基于报告基因的抗HER2单克隆抗体ADCC生物学活性测定方法的建立及应用  

Development and application of ADCC biological activity assay method of anti-HER2 monoclonal antibody based on reporter genes

作　　者：刘春雨[1] 王馨 于传飞[1] 徐刚领 王兰[1] LIU Chun-yu;WANG Xin;YU Chuan-fei;XU Gang-ling;WANG Lan(Division of Monoclonal Antibody,National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 102629,China;Fujian Institute for Medical Device and Pharmaceutical Packaging Material Control,Fuzhou 350001,China)

机构地区：[1]中国食品药品检定研究院单克隆抗体产品室卫生部生物技术产品检定方法及标准化重点实验室,北京102629 [2]福建省医疗器械与药品包装材料检验所,福州350001

出　　处：《药物分析杂志》2019年第1期51-61,共11页Chinese Journal of Pharmaceutical Analysis

基　　金：国家"重大新药创制"科技重大专项资助项目(No.2018ZX09736016-007)

摘　　要：目的:利用转基因细胞法建立抗人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)单抗的抗体依赖细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity,ADCC)生物学活性检测方法。方法:利用Jurkat-hFcγRⅢa-NFAT转基因细胞系作为效应细胞,SKBR3细胞系作为靶细胞,通过荧光素酶检测系统(BioGloTM Luciferase Assay system)进行抗HER2单抗的ADCC生物学活性检测,同时对试验条件进行优化及方法学验证,并将该检测方法用于不同类型的抗HER2单抗ADCC效应的评价。结果:抗HER2单抗在该方法中存在量效关系,且符合四参数方程式:y=(A-D)/[1+(x/C)~B]+D,方法经优化后确定靶细胞为SKBR3细胞,抗体稀释起始工作质量浓度为4 000 ng·mL^(-1),1∶4倍的稀释倍数,效靶比为15∶1,诱导时间为20 h。该方法具有良好的专属性;3次独立检测的板间、日间最大诱导倍数(fold of induction,FI)及半数有效浓度(concentration for 50%of maximal effect,EC50)的RSD均小于10%; 4个不同稀释组回收率样本经3次测定,相对效价分别为(46.27±2.01)%、(71.18±1.55)%、(133.17±9.91)%以及(166.55±15.73)%;对应的回收率分别为(92.53±4.01)%、(94.91±2.07)%、(106.54±7.93)%、(111.04±10.48),RSD均小于10%,且该方法也适用于不同变性程度及各类抗HER2单抗的ADCC效应评价。结论:本研究利用转基因细胞法成功建立抗HER2单抗ADCC生物学活性检测方法,该方法专属性强、重复性好,准确性高,可作为抗HER2单抗ADCC生物学活性的常规检测方法。Objective:To establish method for determining the antibody-dependent cell-mediated cytotoxicity(ADCC) of anti-human epidermal growth factor receptor 2(HER2) monoclonal antibody based on luciferase reporter gene-modified cell assay. Methods:Jurkat-h FcγRⅢa-NFAT transgenic cells and SKBR3 cells were used as effector cells and target cells,respectively. The ADCC activity of anti-HER2 monoclonal antibodies was measured by luciferase detection system(BioGloTM Luciferase Assay system),and the test parameters were optimized,followed by methodology validation. The performance of the established method for ADCC effect detection was further confirmed in assays on different types of anti-HER2 monoclonal antibodies. Results:The ADCC of anti-HER2 monoclonal antibodies showed dose-response pattern as suggested from the data given by the established method,which complied with the following four-parameter equation:y=(A-D)/[1+(x/C)B]+D. The optimized parameters that were determined in the method include the diluted working concentration(4 000 ng·mL^-1) of the antibodies,the dilution rate(1∶4),the ratio of effector cells over target cell(15∶1),and the induction time(20 h). The method also exhibited good specificity confirmed by three parallel tests,in which the RSD of plate-to-plate assay and day-to-day assay of maximum fold of induction(FI),as well as the RSD of the concentration for 50% of maximal effect(EC50),were less than 10%. Four different diluting groups of the recovery rates sample were examined 3 times using the method,the data of which showed mean relative potencies of(46.27±2.01)%,(71.18±1.55)%,(133.17±9.91)% and(166.55±15.73)%,respectively,and the recoveries of(92.53±4.01)%,(94.91±2.07)%,(106.54±7.93)%,(111.04±10.48)%,respectively,with both RSDs less than 10%. Moreover,this method could evaluate the ADCC effect of anti-HER2 monoclonal antibodies with various degrees of degeneration,so it may be adaptable for the majority of anti-HER2 monoclonal antibody classes. Conclusion:In this study,a luciferase re

关 键 词：抗人表皮生长因子受体2单克隆抗体 抗体依赖细胞介导的细胞毒性作用 生物学活性 荧光素酶检测系统 报告基因 

分 类 号：R917[医药卫生—药物分析学]