Enhanced HLA stability of a heteroclitic WT1 vaccine exacts a kinetic penalty in recognition by a natural TCR.

Wilms' tumor 1 (WT1) is aberrantly overexpressed across hematologic and solid malignancies and ranks among top-priority antigens for T cell receptor (TCR)-based immunotherapy. Among WT1 epitopes, the HLA-A∗02:01-restricted nonamer RMFPNAPYL (WT1126-134; RL9) has advanced clinically, including with the naturally selected C4 TCR. Despite encouraging activity in early trials, the molecular basis of C4 recognition and its tolerance to vaccine-motivated anchor modifications remain unclear. Here we report high-resolution crystal structures of C4 bound to HLA-A∗02:01 presenting either the native RL9 peptide or the R1Y variant (YMFPNAPYL; YL9). C4 docks diagonally with a peptide-centric footprint biased toward central and C-terminal positions, contrasting with TCR-mimic antibodies that depend on N-terminal R1 contacts. Although R1Y preserves the peptide backbone, p1Y reorients upon TCR engagement, eliminating the E94 (CDRα3)-p1R salt bridges, remodeling the electrostatic landscape, and weakening interfacial hydrogen-bond/salt-bridge networks. Differential scanning fluorimetry showed that YL9-HLA-A∗02:01 is more thermally stable than RL9-HLA-A∗02:01 (Tm = 54.0 °C ± 0.21 °C versus 48.9 °C ± 0.25 °C). Surface plasmon resonance revealed micromolar binding of C4 to RL9-HLA-A∗02:01 (KD = 13.8 ± 1.53 μM) but weak binding to YL9-HLA-A∗02:01 (KD > 150 μM). Together, these structural and biophysical data clarify the recognition logic of a clinically validated WT1-specific TCR and reveal that an HLA-stabilizing R1Y modification can reduce recognition by C4, even while improving pHLA stability. These findings do not preclude YL9 from eliciting other cross-reactive TCR clonotypes, but highlight the need to evaluate both HLA stabilization and TCR-recognition fidelity during epitope selection and structure-informed TCR engineering in WT1-targeted immunotherapy.