Structure-guided engineering of the affinity and specificity of CARs against Tn-glycopeptides

Significance CAR T cells have shown significant promise in treating hematopoietic cancers but challenges remain, including antigen loss and identification of targets in solid tumors. Aberrant O-linked glycosylation in solid cancers is common, often resulting in expression of cell-surface neoantigens. We used directed evolution to engineer the binding site of an antibody so variants reacted more broadly with tumor-specific glycoprotein epitopes containing Tn (GalNAc-O-S/T). As CARs, these variants showed improved broader activity against mouse and human cancer cells with dysregulated O-glycosylation. Even cancer cells without MUC1, the most studied cancer-associated glycoantigen, were recognized by the engineered CARs. Cancer-specific recognition was retained due to Tn-antigen requirement, but broader activity against the glycoprotein backbone provides opportunities for potent use with minimal antigen-loss escape.