基于报告基因的抗CD3×GPRC5D双特异性抗体生物学活性测定方法建立  

Development and validation of a reporter gene-based bioassay for a bispecific antibody targeting CD3 and GPRC5D

作　　者：李娜 吴振华 梅小芬 柳颖 陈瑶 陈娟 蒋美珠 王海彬 LI Na;WU Zhen-hua;MEI Xiao-fen;LIU Ying;CHEN Yao;CHEN Juan;JIANG Mei-zhu;WANG Hai-bin(BioRay Pharmaceutical Co.,Ltd.,Taizhou 318000,China)

机构地区：[1]浙江博锐生物制药有限公司,台州318000

出　　处：《中国新药杂志》2022年第15期1474-1479,共6页Chinese Journal of New Drugs

摘　　要：目的:建立抗CD3×GPRC5D双特异性抗体(bispecific antibody, BsAb)生物学活性的报告基因测定方法。方法:构建Jurkat/NFAT-Luc转基因细胞株作为效应细胞,MM.1R细胞系作为靶细胞,建立和优化抗CD3×GPRC5D双特异性抗体生物学活性检测方法并进行方法学验证。分别用优化后的报告基因法和PBMC杀伤试验评价不同类型的抗CD3×GPRC5D双特异性抗体生物学活性。结果:报告基因法优化后确定靶细胞为MM.1R细胞,细胞铺板密度为2×10^(4)个·孔^(-1),效靶比1∶1,抗体起始浓度为20μg·mL^(-1),2倍稀释1次后,再6倍梯度稀释,共10个浓度点,诱导时间6 h。方法验证结果说明该方法具有良好的专属性,制备50%,75%,100%,150%和200%理论效价样品,进行准确性、精密度和线性验证,结果显示生物学活性回收率在97.85%~106.51%,变异系数均<10%;相对效价理论值与实测值直线回归分析相关系数为0.997 8。同时,该方法适用于各类抗CD3×GPRC5D双特异性抗体,均有较好的剂量反应曲线,且与PBMC杀伤试验结果具有较好的一致性。结论:本研究成功建立抗CD3×GPRC5D双特异性抗体生物学活性的报告基因检测方法,该方法具有较高的准确性和精密度,且特异性良好,可作为此类双特异性抗体活性评价的常规方法。Objective: To establish a reporter gene assay(RGA) for the determination of the biological activity of anti-CD3×GPRC5 D bispecific antibody(BsAb). Methods: A Jurkat/NFAT-Luc cell line that stably expresses luciferase gene driven by NFAT response element was constructed as effector cell, and MM.1 R cell line was used as the target cell. The RGA was established and optimized, followed by method validation. To confirm the suitability of the method, the biological activity of different types of anti-CD3×GPRC5 D BsAbs was determined by the RGA assay and PBMC cytotoxicity test. Results: The optimized experimental parameters of RGA were determined. The plating density of MM.1 R cell was 2×10^(4) cells·well^(-1) and the ratio of effector to target(E∶T) was 1∶1. The initial concentration of BsAb was 20 μg·mL^(-1), then BsAb was diluted by 1∶2 for once and 1∶6 for 8 times to obtain 10 gradients. The incubation time was 6 h. The specificity of the method is good. The relative activity was calculated and compared with the theoretical titer at 50%,75%,100%,150% and 200%. The recovery rate of the method was between 97.85 % and 106.51%, and the coefficient of variation(CV) values were less than 10%. A good linear relationship between expected relative potency values and the observed potency values was observed(R^(2) value 0.997 8). The biological activity of different types of anti-CD3×GPRC5 D BsAbs was evaluated by this method and the results were consistent with the PBMC killing test. Conclusion: The RGA was successfully established for determination the biological activity of anti-CD3×GPRC5 D BsAb. The qualification results demonstrated that the assay had good linearity, precision and accuracy.

关 键 词：抗CD3×GPRC5D双特异性抗体 生物学活性 报告基因法 

分 类 号：R927[医药卫生—药学]