抗TREM2抗体的制备及生物学活性的初步研究  

Preparation of anti-human TREM2 antibody and preliminary study of its biological activity

作　　者：胡钰清 冯艳平 梁家蓓 郝锋 宁金鹰 李莉[1] HU Yu-qing;FENG Yan-ping;LIANG Jia-bei;HAO Feng;NING Jin-ying;LI Li(College of Pharmacy,Xinjiang Medical University,Urumqi 830017;KYinno Biotechnology Co.,Ltd.,Beijing 100010)

机构地区：[1]新疆医科大学药学院,乌鲁木齐830017 [2]康源博创(北京)生物科技有限公司,北京100010

出　　处：《中南药学》2024年第6期1429-1434,共6页Central South Pharmacy

基　　金：国家重点研发计划项目(No.2018YFC1708303)。

摘　　要：目的制备抗髓系细胞触发受体2(TREM2)抗体并初步探究其生物学活性。方法采用杂交瘤筛选的方法,获得稳定分泌抗TREM2抗体的杂交瘤细胞株,并对杂交瘤细胞株分泌的单克隆抗体进行筛选,选择结合效果最佳的单克隆抗体进行人源化改造得到嵌合抗体;对嵌合抗体进行进一步的人源化改造,采用酶联免疫吸附法(ELISA)及流式细胞术(FACS)检测人源化抗体的抗原抗体结合活性;采用生物膜干涉技术(BLI)检测人源化抗体与人TREM2结合动力学;通过基于报告基因的抗体依赖性细胞介导的细胞毒作用(ADCC)生物学活性测定方法,检测人源化抗体的依赖细胞介导的细胞毒效应;使用免疫缺陷小鼠对人源化抗体进行体内药效实验。结果利用杂交瘤技术成功筛选到稳定表达抗TREM2抗体的杂交瘤细胞株,对杂交瘤细胞株进行进一步人源化改造,获得纯度高于95%的人源化抗体h7B6-11,抗体h7B6-11与TREM2-His蛋白具有高度亲和性,且对293T-TREM2细胞的ADCC活性明显强于对照抗体。体内药效实验结果显示,h7B6-11与程序性死亡受体1(PD1)抗体联合用药抑制肿瘤生长效果明显。结论抗体h7B6-11具有高度亲和力和良好的生物学活性,有望开发成为新一代肿瘤免疫治疗的抗体药物。Objective To prepare antibodies against human triggering receptor expressed on myeloid cells(TREM2)and evaluate their bioactivity.Methods Monoclonal antibodies against human TREM2 were obtained by hybridoma technique,and the monoclonal antibodies with the best binding effect were selected and humanized to obtain chimeric antibodies.Subsequently,the chimeric antibodies were humanized,and the binding activity of the antibody to antigen were determined by enzyme-linked immunosorbent assay and flow cytometry.The binding kinetics of the humanized antibody to human TREM2 was detected by bio-layer interferometry.The cell-dependent cytotoxicity of the humanized antibody was evaluated with an antibody-dependent cell-mediated cytotoxicity(ADCC)biological activity assay based on the reporter gene.In vivo efficacy experiments were conducted in immunodeficient mice.Results The hybridoma cell lines stably expressing antibodies against TREM2 were successfully screened by hybridoma technology.The further humanized antibody,h7B6-11,had a purity over 95%and a high affinity with TREM2-His protein.The ADCC activity of the antibody h7B6-11 against 293T-TREM2 cells was much stronger than that of the control antibody.The in vivo efficacy experiments showed that the combination of h7B6/11 and programmed cell death protein 1(PD1)antibody greatly inhibited the tumor growth.Conclusion The antibodies possess high binding affinity and excellent bioactivity,which may become prospective therapeutic immunity drugs.

关 键 词：髓系细胞触发受体2 杂交瘤 单克隆抗体 人源化抗体 生物学活性 

分 类 号：R943[医药卫生—药剂学] R96[医药卫生—药学]