The GPC3-targeting hYP7 antibody-based gamma/delta TCR-T cell therapy for overcoming antigen mutation and heterogeneity in liver cancer

Antigen mutation and heterogeneous expression remain major barriers to effective antibody- and chimeric antigen receptor (CAR)-based immunotherapies for hepatocellular carcinoma (HCC). Glypican-3 (GPC3) exhibits variable surface density and epitope alteration in advanced or recurrent HCC tumors. To address these clinically relevant obstacles, we developed a modular antibody-based gamma/delta T-cell receptor (gammadeltaAbTCR) platform that integrates antibody specificity with intrinsic TCR-CD3 signaling to enhance tumor recognition under conditions of antigen variation. Using two GPC3 antibodies recognizing distinct epitopes (hYP7 and HN3), we generated four gammadeltaAbTCR constructs-hYP7-hYP7, hYP7-HN3, HN3-hYP7, and HN3-HN3-and evaluated their functional properties in primary human T cells. Among these, hYP7-hYP7 gammadeltaAbTCR-T cells demonstrated the strongest antigen binding and cytotoxic activity against Hep3B and other GPC3⁺ HCC models, particularly those with low or heterogeneous antigen density. In vivo, hYP7-hYP7 T cells showed superior tumor infiltration, persistence, and control of large, established xenografts compared with other configurations. Mechanistic studies revealed that the hYP7-hYP7 gammadeltaAbTCR architecture couples enhanced antigen-binding avidity with coordinated TCR-CD3 and CD30 signaling, leading to potent NF-κB and NFAT activation and rapid induction of caspase-mediated apoptosis in tumor cells. These findings establish hYP7-hYP7 as a lead gammadeltaAbTCR design that overcomes the limitations of antigen heterogeneity and mutation in liver cancer, providing a promising framework for next-generation T-cell therapies targeting liver cancer.