Comprehensive Functional Self-Antigen Screening to Assess Cross-Reactivity in a Promiscuous Engineered T-cell Receptor

T cell receptor therapeutics represent an emerging modality of biologic and cell-based medicines because of the unique nature of these proteins to target intracellular antigens and finely discriminate between healthy and infected or mutated cells. An obstacle to the development of new T cell receptor therapeutics is the difficulty in engineering these proteins for enhanced therapeutic efficacy while avoiding introduction of unexpected off-target autoreactivity. In this study, we demonstrate the application of a functional high-throughput screening assay, Tope-seq, towards detecting cross-reactive epitopes in libraries of >5E+05 unique peptide-coding sequences. We retrospectively analyze an affinity-enhanced engineered T cell receptor, which previously failed clinical trials due to severe off-target toxicity caused by epitope cross-reactivity, by comprehensive functional testing against all genome-coded self-antigens. Using the Tope-seq methodology, we were able to identify the epitope mediating off-target reactivity at a significance threshold of p < 0.01 in first-pass bulk screening. We also identify other potential cross-reactive epitopes of the engineered TCR of-interest, suggesting that the need for assessing promiscuity in TCR based therapeutics is larger than previously appreciated.