基于NFAT信号通路的抗LAG3抗体8F-6生物活性检测

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。

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

Lymphocyte activation gene-3 (LAG3) plays a negative regulatory role in T cell activation and proliferation. 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 antibody 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 cytometry 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 assistance of CHO-K1-FCGRIA cells. In the reaction system containing Daudi/Raji cells, human Staphylococcus aureus enterotoxin E (SEE) and Jurkat-NFAT-Luc2-LAG3 cells, antibody 8F-6 significantly improved the activation effect of Jurkat-NFAT-Luc2-LAG3 cells compared with the control antibody IgG1.