EGFRvⅢ/CAR-T对EGFRvⅢ^+U87胶质瘤细胞和裸鼠移植瘤的特异性杀伤作用  

Specific cytotioxicity of EGFRvⅢ oriented chimeric antigen receptor-engineered T cells on EGFRvⅢ^+ glioma U87 cells and the transplanted tumor in nude mice

作　　者：郑岩 谢甲贝 曹名波 张炳勇 李修岭 韩双印 ZHENG Yan;XIE Jiabei;CAO Mingbo;ZHANG Bingyong;LI Xiuling;HAN Shuangyin(Stem Cell Research Center, People＇ s Hos- pital Affiliated to Zhengzhou University, Zhengzhou 450003, Henan, Chin)

机构地区：[1]郑州大学人民医院干细胞研究中心,河南郑州450003

出　　处：《中国肿瘤生物治疗杂志》2018年第4期334-339,共6页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81372405;No.81772670)~~

摘　　要：目的:制备靶向EGFRvⅢ的第三代CAR-T(EGFRvⅢ/3CAR-T),检测其在体外和体内对EGFRvⅢ+U87胶质瘤细胞的特异性杀伤效应。方法:用磷酸钙沉淀法将三质粒共转染HEK293T细胞包装EGFRvⅢ/3CAR慢病毒(LV-EGFRvⅢ/3CAR),感染健康人CD3+T细胞,Western blotting和流式细胞术检测T细胞中EGFRvⅢ/3CAR的表达水平,51Cr释放法检测EGFRvⅢ/3CART对EGFRvⅢ+U87细胞的体外杀伤效应,ELISA法检测EGFRvⅢ/3CAR+T的IFN-γ分泌水平。构建裸鼠异种胶质瘤移植模型,检测EGFRvⅢ/3CAR-T对移植瘤的体内杀伤活性。结果:EGFRvⅢ/3CAR慢病毒包装成功,滴度值为5×10~6TU/ml。Western blotting在相对分子质量58 000处检测到EGFRvⅢ/3CAR表达,未转导组无相同分子量蛋白表达。流式细胞术检测结果显示EGFRvⅢ/3CAR转导效率平均为52.3%,^(51)Cr释放法检测EGFRvⅢ/3CAR-T特异性杀伤作用与E∶T值(E∶T为4∶1、为8∶1、16∶1、32∶1)呈正比关系。ELISA法检测到细胞因子IFN-γ分泌量为(1 836±148.2)pg/ml,与NT T和GFP+T细胞相比差异有统计学意义(P<0.01)。EGFRvⅢ/3CAR+T细胞的特异性杀伤活性及IFN-γ分泌均依赖于EGFRvⅢ在U87细胞中的表达水平。体内肿瘤生长检测结果显示,注射后3周EGFRvⅢ/3CAR+T组肿瘤体积较GFP+T细胞和PBS组差异有统计学意义(P<0.01)。结论:EGFRvⅢ/3CAR-T在体外和体内均能发挥靶向杀伤EGFRvⅢ+脑胶质瘤细胞的作用,为后续临床试验提供依据。Objective：To prepare the third generation CAR-T cells targeting EGFRvⅢ（EGFRvⅢCAR-T） and to detect its specific killing effect against EGFRvⅢ＋U87 cells in vitro and in vivo. Methods： Human CD3＋T cells were transfected with lentiviral EGFRvⅢ/3 CAR, which was generated by calcium phosphate co-precipitation of three plasmids. The expression of EGFRvⅢ/3 CAR in T cells was detected by Western blotting and flow cytometry. In vitro killing effect of EGFRvⅢ/3 CAR-T cells on EGFRvⅢ＋U87 cells was detected by51 Cr release assay. The secretion of cytokine IFN-γ of EGFRvⅢ/3 CAR-T cells was detected by ELISA. Nude mouse xenograft model was constructed to detect the in vivo cytotoxicity of EGFRvⅢ/3 CAR-T cells on xenograft tumor. Results： The EGFRvⅢ/3 CAR lentivirus was successfully packaged with an average titer of 5×10^6 TU/ml. Western blotting showed that a protein band of approximate 58 000 molecular weight was observed in EGFRvⅢ/3 CAR-T cells but absent in untransfected T cells. Flow cytometry indicated the average transduction efficiency of EGFRvⅢ/3 CAR was 52.3%.^（51） Cr release assay showed that the specific killing effect of EGFRvⅢ/3 CAR-T cells was positively correlated with E/T ratio（E∶T=4∶1, 8∶1, 16∶1, 32∶1）. ELISA showed that cytokine IFN-γ secretion was（1 836±148.2） pg/ml, which was significantly different from that of NT T and GFP＋T cells（P〈0.01）. The specific killing activity of EGFRvⅢ/3 CAR-T cells and IFN-γ secretion were both dependent on the expression level of EGFRvⅢ in U87 cells. The tumor growth monitoring results showed that the tumor volume of EGFRvⅢ/3 CAR-T cell group was significantly different from that of GFP＋T cell group and PBS group around 3 weeks after injection（P〈0.01）. Conclusion： EGFRvⅢ/3 CAR-T cells demonstrated specific antitumor effect against EGFRvⅢ＋U87 cells both in vitro and in vivo, providing basis for immunotherapy of glioma in future clinical use.

关 键 词：EGFRvⅢ 嵌合抗原受体 胶质瘤 U87细胞 免疫治疗 

分 类 号：R739.41[医药卫生—肿瘤] Q785[医药卫生—临床医学]