Specific cytotioxicity of EGFRvⅢ oriented chimeric antigen receptor-engineered T cells on EGFRvⅢ+ glioma U87 cells and the transplanted tumor in nude mice
10.3872/j.issn.1007-385X.2018.04.003
- VernacularTitle: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
- Author:
ZHENG Yan
1
,
2
;
XIE Jiabei
1
,
2
;
CAO Mingbo
1
,
2
;
ZHANG Bingyong
1
,
2
;
LI Xiuling
1
,
2
;
HAN Shuangyin
1
,
2
Author Information
1. (Stem Cell Research Center, People&rsquo
2. s HospitalAffiliated to Zhengzhou University, Zhengzhou 450003, Henan, China
- Publication Type:Journal Article
- Keywords:
EGFRvⅢ;
chimeric antigen receptor;
glioma;
U87 cell;
immunotherapy
- From:
Chinese Journal of Cancer Biotherapy
2018;25(4):334-339
- CountryChina
- Language:Chinese
-
Abstract:
[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 of NTT 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 effectagainstEGFRvⅢ+U87cellsbothinvitro and in vivo, providing basis for immunotherapyofgliomainfuture clinical use.
- Full text:20180403.pdf
