Structure-function profiling identifies determinants of TCR-T cell therapeutic efficacy

Glypican-3 (GPC3) is a promising target in adoptive T-cell therapy for hepatocellular carcinoma (HCC). TCR-A and TCR-B are two recently identified GPC3-specific TCRs recognizing the same HLA-A*02:01-restricted epitope but displaying markedly different therapeutic efficacy. To define the determinants of productive TCR-antigen recognition, we integrated high-resolution structures of both binary pMHC and ternary TCR:pMHC complexes, biolayer interferometry, peptide mutagenesis, target-cell conjugation assays, repetitive antigen challenge (RAC) and in vivo models. Structurally, conformational permissiveness of the immunodominant GPC3(522-530) peptide within HLA-A*02:01’s groove is central for productive TCR docking, with TCR-B presenting slower association but prolonged dwell time. Functional divergence emerged during target-cell engagement and amplified under RAC conditions, where TCR-B sustained cytotoxic activity while TCR-A progressively lost function. This superior functional endurance translated into complete tumor eradication and durable responses in vivo. These findings provide a mechanistic basis and a conceptual framework for the selection and optimization of TCRs for adoptive immunotherapy.