[Study on construction of c-Met specific CAR-T cells and its killing effect on non-small cell lung carcinoma].

Objective: To produce chimeric antigen receptor T cells (CAR-T) targeting human hepatocyte growth factor/c-Met (HGF/c-Met) protein and detect its cytotoxicity against non-small cell lung cancer (NSCLC) cells H1975 in vitro. Methods: The whole gene sequence of c-Met CAR containing c-Met single-chain fragment variable was synthesized and linked to lentiviral vector plasmid, plasmid electrophoresis was used to detect the correctness of target gene. HEK293 cells were transfected with plasmid and the concentrated solution of the virus particles was collected. c-Met CAR lentivirus was transfected into T cells to obtain second-generation c-Met CAR-T and the expression of CAR sequences was verified by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and western blot, and the positive rate and cell subtypes of c-Met CAR-T cells were detected by flow cytometry. The positive expression of c-Met protein in NSCLC cell line H1975 was verified by flow cytometry, and the negative expression of c-Met protein in ovarian cancer cell line A2780 was selected as the control. The cytotoxicity of c-Met CAR-T to H1975 was detected by lactate dehydrogenase (LDH) cytotoxicity assay at 1∶1, 5∶1, 10∶1 and 20∶1 of effector: target cell ratio (E∶T). Enzyme-linked immunosorbent assay (ELISA) was used to detect the release of cytokines such as TNF-α, IL-2 and IFN-γ from c-Met CAR-T co-cultured with H1975. Results: The size of band was consistent with that of designed c-Met CAR, suggesting that the c-Met CAR plasmid was successfully constructed. The results of gene sequencing were consistent with the original design sequence and lentivirus was successfully constructed. CAR molecules expression in T cells infected with lentivirus was detected by western blot and RT-qPCR, which showed c-Met CAR-T were successfully constructed. Flow cytometry results showed that the infection efficiency of c-Met CAR in T cells was over 38.4%, and the proportion of CD8(+) T cells was increased after lentivirus infection. The NSCLC cell line H1975 highly expressed c-Met while ovarian cancer cell line A2780 negatively expressed c-Met. LDH cytotoxicity assay indicated that the killing efficiency was positively correlated with the E∶T, and higher than that of control group, and the killing rate reached 51.12% when the E∶T was 20∶1. ELISA results showed that c-Met CAR-T cells released more IL-2, TNF-α and IFN-γ in target cell stimulation, but there was no statistical difference between c-Met CAR-T and T cells in the non-target group. Conclusions: Human NSCLC cell H1975 expresses high level of c-Met which can be used as a target for immunotherapy. CAR-T cells targeting c-Met have been successfully produced and have high killing effect on c-Met positive NSCLC cells in vitro.目的： 制备靶向c－Met蛋白的嵌合抗原受体T细胞(CAR－T)，并检测其对人非小细胞肺癌(NSCLC)H1975细胞的体外杀伤作用。 方法： 将含c－Met抗体scFv片段的CAR序列插入慢病毒质粒中，用酶切质粒电泳，检测目的基因正确性。质粒转染工具为HEK293细胞，收集浓缩慢病毒，通过慢病毒感染的方式制备第二代c－Met CAR－T，通过实时荧光定量聚合酶链反应(qRT－PCR)、Western blot验证CAR序列的表达，采用流式细胞术检测c－Met CAR－T的阳性率、细胞亚型，流式细胞术验证NSCLC细胞H1975中c－Met蛋白的阳性表达并选择c－Met蛋白阴性表达的卵巢癌细胞A2780作为对照，采用乳酸脱氢酶(LDH)释放法检测c－Met CAR－T在效靶比为1∶1、1∶5、1∶10、1∶20时对H1975细胞的杀伤作用。酶联免疫吸附试验(ELISA)检测c－Met CAR－T在靶抗原刺激下，细胞因子白细胞介素2(IL－2)、肿瘤坏死因子α(TNF－α)和IFN－γ的释放情况。 结果： 成功合成c－Met CAR慢病毒重组质粒，酶切条带位置符合理论值。基因测序结果符合原设计序列，并成功包装慢病毒。Western blot和qRT－PCR结果显示，CAR分子存在于慢病毒感染后的T细胞中，c－Met CAR－T构建成功。c－Met CAR－T阳性率可达38.4%以上，且在慢病毒感染过后CD8(+) T细胞比例有所上调。流式细胞术验证结果显示，NSCLC细胞H1975有较强的c－Met蛋白表达，卵巢癌细胞A2780 c－Met蛋白表达阴性。LDH释放实验提示，c－Met CAR－T的抗肿瘤作用随效靶比的提升而增强，且均高于对照组，当效靶比为20∶1时杀伤率达到51.12%。ELISA检测结果显示，与对照组比较，在靶细胞刺激下c－Met CAR－T能释放出更多的IL－2、TNF－α和IFN－γ，而Non target组无此效应。 结论： c－Met在NSCLC细胞系H1975中表达，可以作为NSCLC免疫治疗的靶点；靶向c－Met的CAR－T能对c－Met阳性肺癌细胞产生较强的体外杀伤作用。.