Abstract
Engineered T cells have shown efficacy in cancer treatment. However, the promiscuity of TCR-engineered T cells may result in recognition of off-target epitopes, causing severe toxicities. A genetic screen of >3,000 proteomic epitopes in the MHCI ligandome uncovered off-target peptides for both, native and affinity-enhanced 1G4 TCR, which target cancer antigen NY-ESO-1/A02-expressing cells. We validated off-target peptides derived from the human proteome recognized by both TCRs, showing that the affinity-enhanced TCR has more off-targets. Multiple off-target epitopes were reactive only in CD8 T cells, not in CD4 T cells. We identified a previously undescribed class of CD8 receptor-dependent off-targets. CD8α-negative cells (CD8α-/-) 1G4 T cells had fewer off-target reactivities, enhancing on-target specificity in vitro and in vivo. We corroborated our findings with the DMF5 TCR, targeting MART1/A02. This research advances our understanding of the distinct roles that CD4 and CD8 proteins play in T cell antigen recognition, potentially leading to more specific, effective, and safer engineered T cell therapies.