SIRPα-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells 2017 Laia Pascual Ponce.pdf

该【SIRPα-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells 2017 Laia Pascual Ponce 】是由【小舍儿】上传分享，文档一共【18】页，该文档可以免费在线阅读，需要了解更多关于【SIRPα-antibody fusion proteins stimulate phagocytosis and promote elimination of acute myeloid leukemia cells 2017 Laia Pascual Ponce 】的内容，可以使用淘豆网的站内搜索功能，选择自己适合的文档，以下文字是截取该文章内的部分文字，如需要获得完整电子版，请下载此文档到您的设备，方便您编辑和打印。:..otarget/Oncotarget,2017,,(),pp:11284-11301ResearchPaperSIRPα-antibodyfusionproteinsstimulatephagocytosisandpromoteeliminationofacutemyeloidleukemiacellsLaiaPascualPonce1,5,,NadineMoritz1,ChristinaKrupka2,3,Jan-HendrikKozik1,KirstenLauber4,MarionSubklewe2,3,Karl-PeterHopfner1,51GeneCenterMunich,DepartmentofBiochemistry,Ludwig-Maximilians-Universit?tMünchen,Munich,Germany2DepartmentofInternalMedicineIII,KlinikumderUniversit?tMünchen,Ludwig-Maximilians-Universit?tMünchen,Munich,Germany3GeneCenterandClinicalCo-operationGroupImmunotherapyattheHelmholtzZentrumMünchen,Munich,Germany4DepartmentofRadiationOncology,KlinikumderUniversit?tMünchen,Ludwig-Maximilians-Universit?tMünchen,Munich,Germany5GraduateSchoolofQuantitativeBiosciencesMunich,Ludwig-Maximilians-Universit?tMünchen,Munich,GermanyCorrespondenceto:Karl-PeterHopfner,email:******@:therapeuticantibody,immunotherapy,CD47,SIRPα,acutemyeloidleukemiaReceived:August02,epted:December12,2016Published:January04,2017ABSTRACTCD47,expressedonavarietyoftumorcells,confersimmuneresistancebydeliveringaninhibitory“don’teatme”ellsviaitsmyeloid--SIRPαaxiswithCD47-directedantibodiesorantibody-,CD47expressiononhealthycellscreatesanantigensinkandpotentialsitesoftoxicity,limitingtheefficacyofCD47-,wefirstcharacterizedCD47expressioninAcuteMyeloidLeukemia(AML)patients(n=213),toinhibittheCD47-SIRPαsignalingpathwayatthetumorsite,wedevelopedaso-calledlocalinhibitorycheckpointmonoclonalantibody(licMAB)bygraftingtheendogenousSIRPαdomaintotheN-terminusofthelightchainofanantibodytargetingCD33,-expressingcellseveninthepresenceofalargeCD33-negativeCD47-positiveantigensink,stimulatephagocytosisofAMLcellsandeliminateAMLcelllinesandprimary,patient--SIRPαaxistotumorantigen-,priseaplethoraofinhibitoryTheuseofmonoclonalantibodies(mAbs)haspathwaysfundamentalforthemaintenanceofeanimportantandpromisingapproachincancerself-toleranceundernormalphysiologicalconditionsimmunotherapy[1].essisbasedon[3,4].Itiswelldocumentedthattumorcellsoftentheirabilitytospecificallybindtoantigensdifferentiallyutilizetheseimmunecheckpointsasamechanismtoexpressedincancercellsandtosimultaneouslyrecruitescapeimmunerecognitionandgainimmuneresistance,immuneeffectorcellsviainteractionwiththeirFcdemonstratingthenecessityoftargetthesemolecularreceptors(FcRs).Thistriggersimmunecellactivation,pathways[5].includingantibody-dependentcellularcytotoxicityCD47isatransmembraneproteinubiquitously()andantibody-,thesignal(ADCP)[2].regulatoryproteinalpha(SIRPα),isexpressedonells,includingmacrophagesanddendriticstrategyfortreatingdifferenttypesofcancersinvolvescells(DCs).UponbindingtoCD47,SIRPαtransmitsaotarget11284Oncotarget:..“don’teatme”signalthatinhibitsphagocytosis,[6,7].However,ithasbeenshownthatHere,wereportthedevelopmentandthedisruptionofCD47-SIRPαinteractionbyablockingcharacterizationofanewantibodyformat,hereafterantibodyagainsthumanCD47()enablescalledalocalinhibitorycheckpointmonoclonalphagocytosisofAcuteMyeloidLeukemiaStemCellsantibody(licMAB).LicMABswerecreatedbygrafting(AMLLSC)andAcuteLymphoidLeukemia(ALL)cellstheendogenousN-terminalIgdomainofSIRPαontoinvitroandinhibitstumorengraftmentinvivo[8,9].thevariablelightchainofaCD33-targetingIgG1Moreover,,similartotherecentlypublishedtumorsinmousestudies[10].Additionally,ahumanized“SIRPabodies”[17].WesuggestthattheresultinglicMABIgG4antiCD47antibody(Hu5F9-G4)waspre-clinicallyblockstheCD47-SIRPαinteractionatthesiteoftumorevaluatedandenteredphaseItrials(NCT02216409)antigen-,bytargetingtheinpatientswithAMLandsolidtumorsin2014[11].tumorantigenCD33,theblockadeofthe“don’teatme”Hu5F9-G4showedapotentenforcementofphagocytosissignalandthereforetheantitumoractivity,isrestrictedofprimaryhumanAMLcellsinvitroandaneliminationtoCD33-,weproposethatofpatient-,theblockadeofCD47--G4havebeenshowntosynergizebyfusingasecondSIRPαdomain(resultinginadoublewithtumor-specificmonoclonalantibodiessuchasSIRPαlicMAB),thusincreasingthelocalSIRPαrituximab,anantiCD20antibody,-HodgkinLymphoma(NHL)andcureSIRPα-antiCD33and2xSIRPα-antiCD33licMABsofxenograftedmice[11,12].wereevaluatedbasedontheirbindingspecificity,Nevertheless,,suchasredbloodcells(RBCs),essfullystimulatephagocytosisofhumanessiblewithinthebloodstream,mightAMLcelllinesandenforceeliminationofhumanAMLcreatealargeantigensinkandpotentialsitesoftoxicitycelllinesandprimary,patient-,-SIRPαreducingthebindingstrengthforCD47[13].[14–16],theendogenousextracellularN-terminaldomainRESULTSofSIRPα,consistingofanimmunoglobulinsuperfamilyV-likefold[15,16],isanattractivealternativetointerfereCD47ishighlyexpressedonbulkAMLcellsandwiththeCD47-SIRPαaxiswhileavoidingthementionedAMLLSCsindependentofdiseasestaterisks[17].Inagreementwiththis,TTI-621,aSIRPα-FcfusionproteinthatusestheendogenousSIRPαdomaintoByexpressingCD47,AMLcellstriggerthe“don’tblocktheCD47-SIRPαaxis,wasdevelopedbyTrilliumeatme”signaltomacrophagesviaSIRPαengagementTherapeuticsandiscurrentlybeingevaluatedinphaseIandcanthereforeescapetheimmunesystembyinhibitingtrialsforhematologicmalignancies(NCT02890368).,previousstudieshaveshownCD33,asialicacid-dependentcytoadhesionthatCD47isexpressedathigherlevelsonAMLLSCsmolecule,isavalidatedtargetinAML[18].ItsexpressionthanontheirnormalcounterpartsanditalsocorrelatesonAMLcellsandAMLLSCs[18,19]es[8].Tofurthercharacterizetargetantigenofchoiceforseveralantibody-basedCD47expressiononAMLcells,weanalyzed213AMLapproaches,includingmAbs,antibody-drugconjugatespatientsamples;182ofwhomwerenewlydiagnosedand(ADC),bispecificT-cellengagers(BiTE),bispecifickiller31ofwhominrelapse(Figure1).cellengagers(BiKE)andsingle-%oftriplebodies(sctbs)[20–27].AlbeitpromisingresultsbulkSSClow/CD45dimAMLcellsfromthe213patientwereobtainedinpre-clinicalandclinicalinvestigationsofsamples(Figure1A).Despiteconsiderableinter-patientseveraltherapeuticformats,noCD33-targetingagenthaveheterogeneity,expressionlevelsofCD47wereconsistentlyeptedbynoneoftheMedicineRegulatoryhigh,resultinginaMedianFluorescenceIntensity(MFI),newCD33-(Figure1B).,CD47isoverexpressedonAMLcells,wesoughttoevaluatetheexpressionlevelofCD47bulkcellsandAMLLSCs[8,28].ExpressionofCD47onAMLLSCs,whicharefoundwithintheCD34+/CD38-partmentofSSClow/[8].Hence,disruptionoftheCD47-SIRPαlevelofCD47wassignificantlyloweronAMLLSCsotarget11285Oncotarget:..withrespecttobulkAMLcells,theexpressionlevelcheckpointmonoclonalantibodies(licMABs),whichpartments(-,respectively)(Figure1C).Furthermore,wetoamAbtargetingCD33,avalidatedtargetantigenincomparedtheexpressionlevelofCD47onbulkandAMLAML[29](Figure2).ThevariablefragmentoftheLSCsatthetimeofinitialdiagnosis(ID),eneratingbulk,norforAMLLSCs,-antiCD33licMAB,the(Figure1D).MFIratiosofCD47expressiononbulkAMLN-terminalIgV-,flexiblepolyglycine-serinelinker(G4S)(IDandrelapse,theN-terminusoftheantiCD33lightchain(Figure2B).respectively).AsecondSIRPαdomainwaslinkedbya(G4S)4linkerCollectively,theseresultsconfirmthatCD47istotheN-terminusoftheSIRPα--antiCD33licMAB(Figure2C).ForMostinterestingly,CD47expressioninAMLpatientsgenerationoftheantiCD33mAb,(Figure2A).GenerationandcharacterizationoflicMABsSIRPα-antiCD33licMAB,2xSIRPα-antiCD33licMABandantiCD33mAbwereproducedinExpi293FTolocallyinterferewiththeCD47-SIRPαaxisatcellsandpurifiedbyProteinAaffinitychromatographythetumorsite,wegeneratedtheso-,,Figure1:CD47ishighlyexpressedonbulkAMLcellsandAMLLSCsirrespectiveofthediseasestate.(A)PercentageofCD47positivecellsofSSClow/CD45dimbulkAMLcellsfrom213AMLpatients.(B)CD47expressionlevels(MFIratio)onSSClow/SSClow/CD45dimbulkAMLcells(n=213).(parisonofCD47MFIratiobetweenSSClow/CD45dimbulkAMLcellsandCD34+/CD38-AMLLSCs(n=213).(parisonofCD47MFIratioatinitialdiagnosis(ID)andrelapseinSSClow/CD45dimbulkAMLcells(n=182ID;=31relapse)andCD34+/CD38-LSCs(n=106ID;=20relapse).StatisticaldifferenceswereassessedbytheMann-WhitneyUtest(p-value<****).otarget11286Oncotarget:..,parethebindingstrengthofcontamination(Figure2A–2C).TwoequimolarbandseachmoleculetoMOLM-13cells,wedeterminedKDwerevisibleinSDS-PAGEanalysisforallmolecules,-antiCD33,2xSIRPα-,,,2xSIRPαandantiCD33lightchain,respectivelynMrange,consistentwithotherCD33-targetingagents(Figure2D).Furthermeasurementsbyfluorescence(Figure3C)[21,24,25].Moreover,°CKDvaluesobtainedforallthreemoleculesconfirmedfortheantiCD33mAb,whereasthemeltingtemperaturethattumorantigen-bindingisnotstronglyinfluencedbywasreducedto66°CwhentheSIRPαdomainwaspresentthepresenceoftheSIRPαdomainoritsinteractionwith(Figure2E).Takentogether,-,μMaffinitiesbetweenCD47andSIRPαhavebeendescribedinpreviousstudies[15,16].LicMABsbindtoCD33withhighaffinityandWehypothesizedthatthehighaffinityforCD33weaklyinteractwithCD47ofthelicMABswouldfacilitatepreferredtumorantigen-bindingoverhealthyCD47-,whichexpressCD47[7],arealikelyantigenandCD47antigens,-andCD47-expressingAMLcelllineMOLM-,aCD33CD33andCD47doublepositivecellsinthepresenceofnegative,CD47positiveB-lineageAcuteLymphoidRBCs,MOLM-13cellsweremixedwitha5-,10-or20-L