基于报告基因的抗HER2单克隆抗体ADCC生物学活性测定方法的建立及应用
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摘要
目的:利用转基因细胞法建立抗人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)单抗的抗体依赖细胞介导的细胞毒作用(antibody-dependent cell-mediated cytotoxicity,ADCC)生物学活性检测方法。方法:利用Jurkat-hFcγRⅢa-NFAT转基因细胞系作为效应细胞,SKBR3细胞系作为靶细胞,通过荧光素酶检测系统(BioGloTM Luciferase Assay system)进行抗HER2单抗的ADCC生物学活性检测,同时对试验条件进行优化及方法学验证,并将该检测方法用于不同类型的抗HER2单抗ADCC效应的评价。结果:抗HER2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,方法经优化后确定靶细胞为SKBR3细胞,抗体稀释起始工作质量浓度为4 000 ng·mL~(-1),1∶4倍的稀释倍数,效靶比为15∶1,诱导时间为20 h。该方法具有良好的专属性;3次独立检测的板间、日间最大诱导倍数(fold of induction,FI)及半数有效浓度(concentration for 50%of maximal effect,EC50)的RSD均小于10%; 4个不同稀释组回收率样本经3次测定,相对效价分别为(46.27±2.01)%、(71.18±1.55)%、(133.17±9.91)%以及(166.55±15.73)%;对应的回收率分别为(92.53±4.01)%、(94.91±2.07)%、(106.54±7.93)%、(111.04±10.48),RSD均小于10%,且该方法也适用于不同变性程度及各类抗HER2单抗的ADCC效应评价。结论:本研究利用转基因细胞法成功建立抗HER2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HER2单抗ADCC生物学活性的常规检测方法。
Objective:To establish method for determining the antibody-dependent cell-mediated cytotoxicity(ADCC) of anti-human epidermal growth factor receptor 2(HER2) monoclonal antibody based on luciferase reporter gene-modified cell assay. Methods:Jurkat-h FcγRⅢa-NFAT transgenic cells and SKBR3 cells were used as effector cells and target cells,respectively. The ADCC activity of anti-HER2 monoclonal antibodies was measured by luciferase detection system(BioGloTM Luciferase Assay system),and the test parameters were optimized,followed by methodology validation. The performance of the established method for ADCC effect detection was further confirmed in assays on different types of anti-HER2 monoclonal antibodies. Results:The ADCC of anti-HER2 monoclonal antibodies showed dose-response pattern as suggested from the data given by the established method,which complied with the following four-parameter equation:y=(A-D)/[1+(x/C)~B]+D. The optimized parameters that were determined in the method include the diluted working concentration(4 000 ng·mL~(-1)) of the antibodies,the dilution rate(1∶4),the ratio of effector cells over target cell(15∶1),and the induction time(20 h). The method also exhibited good specificity confirmed by three parallel tests,in which the RSD of plate-to-plate assay and day-to-day assay of maximum fold of induction(FI),as well as the RSD of the concentration for 50% of maximal effect(EC50),were less than 10%. Four different diluting groups of the recovery rates sample were examined 3 times using the method,the data of which showed mean relative potencies of(46.27±2.01)%,(71.18±1.55)%,(133.17±9.91)% and(166.55±15.73)%,respectively,and the recoveries of(92.53±4.01)%,(94.91±2.07)%,(106.54±7.93)%,(111.04±10.48)%,respectively,with both RSDs less than 10%. Moreover,this method could evaluate the ADCC effect of anti-HER2 monoclonal antibodies with various degrees of degeneration,so it may be adaptable for the majority of anti-HER2 monoclonal antibody classes. Conclusion:In this study,a luciferase reporter gene-modified cell based bioassay for measuring the ADCC of anti-HER2 monoclonal antibodies was successfully establishes,with adequate specificity,high accuracy and good repeatability. This method can be a used as an routine detection method of ADCC of anti-HER2 monoclonal antibodies.
Objective:To establish method for determining the antibody-dependent cell-mediated cytotoxicity(ADCC) of anti-human epidermal growth factor receptor 2(HER2) monoclonal antibody based on luciferase reporter gene-modified cell assay. Methods:Jurkat-h FcγRⅢa-NFAT transgenic cells and SKBR3 cells were used as effector cells and target cells,respectively. The ADCC activity of anti-HER2 monoclonal antibodies was measured by luciferase detection system(BioGloTM Luciferase Assay system),and the test parameters were optimized,followed by methodology validation. The performance of the established method for ADCC effect detection was further confirmed in assays on different types of anti-HER2 monoclonal antibodies. Results:The ADCC of anti-HER2 monoclonal antibodies showed dose-response pattern as suggested from the data given by the established method,which complied with the following four-parameter equation:y=(A-D)/[1+(x/C)~B]+D. The optimized parameters that were determined in the method include the diluted working concentration(4 000 ng·mL~(-1)) of the antibodies,the dilution rate(1∶4),the ratio of effector cells over target cell(15∶1),and the induction time(20 h). The method also exhibited good specificity confirmed by three parallel tests,in which the RSD of plate-to-plate assay and day-to-day assay of maximum fold of induction(FI),as well as the RSD of the concentration for 50% of maximal effect(EC50),were less than 10%. Four different diluting groups of the recovery rates sample were examined 3 times using the method,the data of which showed mean relative potencies of(46.27±2.01)%,(71.18±1.55)%,(133.17±9.91)% and(166.55±15.73)%,respectively,and the recoveries of(92.53±4.01)%,(94.91±2.07)%,(106.54±7.93)%,(111.04±10.48)%,respectively,with both RSDs less than 10%. Moreover,this method could evaluate the ADCC effect of anti-HER2 monoclonal antibodies with various degrees of degeneration,so it may be adaptable for the majority of anti-HER2 monoclonal antibody classes. Conclusion:In this study,a luciferase reporter gene-modified cell based bioassay for measuring the ADCC of anti-HER2 monoclonal antibodies was successfully establishes,with adequate specificity,high accuracy and good repeatability. This method can be a used as an routine detection method of ADCC of anti-HER2 monoclonal antibodies.
引文
[1]CASTAGNOLI L,IORIO E,DUGO M,et al.Intratumor lactate levels reflect HER2 addiction status in HER2-positive breast cancer[J].J Cell Physiol,2019,234(2):1768
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[8]CHENG ZJ,GARVIN D,PAGUIO A,et al.Development of a robust reporter-based ADCC assay with frozen,thaw-and-use cells to measure Fc effector function of therapeutic antibodies[J].J Immunol Methods,2014,18(9):69
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[10]MATAMM,MAHMOODF,SOWELLRT,etal.Effectsof cryopreservation on effector cells for antibody dependent cellmediated cytotoxicity(ADCC)and natural killer(NK)cell activity in(51)Cr-release and CD107a assays[J].J Immunol Methods,2014,40(6):1
[11]PAREKH BS,BERGER E,SIBLEY S,et al.Development and validation of an antibody-dependent cell-mediated cytotoxicityreporter gene assay[J].MAbs,2012,4(3):310
[12]ALVARE RH.Present and future evolution of advanced breast cancer therapy[J].Breast Cancer Res,2010,12(1):68
[13]LEWIS PHILLIPS GD,LI GM,DUGGER DL,et al.Targeting HER-2-positivebreastcancerwithtrastuzumab-DM1,an antibody-cytotoxic drug conjugate[J].Cancer Res,2008,68(22):9280
[14]JUNTTILA TT,LI G,PARSONS K,et al.Trastuzumab-DM1(TDM1)retains all the mechanisms of action of trastuzumab and efficiently inhibits growth of lapatinib insensitive breast cancer[J].Breast Cancer Res Treat,2011,128(2):347
[2]GERECKEKM,WYSSJM,KALAVANOVAI,etal.ErbB transmembrane tyrosine kinasereceptors are differentially expressed throughout the adult rat central nervous system[J].J Comp Neurol,2001,433(l):86
[3]XU ZH,FORD BD.Up regulation of erbB receptors in rat brain after middle cerebral arterial occlusion[J].Neurosci Lett,2005,375(3):181
[4]汤沁,丁倩,林莉,等.针对HER2靶点的抗体药物研究与肿瘤靶向治疗[J].药学学报,2012,47(10):1297TANG Q,DING Q,LIN L,et al.Development of antibody drugs targeting against HER2 for cancer therapy[J].Acta Pharm Sin,2012,47(10):1297
[5]JONATHAN DB,SALVATORE L,EMILIANO C,et al.PIK3CA oncogenic mutations represent a major mechanism of resistance to trastuzumab in HER2/neu overexpressing uterine serous carcinoma[J].Brit J Cancer,2015,113(7):1026
[6]QI WW,LI XX,ZHAN YC,et al.Overexpression of Her-2upregulates FoxM1 in gastric cancer[J].Int J Mol Med,2014,33(6):1531
[7]乔杉杉,王大业.乳腺癌HER2基因的研究进展及其靶点治疗[J].首都医科大学学报,2008,29(1):96QIAO SS,WANG DY.The progressive study of HER2 gene and target therapy in breast cancer[J].J Cap Med Univ,2008,29(1):96
[8]CHENG ZJ,GARVIN D,PAGUIO A,et al.Development of a robust reporter-based ADCC assay with frozen,thaw-and-use cells to measure Fc effector function of therapeutic antibodies[J].J Immunol Methods,2014,18(9):69
[9]MILLER AS,TEJADA ML,GAZZANO SH.Methods for measuring antibody-dependent cell-mediated cytotoxicity in vitro[J].Methods Mol Biol,2014,11(34):59
[10]MATAMM,MAHMOODF,SOWELLRT,etal.Effectsof cryopreservation on effector cells for antibody dependent cellmediated cytotoxicity(ADCC)and natural killer(NK)cell activity in(51)Cr-release and CD107a assays[J].J Immunol Methods,2014,40(6):1
[11]PAREKH BS,BERGER E,SIBLEY S,et al.Development and validation of an antibody-dependent cell-mediated cytotoxicityreporter gene assay[J].MAbs,2012,4(3):310
[12]ALVARE RH.Present and future evolution of advanced breast cancer therapy[J].Breast Cancer Res,2010,12(1):68
[13]LEWIS PHILLIPS GD,LI GM,DUGGER DL,et al.Targeting HER-2-positivebreastcancerwithtrastuzumab-DM1,an antibody-cytotoxic drug conjugate[J].Cancer Res,2008,68(22):9280
[14]JUNTTILA TT,LI G,PARSONS K,et al.Trastuzumab-DM1(TDM1)retains all the mechanisms of action of trastuzumab and efficiently inhibits growth of lapatinib insensitive breast cancer[J].Breast Cancer Res Treat,2011,128(2):347