基于报告基因的抗CD20单克隆抗-省略-ADCC生物学活性测定方法的建立-刘春雨

94ActaPharmaceuticaSinica2015,50(1):94−98CD20ADCC1,1,,,,,,*(,100050):Jurkat/NFAT-luc+FcγRIIIa,WIL2-S,(BioGloTMLuciferaseAssaySystem)CD20ADCC,CD20,:y=(A−D)/[1+(X/C)B]+DWIL2-S,18000ng·mL−1,15,61,6h,8;43,(44.39±3.93)%(72.74±2.78)%(128.28±7.01)%(168.19±2.70)%,10%,(88.78±7.85)%(96.99±3.70)%(102.63±5.61)%(112.12±1.80)%CD20ADCC,,,CD20ADCC:CD20;ADCC;:R917:A:0513-4870(2015)01-0094-05DevelopmentofanovelreportergenemethodfordeterminationofADCCpotencyofanti-CD20monoclonalantibodyLIUChun-yu1,WANGLan1,GUOWei,YUChuan-fei,ZHANGFeng,WANGWen-bo,LIMeng,GAOKai*(NationalInstitutesforFoodandDrugControl,Beijing100050,China)Abstract:ThebiologicalactivityofADCCbyanti-CD20monoclonalantibodywasdeterminedbyBioGloTMLuciferaseAssaySystemusingJurkat/NFAT-luc+FcγRIIIacelllineaseffectorcellandWIL2-Scelllineastargetcell.Thedevelopedmethodwasverifiedforspecificity,precisionandaccuracy.Anti-CD20monoclonalantibodyshowedadose-responsemodebythedevelopedmethod,andthedeterminationresultcompliedwiththefollowingfour-parameterequation:y=(A–D)/[1+(X/C)B]+D.Theoptimizedparametersofthemethodweredeterminedincludingtheantibodiesdilutedconcentration(18000ng·mL−1),dilutionrate(15),theratioofeffectorcellandtargetcell(61),andinductiontime(6h).Thevaluesofeightindependenttestshavepassedastatisticaltestforcurveregressionanalysis,linearorparallelism,whichshowedthemethodpossessedgoodspecificity.Fourdifferentdilutegroupsofrecoveryratessampleweredeterminedfor3times,andtheresultshowedmeanrelativepotenciesof(44.39±3.93)%,(72.74±2.78)%,(128.28±7.01)%and(168.19±2.70)%respectively,withavariationcoefficientoflessthan10%,andtherecoveriesof(88.78±7.85)%,(96.99±3.70)%,:2014-08-21;:2014-09-17.:(2014ZX09304311-001,2012ZX09304010);(2012B6).1.*Tel:[phone],Fax:[phone],E-mail:[e-mail]:CD20ADCC95(102.63±5.61)%and(112.12±1.80)%respectively.AnovelreportergenemethodfordeterminationofbiologicalactivityofADCCbyanti-CD20monoclonalantibodywassuccessfullydeveloped,whichshowedstrongspecificity,goodreproducibilityandhighaccuracy,andmightbeusedroutinely.Keywords:anti-CD20monoclonalantibody;ADCC;biologicalactivityCD2035kD,CD20B,95%B,,CD20,,B[1−4]CD20,CD20B,(CDC)(ADCC)[5]ADCCCD20CD20BCD20,FcFcRγIIIa(CD16),NK,,CD20,CDCCD20ADCCADCC(peripheralbloodmononuclearcell,PBMC)NK[6,7],,ADCC,NKFc(FcR),FcRγIIIa(CD16)NFAT,,FcRγIIIa,FcRγIIIa[8−10],FcγRIIIaNFAT(nuclearfactorofactivatedT-cells)T(Jurkat),WIL2-S(B),CD20ADCC[11]WIL2-S(ATCC);Jurkat/NFAT-luc+FcγRIIIa(Promega),FcγRIIIaNFATT,FcγRIIIaADCC,,NFAT,CD20(FBS)RPMI1640MEM(NEAA)BGIBCO;(BSA)G418Bio-GloTMLuciferaseAssaySystemPromega;SpectraMaxM5SoftMaxMolecularDevices;PLA2.0StegmannADCCWIL2-SJurkat/NFAT-luc+FcγRIIIa,1000r·min−15min0.5%BSARPMI1640(),1×1066×106mL−1,WIL2-S96,25μL;CD2018μg·mL−1,15,9,2;CD20WIL2-S,25μL,Jurkat/NFAT-luc+FcγRIIIa,25μL;25μLCD20(negative),75μL(background,BG);375%CO26h,15min;,75μL,5min,SpectraMaxM5(relativelightunits,RLU),(foldofinduction,FI)FI=RLU(−)/RLU(−);SoftMax,CD20x,96ActaPharmaceuticaSinica2015,50(1):94−98FIy,,CD20:CD20(+)BWIL2-SRajiDaudi;:CD203μg·mL−1,1314,CD2018μg·mL−1,15;:(6×106mL−1),314151617.51101151301;:3456723h,:CD20(−)C8166WIL2-S:NFAT-luc-FcγRIIIaJurkatJurkat/NFAT-luc+FcγRIIIa:HER-2CD20:CD2018μg·mL−150%75%100%125%150%,4,(EC50),100%,:(%)=EC50/EC50×100%,CV(%),:CD2018μg·mL−150%75%100%125%150%,4,:(%)=/×100%,1CD20ADCCSoftMax,CD20x,y,,CD20:y=(A−D)/[1+(X/C)B]+D,S,r20.99,A,D,B,C1EC507.32ng·mL−1(C)2ADCC2.1CD20(+)BWIL2-SRajiDaudi,23CD20B,CD20ADCC,WIL2-S,50.4,RajiDaudi2.5852.6252.2CD201314(3000ng·mL−1)15(18000ng·mL−1),9,3,CD20ADCC,18000ng·mL−1,152.3(6×106mL−1)314151Figure1Dose-responsecurveofanti-CD20mAbADCCbioassayFigure2Optimizationofdose-responsecurvefromthreegroupsofdifferentCD20(+)targetcellsFigure3Optimizationofdose-responsecurvefromthreegroupsofdifferentdilutions:CD20ADCC97617.51101151301,84ADCC,ET=612.4CD20,3456723h75ADCC,67h,,6h33.16,1(6),ADCC3.28,EC50(11.43±0.88)ng·mL−1,CV7.74%,,71Figure4Optimizationofdose-responsecurvefromeightgroupsofdifferentvaluesofET(EffectorcellTargetcell)Figure5Optimizationofdose-responsecurvefromsevengroupsofdifferentinductiontimes,PLA2.0,83.350%75%100%125%150%,4,24(%)(44.39±3.93)%(72.74±2.78)%(128.28±7.01)%(168.19±2.70)%;(88.78±7.85)%(96.99±3.70)%(102.63±5.61)%(112.12±1.80)%,CV(%)10%4,0.9905,Figure6Specificityofanti-CD20mAbADCCbioassayFigure7Repeatabilityresultsofanti-CD20mAbADCCTable2Potencyandrecoveryofanti-CD20mAbADCC(n=4)SampleRelativepotency/%Recovery/%x±sCV/%x±sCV/%50%44.39±3.938.8588.78±7.858.8475%72.74±2.783.8296.99±3.703.81125%128.28±7.015.47102.63±5.615.47150%168.19±2.701.61112.12±1.801.61Table1GroupsofADCCassayforspecificityverificationGroupTargetcellEffectcellmAbSpecificityfortargetcellDilutionbufferJurkat/NFAT-luc+FcγRIIIaAnti-CD20mAbC8166cell(CD20-)Jurkat/NFAT-luc+FcγRIIIaAnti-CD20mAbSpecificityforeffectcellWIL2-SDilutionbufferAnti-CD20mAbWIL2-SJurkat(NFAT-luc-;FcγRIIIa-)Anti-CD20mAbSpecificityformAbWIL2-SJurkat/NFAT-luc+FcγRIIIaDilutionbufferWIL2-SJurkat/NFAT-luc+FcγRIIIaAnti-HER2mAb98ActaPharmaceuticaSinica2015,50(1):94−98b(t=0.033,P0.05),rituximab,CD20(biosi