Enhanced Cytotoxicity against Cholangiocarcinoma by Fifth-Generation Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 and Secreting Anti-PD-L1 scFv

Cholangiocarcinoma (CCA) is a fatal bile duct cancer with high resistance and recurrence rates, with only one fifth of patients eligible for surgical treatment. The disease resists standard chemotherapy and often relapses. Chimeric antigen receptor (CAR) T cell therapy has shown promise for hematological malignancies but faces challenges in solid tumors due to resistance mechanisms like PD-L1 expression, which tumors use to evade the immune system. To address this challenge, we developed fifth-generation CAR T cells targeting integrin αvβ6 that also secrete anti-PD-L1 single-chain variable fragments (scFvs) to target both tumor cells and the PD-1/PD-L1 pathway. We examined integrin αvβ6 and PD-L1 expression in CCA cell lines and engineered T cells to express either fourth-generation CAR T cells targeting integrin αvβ6 (A20 CAR4 T cells) or fifth-generation CAR T cells with anti-PD-L1 scFv secretion (A20 CAR5 T cells). In vitro , A20 CAR5 T cells exhibited less exhaustion and superior long-term functionality compared to A20 CAR4 T cells. In 3D spheroid models of CCA, A20 CAR5 T cells demonstrated enhanced antitumor activity and better infiltration into the spheroid core. These findings suggest that A20 CAR5 T cells have significant potential and warrant further in vivo studies and clinical trials.