Development of CAR-T Therapy Targeting Two Distinct Epitopes of HER2 for Trastuzumab-Resistant Cancer

Abstract Alterations in the human epidermal growth factor receptor 2 (HER2) epitope recognized by trastuzumab-derived chimeric antigen receptors (CARs) may compromise tumor recognition. To diversify HER2 recognition, we generated CARs incorporating the camelid-derived H2H nanobody, the trastuzumab-derived 4D5 single-chain variable fragment, or both binders in two tandem orientations. We evaluated binding competition, murine and human CAR-T cell cytotoxicity, tumor growth, and tumor-infiltrating CAR-T cell phenotypes. H2H did not compete with 4D5 for HER2 binding, and H2H CAR-T cells retained cytotoxicity against targets expressing an engineered HER2 domain IV deletion variant that was poorly recognized by 4D5 CAR-T cells. In mice, H2H-4D5 suppressed the growth of wild-type HER2-expressing B16 tumors more effectively than the reverse tandem configuration, although survival did not differ significantly among groups. In a separate tumor-infiltrating lymphocyte analysis, H2H-4D5 CAR-T cells exhibited lower PD-1/TIM-3 co-expression and TOX expression than 4D5 CAR-T cells. All four human CAR configurations mediated cytotoxicity against breast cancer cells with endogenous HER2 expression. Human H2H-containing CAR-T cells, including both tandem configurations, also retained cytotoxicity against engineered mutant HER2-expressing tumor cells, whereas 4D5 CAR-T cells failed to eliminate these targets. These findings support complementary epitope recognition as a strategy to maintain HER2-directed CAR-T cell activity following disruption of the 4D5-binding region and identify binder orientation as a determinant of antitumor activity and exhaustion-associated phenotypes in the tested models.