Department of Rehabilitation Medicine and Laboratory of Animal Tumor Models, National Clinical Research Center for Geriatrics and Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
Department of Targeting Therapy & Immunology and Laboratory of Animal Tumor Models, Cancer Center and National Clinical Research Center for Geriatrics and Frontiers Science Center for Disease-Related Molecular Network, West China Hospital, Sichuan University, Chengdu, Sichuan, China.
Immunol Invest. 2023 Nov;52(6):735-748. doi: 10.1080/08820139.2023.2232402. Epub 2023 Jul 6.
CAR-T is emerging as an effective treatment strategy for hematologic malignancies, however its effectiveness for treating solid tumors, such as Hepatocellular Carcinoma (HCC) is limited. Here, we screened a variety of CAR-T cells that target c-Met to investigate their potential to induce HCC cell death in vitro.
Human T cells were transduced to express CARs by lentiviral vector transfection. c-Met expression in human HCC cell lines and CARs expression were monitored by flow cytometry. Tumor cell killing was evaluated by Luciferase Assay System Kit. The concentrations of cytokine were tested by Enzyme-linked immunosorbent assays. Knock down and overexpression studies targeting c-Met were conducted to assess the targeting specificity of CARs.
We found that CAR T cells expressing a minimal amino-terminal polypeptide sequence comprising the first kringle (kringle 1) domain (denoted as NK1 CAR-T cells), efficiently killed HCC cell lines that expressed high levels of the HGF receptor c-Met. Furthermore, we report that while NK1 CAR-T cells were efficient at targeting SMMC7221 cells for destruction, and its potency was significantly attenuated in parallel experiments with cells stably expressing short hairpin RNAs (shRNAs) that suppressed c-Met expression. Correspondingly, overexpression of c-Met in the embryonic kidney cell line HEK293T led to their enhanced killing by NK1 CAR-T cells.
Our studies demonstrate that a minimal amino-terminal polypeptide sequence comprising the kirngle1 domain of HGF is highly relevant to the design of effective CAR-T cell therapies that kill HCC cells expressing high levels of c-Met.
嵌合抗原受体 T 细胞（CAR-T）疗法在血液恶性肿瘤的治疗中取得了显著的疗效，但在实体瘤如肝细胞癌（HCC）的治疗中效果有限。本研究旨在筛选靶向 c-Met 的 CAR-T 细胞，探讨其体外诱导 HCC 细胞死亡的潜力。
通过慢病毒载体转染将人 T 细胞转导为表达嵌合抗原受体（CAR）的细胞。通过流式细胞术监测人 HCC 细胞系中 c-Met 的表达和 CAR 的表达。通过荧光素酶检测试剂盒评估肿瘤细胞杀伤情况。通过酶联免疫吸附试验（ELISA）检测细胞因子浓度。通过靶向 c-Met 的敲低和过表达研究评估 CAR 的靶向特异性。
我们发现，表达包含第一个kringle（kringle 1）结构域的最小氨基末端多肽序列的 CAR-T 细胞（命名为 NK1 CAR-T 细胞）能够有效杀伤高表达 HGF 受体 c-Met 的 HCC 细胞系。此外，我们报告称，NK1 CAR-T 细胞能够有效地靶向 SMMC7221 细胞进行杀伤，而在平行实验中，用短发夹 RNA（shRNA）稳定表达抑制 c-Met 表达的细胞，其杀伤能力显著减弱。相应地，在胚胎肾细胞系 HEK293T 中过表达 c-Met 可增强 NK1 CAR-T 细胞对其的杀伤作用。
本研究表明，HGF 的kringle1 结构域的最小氨基末端多肽序列在设计有效杀伤表达高水平 c-Met 的 HCC 细胞的 CAR-T 细胞疗法中具有重要意义。
