ABSTRACT
@#Objective: To explore the gene transduction method of chimeric antigen receptor (CAR) mediated by novel cationic polymer nanocarrier mPEG-P (Asp-AED-g-HFB) (PAEF) and PigyBac transposon system to modify natural killer (NK) cells, providing a new strategy for immunotherapy of cancer cells. Methods: PAEF/DNA (transposase+transposon) complex were prepared. The particle size distribution and surface potential of PAEF/DNA complexes were measured with Nano-ZSE Dynamic Light Scattering System (Malvern Instruments). The DNA encapsulation rate, release and stability of PAEF were evaluated by DNA gel electrophoresis, and then by combiningwithparticlesizeandsurfacepotentialtodeterminethepreferentialN/PratiotoenterNKcells.Thecell cytotoxicity of PAEF/DNA complexes under different N/P ratios was analyzed by CCK-8 cytotoxicity test. Transduction efficiency of NK cells was evaluated by Fluorescence microscopy and Flow cytometry, and the feasibility of PAEF gene transfection vectors was assessed. Results: PAEF could encapsulate DNA to form nano-complexes with the diameter of 100-150 nm, which was suitable to mediate DNA entering into cells. PAEF could completely encapsulate DNA with N/P ratio of 20. In the presence of reducing agent dithiothreitol (DTT), PAEF had a good ability to release DNA. NK-92 cells transfected with PAEF/DNA complex, which was formed at the N/P ratio of 80, attained a significantly higher cell viability than cells of lipofectamine transfection group [(72.50±3.9)% vs (64.03±1.8)%, P<0.05]; Fluorescence microscopic observation showed more fluorescence and higher fluorescence intensity in cells of PAEF/DNA group; Flow cytometry showed the highest transfection efficiency of 83.4%. Conclusions: Nanocarrier PAEF can encapsulate DNA well by electrostatic adsorption, and has good biocompatibility and high efficiency for gene transduction. It provides a good experimental basis for adoptive immunotherapy.
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.