Structural basis of nectin-4 recognition by the antibody–drug conjugate 9MW2821

Abstract

Nectin-4, a membrane protein highly expressed in multiple solid tumors, has become an attractive target for antibody–drug conjugate (ADC) development. We designed and developed 9MW2821, an anti-nectin-4 ADC with an enzymatically cleavable valine–citrulline linker and monomethyl auristatin E as the payload. Although 9MW2821 has shown good efficacy and safety in multiple solid tumors in clinical trials, the interaction between 9MW2821 and nectin-4 at the molecular level remains unclear. In this study, we solved the structure of the antigen-binding fragment (Fab) of 9MW2821 in complex with nectin-4 at 3.26-Å resolution using single-particle cryo-EM. The structure shows that 9MW2821 binds the front β-sheet of nectin-4 D1 through three complementarity-determining region (CDR) loops from the heavy chain and two CDR loops from the light chain. The binding involves extensive hydrogen bonds and hydrophobic interactions. The buried surface area is more than 1600 Ų. Mutagenesis studies revealed that four residues (Q77, E78, H83, and E95) of nectin-4 contributed significantly to the binding of 9MW2821 mAb. The structure also shows that 9MW2821 mAb blocks nectin-4 homodimer formation by competing with the nectin-4 partner D1 domain for part of the nectin-4 surface area. Furthermore, 9MW2821 mAb prevents nectin-4 from interacting with nectin-1 and T-cell immunoglobulin and ITIM domain (TIGIT), enhancing the activation of NK cells. Based on the structure of nectin-4 D1/9MW2821 Fab complex resolved in this study, we have elucidated the molecular mechanism of 9MW2821 in cancer therapy. The findings provide a basis for optimizing future monoclonal antibodies targeting nectin-4.