Abstact

Most neutralizing antibodies against severe acute respiratory syndrome coronavirus 2 target the receptor-binding domain (RBD). However, due to high immune pressure, the RBD accumulates mutations, thus significantly reduce antibody efficacy against emerging variants/subvariants. Although some RBD-targeting antibodies bind conserved epitopes, they usually exhibit weak-to-moderate neutralization. Similarly, antibodies against the N-terminal domain (NTD) or S2 subunit often retain broad binding but generally lack potent neutralization. To address this, we initially identified the broadly reactive nanobody, N103, which exhibited weak neutralizing potency. Structural and functional analyses revealed that N103 targets a conserved NTD epitope and triggers S1 subunit shedding, thereby destabilizing the spike trimer through an allosteric mechanism. Leveraging this insight, we engineered a trispecific antibody combining N103 with antibodies targeting the conserved RBD and S2 epitopes. This design synergistically integrated their distinct binding profiles and mechanisms, achieving potent and broad neutralization against both pseudoviruses and authentic viruses, including the immune-evasive BA.2.86 subvariant. Furthermore, challenge studies in human angiotensin-converting enzyme 2 knock-in mice demonstrated robust in vivo protection. Our findings highlight a cooperative multi-target strategy in which antibodies with limited individual potency can collectively achieve broad and potent neutralization through rational design. This approach provides a promising framework for next-generation antibody therapeutics.