重组抗TNF-α单克隆抗体ADCC生物学活性检测方法的建立
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摘要
目的建立抗TNF-α单克隆抗体抗体依赖细胞介导的细胞毒性(antibody-dependent cell-mediated cytotoxicity,ADCC)生物学活性检测方法。方法以健康人外周血单个核细胞(peripheral blood mononuclear cell,PBMC)作为效应细胞,自主构建膜表面表达TNF-α(membrane-expressed form of TNF-α,m TNF-α)CHO细胞作为靶细胞,通过系列不同浓度的重组抗TNF-α单克隆抗体原研药(m Ab-O)和候选药(m Ab-S)进行ADCC检测,采用四参数拟合计算二者的半数抑制浓度(IC_(50))及m Ab-S的相对活性,并在优化实验条件的基础上进行专属性、精密度验证。结果重组抗TNF-α单克隆抗体的浓度与细胞毒性存在量效关系,符合四参数方程式,曲线在半对数坐标上呈典型S型,R2>0.99。方法经优化确定相同个体来源PBMC作为效应细胞,靶细胞数目4×10~4个/孔,效靶比25∶1,抗体靶细胞孵育时间1 h,诱导时间4 h。该方法具有良好的专属性,对于相同个体来源的PBMC,m Ab-S活性总体与m Ab-O相近(50%~200%),相对活性重复性RSD为4.64%,同一实验人员间隔3个工作日检测结果的RSD为12.50%,2名实验人员2次检测结果的RSD为8.21%,均符合方法验证的可接受标准。结论成功建立了重组抗TNF-α单克隆抗体ADCC生物学活性检测方法,该方法专属性强,精密度好,可作为重组抗TNF-α单克隆抗体与上市原研药相对ADCC生物学活性的评估方法。
Objective To develop a method for determination of antibody-dependent cell-mediated cytotoxicity(ADCC)of recombinant monoclonal antibody(m Ab) against tumor necrosis factor alpha(TNF-α).Methods The ADCC of recombinant m Ab against TNF-α was determined using serially diluted innovator original drug(m Ab-O) drug or its biosimilar candidate(m Ab-S)combined with normal human peripheral blood mononuclear cells(PBMCs)as effector cells and a CHO cell line expressing membrane bound TNF-α as target cells.The median inhibitory concentration(IC_(50))of m Ab and the relative biological activity of m Ab-S were calculated by four-parameter equation.Under the optimized experimental condition,the method was verified for specificity and precision.Results The cytotoxicity of m Ab against TNF-α was in a dose-dependent mode,which was consistent with the result acquired from the four-parameter equation.The dose-response curve was in a typical S shape on semilogarithmic coordinate paper,with a R2 value of more than 0.99.The optimal condition of each critical parameter of this method when using PBMCs from the same individual source as effector cells was as follows: the number of target cells was 4 × 10~4 cells / well,while the ratio of effector cells to target cells was 25 ∶ 1,the incubation time of antibody with target cells was 1 h,and the induction time was 4 h.The method showed good specificity.The ADCC of m Ab-S was similar to that of m Ab-O(50% ~ 200%) using PBMCs from the same individual source as effector cells,while the RSD in reproducibility test on relative activity was 4.64%.However,the RSD of test results by one person in 2 working days at an interval of 3 d was 12.50%,while than by two persons in the same working day was 8.21%,both of which met the acceptance criteria for method validation.Conclusion A method for determination of ADCC of recombinant m Ab against TNF-α was successfully developed,which showed high specificity and precision,and might be used for evaluation of ADCC of the m Ab and commercial innovator original drugs.
Objective To develop a method for determination of antibody-dependent cell-mediated cytotoxicity(ADCC)of recombinant monoclonal antibody(m Ab) against tumor necrosis factor alpha(TNF-α).Methods The ADCC of recombinant m Ab against TNF-α was determined using serially diluted innovator original drug(m Ab-O) drug or its biosimilar candidate(m Ab-S)combined with normal human peripheral blood mononuclear cells(PBMCs)as effector cells and a CHO cell line expressing membrane bound TNF-α as target cells.The median inhibitory concentration(IC_(50))of m Ab and the relative biological activity of m Ab-S were calculated by four-parameter equation.Under the optimized experimental condition,the method was verified for specificity and precision.Results The cytotoxicity of m Ab against TNF-α was in a dose-dependent mode,which was consistent with the result acquired from the four-parameter equation.The dose-response curve was in a typical S shape on semilogarithmic coordinate paper,with a R2 value of more than 0.99.The optimal condition of each critical parameter of this method when using PBMCs from the same individual source as effector cells was as follows: the number of target cells was 4 × 10~4 cells / well,while the ratio of effector cells to target cells was 25 ∶ 1,the incubation time of antibody with target cells was 1 h,and the induction time was 4 h.The method showed good specificity.The ADCC of m Ab-S was similar to that of m Ab-O(50% ~ 200%) using PBMCs from the same individual source as effector cells,while the RSD in reproducibility test on relative activity was 4.64%.However,the RSD of test results by one person in 2 working days at an interval of 3 d was 12.50%,while than by two persons in the same working day was 8.21%,both of which met the acceptance criteria for method validation.Conclusion A method for determination of ADCC of recombinant m Ab against TNF-α was successfully developed,which showed high specificity and precision,and might be used for evaluation of ADCC of the m Ab and commercial innovator original drugs.
引文
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[3]Humira誖.In:adalimumab.North Chicago,IL:Abbott Laboratories,2007.
[4]VICTOR F C,GOTTLIEB A B.TNF-alpha and apoptosis:implications for the pathogenesis and treatment of psoriasis[J].J Drugs Dermatol,2002,1(3):264-275.
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[6]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(1):59-65.
[7]MATA M M,MAHMOOD F,SOWELL R T,et al.Effects of 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,2014,406(1):1-9.
[8]ARORA T,PADAKI R,LIU L,et al.Differences in binding and effector functions between classes of TNF antagonists[J].Cytokine,2009,45(2):124-131.
[9]MITOMA H,HORIUCHI T,TSUKAMOTO H,et al.Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor alpha-expressing cells:comparison among infliximab,etanercept,and adalimumab[J].Arthritis Rheum,2008,58(5):1248-1257.
[10]SHIELDS R L,NAMENUK A K,HONG K,et al.High resolution mapping of the binding site on human Ig G1 for Fc gamma RI,Fc gamma RII,Fc gamma RIII,and Fc Rn and design of Ig G1 variants with improved binding to the Fc gamma R[J].J Biol Chem,2001,276(9):6591-6604.
[11]LIU C Y,WANG L,GUO W,et al.Development of a novel reporter gene method for determination of ADCC potency of anti-CD20 monoclonal antibody[J].Acta Pharm Sin,2015,50(1):94-98.(in Chinese)刘春雨,王兰,郭玮,等.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[J].药学学报,2015,50(1):94-98
[2]DESPLAT-J魪GO S,BURKLY L,PUTTERMAN C.Targeting TNF and its family members in autoimmune/inflammatory disease[J].Mediators Inflamm,2014,2014:628748.
[3]Humira誖.In:adalimumab.North Chicago,IL:Abbott Laboratories,2007.
[4]VICTOR F C,GOTTLIEB A B.TNF-alpha and apoptosis:implications for the pathogenesis and treatment of psoriasis[J].J Drugs Dermatol,2002,1(3):264-275.
[5]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):310-318.
[6]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(1):59-65.
[7]MATA M M,MAHMOOD F,SOWELL R T,et al.Effects of 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,2014,406(1):1-9.
[8]ARORA T,PADAKI R,LIU L,et al.Differences in binding and effector functions between classes of TNF antagonists[J].Cytokine,2009,45(2):124-131.
[9]MITOMA H,HORIUCHI T,TSUKAMOTO H,et al.Mechanisms for cytotoxic effects of anti-tumor necrosis factor agents on transmembrane tumor necrosis factor alpha-expressing cells:comparison among infliximab,etanercept,and adalimumab[J].Arthritis Rheum,2008,58(5):1248-1257.
[10]SHIELDS R L,NAMENUK A K,HONG K,et al.High resolution mapping of the binding site on human Ig G1 for Fc gamma RI,Fc gamma RII,Fc gamma RIII,and Fc Rn and design of Ig G1 variants with improved binding to the Fc gamma R[J].J Biol Chem,2001,276(9):6591-6604.
[11]LIU C Y,WANG L,GUO W,et al.Development of a novel reporter gene method for determination of ADCC potency of anti-CD20 monoclonal antibody[J].Acta Pharm Sin,2015,50(1):94-98.(in Chinese)刘春雨,王兰,郭玮,等.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[J].药学学报,2015,50(1):94-98