Novel Trispecific Neutralizing Antibodies With Enhanced Potency and Breadth Against Pan‐Sarbecoviruses

The ongoing emergence of new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) underscores the urgent need for developing antivirals targeting both SARS-CoV-2 variants and related sarbecoviruses. To this end, we designed novel trispecific antibodies, Tri-1 and Tri-2, engineered by fusing the single-chain variable fragments (scFvs) of a potent antibody (PW5-570) to the Fc region of "Knob-into-Hole" bispecific antibodies (bsAbs) composed of two distinct broad antibodies (PW5-5 and PW5-535). Compared with the parental antibodies, Tri-1 and Tri-2 displayed enhanced binding affinities to the receptor-binding domains of SARS-CoV, SARS-CoV-2 wild type, and Omicron XBB.1.16, with each arm contributed to the overall enhancement. Furthermore, pseudovirus neutralization assays revealed that Tri-1 and Tri-2 effectively neutralized all tested SARS-CoV, SARS-CoV-2 variants, and related sarbecoviruses (Pangolin-GD, RaTG13, WIV1, and SHC014), demonstrating potency and breadth superior to any single parental antibody. Consistently, Tri-1 and Tri-2 were found to effectively neutralize authentic SARS-CoV and SARS-CoV-2 variants with IC₅₀ values comparable to or better than those of parental antibodies. Taken together, this study highlights the potential effectiveness of Tri-1 and Tri-2 as novel formats for harnessing cross-neutralizing antibodies in the development of multivalent agents to combat both current and future SARS-like coronaviruses.