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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!
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