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Archive > Volume 25 Issue 4 > 2018,25(4):334-339. DOI:10.3872/j.issn.1007-385X.2018.04.003 Prev Next
Basic Research
Specific cytotioxicity of EGFRvⅢ oriented chimeric antigen receptor-engineered T cells on EGFRvⅢ + glioma U87 cells and the transplanted tumor in nude mice
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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 de- tected by 51 Cr release assay. The secretion of cytokine IFN-γ of EGFRvⅢ/3CAR-T cells was detected by ELISA. Nude mouse xeno- graft 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 5×10 6 TU/ml. Western blotting showed that a protein band of ap- proximate 58 000 molecular weight was observed in EGFRvⅢ/3CAR-T cells but absent in untransfected T cells. Flow cytometry indi- cated the average transduction efficiency of EGFRvⅢ/3CAR was 52.3%. 51 Cr 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 se- cretion 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 ac- tivity of EGFRvⅢ/3CAR-T cells and IFN-γ secretion were both dependent on the expression level of EGFRvⅢ in U87 cells. The tu-mor 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.
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Project Supported:
Project supported by the National Natural Science Foundation of China (No. 81372405, No. 81772670)
Clc Number:
