CAR-T cells directed toward PD-L1 demonstrate potent, antigen-specific activity against cholangiocarcinoma: A proof...

An immunosuppressive microenvironment driven by tumor and stromal cells expressing programmed death-ligand 1 (PD-L1) contributes to immune evasion and poor prognosis in cholangiocarcinoma. Although antibodies to PD-L1 are used clinically, their benefit is limited by immune exclusion within the local microenvironment. To overcome this, we evaluated engineered T cells directed toward PD-L1 that simultaneously target tumor cells and the immunosuppressive microenvironment. Human donor T cells were transduced with a lentiviral vector encoding a chimeric antigen receptor (CAR) consisting of an anti-PD-L1 scFv, CD4 transmembrane domain, and 4-1BB/CD3ζ signaling domain. The antitumor efficacy of these CAR-T cells was assessed in a murine orthotopic tumor model, and their specificity and effect were validated in human malignant cholangiocytes with varying PD-L1 expression. PD-L1 CAR-T cells retained T cell identity, demonstrated antigen-specific cytotoxicity, and effectively reduced tumor burden in vivo. Cytotoxicity was abrogated in PD-L1 knockout cells, confirming target specificity. PD-L1 CAR-T cells significantly reduced tumor cell viability within multicellular spheroids. Gemcitabine pretreatment upregulated PD-L1 expression and enhanced CAR-T-mediated cytotoxicity. These findings demonstrate the feasibility of second-generation PD-L1 CAR-T cells, demonstrating preclinical efficacy and specificity, and validating a therapeutic strategy that targets the tumor microenvironment for these challenging cancers.