组合型嵌合抗原受体的构建及分析鉴定  

Construction,Analysis and Characterization of Combinvssatorial Chimeric Antigen Receptor

作　　者：林海英[1] 姚玢妍 于艺洁 杨扬 LIN Hai-ying;YAO Bin-yan;YU Yi-jie;YANG Yang(Medicine Biotechnology and Engineering Research Institute,College of Biological Science and Technology,Fuzhou University,Fuzhou 350108,China)

机构地区：[1]福州大学生物科学与工程学院、药物生物技术研究所,福州350108

出　　处：《中国生物工程杂志》2023年第6期12-19,共8页China Biotechnology

基　　金：福建省自然科学基金(2020J01490)资助项目。

摘　　要：目的:构建靶向磷脂酰肌醇蛋白聚糖3(glypican-3,GPC3)的嵌合抗原受体(chimeric antigen receptor,CAR)及靶向上皮细胞黏附分子(epithelial cell adhesion molecule,EPCAM)嵌合抗原共刺激受体(chimeric antigen costimulatory receptor,CCR)共修饰的T细胞,并对其进行体外活性评估。方法:将EPCAM-CCR和GPC3-CAR基因片段克隆入慢病毒载体质粒,酶切、PCR和测序鉴定CCR+CAR pCDH重组载体。分离、激活和扩增人T细胞,利用慢病毒感染并筛选能够稳定表达该组合型嵌合抗原受体人T细胞。通过Western blot、流式细胞术(flow cytometry,FCM)验证CCR+CAR T细胞中CCR+CAR的表达,ELISA检测细胞因子IL-2、INF-γ、IL-4的分泌。结果:成功构建CCR+CAR pCDH慢病毒重组载体。成功分离、激活并扩增人T细胞。CCR+CAR pCDH慢病毒成功感染人T细胞,RT-PCR、Western blot检测也显示其成功表达目的蛋白,同时FCM分析显示CCR+CAR T细胞表面CCR+CAR的表达率为42%左右;ELISA检测CCR+CAR与表达GPC3和EPCAM的HepG2、Huh-7共培,其分泌更高水平的IL-2、INF-γ、IL-4,同时与人正常肝细胞L-02共培未发现CCR+CAR T细胞的有效激活。结论:成功获得组合型CCR+CAR T细胞,并初步探究其在肝癌细胞中的免疫效应,为后续动物体内免疫及实体瘤过继免疫治疗中防脱靶效应奠定基础。Objective:To construct T cells encoding chimeric antigen receptor(CAR)targeting glypican-3(GPC3)and chimeric antigen costimulatory receptor(CCR)targeting epithelial cell adhesion molecule(EPCAM),and analyze their activities in vitro.Methods:Gene fragments encoding EPCAM-CCR and GPC3-CAR was cloned into lentivirus vector plasmid.The recombinant CCR+CAR pCDH vector was identified by enzyme digestion,PCR and sequencing.T lymphocytes were isolated,activated and expanded.A stable combinatorial CAR modified cell line was generated using the lentivirus.Western blot,RT-PCR and flow cytometry(FCM)were used to verify the expression of CCR+CAR in CCR+CAR T cells.The secretion of cytokines IL-2,IL-4 and INF-γwas detected by ELISA.Results:The CCR+CAR pCDH lentiviral recombinant plasmid was successfully constructed.Human T lymphocytes were successfully isolated,activated and expanded.Human T lymphocytes were successfully infected with CCR+CAR pCDH lentivirus.RT-PCR and Western blot showed that the target protein was successfully expressed,and FCM analysis showed that the expression rate of CCR+CAR on CCR+CAR T cells was about 42%.ELISA was used to detect CCR+CAR co-culture with HepG2 and Lu-7 expressing GPC3 and EPCAM,the secretion levels of IL-2,INF-γand IL-4 were higher,and the co-culture of human normal liver cells L-02 did not find effective activation of CCR+CAR T cells.Conclusions:The combined CCR+CAR T cells were successfully obtained,and the immune effect of CAR T combined with the two antigen recognition signals was attempted in hepatocellular carcinoma cells.The result laid a foundation for the immune effects in the subsequent in vitro experiments,and provided a potentially novel approach to augment the off-target effect of CAR-T immunotherapy for solid tumors.

关 键 词：磷脂酰肌醇蛋白聚糖3 上皮细胞黏附分子 嵌合抗原受体 嵌合抗原共刺激受体 

分 类 号：Q812[生物学—生物工程]