AlphaFold 3 prediction of TCR mimic Fv - pMHC complex structures and their validation and optimization

Monoclonal antibody-derived chimeric antigen receptors (CAR) that mimic T‑cell receptors (TCR) on binding to peptide-major histocompatibility complex (pMHC) and activating T‑cell functions hold great promise for the development of effective immunotherapy. This study applied AlphaFold 3 (AF3) to model the quaternary structures of TCR mimic antibody variable fragment (TCRm Fv) complexed with NY-ESO-1_157-165/HLA-A*02:01. Benchmark study suggested that reliable TCRm Fv-pMHC structures were achieved by AF3 with high confidence. Toward NY-ESO-1/A2-specific CAR clones, AF3 prediction revealed their intermolecular geometry resembling the canonical TCR engagement, and binding avidity tests confirmed the peptide-dependent recognition of pMHC. Molecular interactions between Fv and the peptide antigen and its binding groove on MHC were further pinpointed, and the predicted epitopes and paratopes were validated by mutagenesis studies. Aiming to generate TCRm Fv mutants of improved affinity, AF3 aided designs with larger Fv-pHLA contact surface areas and more hydrogen bonds toward the peptide antigen were successfully identified. However, these AF3‑designed mutants failed to deliver enhancement on binding avidity or functional potency in experimental tests. Overall, AF3 is a highly valuable tool for TCRm Fv-pMHC complex modeling, and its combination with force field-based methods will be desirable to aid optimization tasks.