基于报告基因的抗CD20单克隆抗体ADCC生物学活性测定方法的建立

Development of a novel reporter gene method for determination of ADCC potency of anti-CD20 monoclonal antibody

利用Jurkat/NFAT-luc+FcγRIIIa转基因细胞系作为效应细胞,WIL2-S细胞系作为靶细胞,通过荧光素酶检测系统(Bio GloTM Luciferase Assay System)进行抗CD20单抗的ADCC生物学活性检测,并对实验条件进行优化及方法学验证。结果显示抗CD20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。方法经优化确定靶细胞为WIL2-S细胞,抗体稀释浓度为18 000 ng·m L-1,1∶5倍的稀释倍数,效靶比为6∶1,诱导时间为6 h。该方法具有良好的专属性,8次独立实验的回归分析、线性及平行性均通过统计学检验;4个不同稀释组回收率样本经3次测定,相对效价分别为(44.39±3.93)%、(72.74±2.78)%、(128.28±7.01)%和(168.19±2.70)%,变异系数均小于10%,对应回收率分别为(88.78±7.85)%、(96.99±3.70)%、(102.63±5.61)%和(112.12±1.80)%。本研究利用转基因细胞法成功建立抗CD20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CD20单抗ADCC生物学活性的常规检测方法。

The biological activity of ADCC by anti-CD20 monoclonal antibody was determined by BioGlo Tra Luciferase Assay System using Jurkat/NFAT-luc＋FcTRIIIa cell line as effector cell and WIL2-S cell line as target cell. The developed method was verified for specificity, precision and accuracy. Anti-CD20 monoclonal antibody showed a dose-response mode by the developed method, and the determination result complied with the following four-parameter equation： y= （A-D） / [ 1 ＋ （X/C）B] ＋D. The optimized parameters of the method were determined including the antibodies diluted concentration （18 000 ng·mL^-l）, dilution rate （1 ： 5）, the ratio of effector cell and target cell （6 ： 1）, and induction time （6 h）. The values of eight independent tests have passed a statistical test for curve regression analysis, linear or parallelism, which showed the method possessed good specificity. Four different dilute groups of recovery rates sample were determined for 3 times, and the result showed mean relative potencies of （44.39±3.93）%, （72.74±2,78）%, （128.28±7.01）% and （168.19±2.70）% respectively, with a variation coefficient of less than 10%, and the recoveries of （88.78 ±7.85）%, （96.99±3.70）%,（102.63± 5.61）% and （112.12 ± 1.80）% respectively. A novel reporter gene method for determination of biological activity of ADCC by anti-CD20 monoclonal antibody was successfully developed, which showed strong specificity, good reproducibility and high accuracy, and might be used routinely.