Sharma, G.; Teng, F.; Round, J.; Sneddon, S.; Brown, S.; Sivasothy, S.; Holt, R.

T cell receptor therapeutics are an emerging modality of biologic and cell-based medicines with the unique ability 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 apply a functional high-throughput screening assay, Tope-seq, to detecting cross-reactive epitopes in libraries of >5 x 105 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 identified 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.