Dual Epitope Engagement Enables Broad-Spectrum Neutralization of SARS-CoV-2 Variants by Bispecific Antibody 9A6-6C3

Given the continuously mutating nature of SARS-CoV-2, the sensitivity of most monoclonal therapeutic antibodies has decreased to the mutants. Bispecific antibodies (BsAbs), with their unique antiviral mechanisms that enable simultaneous binding to two epitopes, offer a distinct advantage in combating the continuous viral mutation by preventing immune escape and enhancing viral neutralization. In this study, we synthesized 67 bsAbs based on the epitope distribution from antibodies isolated using single B-cell cloning from convalescent patients and phage display, 11 of which showed superior neutralization of WA1/2020 compared to their parent antibodies. One bispecific antibody (9A6-6C3), exhibiting 100-fold greater neutralizing activity than its parent antibodies, efficiently neutralized various SARS-CoV-2 variants (IC50 < 100ng/mL). Structural analysis indicates that 9A6 binds to the H-RBD epitope, encountering spatial conflict with the NTD of neighboring S monomer, while 6C3 is capable of binding to a conserved loop on S2. In vitro evidence demonstrates that 9A6-6C3 promotes the disassembly of the S protein, exposing S2, which likely contributes to its broad-spectrum neutralizing activity. In summary, we discovered a potential broad-spectrum mechanism and presented an epitope design strategy for bsAbs, offering valuable insights for the design and development of bsAbs in the fight against COVID-19.