基于NFAT信号通路的抗LAG3抗体8F-6生物活性检测

Biological activity detection of the anti-LAG3 antibody 8F-6 based on a nuclear factor of activated T-cell(NFAT)signaling pathway

淋巴细胞活化基因3(LAG3)在T细胞活化和增殖过程中起负调控作用,通过单克隆抗体封闭LAG3分子可以增强T细胞对肿瘤的杀伤力,因此开发抗LAG3抗体的生物活性检测平台对免疫治疗具有重要意义。前期利用杂交瘤融合及抗体人源化改造技术,得到了一株全人源化的抗LAG3单克隆抗体8F-6,并基于活化T细胞核因子(NFAT)信号通路构建了Jurkat-NFAT-Luc2-LAG3和Jurkat-NFAT-Luc2-LAG3-CD3zeta细胞。本文利用所构建的细胞,通过酶联免疫吸附测定法(ELISA)和流式细胞技术对抗体8F-6进行亲和力及阻断效果检测。结果表明,抗体8F-6与LAG3分子具有良好的亲和力,可以阻断主要组织相容性复合体II(MHC II)分子与LAG3分子的结合;在报告细胞检测平台中,抗体8F-6在CHO-K1-FCGRIA细胞的协助下可以激活Jurkat-NFAT-Luc2-LAG3-CD3zeta细胞;在Daudi/Raji细胞、人金黄色葡萄球菌肠毒素E(SEE)和Jurkat-NFAT-Luc2-LAG3细胞的反应体系中,相比于对照抗体IgG1,抗体8F-6可以明显提高Jurkat-NFAT-Luc2-LAG3细胞的激活效果。

Lymphocyte activation gene-3(LAG3)plays a negative regulatory role in T cell activation and prolifera-tion.Blocking LAG3 molecules with monoclonal antibodies can enhance the cytotoxicity of T cells toward tumors.Bioactivity detection platforms for anti-LAG3 antibody are therefore of great significance for immunotherapy.In our earlier work,a fully humanized anti-LAG3 monoclonal antibody 8F-6 was obtained by hybridoma fusion and anti-body humanization technology.Jurkat-NFAT-Luc2-LAG3 and Jurkat-NFAT-Luc2-LAG3-CD3zeta cells were constructed based on a nuclear factor of activated T-cell(NFAT)signaling pathway.In this study,the affinity and blocking effect of antibody 8F-6 were detected by enzyme-linked immunosorbent assay(ELISA)and flow cytome-try using the previously constructed cells.The results showed that antibody 8F-6 had good affinity for the LAG3 molecule,and could block the binding of major histocompatibility complex II(MHC II)to LAG3.In the reporter cell detection platform,antibody 8F-6 could activate Jurkat-NFAT-Luc2-LAG3-CD3zeta cells with the assis-tance of CHO-K1-FCGRIA cells.In the reaction system containing Daudi/Raji cells,human Staphylococcus au-reus enterotoxin E(SEE)and Jurkat-NFAT-Luc2-LAG3 cells,antibody 8F-6 significantly improved the activa-tion effect of Jurkat-NFAT-Luc2-LAG3 cells compared with the control antibody IgG1.