mimetic cofactor activity of a bispecific antibody topdfWord文件下载.docx

mimetic cofactor activity of a bispecific antibody topdfWord文件下载.docx

《mimetic cofactor activity of a bispecific antibody topdfWord文件下载.docx》由会员分享，可在线阅读，更多相关《mimetic cofactor activity of a bispecific antibody topdfWord文件下载.docx（4页珍藏版）》请在冰豆网上搜索。

YoshikiKawabe2;

TomoyukiIgawa2;

HiroyukiTsunoda1;

KeijiNogami3;

MidoriShima3;

KunihiroHattori11ResearchDivision,ChugaiPharmaceuticalCo.,Ltd.,Kamakura,Kanagawa,Japan;

2ResearchDivision,ChugaiPharmaceuticalCo.,Ltd.,Gotemba,Shizuoka,Japan;

3DepartmentofPediatrics,NaraMedicalUniversity,Kashihara,Nara,JapanSummaryEmicizumab,ahumanisedbispecificantibodyrecognisingfactors（F）

IX/IXaandX/Xa,canaccelerateFIXa-catalysedFXactivationbybridg-ingFIXaandFXinamannersimilartoFVIIIa.However,detailsoftheemicizumab–antigeninteractionshavenotbeenreportedsofar.Inthisstudy,wefirstshowedbysurfaceplasmonresonanceanalysisthatemicizumabboundFIX,FIXa,FX,andFXawithmoderateaffin-ities（KD=

1.58,

1.52,

1.85,and0.978µ

M,respectively）.Wenextshowedbyimmunoblottinganalysisthatemicizumabrecognisedtheantigens’epidermalgrowthfactor（EGF）-likedomains.Wethenper-formedKD-basedsimulationofequilibriumstatesinplasmaforquantitativelypredictingthewaysthatemicizumabwouldinteractwiththeantigens.ThesimulationpredictedthatonlyasmallpartofplasmaFIX,FX,andemicizumabwouldformantigen-bridgingFIX–emicizumab–FXternaryplex,ofwhichconcentrationwouldformabell-shapedrelationshipwithemicizumabconcentration.The

bell-shapedconcentrationdependencywasreproducedbyplasmathrombingenerationassays,suggestingthattheplasmaconcen-trationoftheternaryplexwouldcorrelatewithemicizumab’sco-factoractivity.Thesimulationalsopredictedthatat10.0–100µ

g/mlofemicizumab–levelsshowninapreviousstudytobeclinicallyeffec-tive–themajorityofplasmaFIX,FX,andemicizumabwouldexistasmonomers.Inconclusion,emicizumabbindsFIX/FIXaandFX/FXawithmicromolaraffinitiesattheirEGF-likedomains.TheKD-basedsimu-lationpredictedthattheantigen-bridgingternaryplexformedincirculatingplasmawouldcorrelatewithemicizumab’scofactoractiv-ity,andthemajorityofFIXandFXwouldbefreeandavailableforothercoagulationreactions.KeywordsCoagulationfactors,drugdesign,factorVIII,haemophiliatherapyCorrespondenceto:

Financialsupport:

TakehisaKitazawaThisstudywassupportedbyChugaiPharmaceuticalCo.,Ltd.ResearchDivisionChugaiPharmaceuticalCo.,Ltd.Received:

January15,

xx200Kajiwara,KamakuraAcceptedafterminorrevision:

March25,

xxKanagawa247–8530,JapanEpubaheadofprint:

April28,

xxTel.:

+81467472260,Fax:

+81467467795s:

//doi.org/10.1160/TH17-01-0030E-mail:

kitazawatkh@chugai-pharm.co.jpThrombHaemost

xx;

117:

1348–1357Institutionwheretheworkwascarriedout:

ResearchDivision,ChugaiPharmaceuticalCo.,Ltd.SupplementaryMaterialtothisarticleisavailableonlineat.thrombosis-onlineZZZ.IntroductionRoutineadministrationofexogenousfactor（F）

VIIIforbleedingprophylaxisinpatientswithhaemophiliaA–apracticewidespreadmainlyindevelopedcountries–improvespatients’qualityoflifebyreducingbleedingepisodesandbleeding-relatedplications（1,2）.However,thetreatmentstillhassomedrawbacks,includingim-perfectcontrolofbleedinginpatientswhodevelopalloantibodiestoFVIIIandthenecessityofadministrationviafrequentintra-venousinjections（2,3）.Tooveresuchdrawbacks,weex-ploredtheideaofcreatinganantibodyfunctioningasaFVIIIco-

factor,whichwebelievedwouldbeapromisingapproachbecauseIgGantibodiesgenerallyhavealonghalf-life,highsubcutaneousbioavailability,andamolecularstructureorantigenicitydifferentfromthatofFVIII（4）.Inthe1990s,severalpioneeringresearchersrevealedthewaysthatFVIIIainteractswithbothFIXaandFX,whichwouldfacilitateaccelerationofFIXa-catalysedFXactivation（5–7）.Theseinteractionswerefurtherunveiledthroughthe2000sand

xxs（8–10）,andconsequentlyFVIIIaisnowknowntohavemultiplecontactswithbothFIXaandFX（Figure1A）.TheideabehindourFVIIIa-mimeticcofactorantibodywastocreateananti-FIXa/FXbispecificantibodythatplacesthetwoantigensLicenseterms:

CC-BY-NC-ND（s:

//creativemons.org/licenses/by-nc-nd/

4.0）©

Schattauer

xxKitazawaetal.Emicizumab’scofactoractivitydependsonitsantigen-bridgingability1349Figure1:

SchematicillustrationsoftheinteractionsofFVIIIaoremicizumabwithFIX/FIXaandFX/FXa.A）

InteractionsofFVIIIawithFIXaandFXre-portedpreviously（5–10）.B）

Interactionsofemi-cizumabwithFIX/FIXaandFX/FXa.Theillus-trationsdonotnecess-arilyindicateprecisemolecularstructures.（FIXaandFX）

inaspatiallyappropriatepositiontoaccelerateFIXa-catalysedFXactivationinthesamemannerthatFVIIIashoulddo（4）.Afterlongresearch,weeventuallyidentifiedapo-tenthumanisedbispecificantibody,termedACE910oremicizu-mab（11）.Aswehadintended,emicizumabexertedapotentFVIIIa-mimeticcofactoractivityinvitroandinvivo（11–13）,andableedingpreventiveeffectataround10–100µ

g/mlofplasmaemi-cizumabinthepatientpartofthephase1clinicalstudy（14）.Asisobvious,however,emicizumabisnotthesameasFVIII/FVIIIaandthushassomecharacteristicsdifferentfromthoseof

FVIII/FVIIIa.Weconsideredthedifferenceintheirbindingprop-ertiesasoneofthenon-negligiblepointstobetakenintoaccount.Inthisstudy,weelucidatedemicizumab’sbindingaffinities（KD）

tothebothantigensbyusingsurfaceplasmonresonance（SPR）

analysisandexploredepitopesontheantigensbyusingimmuno-blottinganalysis.WethenusedthedeterminedKDvaluestosimu-lateequilibriumstatesinplasmainordertopredictthewaysthatemicizumabwouldinteractwiththeantigensquantitatively,andtodiscusshowemicizumabandtheantigenswouldbehave.©

xxLicenseterms:

4.0）

1350Kitazawaetal.Emicizumab’scofactoractivitydependsonitsantigen-bridgingabilityMaterialsandmethodsAntibodiesandcoagulationfactorsEmicizumab（ahumanisedbispecificIgG4antibodyrecognisingFIX/FIXaandFX/FXa）

wasrebinantlyproducedfromaChi-nesehamsterovarycellline（15）.Anti-FIX/FIXaoranti-FX/FXamonospecifictwo-armedIgG4antibodieseachhavingoneoftheantigenbindingfragmentsofemicizumab（emicizumabFab）

weretransientlyexpressedinHEK293cellsandpurified（11）.ExceptforFVIIIusedinSuppl.Table1（availableonlineat.thrombosis-onlineZZZ）,allcoagulationfactorsusedwerehumanplasma-de-rived（EnzymeResearchLaboratories,SouthBend,IN,USA）.SPRanalysisWeanalysedtheinteractionsofFIX,FIXa,FX,andFXawiththecorrespondingvariableregionofemicizumabbyusingaBiacoreT200SPRsystem（GEHealthcare,Uppsala,Sweden）.First,weim-mobilisedMabSelectSuReLigand（rebinantProteinA;

GEHealthcare）

ontoaCM4sensorchip（GEHealthcare）

thathadbeenpre-activatedwithNHSandECD,andpre-deactivatedwithethanolamine-HClusinganAmineCouplingKit（GEHealthcare）.Tocapturethetestantibodiesonthesensorchip,weinjectedtheanti-FIX/FIXaoranti-FX/FXamonospecifictwo-armedIgG4anti-bodyhavingeitheroftheemicizumabFabintoflowcell2,andna-talizumab（Biogen,Cambridge,MA,USA）

ascontrolhumanisedIgG4antibodyintoflowcell

1.Wenextinjectedeachanalyte（0asbaseline,80,160,320,640,or1280nMofFIX,FIXa,FX,orFXa）,whichhadbeendissolvedinrunningbuffer（10mMHEPES,150mMNaCl,0.05vol%SurfactantP20,

2.5mMCaCl2

[pH

7.4]）,intobothflowcellsonthesensorsurfaceataflowrateof30µ

l/minute（min）

tomonitortheassociationphasefor120sec-onds（s）

andthenthedissociationphasefor30swiththerunningbuffer.Thedatawereanalysedbythe1:

1bindingmodelintheBia-coreT200Evaluationsoftware（version

1.0,GEHealthcare）.

andperformedSDS-PAGEinanon-reducedcondition.Forim-munoblottinganalyses,weusedtheanti-FIX/FIXaoranti-FX/FXamonospecifictwo-armedIgG4antibodieshavingeitheroftheemi-cizumabFabsasthetestantibody,anddetectedtheantigen–anti-bodybindingbyhorseradishperoxidase-labelledrebinantPro-teinL（Actigen,Cambridge,UK）

andaperoxidaseimmunoblottingsubstrate（NacalaiTesque,Kyoto,Japan）.ELISAWecoatedwellsof96-wellimmunoplates（ThermoFisherScientific,Waltham,MA,USA）

withFVII,FIX,FX,FXII,orProteinC（EnzymeResearchLaboratories）

at1µ

g/mlinphosphatebufferedsaline.Afterwashingthewellswithtrisbufferedsalinecontaining1mMCaCl2and0.05vol%Tween®

20（Bio-Rad,Hercules,CA,USA）

（TBS-Ca/tween）,weblockedthewellswith30mg/mlbovineserumalbumin（BSA）

dissolvedinTBS-Ca/tween（TBS-Ca/tween/BSA）.Aftertheblocking,weappliedvariedconcentrationsofemicizumabdilutedwithTBS-Ca/tween/BSAtothewellsforthereactionofthecoatedtestprotein-emicizumabinteraction.AfterwashingthewellswithTBS-Ca/tween,weappliedanalkalinephosphatase-labelledmousehuman-γ4-chain-specificantibody（SouthernBiotech,Bir-mingham,AL,USA）

dilutedwithTBS-Ca/tween/BSA.Afterwash-ingthewellsagainwithTBS-Ca/tween,weappliedphosphatasesubstrate（Kirkegaardaprecursorofemicizumab）

reportedpre-viously（4）.Antigens’domainsrecognisedbyemicizumabNext,wedeterminedbyimmunoblottinganalysisthedomainineachantigenthatemicizumabrecognises.SincehBS23hadrecog-nisedthelightchainsofFIX/FIXaandFX/FXa（4）,wefocusedontherespectivelightchains.ForFIX/FIXa,weappliedplasma-de-rived