Fourth-generation multi-costimulatory chimeric antigen receptor T cells targeting CD133 and integrin αvβ6 exert potent antitumor activity in cholangiocarcinoma

Cholangiocarcinoma (CCA) is an aggressive and highly metastatic malignancy with limited responsiveness to conventional chemotherapy, underscoring the need for innovative therapeutic strategies. This study aimed to develop and evaluate fourth-generation chimeric antigen receptor (CAR) T cells (CAR4; a multi-costimulatory CD28/4–1BB/CD27/CD3ζ design without a cytokine or other transgene payload) targeting CD133 and integrin αvβ6 to treat CCA. Two parallel CAR T-cell products, each incorporating a single antigen-recognition domain, were generated and evaluated independently rather than as one bispecific construct. We transduced activated human T lymphocytes with lentiviral constructs encoding anti-CD133 CAR4 and an integrin-targeted CAR (A20 CAR4), confirmed CAR expression by flow cytometry, then expanded them in vitro and evaluated therapeutic efficacy after intratumoral administration in a CCA xenograft nude mouse model. CAR4 T-cell treatment produced dose-dependent suppression of tumor growth and significant prolongation of survival compared with control and untreated groups. High-dose anti-CD133 and anti-integrin CAR4 T cells achieved greater tumor inhibition and numerically extended median survival beyond that observed with cisplatin. Histopathological analysis revealed no treatment-related abnormalities in heart, liver, spleen, or kidney tissues, and lung metastasis incidence was markedly reduced in high-dose CAR-treated groups. Quantitative real-time PCR analysis demonstrated coordinated downregulation of genes associated with angiogenesis (VEGF), matrix remodeling (MMP9), inflammatory signaling (COX-2, mPGES-1, TNF-α, IL-6), and immunosuppressive pathways (TGF-β, IL-10). These findings indicate that fourth-generation CAR T cells targeting CD133 and integrin αvβ6 exert potent antitumor activity in this model and that their activity is associated with transcript-level downregulation of tumor microenvironment-related genes, suggesting potential modulation of the tumor microenvironment that will require protein-level and functional confirmation. Antigen-dependent cytotoxicity was not directly assessed in vitro in the present study. Together, these data support further translational development of multi-costimulatory CAR T-cell platforms for CCA.