Structure-guided bispecific antibody engineering confers broad protection against KP.3.1.1 and sarbecoviruses

The emergence of SARS-CoV-2 variants like KP.2 and KP.3.1.1 challenges COVID-19 countermeasures. Bispecific antibodies (bsAbs) targeting two antigens show promise, but the impact of scFv sequence arrangement on functionality remains unclear. Using neutralizing mAbs 7F3 (Omicron XBB.1-specific) and GW01 (cross-neutralizing), we engineered four bsAbs with diverse scFv configurations. Despite similar RBD binding affinities, only G7-Fc neutralized all 22 tested pseudoviruses, including KP.2, with 18-fold enhanced potency over S309. Cryo-EM revealed that G7-Fc uniquely stabilizes a BA.2.86 spike trimer-dimer apex-to-apex configuration, blocking RBD-ACE2 interaction via two conserved epitopes distinct from S309. Structure-guided A227S/T and Y229N mutations restored G7-Fc activity against KP.3.1.1. In hACE2-transgenic mice, G7-Fc demonstrated robust prophylactic and therapeutic efficacy against EG.5. This study systematically evaluates how scFv positioning affects bsAb performance, demonstrating rapid antibody optimization through structural insights. G7-Fc's unique trimer-dimer engagement and conserved epitope targeting provide a framework for designing potent, variant-resistant bsAbs against emerging SARS-CoV-2 strains.