中国肿瘤生物治疗杂志2018，Vol.25Issue(4)：334-339,6.DOI:10.3872/j.issn.1007-385x.2018.04.003
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
摘要
Abstract
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 EGFRv Ⅲ/3CAR,which was generated by calcium phosphate co-precipitation of three plasmids.The expression of EGFRvⅢ/3CAR in T cells was detected by Western blotting and flow cytometry.In vitro killing effect of EGFRvⅢ/3CAR-T cells on EGFRvⅢ+ U87 cells was detected by 51Cr release assay.The secretion of cytokine IFN-γ of EGFRvⅢ/3CAR-T cells was detected by ELISA.Nude mouse xenograft model was constructed to detect the in vivo cytotoxicity of EGFRvⅢ/3CAR-T cells on xenograft tumor.Results:The EGFRvⅢ/ 3CAR lentivirus was successfully packaged with an average titer of 5 × 106 TU/ml.Western blotting showed that a protein band of approximate 58 000 molecular weight was observed in EGFRvⅢ/3CAR-T cells but absent in untransfected T cells.Flow cytometry indicated the average transduction efficiency of EGFRvⅢ/3CAR was 52.3％.51Cr release assay showed that the specific killing effect of EGFRvⅢ/3CAR-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 ofNT T and GFP+ T cells (P＜0.01).The specific killing activity of EGFRvⅢ/3CAR-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/3CAR-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/3CAR-T cells demonstrated specific antitumor effect against EGFRvⅢ+ U87 cells both in vitro and in vivo,providing basis for immunotherapy ofglioma in future clinical use.关键词
EGFRvⅢ/嵌合抗原受体/胶质瘤/U87细胞/免疫治疗Key words
EGFRvⅢ/chimeric antigen receptor/glioma/U87 cell/immunotherapy分类
医药卫生引用本文复制引用
郑岩,谢甲贝,曹名波,张炳勇,李修岭,韩双印..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[J].中国肿瘤生物治疗杂志,2018,25(4):334-339,6.基金项目
国家自然科学基金资助项目(No.81372405,No.81772670).Project supported by the National Natural Science Foundation of China (No.81372405,No.81772670) （No.81372405,No.81772670）
