靶向核抗原的嵌合抗原受体T细胞构建及其分泌IFN-γ功能验证

Construction and IFN-γ secretion verification of chimeric antigen receptor T cells targeting nuclear antigen

目的构建靶向实体瘤微环境嵌合抗原受体T细胞(CAR-T细胞),诱导微环境中IFN-γ的表达,以达到抗肿瘤的目的。方法筛选核抗原特异性单克隆抗体,通过基因工程技术构建嵌合抗原受体(CAR);将CAR亚克隆到慢病毒载体后转染HEK293细胞,包装成表达CAR的慢病毒,转导HEK293F细胞后,以流式细胞术检测CAR表达细胞比例判断转导效率;将CAR表达慢病毒转导活化T细胞后形成CAR-T细胞,采用流式细胞术检测在不同抗原(核小体抗原、Brefeldin A、二者联合)诱导下或核小体抗原单独诱导0、2、48 h时CAR-T细胞分泌IFN-γ情况。结果在培养10~15 d时,表达CAR的HEK293F细胞比例达53.04%~87.17%。在核小体抗原、Brefeldin A、二者联合组诱导下,分泌IFN-γ的T细胞比例分别为0.028%、0.034%、3.570%;核小体抗原诱导0、48 h时检测不到分泌IFN-γ的CAR-T细胞,2 h时分泌IFN-γ的CAR-T细胞最多。结论成功构建靶向核抗原的CAR-T细胞,且该细胞具有IFN-γ分泌功能。

Objective To construct chimeric antigen receptor T cells(CAR-T cells)targeting solid tumor microenvironment to induce interferon-γ(IFN-γ)expression in microenvironment,and to achieve the purpose of anti-tumor effect.Methods The monoclonal antibody specific to nuclear antigen was screened out,the chimeric antigen receptor(CAR)was constructed by gene engineering technology,and was subcloned to lentivirus vector,which was then used to transfect into HEK293 cells.The lentivirus was packaged to express CAR,and was used to transduce into HEF293F cells.The transduction efficacy was analyzed by flow cytometry.The CAR-T cells were constructed after the lentivirus re-expressing CAR was transduced into the active T cells.We analyzed IFN-γ secretion of CAR-T cells after incubation for 0,2,and 48 h with nucleosome antigen,or with different antigens(nucleosome antigen,Brefeldin A,and both of them)by flow cytometry.Results The ratio of CAR expressing HEK293F cells reached 53.04%-87.17%after incubation for 10-15 h,that of IFN-γsecretion of CAR-T cells reached 0.028%,0.034%,and 3.57%in the presence of nucleosome antigen,Brefeldin A,and the combination of them.The IFN-γsecretion of CAR-T cells reached the peak at 2 h,and could not be detected at 0 and 48 h.Conclusion The CAR-T cells targeting nuclear antigen are successfully constrcuted with the function of IFN-γ secretion.