基于实验设计的抗PD-1/PD-L1单抗报告基因抗体依赖细胞介导的细胞毒效应生物学活性优化及验证方法的建立

doi:10.11669/cpj.2019.24.002

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摘要目的利用实验设计(design of experiment,DOE)建立抗程序性死亡因子1(programmed cell death 1,PD-1)及其配体(programmed cell death-ligand 1,PD-L1)单抗基于报告基因的抗体依赖细胞介导的细胞毒效应(antibody-dependent cell-mediated cytotoxicity, ADCC)生物学活性优化及验证方法。方法利用Jurkat-hFcγRⅢa-NFAT转基因细胞系作为效应细胞,分别以293FT-PD-1细胞系和CHO-PD-L1细胞系作为靶细胞,通过荧光素酶检测系统(BrightGlo^TMLuciferase Assay system)建立抗PD-1/PD-L1单抗的ADCC生物学活性检测方法,并运用DOE方法进行试验优化和方法学验证。结果抗PD-1/PD-L1单抗在该方法中存在量效关系,且符合四参数方程:y=(A-D)/+D,方法经优化后确定抗体量效范围分别为10 000~4.833 ng·mL^-1和2 000~0.488 ng·mL^-1,效靶比(effect target ratio, E∶T)为分别为6∶1和3∶1,诱导时间均为20 h。该方法具有良好的专属性;抗PD-1单抗4个不同稀释组回收率样本经3次检测,相对效价分别为(51.74±2.22)%、(77.12±3.14)%、(118.71±2.83)%和(156.20±12.99)%;对应的回收率分别为(103.49±4.44)%、(102.83±4.19)%、(94.96±2.26)%和(104.14±8.66)%,抗PD-L1单抗4个不同稀释组回收率样本经3次检测,相对效价分别为(54.32±4.75)%、(75.24±4.25)%、(127.40±2.43)%和(156.82±3.27)%;对应的回收率分别为(108.64±9.51)%、(100.33±5.67)%、(101.92±1.94)%和(104.55±2.18)%,上述结果的RSD均小于10%。结论本实验利用DOE设计成功建立基于报告基因的抗PD-1/PD-L1单抗ADCC生物学活性的优化及验证方法,该方法专属性强、重复性好,准确性高,可作为抗PD-1/PD-L1单抗ADCC生物学活性的评价方法。

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	刘春雨
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	黄璟
	王兰

关键词 ：实验设计, 抗PD-1/PD-L1单抗, 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, 报告基因, 优化, 验证

Abstract：OBJECTIVE To develop a novel optimization and validation method based on design of experiment(DOE) for the antibody-dependent cell-mediated cytotoxicity (ADCC) potency of anti-programmed cell death 1 and anti-programmed cell death-ligand 1 (PD-1/PD-L1) monoclonal antibodies using reporter genes. METHODS Jurkat-hFcγRⅢa-NFAT transgenic cell line was used as effector cells, 293FT-PD-1 cell line and CHO-PD-L1 cell line were used as target cells, respectively. The ADCC potency for anti-PD-1/PD-L1 monoclonal antibodies was detected with Luciferase detection system (BrightGlo^TM Luciferase Assay system), then the method was optimized and validated based on DOE. RESULTS The anti-PD-1/PD-L1 monoclonal antibodies showed a dose-response relationship and the determination result complied with the following four-parameter equation: y=(A-D)/+D. The method was optimized and the testing parameters were determined as follows: the working concentration of anti-PD-1 monoclonal antibody was 10 000 ng·mL^-1 to 4.833 ng·mL^-1 and that of anti-PD-L1 was 2 000 ng·mL^-1 to 0.488 ng·mL^-1, the ratio of effector cells and target cells for anti-PD-1/PD-L1 monoclonal antibodies were 6∶1 and 3∶1, and the induction time for both of these antibodies was 20 h. The method possessed good specificity. The recovery rate test samples in the four different dilution groups were determined for 3 times, and the results showed that the relative potencies of anti-PD-1 monoclonal antibody were (51.74±2.22)%, (77.12±3.14)%, (118.71±2.83)% and (156.20±12.99)%, and the recoveries of which were (103.49±4.44)%, (102.83±4.19)%, (94.96±2.26)% and (104.14±8.66)%, respectively. While as for anti-PD-L1 monoclonal antibody, the relative potencies were (54.32±4.75)%, (75.24±4.25)%, (127.40±2.43)%, (156.82±3.27)% and the recoveries were (108.64±9.51)%, (100.33±5.67)%, (101.92±1.94)% and (104.55±2.18)%, respectively. The RSDs of the above results were all less than 10%. CONCLUSION A novel optimization and validation method based on DOE for detecting ADCC potency of anti-PD-1/PD-L1 mAb is successfully developed. This detecting method based on reporter gene shows high specificity, good reproducibility and high accuracy, and might be used in the evaluation of ADCC potency of anti-PD-1/PD-L1 mAb.

Key words： DOE anti-PD-1/PD-L1 mAb ADCC potency reporter gene optimization validation

收稿日期: 2019-08-08

PACS:

R944

基金资助:国家“重大新药创制”科技重大专项项目资助(2018ZX09101001-003)

通讯作者: 王兰,女,研究员研究方向:抗体类生物治疗药物质量控制 Tel:(010)53852159 E-mail:wanglan@nifdc.org.cn

作者简介: 刘春雨,女,副研究员研究方向:抗体类生物治疗药物质量控制;于传飞,男,副研究员研究方向:抗体类生物治疗药物质量控制

引用本文:

刘春雨, 于传飞, 崔永霏, 肖启东, 黄璟, 王兰. 基于实验设计的抗PD-1/PD-L1单抗报告基因抗体依赖细胞介导的细胞毒效应生物学活性优化及验证方法的建立[J]. 中国药学杂志, 2019, 54(24): 2010-2017. LIU Chun-yu, YU Chuan-fei, CUI Yong-fei, XIAO Qi-dong, HUANG Jing, WANG Lan. Development of a Novel Optimization and Validation Method Based on DOE for the ADCC Potency of Anti-PD-1/PD-L1 Monoclonal Antibodies Using Reporter Genes. Chinese Pharmaceutical Journal, 2019, 54(24): 2010-2017.

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http://journal11.magtechjournal.com/Jwk_zgyxzz/CN/10.11669/cpj.2019.24.002 或 http://journal11.magtechjournal.com/Jwk_zgyxzz/CN/Y2019/V54/I24/2010

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