S2H97

Members of the Sarbecovirus subgenus of Coronaviridae have twice caused deadly threats to humans. There is increasing concern about the rapid mutation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which has evolved into multiple generations of epidemic variants in 3 years. Broad neutralizing antibodies are of great importance for pandemic preparedness against SARS-CoV-2 variants and divergent zoonotic sarbecoviruses. Here, we analyzed the structural conservation of the receptor-binding domain (RBD) from representative sarbecoviruses and chose S2H97, a previously reported RBD antibody with ideal breadth and resistance to escape, as a template for computational design to enhance the neutralization activity and spectrum. A total of 35 designs were purified for evaluation. The neutralizing activity of a large proportion of these designs against multiple variants was increased from several to hundreds of times. Molecular dynamics simulation suggested that extra interface contacts and enhanced intermolecular interactions between the RBD and the designed antibodies are established. After light and heavy chain reconstitution, AI-1028, with five complementarity determining regions optimized, showed the best neutralizing activity across all tested sarbecoviruses, including SARS-CoV, multiple SARS-CoV-2 variants, and bat-derived viruses. AI-1028 recognized the same cryptic RBD epitope as the parental prototype antibody. In addition to computational design, chemically synthesized nanobody libraries are also a precious resource for rapid antibody development. By applying distinct RBDs as baits for reciprocal screening, we identified two novel nanobodies with broad activities. These findings provide potential pan-sarbecovirus neutralizing drugs and highlight new pathways to rapidly optimize therapeutic candidates when novel SARS-CoV-2 escape variants or new zoonotic coronaviruses emerge. IMPORTANCE The subgenus Sarbecovirus includes human SARS-CoV, SARS-CoV-2, and hundreds of genetically related bat viruses. The continuous evolution of SARS-CoV-2 has led to the striking evasion of neutralizing antibody (NAb) drugs and convalescent plasma. Antibodies with broad activity across sarbecoviruses would be helpful to combat current SARS-CoV-2 mutations and longer term animal virus spillovers. The study of pan-sarbecovirus NAbs described here is significant for the following reasons. First, we established a structure-based computational pipeline to design and optimize NAbs to obtain more potent and broader neutralizing activity across multiple sarbecoviruses. Second, we screened and identified nanobodies from a highly diversified synthetic library with a broad neutralizing spectrum using an elaborate screening strategy. These methodologies provide guidance for the rapid development of antibody therapeutics against emerging pathogens with highly variable characteristics.

An ideal therapeutic anti-SARS-CoV-2 antibody would resist viral escape^1-3, have activity against diverse sarbecoviruses^4-7, and be highly protective through viral neutralization^8-11 and effector functions^12,13. Understanding how these properties relate to each other and vary across epitopes would aid the development of therapeutic antibodies and guide vaccine design. Here we comprehensively characterize escape, breadth and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD). Despite a trade-off between in vitro neutralization potency and breadth of sarbecovirus binding, we identify neutralizing antibodies with exceptional sarbecovirus breadth and a corresponding resistance to SARS-CoV-2 escape. One of these antibodies, S2H97, binds with high affinity across all sarbecovirus clades to a cryptic epitope and prophylactically protects hamsters from viral challenge. Antibodies that target the angiotensin-converting enzyme 2 (ACE2) receptor-binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency. Nevertheless, we also characterize a potent RBM antibody (S2E12⁸) with breadth across sarbecoviruses related to SARS-CoV-2 and a high barrier to viral escape. These data highlight principles underlying variation in escape, breadth and potency among antibodies that target the RBD, and identify epitopes and features to prioritize for therapeutic development against the current and potential future pandemics.