Development of a T-cell receptor mimic antibody targeting a novel Wilms tumor 1-derived peptide and analysis of its specificity.

Wilms tumor 1 (WT1) is an intracellular tumor-associated antigen that remains inaccessible to antibodies. Recently, T-cell receptor (TCR) mimic antibodies (TCRm-Abs), which recognize peptides loaded on human leukocyte antigen (HLA) with higher specificity and affinity than TCR, have been developed as a new antibody class that can target intracellular antigens. To expand the therapeutic targets in tumors with WT1, we developed TCRm-Abs targeting a novel HLA-A*02:01-restricted peptide, WT1C (ALLPAVPSL), and validated their specificity using multiple techniques. Screening of these antibodies by ELISA with a panel of peptide/HLA complexes and by glycine scanning of peptide-pulsed T2 cells identified one specific clone, #25-8. Despite the low risk for eliciting broad cross-reactivity of this TCRm-Ab, analysis of a panel of cell lines, in conjunction with exogenous expression of either or both the HLA-A*02:01 and WT1 genes in HeLa cells, revealed that #25-8 reacts with WT1C but also with unknown peptides in the context of HLA-A*02:01. This potentially dangerous cross-reactivity was confirmed through analysis using chimeric antigen receptor T-cells carrying the single-chain variable fragment of #25-8, which targets WT1-negative HeLa/A02 cells. To determine the cross-reactive profiles of #25-8, we applied the PresentER antigen presentation platform with the #25-8-recognition motif, which enables the identification of potential off-target peptides expressed in the human proteome. Our results demonstrate the potential of TCRm-Abs to target a variety of peptides in the context of HLA but also depict the need for systematic validation to identify the cross-reactive peptides for the prediction of off-target toxicity in future clinical translation.