Multi-tiered approach to detect autoimmune cross-reactivity of therapeutic T cell receptors

T cell receptor (TCR)–engineered T cell therapy using high-affinity TCRs is a promising treatment modality for cancer. Discovery of high-affinity TCRs especially against self-antigens can require approaches that circumvent central tolerance, which may increase the risk of cross-reactivity. Despite the potential for toxicity, no standardized approach to screen cross-reactivity has been established in the context of preclinical safety evaluation. Here, we describe a practical framework to prospectively detect clinically prohibitive cross-reactivity of therapeutic TCR candidates. Cross-reactivity screening consisted of multifaceted series of assays including assessment of p-MHC tetramer binding, cell line recognition, and reactivity against candidate peptide libraries. Peptide libraries were generated using conventional contact residue motif–guided search, amino acid substitution matrix–based search unguided by motif information, and combinatorial peptide library scan–guided search. We demonstrate the additive nature of a layered approach, which efficiently identifies unsafe cross-reactivity including one undetected by conventional motif-guided search. These findings have important implications for the safe development of TCR-based therapies.