Facile method for screening clinical T cell receptors for off-target peptide-HLA reactivity

T cell receptors (TCRs) exhibit varying degrees of cross-reactivity for peptides presented by the human leukocyte antigen (HLA). In engineered T cell therapies, TCR affinity maturation is a strategy to improve the sensitivity and potency to often a low-density peptide-HLA (pHLA) target. However, the process of affinity maturation towards a known pHLA complex can introduce new and untoward cross-reactivities that are difficult to detect and raises significant safety concerns. We developed a yeast-display platform of pHLA consisting of ~100 million different 9mer peptides presented by HLA-A*01 and used a previously established selection approach to validate the specificity and cross-reactivity of the A3A TCR, an affinity-matured TCR against the MAGE-A3 target (EVDPIGHLY). We were able to identify reactivity against the titin peptide (ESDPIVAQY), to which there is now known clinical toxicity. We propose the use of yeast-display of pHLA libraries to determine cross-reactive profiles of candidate clinical TCRs to ensure safety and pHLA specificity of natural and affinity-matured TCRs.