Sebas&anSpindeldreherProjectcoordinator&WP3co-leader

onbehalfofWP3partners

ABIRISK-EIPOpenSymposiumNovember14th-16thLisbon,Portugal

WP3 Evaluation of different T cell assay formats

•  Inves&ga&onoftheclinicalrelevanceofbiopharmaceu&cal-associatedimmunogenicity…

•  Evalua&onofthepredic&vevalueofexis&ngtoolsandnewlydevelopedexvivomethods,alongwithinves&ga&onsintotheimmunologicalmechanismsthatformthebasisofthedevelopmentofan&-drugan&bodies….

•  Providedata-drivenfeed-backtoregulatorsandhealthcareprofessionals.

ABIRISKobjec&ves

•  Inves&ga&onoftheclinicalrelevanceofbiopharmaceu&cal-associatedimmunogenicity…

•  Evalua&onofthepredic&vevalueofexis&ngtoolsandnewlydevelopedexvivomethods,alongwithinves&ga&onsintotheimmunologicalmechanismsthatformthebasisofthedevelopmentofan&-drugan&bodies….

•  Providedata-drivenfeed-backtoregulatorsandhealthcareprofessionals.

ABIRISKobjec&ves

Workpackage3.1:Evalua&onofdifferentTcellassayapproaches

Novar&sSebas&anSpindeldreherAneSeKarleHannahMorganVerenaRombach-Riegraf*

INSERMUMR996MarcPallardyIsabelleTurbicaSophieTourdot*

INSERMUMR872Sébas&enLacroix-DesmazesAnastasPashovNimeshGuptaSandrineDelignat

CEABernardMaillèreMoustafaHamzeSylvainMeunierMariedeBouraineAmelieGoudetPierreBonnesoeur

DRK-BSDPeterMilanovStefanieRoth

SANOFIVincentMikolCatherinePradesLaurentDuhauMagaliAgnelStephaneCoren

NovoNordiskChris&anRossPedersenS&neLouiseReedtz-Runge*AnneMånssonKvarnhammar*

BayerSu-YiTsengPascaleBuchmannHans-WernerVohr*JeanneSeLo*MargretLeineweber*

MERCKSimonaRivaYvesFomekongNanfackDanielKramer*

SciCrossPierreDönnes

WP3partners

*LebABIRISKconsor&um

InvitroTcellassaysprovidedbyselectedEuropeanbasedCROs

•  An&topeEpiScreenTM–sponsoredbyMerck•  LonzaEpiBaseTM–sponsoredbyNovar3s•  Pla&neImmuno’lineTM–sponsoredbyCEA•  ProImmuneREVEAL®–sponsoredbySanofi

•  Thisstudywasnotdonetoiden&fythebestCRObuttounderstandhowrobusttheTcelldatais

•  Datainthispresenta&onisblindedbutifyouknowtheassaysprovidedbytheCROsyouwillbeabletoiden&fythedata

•  CROsreceivediden&calbatchesoftestitemswithSOPhowtohandlethem(freezethawcycles,etc.)

•  NotallCROswereblindedbutwereaskedtoapplytheirstandardassayformatandnottoop&mize

Consideredthat...

Testar&cles

MAb Type Target Adm.route

Indica&ons ADAincidence1-6

Infliximab ChimericAb(IgG1)

TNF-α i.v. Crohn’s,RA,Cutaneoussystemicsclerosis,Ankylosingspondyli&s

7-61%

Rituximab ChimericAb(IgG1)

CD20 i.v. Non-Hodgkin’slymphoma,SLE,Vasculi&s,PrimarySjögren’ssyndrome,Severepemphigus,RA

0-50%

Adalimumab HumanAb(IgG1)

TNF-α s.c. RA,Crohn’s,PsO,PsA 2.6-50%

Natalizumab ChimericAb(IgG4)

VLA-4Integrin

i.v. MS,Crohn’s 9%

Rebif® Cytokine IFNAR s.c. MS 12-28%

Betaferon® Cytokine IFNAR s.c. MS 16.5–47%

1DellucSetal.FASEBJ,2011;2BakerMetal.Self/Nonself,2010;3SauerbornM.HandbookofTherapeu&can&bodies2ndedi&on,2014;4BertoloSoetal.JNeurol,2004;5ZisapelMetal.JRheumatol,2015;6HsuLetal.ExpertRevClinImmunol,2013

•  Assayvalida&on–  Robustnessandconsistencyofdata–  Rankingoftestitemsrela&vetoan&genicityriskorimmunogenicitypoten&al

•  Biologicalvalida&on–  DoinvitroassayswithheathydonorcellsreflectinvivoTcellresponsesintreatedpa&ents?

•  Clinicalvalida&on–  Predic&ngclinicalincidence/outcome

•  Isclinicalvalida&onpossibleatall?•  Tcelldataconsistency?

Evalua&on/Valida&on?

Tcellassayformats

§  Short-termTcellassays Providers1,3,4

Control

+protein

Measure of the frequency of IFNγ secreting cells: IFNγ ELISPOT

§  Long-termTcellassays

-  Onepriming-  2res&mula&onrounds(precursoramplifica&on)

Provider2

Prolifera&onMeasureofcellprolifera&on:CFSE+,3HorEdU+Measureofcytokinesecre&on:IL-2ELISPOT

Tcellspecificityisassesseddirectlyaberashortculturestep7-10days

Tcellsareamplifiedinvitrobys&mula&onwithproteinduring3-4weeks

Assayparameter Provider1 Provider2 Provider3 Provider4Testedan&gens Infliximab,adalimumab,rituximab,natalizumab,Betaferon®andRebif®

Noofdonors 50 16 50 50

Cells Ag-loadedDC(matura&ons&mnotspecified)+CFSE-labelledCD8-depeltedPBMC

Ag-loadedDC(maturedwithLPS)+CD4Tcells

Ag-loadedDC(maturedwithTNFα+IL-1β)+CD4Tcells

Ag-loadedDC(maturedTNFα)+CD4Tcells

Readout CFSEFACS

IFN-γ ELISPOT

EdUFACS

Thymidineincorpora&onandIL-2ELISPOT

Dataevalua&on Posi&veif%s&mula&on≥0.5%and2SEMabovebackground

Posi&vewhenspotcount≥2xbackgroundandminimaldifferenceof25spots

Posi&veifSI≥2andsignificantvscontrol(p<0.05)

Posi&veifSI≥2andsignificantvscontrol(p<0.05)

Ranking Rankingbasedondonorfrequencyandmagnitude

Rankingbasedonprecursor&donorfrequency

Rankingbasedondonorfrequencyandmagnitude

Rankingbasedondonorfrequency

ComparisonofdifferentTcellassayapproaches

10

Resultsprovider1

•  “Overall,takingintoaccountthelownumbersofrespondingdonors,thelowlevelsof%s&mula&onandlackofsignificantresponses,thedatasuggeststhatthesetestproteinsareunlikelytobestronglyan&genic.However,externalfactorssuchaslengthand/orconcentra&onofexposure,repeatedexposureevents,andmode(s)ofac&onmayaffectresponseselicitedinvivo.”

PrecursorfrequencymAbs

Resultsprovider2

Precursorfrequencyinterferon

•  “Incontrasttothethreean&bodiesRituximab,InfliximabandNatalizumabwhicharelessimmunogenic,thean&bodyAdalimumabappearstobemoderatelyimmunogenic.RituximabisnotsignificantlydifferentfromtheAdalimumabbutisalsosimilartothean&bodiesInfliximabandNatalizumab.”

•  “Onthebasisofthesedata,bothformsofIFN-βwouldhavebeenconsideredasmoleculeswithmoderateriskofimmunogenicity”

Resultsprovider3

Product Number of responding

donors

Frequency of responding donors (%)

Mean SI of responding

donors

KLH 50 100 15.16

Adalimumab 9 18 2.59

Rituximab 8 16 3.65

Infliximab 7 14 2.95

Natalizumab 5 10 2.77

Betaferon 4 8 2.47

Rebif 2 4 2.17

•  “Rituximab,adalimumabandinfliximabarehigherriskcomparedtonatalizumab.”

•  “DirectcomparisonoftheIFNβproductssuggeststhatBetaferon®isathigherriskthanRebif®.”

Resultsprovider4

•  Alltestitems<10%cut-off,sugges&ngthattheyallfallintothe‘lowrisk’categoryforpoten&alclinicalimmunogenicity(basedonhistoricdatawiththisassay)

•  However,β-IFNsandan&-α-TNFsmayhaveaffectedtheoutcomeduetodirecteffectsonDCviabilityand/ormatura&on

Comparisonofresponsesacrossallassays

Comparisonofranking

Infliximab Rituximab Adalimumab Natalizumab Betaferon® Rebif®

Provider 1 1 3 2 4 2 1

Provider 2 3 2 1 4 1 1

Provider 3 3 1 1 4 1 2

Provider 4 1 2 3 4 1 1

Colourcodingindicatesranking,fromhightolow

Rankingonthisslidedoesnotnecessarilyreflectsta&s&callysignificantdifferences!

•  Allcompoundstestedhavedemonstratedimmunogenicityinclinicalinvivostudies,butonlyoneassaycouldshowstronginvitroimmunogenicity

–  Assaysnotsensi&ve/accurateenoughtodifferen&ate

•  Nogoodcorrela&onintermsofrankingbetweendifferentassays

•  UnderstandingMoAofthecompoundsisessen&al

–  βIFNsnotsuitabletouseinDC:Tcellassaysduetotheirinterac&onwithDCs

–  An&-αTNFspossiblyinterferewiththeDC matura&onwhenTNFαisusedformatura&on

Overallconclusions

•  Thecomparisonand“indirectearlyvalida&on”ofselectedpredic&veimmunogenicitytoolswasoneoftheini&alkeygoalsofABIRISK

•  Thisprojecthasbeenlogis&callycarriedoutaccordingtoplannedstrategy

•  Thisdatademonstratesalackofcorrela&onbetweenthedifferentassaysusedinthisproject(NB,noop&miza&onswereallowed)

•  However,lowandhighresponsescouldbedifferen&atedconsistentlyandmatchedclinicalexperience,althoughsomeassaysfailedtopredictahighriskatall.

•  Thereisaneedforgloballyacceptedreferencestandardsandqualitycontrolstoensurecomparableperformanceofsuchassays;notjuststrongan&genssuchasKLH.

Generalconclusions

Thankyou!