Next-generation CAR T cells targeting integrin αvβ6 and PD-L1 to enhance immunotherapy for cholangiocarcinoma.

Cholangiocarcinoma (CCA) is an aggressive epithelial malignancy characterized by poor prognosis, limited treatment options, and high recurrence rates even after surgery. Chimeric antigen receptor (CAR) T cell therapy has achieved notable success in hematologic malignancies, but its efficacy in solid tumors such as CCA is hindered by the immunosuppressive tumor microenvironment, particularly through PD-1/PD-L1 axis. To address these barriers, we developed a sixth-generation CAR T cell, A20 CAR6, incorporating the A20 peptide, a high-affinity ligand for integrin αvβ6-a tumor-associated antigen frequently overexpressed in CCA. Beyond antigen targeting, A20 CAR6 T cells are engineered to secrete a bispecific protein engager (BiPE) that binds PD-L1 on tumor cells and CD3 on T cells. This dual-function design aims to neutralize PD-L1-mediated immune suppression and recruit both CAR and bystander T cells to enhance tumor killing. Compared with conventional fourth-generation A20 CAR4 T cells lacking BiPE secretion, A20 CAR6 T cells exhibited superior cytotoxicity, cytokine production, and proliferation against integrin αvβ6/PD-L1 CCA cells. Notably, the secreted αPD-L1/αCD3 BiPE augmented CAR T cell activity and redirected non-engineered T cells to target tumor cells, amplifying the overall anti-tumor response. These findings suggest that A20 CAR6 T cells represent a promising next-generation immunotherapy with the potential to overcome key resistance mechanisms in CCA and improve treatment outcomes.