Enhancement of CAR‐T cell activity against cholangiocarcinoma by simultaneous knockdown of six inhibitory membrane proteins

BACKGROUND: Existing treatments for cholangiocarcinoma have poor efficacy. However, chimeric antigen receptor‐T (CAR‐T) cells are emerging as a potential therapeutic strategy. Solid tumors possess multiple adverse factors in an immunosuppressive microenvironment that impair CAR‐T cell infiltration and function. This study aimed to improve the function of CAR‐T cells through knock down immune checkpoints and immunosuppressive molecular receptors. METHODS: We evaluated the expression of epidermal growth factor receptor (EGFR) and B7 homolog 3 protein (B7H3) antigens in cholangiocarcinoma tissues using immunohistochemistry and screened specific immune checkpoints in the cholangiocarcinoma microenvironment via flow cytometry. Subsequently, we engineered CAR‐T cells targeting EGFR and B7H3 antigens. We simultaneously knocked down immune checkpoints and immunosuppressive molecular receptors in CAR‐T cells by constructing two clusters of small hairpin RNAs and evaluated the engineered CAR‐T cells for antitumor activity both in vitro, using tumor cell lines and cholangiocarcinoma organoid models, and in vivo, using humanized mouse models. RESULTS: We observed high expression of EGFR and B7H3 antigens in cholangiocarcinoma tissues. EGFR‐CAR‐T and B7H3‐CAR‐T cells demonstrated specific anti‐tumor activity. We found an abundance of programmed cell death protein 1 (PD‐1), T cell immunoglobulin and mucin domain‐containing protein 3 (Tim‐3), and T cell immunoglobulin and ITIM domain (Tigit) on infiltrated CD8(+) T cells in the cholangiocarcinoma microenvironment. We then decreased the expression of these 3 proteins on the surface of CAR‐T cells, named PTG‐scFV‐CAR‐T cells. Furthermore, we knocked‐down the expression of transforming growth factor beta receptor (TGFβR), interleukin‐10 receptor (IL‐10R), and interleukin‐6 receptor (IL‐6R) of PTG‐scFV‐CAR‐T cells. Those cells, named PTG‐T16R‐scFV‐CAR‐T cells, potently killed tumor cells in vitro and promoted apoptosis of tumor cells in a cholangiocarcinoma organoid model. Finally, the PTG‐T16R‐scFv‐CAR‐T cells showed greater inhibitory effect on tumor growth in vivo, and were superior in prolonging the survival of mice. CONCLUSIONS: Our results revealed that PTG‐T16R‐scFV‐CAR‐T cells with knockdown of sextuplet inhibitory molecules exhibited strong immunity against cholangiocarcinoma and long‐term efficacy both in vitro and in vivo. This strategy provides an effective and personalized immune cell therapy against cholangiocarcinoma.