Affinity-enhanced T-cell receptors for adoptive T-cell therapy targeting MAGE-A10: strategy for selection of an optimal candidate

Abstract

Circulating T-cells that have passed thymic selection generally bear T-cell receptors (TCRs) with sub-optimal affinity for cancer-associated antigens, resulting in a limited ability to detect and eliminate tumor cells. Engineering TCRs to increase their affinity for cancer targets is a promising strategy for generating T-cells with enhanced potency for adoptive immunotherapy in cancer patients. However, this manipulation also risks generating cross-reactivity to antigens expressed by normal tissue, with potentially serious consequences. Testing in animal models might not detect such cross-reactivity due to species differences in the antigenic repertoire. To mitigate the risk of off-target toxicities in future clinical trials, we therefore developed an extensive <i>in vitro</i> testing strategy. This approach involved systematic substitution at each position of the antigenic peptide sequence using all natural amino acids to generate a profile of peptide specificity ("X-scan"). The likelihood of off-target reactivity was investigated by searching the human proteome for sequences matching this profile, and testing against a panel of primary cell lines. Starting from a diverse panel of parental TCRs, we engineered several affinity-enhanced TCRs specific for the cancer-testis antigen MAGE-A10. Two of these TCRs had affinities and specificities which appeared to be equally optimal when tested in conventional biochemical and cellular assays. The X-scan method, however, permitted us to select the most specific and potent candidate for further pre-clinical and clinical testing.