Chimeric Antigen Receptor T(CAR-T) cells represent a transformative approach in immunotherapy, using engineered T lymphocytes to target cancer cells. The antigen-binding domain of CARs is typically derived from a single-chain variable fragment(scFv), providing target specificity. HER2 is one of the target of CAR-T cells, as HER2 overexpression has been observed in various malignancies, including breast cancer, ovarian cancer, and others. Trastuzumab, a monoclonal antibody targeting HER2, has demonstrated efficacy in the treatment of HER2⁺ cancers by binding to domain IV of the HER2 protein. However, tumors with mutations in the trastuzumab binding site become resistant to trastuzumab, posing a significant therapeutic challenge. This challenge also applies to trastuzumab-derived CAR-T cells(4D5 CAR-T).
To address this issue, we engineered CAR-T cells utilizing distinct nanobody-based antigen-binding domains derived from camelid antibodies. Specifically, we developed CAR-T cells using nanobodies, including a single nanobody (H2H) and tandem constructs combining the scFv 4D5 with different orientations (4D5-H2H and H2H-4D5). The tandem CARs were designed to broaden HER2 recognition, enhance anti-tumor efficacy, and overcome resistance mechanisms associated with mutations in individual binding sites.
Our results show that H2H CAR-T cells exhibit superior cytotoxicity in vitro, maintaining efficacy in trastuzumab-resistant models. In an in vivo assessment, the H2H-4D5 CAR-T cells showed enhanced antitumor effects, reduced exhaustion-associated populations, and an increased percentage of stem-like T cell populations compared to H2H CAR-T cells. These findings highlight the potential of the H2H domain and the H2H-4D5-CAR-T construct as promising therapeutic strategies for targeting HER2⁺tumors, particularly those resistant to existing treatments.