Specific cytotoxicity of epidermal groth factor variantIII oriented chimeric antigen receptor-engineered T cells on SMMC-7721

Abstract

Objective To observe the specific killing effect of anti-epidermal groth factor variantⅢ (EGFRvⅢ) chimeric antigen receptor modified T cells (EGFRvⅢ/2CAR-T) on liver cancer cell SMMC-7721. Methods Expression cassette of EGFRvⅢ/2CAR (1 374 bp) was inserted into XbaⅠ and BamHⅠ sites of piggybac transposon vector PB513B-1 (PB-EGFRvⅢ/2CAR), the latter together with transposase plasmid was then transducted into human peripheral blood T cells by electroporation (1×20 ms, 840 V). The expression of EGFRvⅢ/2CAR on T cells was detected by flow cytometry. The expression of EGFRvⅢ in SMMC-7721 was detected by Western blotting. After EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721, the killing effect was detected by lactate dehydrogenase (LDH) release test and cytokine interferon (IFN)-γ secretion. Results The recombinant vector PB-EGFRvⅢ/2CAR was confirmed by XbaⅠ and BamHⅠ enzyme digestion, electrophoresis and DNA sequencing analysis. The expression of EGFRvⅢ/2CAR on T cells was detected by Flow cytometry (55.5%). Western blotting analysis showed that a protein band of approximate 145 000 molecular weight was observed in SMMC-7721 cells, which corresponds to the calculated molecular weight of EGFRvⅢ. EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721 for 24 h, LDH release test showed that tumor specific killing was positively correlated with E∶T ratio (E∶T=1∶1, 5∶1, 10∶1, 15∶1, 20∶1); enzyme linked immunosorbent assay (ELISA) showed that cytokine IFN-γ secretion was (1 060.1±89.0) pg/ml, which was significantly higher than control group (P=0.000). Conclusion Anti-EGFRvⅢ CAR-T cells can kill specifically EGFRvⅢ+ liver cancer cell SMMC-7721, providing research basis for immunotherapy of hepatic cancer. Key words: Epidermal groth factor variantⅢ; Chimeric antigen receptor; Immunotherapy; PiggyBac transposon