McCarthy, J. V.; Heroven, A. C.; Ifashe, K.; Fellermeyer, M.; Hudson, D.; Cioaca, R.; Quastel, M. N.; Yang, Y.; Cagiada, M.; Dragonov, S. D.; Thorpe, C. J.; Pinto-Fernandez, A.; Greenshields-Watson, A.; Gillespie, G. M.; Deane, C. M.; Fernandes, R. A.

T cell receptor (TCR) affinity enhancement can introduce off-target cross-reactivity with life-threatening consequences, as illustrated by the MAGE-A3-specific A3A TCR, which caused fatal cardiotoxicity through recognition of a Titin-derived peptide. Here, we reconstructed the crossreactivity landscape by reverse-engineering A3A toward its wild-type precursor, generating intermediate variants in which engineered CDR2 residues are systematically reverted to the wild-type sequence. Reverting just two engineered residues yields a receptor, v9, that retains MAGE-A3 cytotoxicity comparable to A3A while eliminating Titin and other acquired cross-reactivities. Structurally, these substitutions reduce CDR2-MHC contacts and disrupt an intra-TCR CDR2-CDR3{beta} interaction, propagating conformational changes across CDR3 loops that reshape peptide engagement without altering docking geometry. These results demonstrate that mutations outside the peptide-contacting CDR3 loops can allosterically reconfigure antigen specificity and establish simple stepwise reverse engineering to wild-type as a strategy for correcting TCR cross-reactivity.