重组抗TNF-α单克隆抗体ADCC生物学活性检测方法的建立  

Development of a method for determination of antibody-dependent cell-mediated cytotoxicity of recombinant monoclonal antibody against TNF-α

作　　者：吕丽娟 陈保汉 周冬梅 徐军 孙文正 杨彬 LV LI-juan CHEN Bao-han ZHOU Dong-mei XU Jun SUEN Wen-zheng YANG Bin(Sunshine Lake Pharma Co. , Ltd. , Dongguan 523867, Guangdong Province, Chin)

机构地区：[1]广东东阳光药业有限公司,广东东莞523867 [2]湖北广济药业股份有限公司,湖北武穴435400

出　　处：《中国生物制品学杂志》2017年第3期293-297,共5页Chinese Journal of Biologicals

基　　金：广东省引进创新科研团队计划资助项目(201101Y0104990178)

摘　　要：目的建立抗TNF-α单克隆抗体抗体依赖细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicity,ADCC)生物学活性检测方法。方法以健康人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)作为效应细胞,自主构建膜表面表达TNF-α(membrane-expressed form of TNF-α,m TNF-α)CHO细胞作为靶细胞,通过系列不同浓度的重组抗TNF-α单克隆抗体原研药(m Ab-O)和候选药(m Ab-S)进行ADCC检测,采用四参数拟合计算二者的半数抑制浓度(IC_(50))及m Ab-S的相对活性,并在优化实验条件的基础上进行专属性、精密度验证。结果重组抗TNF-α单克隆抗体的浓度与细胞毒性存在量效关系,符合四参数方程式,曲线在半对数坐标上呈典型S型,R2>0.99。方法经优化确定相同个体来源PBMC作为效应细胞,靶细胞数目4×10~4个/孔,效靶比25∶1,抗体靶细胞孵育时间1 h,诱导时间4 h。该方法具有良好的专属性,对于相同个体来源的PBMC,m Ab-S活性总体与m Ab-O相近(50%~200%),相对活性重复性RSD为4.64%,同一实验人员间隔3个工作日检测结果的RSD为12.50%,2名实验人员2次检测结果的RSD为8.21%,均符合方法验证的可接受标准。结论成功建立了重组抗TNF-α单克隆抗体ADCC生物学活性检测方法,该方法专属性强,精密度好,可作为重组抗TNF-α单克隆抗体与上市原研药相对ADCC生物学活性的评估方法。Objective To develop a method for determination of antibody-dependent cell-mediated cytotoxicity （ADCC） of recombinant monoclonal antibody （mAb） against tumor necrosis factor alpha （TNF-α）. Methods The ADCC of recombinant mAb against TNF-α was determined using serially diluted innovator original drug （mAb-O） drug or its biosimilar candidate （mAb-S） combined with normal human peripheral blood mononuelear cells （PBMCs） as effeetor cells and a CHO cell line expressing membrane bound TNF-α as target cells. The median inhibitory coneentration（IC50） of mAb and the relative biological activity of mAb-S were calculated by four-parameter equation. Under the optimized experimental condition, the method was verified for specificity and precision. Results The cytotoxieity of mAb against TNF-α was in a dose-dependent mode, which was consistent with the result acquired from the four-parameter equation. The dose-response curve was in a typical S shape on semilogarithmic coordinate paper, with a R2 value of more than 0. 99. The optimal condition of each critical parameter of this method when using PBMCs from the same individual source as effector cells was as follows： the number of target cells was 4 x 104 cells/well, while the ratio of effeetor cells to target cells was 25 ： 1, the incubation time of antibody with target cells was 1 h, and the induction time was 4 h. The method showed good specificity. The ADCC of mAb-S was similar to that of mAb-O （50% - 200% ） using PBMCs from the same individual source as effector cells, while the RSD in reproducibility test on relative activity was 4. 64%. However, the RSD of test results by one person in 2 working days at an interval of 3 d was 12. 50%, while than by two persons in the same working day was 8. 21%, both of which met the acceptance criteria for method validation. Conclusion A method for determination of ADCC of recombinant mAb against TNF-c~ was successfully developed, which showed high specificity and precision, and might be used for

关 键 词：肿瘤坏死因子 单克隆抗体 抗体依赖细胞介导的细胞毒性 生物学活性 

分 类 号：Q511[生物学—生物化学] R392.33[医药卫生—免疫学]