抗OX40/EGFR双特异性抗体的制备和活性鉴定  

Construction and activity identification of anti-OX40/anti-EGFR bispecific antibody

作　　者：晋瑞娜 边海波 张晓敏 杨帆 王晚萍 Jin Ruina;Bian Haibo;Zhang Xiaomin;Yang Fan;Wang Wanping(Department of Central Laboratory,Changzhi People s Hospital,Changzhi 046000,Shanxi Province,China;Department of Respiratory and Critical Care Medicine,Changzhi People s Hospital,Changzhi 046000,Shanxi Province,China)

机构地区：[1]长治市人民医院中心实验室,山西长治046000 [2]长治市人民医院呼吸与危重症学科,山西长治046000

出　　处：《首都医科大学学报》2024年第2期296-301,共6页Journal of Capital Medical University

基　　金：山西省自然科学基金面上项目(20210302124004);山西省卫健委项目(2022024)。

摘　　要：目的构建同时靶向表皮生长因子受体(epidermal growth factor receptor,EGFR)和OX40的双特异性抗体,并在体外初步验证其对T细胞的特异性激活作用。方法构建抗OX40/EGFR双特异性抗体的真核表达载体,转染293F细胞进行表达和纯化。构建外源表达OX40和EGFR的HEK293T细胞,利用流式细胞术分析双抗与靶蛋白表达细胞的结合活性。并利用Jurkat-OX40-NF-κB-GFP报告细胞,验证双特异性抗体对报告细胞的激活活性。通过酶联免疫吸附法(enzyme-linked immunosorbent assay,ELISA)检测外周血单个核细胞(peripheral blood mononuclear cell,PBMC)的白细胞介素-2(interleukin-2,IL-2)和γ-干扰素(interferon-γ,IFN-γ)分泌,进一步验证抗OX40/EGFR双抗对原代T细胞的激活。结果成功构建并且纯化了抗OX40/EGFR双特异性抗体,并且验证了该双抗可以特异性地结合表达EGFR和OX40的HEK293T细胞。在表达EGFR的A549肺癌细胞介导的交联作用下,该双特异性抗体较OX40单抗更强地激活OX40-NF-κB报告细胞,并且促进PBMC分泌IL-2和IFN-γ细胞因子。结论抗OX40/EGFR双特异性抗体构建成功,可同时识别OX40和EGFR受体并激活肿瘤特异性T细胞。Objective To construct a bispecific antibody targeting epidermal growth factor receptor(EGFR)and OX40 and evaluate the function for tumor-specific T cell activation.Methods The gene of anti-OX40/anti-EGFR bispecific antibody was cloned into eukaryotic expression vector,and then the constructed vector were transfected to 293F cells for the bispecific antibody purification.The binding activity of anti-OX40/anti-EGFR bispecific antibody with the cells expressing target proteins were detected by flow cytometry.To identify the activation of T cells mediated by anti-OX40/anti-EGFR bispecific antibody,the activation of NF-κB signal activation was evaluated by Jurkat-OX40-NF-κB-GFP reporter cells and the activation of primary T cells was detected by interleukin-2(IL-2)and interferon-γ(IFN-γ)secretion of peripheral blood mononuclear cell(PBMC).Results Anti-OX40/anti-EGFR bispecific antibody was successfully constructed and purified,and its binding ability to HEK293 cells expressing OX40 and EGFR was verified.Jurkat-OX40-NF-κB-GFP reporter cells were activated by the bispecific antibody with the crosslinking of A549 cells.Further,the anti-OX40/anti-EGFR bispecific antibody promoted the secretion of IL-2 and IFN-γof PBMC.Conclusion Anti-OX40/anti-EGFR bispecific antibody was successfully constructed which could specifically recognize OX40 and EGFR and activate tumor specific T cells.

关 键 词：OX40 表皮生长因子受体 双特异性抗体 癌症 T细胞激活 

分 类 号：R730.51[医药卫生—肿瘤]