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

作者：Yidan Qiao, Jie Chen, Xuemei Wang, Shumei Yan, Jizhou Tan, Baijin Xia, Yongjian Chen, Keming Lin, Fan Zou, Bingfeng Liu, Xin He, Yiwen Zhang, Xu Zhang, Hui Zhang, Xiang-yuan Wu, Lijuan Lu · 发表于：癌症：英文版 · 年份：2023 · DOI：10.1002/cac2.12452 · 被引用次数：52 · 研究领域：CAR-T cell therapy research、Monoclonal and Polyclonal Antibodies Research、Immunotherapy and Immune Responses

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: 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-...