Mapping specificity and cross-reactvity of wild-type and engineered T cell receptors 4308

Abstract Description T cells identify cancer cells via their T cell receptor (TCR), which recognises peptides presented by the major histocompatibility complex (pMHC). Deciphering the principles of TCR-pMHC interactions and the origins of cross-reactivity remains a major challenge for developing efficient TCR-based immunotherapies. Here, we developed a workflow to characterise the binding landscape and cross-reactivity of tumour-reactive TCRs through unbiased affinity-based selections of pMHC yeast display libraries, coupled with computational, functional and structural analysis. Peptide enrichment analysis of Tebentafusp, an affinity-enhanced TCR, revealed conserved motifs strikingly similar to its known agonist peptide, gp100. Yet, Tebentafusp displayed reactivity to off-target peptides with substitutions only tolerated at P6. In contrast, TIL-011, an orphan TCR isolated from a lung cancer biopsy, recognised two distinct peptide motifs, highlighting the positive role of cross-reactivity in tumour antigen recognition. Using novel in silico workflows, we predicted and confirmed binding to several wild-type candidate peptides. Consistent with the peptide motif analysis, structural predictions demonstrated that the TIL-011 TCR exclusively binds the C-terminus of the wild-type peptides, permitting N-terminus promiscuity. By deconvoluting the complexities of TCR-pMHC interactions, we can use this approach to de-risk candidate therapeutic TCRs and deorphanise TCRs to identify novel antigen targets. Funding Sources CRUK Oxford Centre’s DPhil in Cancer Science programme CAMS Oxford Institute, CAMS Innovation Fund for Medical Sciences (CIFMS) 2018-I2M-2-002 Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)