Novel domain-targeted anti-Trop-2 monoclonal antibodies to elicit therapeutic synergy against multiple human cancers

Background: Trop2 is a calcium signal transducer that drives tumor cell growth. Trop2 is overexpressed in the majority of carcinomas, where it associates with worse prognosis. The Trop2 extracellular domain contains a Nterminal cysteinerich globular domain followed by a stemlike cysteineless region that connects to the transmembrane domain. Trop2 engages in homophylic interactions between adjacent cells and interacts with tightjunction proteins, which may severely affect accessibility by therapeutic monoclonal antibodies (mAb). Available antiTrop2 mAb mostly recognize a single immunodominant epitope between the globular and stem regions, and have limited or no therapeutic efficacy. In order to untap the potential of antiTrop2 immunotherapy we generated novel antiTrop2 mAb with tailored specificity towards the globular versus stem regions. Methods: BalbC mice were immunized with soluble human Trop2 produced in human 293 and murine L cell lines and in baculovirus expression system. Domaintargeted antiTrop2 mAb were selected by flow cytometry using live 293 transfectants.Therapeutic potential was assessed in human cancer xenografts in murine models. mAb mode of action was investigated by Western blot, livecell imaging and in†vitro†ADCC and cancer cell growth inhibition assays. Results: mAb were identified that were differentially directed against the Trop2 stem versus globular regions. These mAb efficiently bound Trop2 expressing cancer cells and inhibited cell growth in†vitro. In†vivo¨†naked antiglobular OXG64 and antistem OXS55 mAb were most effective in inhibiting the growth of colon, ovary, breast and prostate cancers as xenografts in nude mice. NSG mice and in†vitro†mechanism profiling indicated efficient ADCC, together with efficient internalization for ADC development. Differential efficacy for established tumors versus isolatedcell models of metastatic dissemination was shown. Most remarkably, OXG64/ OXS55 coadministration demonstrated sinergic growth inhibition in†vivo. Conclusions: The OXG64 and OXS55 antiTrop2 mAb are novel domaintargeted, sinergic therapeutic mAb for gamechanging anticancer immunotherapy.