Construction of HER2-specific CAR-T cells and in vitro analysis of their activity to suppress tumor cell growth / 生物工程学报

CAR-T cell therapy that targets surface antigens to kill tumor cells specifically has recently become another cornerstone in tumor immunotherapy. In this study, a lentiviral expression plasmid of CAR targeting human epidermal growth factor receptor 2 (HER2) was constructed by genetic engineering. The recombinant plasmid was co-transfected with other packaging plasmids into HEK293T cells by calcium phosphate precipitation to generate lenti-car, which are CAR lentiviral particles. HER2-specific CAR-T cells were obtained by transducing human peripheral blood mononuclear cells with lenti-car. Their specific inhibitory effects on HER2-positive and HER2-negative tumor cells were analyzed in vitro. The constructed CAR-T cells were specifically activated by HER2-expressing tumor cells as indicated by secretion of IFN-γ and IL-2. The inhibitory rate on HER2-positive SK-OV-3 cell line was (58.47±1.72)%, significantly higher than that on the mock-treated control group (P<0.05). The inhibitory rate on HER2-negative K562 cell lines was (11.74±2.37)%, which was not significantly different from that on the control group (P>0.05). Furthermore, when we transfected a HER2-expressing vector into K562, the inhibitory rate increased to (30.41±7.59)%, which was higher than that on HER2-negative K562 (P<0.05). Thus, the constructed second-generation HER2-specific CAR-T cells specifically suppressed growth of tumor cells overexpressing HER2 protein, suggesting that HER2-specific CAR-T cells might prove useful for immunotherapy of HER2-positive cancer.

Effect of 20 and 22 amino acid on superantigen activity of staphylococcal enterotoxin C2 / 中国免疫学杂志

Objective:To investigate the improved superantigen activity of SEC2(T20L/G22E) compared with recombinant staphylococcal enterotoxins C 2 ( rSEC2 ).Methods: The proliferation of spleen lymphocytes and T-cell subpopulations induced by rSEC2 and SEC2(T20L/G22E) were examined by WST-1 and flow cytometry separately,and the gene expression of cytokines and Vβspecificities were quantified by real-time PCR.Results: WST-1 and Flow cytometry assays showed that the superantigen activity of SEC2(T20L/G22E) was improved due to enhanced T-cell stimulating potency,resulting in massive activation of T-cells,particularly CD4+and CD8+T-cells.Quantitative real-time PCR assay showed that despite similar Vβspecificities induced by rSEC 2 and SEC2 (T20L/G22E),the quantities of activated T-cells bearing specific Vβwere different,and SEC2(T20L/G22E) could stimulate more gene expression of associated cytokines simultaneously.Conclusion: The results strongly suggested that the increased SEC 2 ( T20L/G22 E)-TCR-binding affinity contributed to more T-cells activation and cytokine release ,which elicit powerful immune activition.

Effects of Zn2+and Zn2+-coordinating glutamic acid residues on the superantigenic activities of staph-ylococcal enterotoxin C2 / 中华微生物学和免疫学杂志

Objective To investigate the role of Zn 2+and Zn2+-coordinating glutamic acid residues ( 71Glu and 80Glu ) in the superantigenic activities of staphylococcal enterotoxin C2 ( SEC2 ) .Methods Over-lap PCR was used to amply genes encoding recombinant mutant proteins rSEC 2 ( E71A), rSEC2 (E80A) and rSEC2 (E71A/E80A).The mutant proteins were expressed in E.coli BL21 (DE3) and puri-fied by affinity chromatography .The differences of biological activities between rSEC 2 and its mutants were compared in vitro.The effects of Zn 2+on the superantigenic activities of rSEC 2 were evaluated by analyzing the proliferation of splenic lymphocytes in the presence or absence of ethylenediaminetetraacetic acid ( EDTA) .Results The substitution of glutamic acid residue at position 71 and 80 by alanine residue had no significant effects on the superantigenic activities and the conformational stability of rSEC 2 mutants.How-ever, traces of Zn2+(10μmol/L) could significantly enhance rSEC2-induced proliferation of splenic lympho-cytes, and only certain amount of Zn 2+could completely restore rSEC2-induced proliferation in the presence of EDTA.Conclusion This study indicated that mutations at Zn 2+-coordinating glutamic acid residues had no significant effects on the conformational stability and the superantigenic activities of SEC 2.Zn2+might play a role in regulating the superantigenic activities of SEC 2 through some indirect ways .

Enhanced SEC2 mutants and their superantigen activities / 生物工程学报

As a superantigen protein, Staphylococcal enterotoxin C2 (SEC2) activates the immune system effectively even in extremely low concentrations, and this property could be applied in adjuvant therapy against tumors and infectious diseases. In order to enhance the superantigen activity of SEC2, the residues at position 102-106 of native SEC2 were substituted for WWH, WWT and WWP by over-lap PCR, and three mutants named ST-1, ST-2 and ST-3 were obtained. Stimulating activity to murine lymphocytes proliferation and inhibiting activity to tumor cell growth of the three mutants were significantly improved compared with the native SEC2. Febrile activities of ST-1 and ST-3 were comparable with the native SEC2, but ST-2 showed markedly increased febrile activity than native SEC2. Moreover, the levels of IL-2, IFN-gamma and TNF-alpha secreted by T cells stimulated with the three mutants were significantly improved, which might be the possible reason for enhanced tumor cell growth inhibition activities. Furthermore, mVbeta8.2 gene transcription levels of murine splenocytes stimulated by the three mutants were dramatically increased compared with native SEC2, suggesting their increased affinities to TCR mVbeta8.2 molecular, which might be the main reason for their enhanced superantigen activities.

Fusion Immunotoxin Anti-HER-2-scFv-SEC2 Expressed in E.coli With an Improved Expression Vector pASK75-EX: Its Construction and Functioning / 生物化学与生物物理进展

A tumor-targeting recombinant fusion immunotoxin B-L-SEC2 was constructed by fusing staphylococcal enterotoxin C2 (SEC2) and an anti-HER-2 single-chain Fv B1 through a peptide linker, and expressed in E. coli strain BL21 (DE3) with an improved expression vector pASK75-EX as inclusion body. The denatured inclusion body was purified with Ni-NTA chelate agarose, and then re-natured by dialysis. FACS and MTT assays indicated that the re-natured fusion immunotoxin B-L-SEC2 could target the HER-2 over-expressing breast tumor cell SK-Br-3 in vitro, and inhibit the growth of SK-Br-3.