The therapeutic antibody sacituzumab induces trophoblast cell-surface antigen-2 conformational rearrangement.

Sacituzumab govitecan (SG) is a therapeutic antibody-drug conjugate globally approved for the treatment of breast cancer. SG targets the trophoblast cell-surface antigen-2 (Trop2) at the surface of cancer cells to deliver the cytotoxic topoisomerase I inhibitor SN-38 to the tumor microenvironment. SN-38 is covalently linked to the humanized monoclonal antibody (mAb) sacituzumab via a hydrolyzable linker. Here, we describe the 1.56-Å X-ray crystal structure and stoichiometry of the human Trop2 ectodomain in complex with a sacituzumab (hRS7) antigen-binding Fab fragment. The complex reveals a 2:2 stoichiometry where two sacituzumab Fabs bind across the two Trop2 dimer subunits, inducing a conformational change compared to the apo-structure. Cryo-electron microscopy (cryoEM) and size-exclusion chromatography in combination with multi-angle light scattering (SEC-MALS) analysis of the intact sacituzumab mAb bound to the Trop2 ECD reveals a complex whereby sacituzumab engages two Trop2 dimers in a 2:4 stoichiometry.