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

刘春雨, 于传飞, 崔永霏, 肖启东, 黄璟, 王兰

中国药学杂志 ›› 2019, Vol. 54 ›› Issue (24) : 2010-2017.

PDF(2083 KB)
PDF(2083 KB)
中国药学杂志 ›› 2019, Vol. 54 ›› Issue (24) : 2010-2017. DOI: 10.11669/cpj.2019.24.002
单克隆抗体药物分析方法验证专栏

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

  • 刘春雨, 于传飞, 崔永霏, 肖启东, 黄璟, 王兰
作者信息 +

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

  • LIU Chun-yu, YU Chuan-fei, CUI Yong-fei, XIAO Qi-dong, HUANG Jing, WANG Lan
Author information +
文章历史 +

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(BrightGloTMLuciferase 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。

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 (BrightGloTM 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.

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

Key words

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

引用本文

导出引用
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 https://doi.org/10.11669/cpj.2019.24.002
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[J]. Chinese Pharmaceutical Journal, 2019, 54(24): 2010-2017 https://doi.org/10.11669/cpj.2019.24.002
中图分类号: R944   

参考文献

[1] ISHIDA Y, AGATA Y, SHIBAHARA K, et al. Induced expression of PD-1, a novel member of the immunoglobulin gene superfamily, upon programmed cell death [J]. EMBO J, 1992,11(11):3887-3895.
[2] KEIR M E, BUTTE M J, FREEMAN G J, et al. PD-1 and its ligands in tolerance and immunity [J]. Annu Rev Immunol, 2008,26:677-704.
[3] DONG H, ZHU G, TAMADA K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion [J]. Nat Med, 1999,5(12):1365-1369.
[4] FREEMAN G J, LONG A J, IWAI Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation [J]. J Exp Med, 2000,192(7):1027-1034.
[5] ANSELL S M. Nivolumab in the treatment of hodgkin lymphoma [J]. Clin Cancer Res, 2017,23(7):1623-1626.
[6] WOLCHOK J D, KLUGER H, CALLAHAN M K, et al. Nivolumab plus ipilimumab in advanced melanoma [J]. N Engl J Med, 2013,369(2):122-133.
[7] OZONO S. Nivolumab (anti-PD-1 antibody; ONO-4538/BMS-936558) in renal cancer [J]. Gan To Kagaku Ryoho, 2014,41(9):1077-1080.
[8] DEEKS E D. Nivolumab: a review of its use in patients with malignant melanoma [J]. Drugs, 2014,74(11):1233-1239.
[9] POOLE R M. Pembrolizumab: first global approval [J]. Drugs, 2014,74(16):1973-1981.
[10] MARKHAM A. Atezolizumab: first global approval [J]. Drugs, 2016,76(12):1227-1232.
[11] INMAN B A, LONGO T A, RAMALINGAM S, et al. Atezolizumab: a PD-L1-blocking antibody for bladder cancer [J]. Clin Cancer Res, 2017,23(8):1886-1890.
[12] HAYES J M, WORMALD M R, RUDD P M, et al. Fc gamma receptors: glycobiology and therapeutic prospects [J]. J Inflamm Res, 2016,9:209-219.
[13] CRESCIOLI S, CORREA I, KARAGIANNIS P, et al. IgG4 Characteristics and functions in cancer immunity [J]. Curr Allergy Asthma Rep, 2016,16(1):7-11.
[14] BEERS S A, GLENNIE M J, WHITE A L. Influence of immunoglobulin isotype on therapeutic antibody function [J]. Blood, 2016,127(9):1097-1101.
[15] BARNHART B C, QUIGLEY M. Role of Fc-FcgammaR interactions in the antitumor activity of therapeutic antibodies [J]. Immunol Cell Biol, 2017,95(4):340-346.
[16] PAREKH B S, BERGER E, SIBLEY S, et al. Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay [J]. mAbs, 2012,4(3):310-318.
[17] CHUN-YU L, XIN W, CHUAN-FEI Y. Development and application of ADCC biological activity assay method of anti-HER2 monoclonal antibody based on reporter genes [J]. Chin J Pharm Anal(药物分析杂志),2019,39(1):51-61.
[18] LEE J I, ZHANG L, MEN A Y, et al. CYP-Mediated therapeutic protein-drug interactions: clinical findings, proposed mechanisms and regulatory implications [J]. Clin Pharmacokinet, 2010,49(5):295-310.
[19] MILLER A S, TEJADA M L, GAZZANO-SANTORO H. Methods for measuring antibody-dependent cell-mediated cytotoxicity in vitro [J]. Methods Mol Biol, 2014,1134:59-65.
[20] MATA M M, MAHMOOD F, SOWELL R T, et al. Effects of cryopreservation on effector cells for antibody dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell activity in (51)Cr-release and CD107a assays [J]. J Immunol Methods, 2014,406:1-9.
[21] RAJU T S. Terminal sugars of Fc glycans influence antibody effectorfunctions of IgGs [J]. Curr Opin Immunol, 2008, 20(4):471-478.
[22] PEREIRA N A, CHAN K F, LIN P C, et al. The “less-is-more” in therapeutic antibodies: afucosylated anti-cancer antibodieswith enhanced antibody-dependent cellular cytotoxicity [J]. mAbs, 2018, 10(5):693-711.

基金

国家“重大新药创制”科技重大专项项目资助(2018ZX09101001-003)
PDF(2083 KB)

356

Accesses

0

Citation

Detail

段落导航
相关文章

/