The ongoing COVID-19 pandemic, caused by SARS-CoV-2, represents a significant global health crisis. While convalescent antibodies and several vaccines have shown efficacy against the original SARS-CoV-2 strain, the emergence of numerous variants poses a significant challenge. These variants exhibit mutations that allow them to escape neutralization by some potent antibodies and reduce the effectiveness of existing vaccines and antibody treatments. For instance, vaccines like Moderna's mRNA-1273, Pfizer's BNT162b2, and Novavax's NVX-CoV2373 demonstrate reduced protection against certain epidemic variants compared to the original strain. Therefore, the development of broadly protective vaccines and therapies capable of neutralizing both existing and future variants is crucial. Broadly neutralizing antibodies could serve as both therapeutics and prophylactics, and their identification can guide the design of pan-sarbecovirus vaccines by highlighting highly conserved antigenic determinants. The viral spike glycoprotein plays a central role in SARS-CoV-2 transmission, mediating viral entry into host cells. Both SARS-CoV-2 and SARS-CoV utilize the angiotensin-converting enzyme 2 (ACE2) receptor for entry, with the receptor-binding domain (RBD) of the spike protein mediating this interaction. Many previously identified neutralizing antibodies target the receptor-binding motif (RBM) within the RBD, blocking ACE2 binding. However, these antibodies often exhibit limited breadth of neutralization or fail to neutralize variant strains. Two classes of cross-neutralizing antibodies, CR3022 and S309, have been identified, recognizing conserved epitopes outside the RBM. However, S309's neutralization is impaired by mutations present in some variants, while CR3022's neutralization of these variants is yet to be fully determined. This highlights the need for antibodies targeting highly conserved, variant-resistant epitopes.

Numerous studies have explored the development and characterization of neutralizing antibodies against SARS-CoV-2. Early work focused on antibodies derived from convalescent patients, demonstrating their efficacy in treating COVID-19. These studies provided valuable insights into the mechanisms of antibody neutralization, identifying epitopes within the RBD that are crucial for viral entry. The structures of several potent neutralizing antibodies have been elucidated, revealing their binding modes and interactions with the viral spike protein. However, a limitation of many of these antibodies is their limited cross-reactivity and susceptibility to escape mutations arising in circulating variants. Studies exploring cross-neutralizing antibodies, such as those targeting epitopes outside the main receptor-binding motif, have offered promising avenues for broader protection. These studies have highlighted the need for a multi-pronged approach involving antibodies with different neutralization mechanisms and epitopes to tackle the ongoing challenge of variant emergence.

To identify cross-neutralizing antibodies, the researchers employed an immunization strategy involving mice. Two immunization approaches were used: (1) immunization with the SARS-CoV-2 spike protein alone, and (2) combined immunization with SARS-CoV-2 spike, SARS-CoV spike, and MERS-CoV RBD. Hybridomas were generated and screened for antibodies exhibiting cross-reactivity against both SARS-CoV-2 and SARS-CoV. The neutralization potency of the resulting monoclonal antibodies (mAbs) was evaluated using lentiviral and vesicular stomatitis virus (VSV) pseudotyping systems, as well as with authentic SARS-CoV-2 virus. Surface plasmon resonance (SPR) analysis determined the binding affinities of the antibodies to both SARS-CoV-2 and SARS-CoV RBD and spike trimers. Blocking assays assessed the antibodies' ability to inhibit ACE2-RBD binding. High-resolution crystal structures of Fab-RBD complexes were determined to elucidate the binding mechanisms. The antibodies' resistance to mutations in circulating SARS-CoV-2 variants was assessed using both pseudotyped viruses and authentic virus. Finally, cryo-electron microscopy (cryo-EM) and high-performance liquid chromatography (HPLC) were used to investigate the mechanism of neutralization by the identified antibodies. Detailed protocols for hybridoma generation, protein expression and purification, neutralization assays, SPR analysis, crystallography, cryo-EM, and HPLC are provided in the supplementary information.

The study successfully identified two potent cross-neutralizing antibodies, 7D6 and 6D6. These antibodies demonstrated sub-picomolar affinities for the SARS-CoV-2 RBD and effectively neutralized authentic SARS-CoV-2 virus. Structural analysis revealed that both 7D6 and 6D6 bind to a cryptic site on the RBD, distinct from the RBM and other known antibody binding sites. This cryptic epitope is highly conserved across various Sarbecovirus isolates. Notably, binding of 7D6 and 6D6 to the RBD clashes sterically with the adjacent N-terminal domain (NTD) of the spike protein. This steric clash likely contributes to their neutralizing mechanism, potentially by inducing spike destabilization and shedding of the S1 subunit. Functional studies confirmed that 7D6 and 6D6 displayed robust neutralization activity against various circulating SARS-CoV-2 variants, including Alpha (B.1.1.7), Beta (B.1.351), and Gamma (P.1). In contrast, a control antibody, REGN10933, exhibited significantly reduced activity against Beta and Gamma variants. The identified antibodies were classified into Class 1 based on their binding location and mode of action, distinct from other classes of antibodies that target the RBM or other epitopes on the RBD. Cryo-EM studies confirmed that 7D6 and 6D6 binding leads to the dissociation of the spike trimer into smaller components. Furthermore, an in vitro shedding assay indicated that these antibodies induce S1 shedding from the spike protein.

The identification of 7D6 and 6D6, which target a highly conserved cryptic site on the RBD and exhibit resistance to mutations in circulating SARS-CoV-2 variants, addresses the critical need for broadly neutralizing antibodies. The unique binding mechanism involving steric clash with the NTD and potential S1 shedding represents a novel approach to neutralizing SARS-CoV-2. This contrasts with other known antibodies which primarily neutralize by blocking ACE2 binding. The finding that these antibodies demonstrate potent neutralization of various circulating variants underscores their therapeutic potential. Further development of these antibodies, including humanization and affinity maturation, could pave the way for effective therapeutic antibody cocktails, or as adjuncts to existing cocktails, against COVID-19. The conserved nature of the identified epitope also makes it an attractive target for the development of pan-sarbecovirus vaccines. Future work should focus on optimizing these antibodies for clinical translation and exploring their prophylactic efficacy in pre-clinical animal models.

This study successfully identified two potent cross-neutralizing antibodies, 7D6 and 6D6, that target a conserved cryptic site on the SARS-CoV-2 RBD. These antibodies display potent neutralization activity against various SARS-CoV-2 variants, and their unique mechanism involving steric hindrance and potential S1 shedding offers a promising approach for COVID-19 treatment and prevention. This discovery holds significant implications for developing effective therapeutic antibodies and broadly protective vaccines.

The study primarily focuses on *in vitro* experiments. Further *in vivo* studies are necessary to validate the protective efficacy of these antibodies. While the antibodies showed resistance to mutations present in currently circulating variants, the emergence of future variants with mutations within the 7D6/6D6 epitope cannot be entirely ruled out. The mechanistic understanding of spike destabilization and S1 shedding requires further investigation, potentially using advanced structural biology techniques. Finally, the study was conducted in mice, and further investigation is needed to assess the efficacy and safety of these antibodies in humans.