattisai et al., 2021;Wang et al., 2021aWang et al., , 2021bZhou et al., 2021). Although the Gamma variants remained susceptible by NT-193, the SPR analysis showed that NT-193 Fab bound the RBD variant with Gamma mutations (K417T/E484K/N501Y) at high (K D = 3.0 3 10 À8 M) but reduced affinity compared with the original RBD (K D = 1.2 3 10 À9 M), suggesting the hydrophobic K417-Y104 interaction somewhat contributes to NT-193 binding. These data underscore the resistance of NT-193 to emerging  See also Figures S4 and S5 and Tables S1 and S2.

OPEN ACCESS
Article mutations that allow viral escape from previously isolated RBS antibodies in the clinical stage.

Prophylactic and therapeutic use of NT-193 protects against SARS-CoV-2 infection in hamsters
The Syrian hamster model has been used to assess prophylactic and therapeutic activities of several monoclonal antibodies against CoV2 infection (Baum et al., 2020a;Rogers et al., 2020). This model recapitulates non-lethal but severe forms of CoV2 infection with pneumonia and body weight loss of infected animals (Imai et al., 2020). Using this Syrian hamster model, we examined the prophylactic and therapeutic efficacy of NT-193 monotherapy. Previously isolated monoclonal antibodies provided prophylactic or therapeutic effects when given a >0.25-0.5 mg/kg dosage or a >4 mg/kg dosage, respectively (Andreano et al., 2021;Baum et al., 2020a;Rogers et al., 2020). In this study, the hamsters were treated with 5 or 1.25 mg/kg of IgG3 antibodies at 2 h before (prophylaxis) or 1 day after (therapy) intranasal challenge with CoV2 (Wuhan strain) ( Figure 6A). Prophylaxis with  Values represent mean ± SD. The representative data from three independent experiments are shown (E and F). See also Figure S6. monotherapy prevented severe body weight loss in the hamsters until day 6 after infection ( Figure 6B). The prophylactic effects were more pronounced in the 5 mg/ kg group than in the 1.25 mg/kg group, and the effects became evident as early as 2 days after infection. The difference in body weight between treated and untreated groups further widened at later time points, resulting in reduced viral genomes and antigens in the lungs at day 6 after infection ( Figure 6C). Similar to prophylaxis, all therapeutic treatments, including the groups at the 1.25 mg/kg dosage, prevented body weight loss in hamsters by day 6 after infection (Figure 6D) and reduced the viral genomes and antigens detected in the lungs ( Figure 6E). Although therapeutic efficacy was slightly attenuated compared with prophylactic efficacy, the efficacy of NT-193 IgG3 at the 1.25 mg/kg dosage was comparable to that provided by REGN10933 IgG3 of the same dosage, which is one of the most potent neutralizing and protective antibodies to the original CoV2 strain (Figures 6F and 6G) (Baum et al., 2020b). In addition, despite better neutralizing activity of the IgG3 subclass in vitro, therapeutic efficacies of the IgG3 and IgG1 subclasses were equivalent under the challenge condition that induced more profound weight loss (<85% of initial body weight) than previous studies (Baum et al., 2020a;Imai et al., 2020;Rogers et al., 2020). Dashed lines indicate the humane endpoint (75% weight change). The combined data from more than two independent experiments (n = 5-12 per group) are shown. Statistical significance is indicated above the columns; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 (two-tailed Mann-Whitney test).

DISCUSSION
We showed here a RBS antibody that acquired neutralization potency and breadth for SARS-related coronaviruses, including emerging CoV2 VOCs. Several lines of evidence demonstrated that this antibody acquired broad neutralization activity by two strategies. The first strategy is coordinated antigen recognition by heavy and light chains. Cross-reactivity is mainly acquired through recognition of conserved sites by heavy chains, and neutralization potency is achieved through broad masking of the RBS by light chains. Such functional compartmentalization in heavy and light chains probably permits the breadth and potent neutralization by a single antibody clone, despite these characters being incompatible in most neutralizing RBS antibodies. X-ray crystallography revealed that contact residues for high-affinity binding by NT-193 are relatively conserved in clade 1b (CoV2 related) and clade 1a (CoV1 related) sarbecoviruses, probably because of the necessity for formation of a closed prefusion trimer and/or ACE2 binding. Such a conformational constraint likely makes the key footprints of NT-193 the vulnerable sites for emerging CoV2 variants and circulating CoV1-related viruses in animal reservoirs as the next pandemic potential (Hu et al., 2017). The second strategy of NT-193 for acquiring robust crossneutralization activity is the avidity effect by IgG3 switching. The avidity effect increased CoV1 neutralization activity solely by switching from IgG1 to IgG3, despite the similarity of apparent Fab affinities to RBD. We speculate that IgG3 switching potentiates neutralization activity of NT-193 through the long and flexible hinge of the IgG3 subclass, as is the case for HIV-1 antibodies (Richardson et al., 2019;Scharf et al., 2001). Supporting this, we observed that increased amounts of NT-193 IgG3 bound to RBD antigens that were sparsely coated on plates. In addition, NT-193 recognizes a cryptic epitope that is only accessible by antibody under the open configuration of RBD. Transition between open and closed configurations of RBD generates heterogenous S-trimer structures with different combinations of open and closed RBD conformations. During the steady state, CoV1 S trimers have higher frequencies of open configuration compared with CoV2 S trimers (Walls et al., 2019). In addition, the numbers of S trimers present on the individual virion are different between CoV2 (24 ± 9 per virion)  and CoV1 (65 per virion) (Beniac et al., 2006). Therefore, the difference in the open/closed RBD conformation and S-trimer density between CoV1 and CoV2 could affect inter-and/or intra-S-trimer cross-linking by  In vitro escape mutation screening identified G504 as another key footprint of  Recently, the in vitro evolution approach with random mutagenesis and selection by yeast display libraries was applied to generate an anti-SARS-related virusneutralizing antibody, ADG-2 (Rappazzo et al., 2021). This engineered process elevated the potency and breadth against SARS-related virus neutralization, and coincidently, the binding mode of this antibody depended on G504. G504V mutation is infrequently detected (<0.001%) among CoV2 sequences deposited in the GISAID database. Together with attenuated ACE2 binding and replication of G504V mutants, G504 mutations likely lose the competitive advantage over circulating CoV2. The low-resolution cryoelectron microscopy (cryo-EM) structure showed that ADG-2 recognizes only a small portion of the ACE2 binding site. Therefore, although NT-193 and ADG-2 share G504 as the key footprint, it is important to stress that the NT-193 binding mode differs from that of ADG-2, including coordinated binding by heavy and light chains and overall ACE2 mimicry for the binding angle. Furthermore, NT-193 uses the near-germline heavy chain, making NT-193-like antibodies more attractive targets of vaccines, because poorly mutated antibodies can be elicited by immunization with less labor and time than heavily mutated antibodies. In addition, NT-193 may leave more room for potentiating the neutralizing activity by introducing additional affinity-enhancing mutations during affinity-based maturation of humoral immune responses.
NT-193 was originally isolated from humanized mice, but the NT-193-like antibodies sharing structural and functional characters are potentially elicited in COVID-19 patients by following reasons. First, IgG3 antibodies are elicited in severe COVID-19 patients during the acute phase (Amanat et al., 2020;Zohar et al., 2020). Second, the humoral response in severe patients is characterized by the robust extrafollicular responses that harbor IGVH4-34-encoded antibodies at elevated frequencies (Woodruff et al., 2020). Third, IGHV4-34-encoded monoclonal antibodies with CoV1/CoV2 cross-reactivity were recovered from COVID-19 patients and deposited in the database (Raybould et al., 2021). Even though IGHV4-34-encoded antibodies are present in extrafollicular response in COVID-19 patients, infrequent recovery of the similar antibodies from memory B cell repertoires suggests that IGHV4-34-encoded antibodies are less competitive than those with convergent V H genes that are frequently recovered from memory repertoires across multiple individuals (Andreano et al., 2021;Robbiani et al., 2020;Yuan et al., 2020b). Rational design of vaccine formulations and adjuvants may be required to overcome such possible roadblocks that prevent IGHV4-34-encoded antibodies from recruitment into memory B cell repertoires.
Neutralizing RBS antibodies are frequently encoded by convergent V H genes that share similar binding modes and footprints (Robbiani et al., 2020;Yuan et al., 2020b). Such focused antibody responses could increase the risk of generating antibody escape mutations because of limited diversity. Indeed, persistent infection in an immunocompromised COVID-19 patient generated several variants, including the one with the E484K mutation (Choi et al., 2020), allowing escape from convergent neutralizing antibodies (Gaebler et al., 2021). The emerging VOCs possess the E484K mutation (Tegally et al., 2021), raising the concern of viral escapes from vaccine-induced immunity and therapeutic antibodies. Therefore, countermeasures to boost resistance to escape mutations need to be prepared. We believe that NT-193 is one of the candidates possessing such activities via its distinct binding mode that is not used by convergent antibodies. Further analysis of NT-193 will guide the development of broad protective interventions against emerging CoV2 variants and SARS-related viruses with pandemic potential.

LIMITATIONS OF THE STUDY
A limitation of this study is that there was no apparent difference in the therapeutic activities of NT-193 between the IgG1 and the IgG3 subclasses against the original CoV2 strain. This point needs to be further investigated using either CoV1 or WIV-1 for challenge, because more robust enhancement of neutralization was observed by using these viruses. In addition, it is important to assess the therapeutic activities under the VOC-challenged model. Optimal therapeutic protection by  in vivo could be regulated by the IgG Fc effector functions, in addition to the neutralization activity in vitro , but this point remains to be clarified.

STAR+METHODS
Detailed methods are provided in the online version of this paper and include the following:

ACKNOWLEDGMENTS
We thank Akira Dosaka, Eriko Izumiyama, and Ryoko Itami at NIID for technical support. We also thank the beamline staff at Photon Factory and Swiss Light Source for supporting data collection. This work was supported by Japan

DECLARATION OF INTERESTS
A.S. is an employee of Shionogi & Co., Ltd. M.O. is a CEO, employee, and shareholder of Trans Chromosomics, Inc. These authors acknowledge a potential conflict of interest and attest that the work contained in this report is free of any bias that might be associated with the commercial goals of the company. T.O., Y. Adachi, M.O., T.H., K.M., and Y.T. declare that an intellectual property application has been filed using the data presented in this paper. The other authors declare that they have no competing interests.

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Pinto (Continued on next page) Japan). CoV2 antigens were detected using a standard immunoperoxidase method, and a rabbit anti-CoV2 N antibody was used as the primary antibody (Nagata et al., 2021). Nuclei were counterstained with hematoxylin for 10 s. Scores were determined based on the percentage of N antigen-positive cells, as determined by immunohistochemistry in each group using the following scoring system: 0, no antigen positive cells; 1, antigen positive cells were occasionally observed in each cut sections (1-3 antigen-positive areas per section were observed in the high magnification); 2, scattered positive cells were observed (4-9 antigen-positive areas per section were observed in the high magnification); 3, many positive cells were diffusely and widely observed (more than 10 antigen-positive areas per section were observed in the low magnification). Mean scores from all lung sections (5-10 lung sections/animal) in each animal were calculated.

Study approval
Animal procedures were approved by the Animal Ethics Committee of the National Institute of Infectious Diseases, Japan, and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee. In vitro escape mutation screening experiments for CoV2 were performed at the Biosafety Level-3 facility of the Research Center for Zoonosis Control, Hokkaido University, and the National Institute of Infectious Diseases following the institutional guidelines.

QUANTIFICATION AND STATISTICAL ANALYSIS
Statistical analysis for virus titers in culture, body weight change and viral antigens in hamsters are described in the relevant figure legends. Each dot represents the numbers from individual experiment (virus titers) or the result from individual hamster. Values represent mean ± SD. Data were analyzed using Prism (GraphPad) by a two-tailed Mann-Whitney test. Significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Further statistical details for each plot can be found in the corresponding figure legends.