biorxiv.org
Reverse engineering the fatally cross-reactive A3A TCR to decouple potency and specificity
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 CDR2α 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 CDR2α-MHC contacts and disrupt an intra-TCR CDR2α-CDR3β 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. ### Competing Interest Statement The authors have declared no competing interest. Chinese Academy of Medical Sciences (CAMS) Innovation Fund for Medical Science (CIFMS), China, 2018-I2M-2-002 and 2024-I2M-2-001-1 Cancer Research UK (CRUK) DPhil in Cancer Science Award, C2195/A31310, CANCTA-2024/100004 Wellcome Trust, https://ror.org/029chgv08, 227585/Z/23/Z, 227388/Z/23/Z Cancer Research UK, DRCCIP-Nov23/100004 Engineering and Physical Sciences Research Council, https://ror.org/0439y7842, EP/S024093/1 Biotechnology and Biological Sciences Research Council, BB/T008784/1 Wellcome Trust Core Award, 203141/Z/16/Z Novo Nordisk Foundation Postdoctoral Fellowship, NNF23OC0082912 Gates Foundation, INV-055780 CSC-COI MD/PhD High-level Medical Innovative Talent Scholarship, 2024-I2M-2-001-1 ARISE Fellowship - Marie Skłodowska-Curie Actions, 945405
