摘要
目的利用报告基因法建立抗人类表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)单抗的抗体依赖性细胞吞噬作用(antibody-dependent cell-mediated phagocytosis,ADCP)生物学活性检测方法。方法以Jurkat/NFAT/CD32a-FcεRIγ转基因细胞系作为效应细胞,SKBR3细胞系为靶细胞,通过荧光素酶检测系统建立抗HER2单抗的ADCP测活方法,并运用实验设计(design of experiment,DOE)方法进行实验优化和方法学验证。结果该方法量效关系符合四参数方程:y=(A-D)/[1+(x/C)^(B)]+D,方法优化确定量效范围为400~3.959 ng·mL(-1),靶细胞接种密度为每孔1.5×10^(4)个,效靶比为5∶1,诱导时间为7 h。该方法具有良好的专属性;不同稀释组样本百分相对效价分别为(55.04±1.35)%、(78.10±6.33)%、(102.69±5.21)%、(133.32±2.58)%和(152.87±8.11)%;回收率在101%~110%,相对标准偏差(RSD)均小于10%。结论本研究成功建立抗HER2单抗ADCP生物学活性方法,该方法专属性强、重复性好,准确性高,可作为抗HER2单抗ADCP生物学活性的评价方法。
OBJECTIVE To develop a detecting method for the antibody-dependent cell-mediated phagocytosis(ADCP)potency of anti-human epidermal growth factor receptor 2(HER2)monoclonal antibodies based on reporter genes.METHODS Using SKBR3 cell line as target cell and Jurkat/NFAT/CD32 a-FcεRIγtransgenic cell line as effector cell,the ADCP potency for anti-HER2 monoclonal antibodies was detected with luciferase detection system(BrightGlo^(TM)luciferase assay system),then the method was optimized and validated based on design of experiment(DOE).RESULTS The anti-HER2 monoclonal antibodies showed a dose-response relationship and the determination result complied with the following four-parameter equation:y=(A-D)/[1+(x/C)^(B)]+D.The method was optimized and the testing parameters were determined as follows:the working concentration of anti-HER2 monoclonal antibody was 400 to 3.959 ng·mL(-1),the target cell density was 1.5×10^(4)per well,the ratio of effector cells and target cells was 5∶1,and the induction time was 7 h.The method was proved to possess good specificity.The results showed that the relative potencies from different groups were(55.04±1.35)%,(78.10±6.33)%,(102.69±5.21)%,(133.32±2.58)%and(152.87±8.11)%,respectively,and their recoveries were from 101%to 110%.The RSDs of the above results were all less than 10%.CONCLUSION A method for detecting ADCP potency of anti-HER2 mAb is successfully developed,which shows high specificity,good reproducibility and high accuracy,and might be used in the evaluation of ADCP potency of anti-HER2 mAb.
作者
刘春雨
于传飞
付志浩
崔永霏
杨雅岚
王兰
LIU Chun-yu;YU Chuan-fei;FU Zhi-hao;CUI Yong-fei;YANG Ya-lan;WANG Lan(Division of Monoclonal Antibody,National Institutes for Food and Drug Control,NHC Key Laboratory of Research on Quality and Standardization of Biotech Products,NMPA Key Laboratory for Quality Research and Evaluation of Biological Products,Beijing 102629,China)
出处
《中国药学杂志》
CAS
CSCD
北大核心
2022年第23期1991-1997,共7页
Chinese Pharmaceutical Journal
基金
国家“重大新药创制”科技重大专项项目资助(2018ZX09736-016-007)
中山市重大科技专项项目资助(210204163866513)。
