Antibody-DrugConjugateBioanalyticalAssayDevelopment:ImmunogenicityChallenges

November16,2016

PresentedbyCorinnaFiorotti,Ph.D.

2

PresentationOverview

ADCOverview

ADCAssays

ADCImmunogenicityØ RiskAssessments

Ø TestingStrategy

NeutralizingAntibodyDevelopment&Validation

Ø CriticalReagents

Conclusions

3

AntibodyDrugConjugates:WhatareThey?

• Monoclonalantibodiescovalentlyboundtocytotoxicsmall-moleculedrugs viastablelinkerswithlabilebonds

• Designedtoprovidetargetedcancertherapybypreferentiallybindingantigensover-expressedontumorcells,whileatthesametime,minimizingsystemictoxicity

• ADCtherapeuticinvivoisacomplexandchangingmixture

Anunderstandingofthismixture,includingtheprevalentDARspecies,isnecessaryforthedevelopmentofbioanalyticalassayswhichareusedtodetermineoverallsafety,efficacyandtoxicityofthetherapeutic

ConjugationChemistryØ Lysine,Cysteine-basedØ Engineeredcysteineresidues

Ø DeterminestheinitialprevalentDARspecies(importantforADAandlabeling)

Ø Caninhibit/enhancethebindingpropertiesofthemAb

Ø Canaffecttertiarystructure

Ø Canpotentiallyinduceananti-therapeuticantibodyresponse

LinkersØ Importantdeterminantofsafety,specificity,potencyand

activityofADCs

Ø Designedtobestableinthebloodstream

Ø CandeconjugateinvivoresultingincomplexDARspecies

Ø Chemicallycleavablelinkers

Ø Enzymaticallycleavablelinkers

Ø Non-cleavable linkers

Ø Playsaroleinimmunogenicity(neo-epitope)

ConjugationChemistryand Linkers

WhyDoesItMatter?

5

ImmunogenicityConsiderations

Biologicaltherapeuticshavethepotentialtoelicitanti-drugantibodies(ADAs)andgenerateunwantedimmunogenicity

Ø Non-neutralizingantibodies(NNab)

Ø Neutralizingantibodies(Nab)

ADAscanresultinarangeofeffectsfrombenignandasymptomatictoalteredpharmacokineticsand/orpharmacodynamicsandadverseclinicalsequelae

Immunogenicityriskmanagementisapivotalpartofthedrugdevelopmentprocess

Majorconcernsfromaclinicalperspective:

Crossreactwithendogenousproteintoinduceadverseaffects

Effectbioavailability,alterPK/PD(increasedordecreasedrateofclearance);NeutralizebiologicaleffectsandcompromisefurthertherapySAFETY EFFICACY

Anti-DrugAntibodies

Ø Separatelydivide&characterizefactorsthatcanaffectthelikelihood andmagnitudeofADAresponsesfromthesubsequentconsequences ofthatresponse

[Howmanypatientsarelikelytomountanimmuneresponse?] X[Whathappenstothepatientiftheymountanimmuneresponse?]

Ø Identifyriskfactorstoclassifythetherapeuticintohigherorlowerriskcategories

Ø ChallengesarisefromtheheterogeneousnatureofADCsandtheircomplexinvivobehavior

Ø ImmunogenicitystrategiesforADCsshouldincludearisk-basedassessmentandassaysthatareconsistentwithindustrybestpracticesandguidancedocumentsbutwithmodificationandadditionalcharacterizationtobetterunderstandtheADAspecificity

AssessingImmunogenicityRisk

Intrinsic:Product-relatedü Degreeofhomologywithendogenouscounterpart(s)ü Presenceofnon-humanaminoacidsequenceü Posttranslationmodification(glycosylationdifferences)ü Alterationstothemolecularstructure(aggregation,oxidation,deamidation)ü Modificationsthatintroduceuniqueepitopesü Prevalenceofpre-existingADAorcross-reactiveantibodiesthatcouldelicita

rapidrecallü Manufacturingprocess(impurities)ü Formulationü Doseandfrequencyoftherapy

Ø Hydrophobicity ofthecytotoxicdrug

Ø InternalizationofimmunecomplexescontainingADAandthecytotoxicdrugbynontargettissuescouldresultintoxicity

Ø Priorexposuretoatherapeuticcontainingthesamelinkerorsmallmoleculedrug

Ø Conjugationofahapten-likelinkerandsmall-moleculedrugisauniquepotentialimmunogenicityriskitself

Ø Epitopespreadeffectingdomainimmunetolerance

Ø CouplingatoxinviaalinkertoanmAbmayinduceneo-epitopesatthesiteofattachment

Ø Immunosuppressed oncologypatientpopulation

Ø Intravenousrouteofadministration

Ø ADAnotexpectedtocross-react withendogenousimmunoglobulinmolecules

Ø ADAsagainstthemAb maybemorelikelytoaffectefficacy;anti-cytotoxin ADAs,onceinternalized,areacidifiedandcatabolizedwithinthelysosomeandareexpectedtolosetheirneutralizingpotential

ImmunogenicityRiskAssessmentforADCs

Extrinsic:Patient/Treatment-relatedü Hostimmunecompetenceü Concomitant-medsü Previousexposuretosimilarproductsü Geneticpredispositionü Ageofpatientü Routeofadministration

8

ThelimiteddataonimmunogenicityofADCscomefromthetwoADCsontheUSmarket,Adcetris®(brentuximabvedotin)andKadcyla ®(ado-trastuzumabemtansine)andtheonediscontinuedADC,Mylotarg ®(gemtuzumabozogamicin)

ADAincidence:Ø 5.3%foradotrastuzumabemtansinewithADAsprimarilyagainstlinker-drugand/orneoepitopesinthemAb.

NodatahavebeenreportedontheneutralizingactivityoftheADAs.Ø 37%forbrentuximabvedotinallADAsdirectedagainstthechimericmAbcomponentoftheADC

Neutralizingantibodiesweredetectedin62% oftheADA-positivepatients.

Forgemtuzumabozogamicin,ADAswerenotdetectedinanypatientsenrolledinPhaseIIclinicalstudies.TwopatientsinaPhaseIstudydevelopedantibodiesagainstthecalicheamicin/linkerdomain.

ClinicalImmunogenicityDataforADCs

9

Anti-ADCAntibodyAssay:KeyConsiderations

Ø Onceappropriatereagentsbecomeavailable,domainspecificityassayscanbeimplemented

Ø Laterindevelopment,thefocuscanbeonclinicallyrelevantmeasurements

Ø Considerlinker(pH-labilelinker)ifanacid-dissociationpretreatmentimplemented

Ø Twopositivecontrolswithspecificitytoeithertheunconjugatedantibodyorthecytotoxin portionsØ Initially,reagentavailabilitylimitationsmayallowonlythemeasurementofADAforthewholemolecule

ImmunogenicitycouldincludeADAtotheantibody,linker,drugorepitopesinvolvingmultipleADCcomponents

TotalADAAssaysØ ScreenØ Confirm(Igdepleteand/or

excessdrug)Ø Quasi-quantitative

CharacterizationØ DomainspecificityØ IsotypeØ Subclass

NeutralizingAssaysØ Quasi-quantitativeor

qualitative

TieredImmunogenicityTestingStrategyforAnti-DrugAntibodiesAgainstADCs

NAbAssayPrinciple

12

Anti-ADCNeutralizingAntibodyAssay:EffectsofNAbonCellularResponse

CompetitiveLigandBindingELISA NeutralizingAntibodyBioassay

13

ThedomainsoftheADCmaybeinvolvedinsequentialstepsofthedrugfunctionalpathwayleadingtoitsfinalactivity (bindingtotargetfollowedbyinternalizationanddrugdeliverytocytosolicmicrotubules).

Selectionofalatestepofthedrugsignalcascade(celldeath)astheNAbassayreadoutwouldallowdetectionoftheapparentpotencyofallthedomainsinvolved.

TheinhibitoryeffectofNAbswouldthenindicatebindingtooneormoreofthedomains.

Asingleassaythereforecouldbesufficientforassessingalldomainspecificanti-drugNAbactivity.

Ø Generally,theuseofasingleNAbassayispreferredunlessamoredetailedevaluationofdomainspecificNAbisjustified.

InformationfromADAanalysisincludingdomainspecificitymaybeutilizedwhendeterminingtheneedfordomainspecificNAbactivityanalysis.

EvaluationofNAbsAgainstADCs

14

Availabilityofcriticalreagentsisofkeyimportanceforestablishingarobustandreliableperformanceofthebioanalyticalassay

Creatinganti-idiotypeantibodies againstthecarrierantibodyorthecytotoxicdrug

Conjugationwithkeyholelimpethemocyanin(KLH)oranadjuvantmayberequiredforantibodycreation

LabelingofADCsforcaptureand/ordetectionposeschallenges

ADC:CriticalReagentConsiderations

Obtainwell-characterizedreagentsorcharacterizethemin-housetoensurelot-tolotconsistencyandlong-termsupply

Securesufficientquantitiesofthelotsothatthereagentinventorycancovertheentirestudy

Establishandimplementanappropriatewayofassessingandbridgingdifferentreagentlots

Characterizationofreagentsshouldinclude:

Ø Anearlyassessmentofbindingproperties(cross-reactivitytomatrixcomponents)

Ø Bindingkineticsandanyalteredbindingissues(labelingorstorage)

Ø Biophysicalcharacterizationofreagentsshouldincludeidentity,concentration,storageconditionsandstabilityinformation

ü Isthedrugahigh-riskmolecule?

ü Chooseassayformat(s)ü UnderstanddrugMOA

biology,structureü Identifymeasurable

assayoutputrepresentingMOA(late/early)

ü Testspeciesserumthatrepresentsthematrixofthebiologicalsamples

ü Reagentidentification/resupply

ü Cellbanks/mycoplasmaü Drugdoseinmatrixü Chosedrugandligand

concentrationsü ObtainNabPCü ChoseNabPC

concentrationü Plateeffectü Cellpassagetestingü DOEfactorialusedto

optimizeconditions

ü MRDusingindividualsera

ü Cutpointassessmentü Matrixinterferenceü Sensitivityassessmentü Intra-/Interassay

precisionofcontrolsü DrugToleranceü Solubletarget

interferenceü Robustness

ü Reportwritingü Transfermeetingwith

CROü Criticalreagents

transferredü Transferexperimentsü ValidationatCROü Sponsorsupportto

CROü Datareviewü Sampleanalysisü Reports

ResearchPhase

Feasibility/Development

Qualification/Validation Transfer

LifecycleofaCell-BasedNAbAssay

ü Drugdose

ü Effectofmatrixü Cellpassagenumberü Cellcultureincubationtimeandconfluence

ü Optimalcellnumber/wellü Pre-platedvs.freshlyplatedcells

ü FBSselection/HIü BufferSelectionü Incubationtimeandtemperature

ü Trypsinizationü Washingsteps

ü Samplepreparation/pre-treatmentü Useoffactorialsforrobustnesstest

BioanalyticalMethod

ResponseMeasures(Y)S/NRatio,%recovery,EC50,backgroundsignal,%CV

ControllableFactors(X)cellnumber,[serum],

incubationtime

Factorialdesignallowsexperimentationonmanyfactorssimultaneously:

Ø Statisticalsoftwareforanalysis

Ø Interactionsoffactorsareidentified

Aseriesoftestsinwhichpurposefulchangesaremadetoinputfactorstoidentifycausesforsignificantchangesintheoutputresponses

DesignofExperiments(DOE)forNAbBioassayOptimization

ParameterEvaluationandSelection

CutPointDeterminationü Establishedusingsamplesfromtreatment-naïvesubjectsü 30samplestestedon3differentdaysbytwoanalysts,

usingsuitablestatisticalmethodsü 1%false-positiverateisacceptable

AssaySensitivityü Purifiedpositivecontrol

AssaySpecificityü DrugshouldbeblockedbyaspecificNAbresponse,versus

alternativestimuli,whichshouldnotbeblockedbyaspecificNAbresponse

ü Addtestmatrixdirectlytothebioassayintheabsenceofdrug

Precision

ü Intra/Inter-assayprecisionofpositiveandnegativecontrolsü Inter-analystprecisionofpositiveandnegativecontrols

Robustnessü Useoffactorialsforrobustnesstest(pushthesystem)

Reproducibility

ValidationConsiderationsforBioassays

DrugToleranceü Dependonthesurrogatepositivecontrol

MatrixInterference/Selectivityü Severalindividualmatrixsamples,(spiked/unspiked)ü Robustsignaltonoiseinthepresenceoftestmatrix

PCDilutionalLinearity/Prozone

StabilityAssessmentsü Mocksamples(PCspikedintomatrix)ü Cellline

18

TheConclusionsTherearedistinctchallengestothebioanalyticaldevelopmentofADCassaysrequiringsmallmoleculeandantibodybioanalyticalmethodsimplementedinparallel

Ø TheassayformatsandassayreagentsforADCbioanalysismayevolveasADCdrugcandidatesprogressfromearlytolatestagesofdrugdevelopment

ImmunogenicityassaysshouldbecapableofdetectingantibodiesdirectedagainstalldomainsØ DomaincharacterizationmaybehelpfulinunderstandingtheimmuneresponseelicitedbyADCs

andinformthedesignoffuturebiotherapeutics

Cell-basedassaysarewell-suitedfordetectingNAbsbecausetheymimicthemechanismbywhichtheNAbsandthedruginteractinvivo

EachNAbassayisuniqueandcomplex, andacarefulandthoroughassessmentandvalidationofmultiplefactorsincludingreagents,assaycontrols,matrixinterferenceandconditionswillhelptoensureasensitive,specificandrobustassay

Thecriticalreagentsarecrucialfactors;sufficientleadtimeandeffortmustbefactoredintomeetingprojectmilestones

CollectionofclinicaldatawillincreaseourunderstandingofADCsandhelptoidentifythemainspeciesinfluencingPKandimmunogenicbehavior

ThankYou!

20

Anti-ADCAssay:ScreeningandSpecificityAssays

21

Anti-ADCAssay:DomainCharacterizationStrategies

DrugResponsiveCellLineü Potencyassaycelllineisastartingpointü Shouldyieldaspecificresponsetothedrugü Maybenaturaloranengineeredestablishedcelllineü Cellculturingconditionsareanimportantparameterü Robustsignaltonoiseinthepresenceoftestmatrixü Canthecellsbefrozeninplatesü Randomization(template)ü Monitorpassage#andperformanceü DifferentlotsofFBSandassayperformanceü Serumdeprivation/definedmedium

DrugConcentrationü Shouldbeaconcentrationthatyieldsaround60-

70%maxresponse

PositiveControlAntibodiesü Generallypurifiedfromhyper-immunizedanimalantiseraü Shouldneutralizethebiologicalactivityofthetherapeuticü Specificitytoeithertheunconjugatedantibodyorthe

cytotoxinportions

DevelopmentConsiderationsforNeutralizingAntibodyBioassay

MRDassessmentandmatrixinterferenceAssayshouldbereproducible,specific,robust,andresultseasilyinterpretableTechnologyreadilyavailableatCRO

AssayEndpointü Measurableandrelevantassayendpoint

Ø Responseshouldassesssomeaspectofdrug’sMOAØ UtilizeearlyorlatesignalingeventtriggeredbythedrugØ Specificresponsetodrug

Reagentsü Therapeuticü Endogenousprotein(ifapplicable)ü Positivecontrolthatneutralizesdrugactivityü Reagentresupplyandcharacterization

AssayMethodology:SolidPhaseExtractionandAcidDissociation

SerumsampleorPositivecontrol

1.

Incubateserumsamplewithbiotinylateddrug

2.

TransfersampletoblockedSAplate

3.

Addacidtodissociateantibodies

Transfertoneutralizationplate

4.

Immunecomplex

ADA

Streptavidin

Biotinylateddrug

AssayMethodology:Dialysis

NeutralizedSample DialyzeagainstAssaybuffer

Restoresconditionstoallowforsampletobeusedincell-basedbioassays