TCR-like MAGE-A4/HLA-A*02:01 CAR-NK cells enhance antitumor effector function and control melanoma xenografts

Chimeric antigen receptor (CAR)-cell-based therapies have demonstrated remarkable clinical success in hematologic malignancies; however, their application in solid tumors remains limited, largely due to the scarcity of suitable surface antigens. TCR-like CARs offer a promising strategy to overcome this barrier by targeting peptide-HLA complexes derived from intracellular tumor-associated antigens. In this study, we investigated NK cells expressing a TCR-like CAR directed against the MAGE-A4 peptide presented by HLA-A*02:01 on tumor cells. The CAR construct incorporated CD3ζ and GITR intracellular signaling domains and was assessed in both NK-92 cells and primary peripheral blood NK cells. Our results demonstrated that MAGE-A4 CAR expression enhanced the metabolic fitness of NK-92 cells, as evidenced by increased glycolysis, basal respiration, and ATP production. Functionally, CAR-engineered NK cells exhibited significantly enhanced cytotoxicity against MAGE-A4⁺ tumor cell lines (A375, U2OS, and U266), but not against MAGE-A4⁻ targets (HCT116), confirming antigen specificity. Enhanced tumor cell killing was associated with increased CD107a degranulation, elevated granzyme B and perforin expression, and augmented TNF-α and IFN- γ production. In vivo, MAGE-A4 CAR NK-92 cells demonstrated potent antitumor activity in an A375 xenograft model. Consistent with these findings, primary NK cells expressing the MAGE-A4 CAR also displayed enhanced antitumor activity and cytokine secretion in vitro. Collectively, these findings demonstrate that TCR-like targeting of the intracellular tumor antigen MAGE-A4 using CAR-engineered NK cells enhances metabolic fitness, effector function, and antitumor activity, highlighting the potential of this approach as a promising immunotherapeutic strategy for MAGE-A4⁺ solid tumors.