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Immunobiology
j ou rn a l h o m e pa g e :w w w . e l s e v i e r . c o m / l o c a t e / i m b i o
Eculizumab treatment during pregnancy does not affect the complement system activity of the newborn
Randi Fykse Hallstensen
a, Grethe Bergseth
b,1, Stian Foss
c,d,e,1, Steinar Jæger
a,
Tobias Gedde-Dahl
f, Jan Holt
g, Dorte Christiansen
b, Corinna Lau
b, Ole-Lars Brekke
b,h, Elina Armstrong
i, Vedran Stefanovic
j, Jan Terje Andersen
c,d, Inger Sandlie
c,d,e,
Tom Eirik Mollnes
b,d,h,k,∗aDivisionofInternalMedicine,NordlandHospital,Bodø,Norway
bResearchLaboratory,NordlandHospital,Bodø,Norway
cCentreforImmuneRegulation,UniversityofOslo,Norway
dDepartmentofImmunology,OsloUniversityHospitalRikshospitaletandUniversityofOslo,Norway
eDepartmentofBiosciences,UniversityofOslo,Norway
fDepartmentofHematology,OsloUniversityHospitalRikshospitalet,Oslo,Norway
gDivisionofPediatrics,NordlandHospital,Bodø,Norway
hFacultyofHealthSciences,UniversityofTromsø,Norway
iCoagulationDisorderUnit,DepartmentofHematology,HelsinkiUniversityCentralHospitalComprehensiveCancerCenter,Helsinki,Finland
jDepartmentofObstetricsandGynecology,HelsinkiUniversityCentralHospital,Helsinki,Finland
kCentreofMolecularInflammationResearch,NorwegianUniversityofScienceandTechnology,Trondheim,Norway
a r t i c l e i n f o
Articlehistory:
Received1November2014
Receivedinrevisedform4November2014 Accepted4November2014
Availableonline13November2014
Keywords:
Complement Therapy Placenta Eculizumab
Paroxysmalnocturnalhemoglobinuria
a b s t r a c t
EculizumabisahumanizedIgG2/4chimericanti-complementC5antibodyusedtotreatpatientswith paroxysmalnocturnalhemoglobinuria(PNH)oratypicalhemolyticuremicsyndrome.Theaimofthis studywastoevaluatewhetherornotthecomplementactivityinnewbornsfrompregnantwomenwho receiveeculizumabisimpaired.Anoveleculizumab-C5complex(E-C5)specificassaywasdeveloped andrevealedthattwonewbornscarriedonly6–7%oftheE-C5detectedintheireculizumab-treatedPNH mothers.Serumfromthepregnantwomencompletelylackedterminalcomplementpathwayactivity, whereasthecomplementactivityintheserumofthenewbornswascompletelynormal.Datafromthe pregnantwomenandtheirnewbornswerecomparedwiththatofhealthyage-matchedfemalecontrols andhealthynewborns,aswellasanon-treatedpregnantwomanwithPNHandhernewborn.These allshowednormalcomplementactivitywithoutdetectableE-C5complexes.Furthermore,absenceof eculizumaborE-C5inthenewborncouldnotbeexplainedbylackofeculizumabbindingtotheneonatal Fcreceptor(FcRn),aseculizumabboundstronglytothereceptorinvitro.Inconclusion,despitebinding toFcRnneithereculizumabnorE-C5accumulatesinfetalplasma,andeculizumabtreatmentduring pregnancydoesnotimpairthecomplementfunctioninthenewborn.
©2014TheAuthors.PublishedbyElsevierGmbH.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/3.0/).
Introduction
Complementispartoftheinnateimmunesystem,recogniz- ing microbes and thus protecting the host against invasion of pathogens. Although discovered more than hundredyears ago, asa factorinserumthatinducedlysisandkillingofbacteriain
∗ Corresponding author at: Research Laboratory, Nordland Hospital,Bodø, Norway.Tel.:+4790630015;fax:+4723073510.
E-mailaddress:t.e.mollnes@medisin.uio.no(T.E.Mollnes).
1 Theseauthorscontributedequallytothestudy.
thepresence of antibodies,it is only during the last couple of decadesthat the functional role of complement in humandis- easeshasbeenexplored.Previouslyregarded asapuredefense system,complementisnowappreciatedascruciallyimportantfor tissuehomeostasisbyclearancemechanisms,tissueregeneration andrepair(Ricklinetal.,2010).Themaineffectorsofcomplement activationtoservethesefunctionsareinductionofanumberof downstreaminflammatorynetworksaswellasphagocytosisand celllysis (Walport, 2001).Complement activationproductslike theanaphylatoxinsC3aandC5aarepotentinflammatoryinduc- ers(Klosetal.,2009).AssemblyoftheterminalC5b-9complement complex (TCC) on a lipid membrane leads to insertion of the
http://dx.doi.org/10.1016/j.imbio.2014.11.003
0171-2985/© 2014 The Authors. Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/
licenses/by-nc-nd/3.0/).
membraneattackcomplex(MAC),whichinsublyticdosesinduces inflammation(Morgan,1989;Teglaetal.,2011),orinthecaseof redbloodcellsandneisserialbacterialeadstolysis(Bhakdi,1991).
WhentheTCCis formedinthefluid phase, thesolublesC5b-9 complexisgenerated,whichisausefulindicatorofcomplement activation.
Complement activation is a double-edged sword.By induc- ing inflammation and cell lysis as response to both microbes and damaged host structures, there is a potential for comple- ment to damage self-tissue (Mollnes et al., 2002). In order to avoidself-damage,thereisanabsoluteneedforstrictcontrolby a number of fluid-phaseand membrane-bound regulatorypro- teins(Meri,2007;Sjobergetal.,2009;ZipfelandSkerka,2009).
Many of the complement components are spontaneously acti- vatedintheabsenceofactivatingstimuli.Thus,thelackofsuch a regulatory protein per se is sufficient for a pathologic com- plementactivation leading totissue damage in variousdisease conditions.
Geneticdeficiencies ormutationsof regulatoryproteinslike factorH,factorIandmembranecofactorprotein(CD46)areasso- ciated with diseases like atypical hemolytic uremic syndrome (aHUS)(Leetal.,2010),membranoproliferativeglomerulonephris- tis(MPGN)typeII(densedepositsdisease)(Servaisetal.,2012) andage-relatedmaculardegeneration(Shawetal.,2012).Parox- ysmalnocturnalhemoglobinuria(PNH)isadiseasecharacterized byspontaneouscomplement-mediatedredbloodcelllysisdueto asomaticmutationinthePIG-Agenecodingfortheglycosylphos- phatidylinositolanchor,whichisrequiredfortheattachmentof complementregulatorsdecayacceleratingfactor(DAF;CD55)and CD59(Takedaet al.,1993).ThephenotypeofPNH issimilarto geneticdeficiencyofCD59(Yamashinaetal.,1990),suggestingthat thisprotein,whichprotectsagainstinsertionofC8andC9intothe TCCand,thus,againstcelllysis,iscrucialforthepathogenesisof PNH.
Efficient therapeutic alternatives for these conditions are limited.Eculizumab(Soliris®),ahumanizedIgG2/4kappaanti-C5 antibodyistheonlyspecificcomplement inhibitorapprovedby theFoodandDrugAdministration(FDA)(Dmytrijuketal.,2008).
It blocksthe enzymaticcleavage ofC5toC5a and C5b,leaving theinitialactivationofthethreecomplementpathwaysuptoand includingC3intact.Eculizumabisinroutineclinicaluseontheindi- cationofPNHandaHUS.Manyyearsofexperiencehaveshownthat eculizumabreducestheneedforbloodtransfusionsandimproves theprognosisinpatientswithPNH(Parker,2009;Brodsky,2009;
Kellyetal.,2011;Luzzattoetal.,2011;RothandDuhrsen,2011;
Palmeiraetal.,2012;Hillmenetal.,2013;Varela,2013;Heitlinger, 2013).Recently,eculizumabwasalsodocumentedtobeeffective andwithoutadverseeffectsinchildren(Reissetal.,2014).Thus, treatmentwitheculizumabisgenerally safe,themainconcerns beinghighcostsandasmallriskofneisserialinfection,requiring vaccinationpriortotreatment.Recently,itwassuggestedthatinhi- bitionofC3wouldreducelysismorethanC5inhibition,sincesome PNHcellsareopsonizedwithC3fragmentsandundergoextravas- cularhemolysis(Risitanoetal.,2014).WhetherC3orC5shouldbe thetargetforsystemiccomplementinhibitionoveralongperiod oftimeinPNHpatientswilldependonrelativetheriskofinfec- tionsduetolackofC3opsonisationofpathogensascomparedto thebenefitonthehemolysis.
LittleisknownaboutpossiblepassageofeculizumaborE-C5 over thehumanplacentaand, consequently,howsuchpassage couldaffectthecomplement systemofthenewborn.Thisis of particularimportanceinPNHsincewithouttreatment,theriskof complicationsisincreasedforboththepregnantwomanandher childduringpregnancyanddelivery(deGuibertetal.,2011).Only afewcaseshavebeenexaminedwithrespecttotheamountoffree eculizumabinpregnantwomenandtheirnewborns,reviewedby
Kellyetal.(Kellyetal.,2010).Offournewbornsfromthreemoth- erswiththerapeuticlevels,eculizumabwasundetectedintwoand detectedinlowamountsintwo(twins).
ThereisaneedforsensitiveandspecificmethodstodetectE-C5, combinedwithnovelfunctionalassaysforevaluationofthetotal complementactivity,inordertostudythebalancebetweenfree eculizumabandfreeC5.Thus,theaimsofthepresentstudywereto establishsuchassays,andtoinvestigatewhetherornoteculizumab giventopregnantwomenaffectsthecomplementsystemofthe newborn.
Methods
Patients
Threepregnant women withPNH and theirnewborns were included. Two of the patients were treated with eculizumab.
Patient1(P1),born1981,wasdiagnosedwithPNHin2007dur- ing the sixth week of her first pregnancy. Fluorescein-labeled proaerolysin(FLAER)negativemonocytesandgranulocyteswere 80%,lactatedehydrogenase(LD)840Units/L(U/L)andhemoglobin (Hb)11.2g/dL.Lowmolecularweightheparin(LMWH)wasstarted asthrombosisprophylaxis.Emergencycesareansectionwasper- formedat35thgestationalweekduetofetaldistress.TheApgar scorewas5 andplacentashowedimmaturitywithoutthrombi.
Duringhernextpregnancyin2010,shewastreatedwithLMWH from week nine. FLEAR negative monocytes and granulocytes were 91% and 94%, respectively. Hb fell gradually to 7g/dL and LD increased to3300U/L. Fatigue gradually increased.The patient received meningococcal vaccine and was started with eculizumabduringweek34(600mg/weekforfourweeks,there- after900mgeverysecondweek).Thelastinfusioninthepregnancy wasgiven sixdays beforedelivery. Sheimprovedclinically, Hb increased to 9g/dL and LD decreased to 300U/L. At 41 gesta- tional week she delivered a healthy child. Vaginal birth was complicated by postpartum hemorrhage (the lowest postpar- tumHblevelwas5.8g/dL)and shereceivedfourredbloodcell units.
Patient2(P2),born1986,wasdiagnosedwithclassicalPNHin 2009.Initiallyflowcytometryofredcellsshowed30%CD59neg- ativecells(typeIII)and47%typeIIcells.In2012,FLAERshowed 92%cloneofGPInegativegranulocytes.Despitechronichemoly- sisandLDranging400–600U/L,Hblevelswereclosetonormal (11–12g/dL)andshedidnotneedanytransfusions.Aspirinwas usedforprimarythromboprophylaxis.Herfirstpregnancyresulted in earlyspontaneousmiscarriageatweek10 inFebruary 2013.
AspirinhadbeenswitchedtoLMWHprophylaxissincethedetec- tionofthepregnancy.LDandHbremainedstable.Sheconceived againsixmonthslater.LMWHwasusedforprophylaxisandshe startedwitheculizumabduringhersecondtrimesterinNovember 2013,initiallydosed600mgonceweeklyforfourdosesfollowed by900mgeverytwoweeks.Inadditiontoreceivingmeningococ- calvaccine priorstarting eculizumab,continuousoral penicillin prophylaxiswasalsoused.Therewerenocomplicationsduring pregnancy,Hbwas10–12g/dLandLDdecreasedtonormallevels.
Deliverywaselectivelyinducedduetohyperglycemiainpregnancy week39,April2014.Ahealthybabywasdeliveredbyemergency cesareansectionduetoobstructedlabor.Bloodlossduetouterine atonywas2500mLandthepatientreceivedtwounitsofPRBCs, eightunitsof plateletsandthree unitsofplasmaonthedayof delivery.Shehadreceivedeculizumabonschedule9dayspriorto deliveryandreceivedthenextdosethedaybeforedischarge.Her Hbwas9.6g/dLandLDwas387U/Latdischarge.Twelvedaysafter dischargethepatientdevelopeduterineinfectionandamesenteric veinthrombosiswasdiagnosed.ProphylacticLMWHwasincreased
totherapeuticdosewithplansoflong-termanticoagulationwith warfarin.Eculizumabwasplannedtocontinueforatleast3months post-partum.
Patient3(P3)wasnottreatedwitheculizumab.Shewasborn 1981anddiagnosedwithclassicalPNHin1998.Clonesizefrom granulocyteshasbeen50–60%.Shehada historyof pulmonary embolismtwoyearsafterthePNHdiagnosisandwarfarinanticoag- ulationwasthenstarted.Shehasnotrequiredtransfusions,buthas beenirondeficient.Herobstetrichistoryincludesaspontaneous earlymiscarriagein2008andtwopriorsuccessfulpregnanciesand deliveriesin2009and2011,forwhichsherequiredmultipleredcell transfusions.HermostrecentpregnancywasdetectedinJuly2013.
Eculizumabtreatmentwasoffered,butshedeclinedasherHblevels stayedat10–12g/dLandtherewerenosignsofincreasedhemoly- sis(LD350–450U/L).ShereceivedLMWHattreatmentdoses.She had anuncomplicated vaginal termdeliveryof a healthybaby.
Therewerenosignsofincreasedhemolysisandthepuerperium wasuneventful.
Bloodsamplingandpreparation
SerumsampleswereobtainedfromP1andP2beforestarting eculizumabtreatmentandatdelivery.SerumfromP3wasobtained duringpregnancyandatdelivery.Bloodsamplesfromtheumbilcal cordswerecarefullyobtainedbyneedlepunctureofaplacentalvein toavoidcontaminationfrommothers’bloodandWharton’sjelly.
Serumwaspreparedbydrawingwholebloodintoemptytubes, clottingwasallowedfor60min,followedby15mincentrifuga- tionat3220×g.Thesampleswereimmediatelystoredinmultiple aliquotsat−80◦Candrepeatedthawingandfreezingwasavoided.
Reagents
Chemicalsandbuffers
2,2-Azino-di(3-ethylbenzthiosolinesulphonate)(ABTS),ethyl- enediaminetetra-aceticacid(EDTA) and polyethylenesorbitan monolaurate(Tween20)wereobtainedfromSigma-Aldrich(St.
Louis,MO). Na2CO3·H2O,NaHCO3, KH2PO4, Na2HPO4,NaCl and NaC2H3O2 were obtained from Merck (Darmstadt, Germany).
Phosphate-bufferedsaline(PBS) consistedof 0.068Mphosphate buffer,pH7.2,containing0.077MNaCl.Coatingbufferconsistedof 0.05Mcarbonatebuffer,pH9.6.AntigensandantibodiesforELISA analysesweredilutedinPBScontaining0.2%Tween20.Washing bufferconsistedofPBScontaining0.1%Tween20.Substratebuffer consistedof0.15Msodium-acetatebuffer,pH4.0.
Antibodiesandproteins
Monoclonalantibody against human C5was obtained from Quidel(SanDiego,CA).Horseradishperoxidase(HRP)-conjugated mouseanti-humanIgG4antibody(cloneHP6025)wasobtained fromSouthernBiotech(Birmingham,AL).Eculizumab(Soliris®),a humanizedmonoclonalantibody(IgG2/4)inhibitingcleavageofC5, wasobtainedfromAlexionPharmaceuticals(Cheshire,CT).Purified humancomplementproteinC5wasobtainedfromQuidel.
Functionalcomplementactivityassay
FunctionalcomplementactivitywasmeasuredusingtheCom- plementsystemScreenWieslab® (Euro DiagnosticaAB,Malmö, Sweden)detectingcomplement activationthrough theclassical andalternative pathways, withthecompleteterminal pathway activationasreadout.Thus,sampleswithoutC5activitywillgive zero%activityinthisassay,duetoblockingoftheterminalpathway, althoughtheactivityoftheinitialpathwaysuptoandincludingC3 isintact.
Enzyme-immunoassayforthedetectionofeculizumab-C5 complexes
AnewsandwichELISAtomeasureeculizumab-C5(E-C5)com- plexeswasdesignedbyusinganti-C5andanti-IgG4ascaptureand detectionantibodies,respectively,andusinginvitro-generatedE- C5complexesasstandard.ThestandardofE-C5complexeswas madeinvitrobyaddingeculizumabtoanormalserumpoolfrom20 healthyblooddonors.EculizumabwasdilutedinPBStwofoldin10 stepsstartingat1mg/mL.FiveLfromeachdilutionwereaddedto newtubes,eachcontaining95Lofthestandardserumpool.Opti- maldilutionsofthereagentsinthedifferentstepswerefoundafter carefultitration.NuncMaxisorp®96-wellsImmunoplates(Thermo ScientificNuncA/S,Roskilde,Denmark)werecoatedovernightat 4◦C with a monoclonalantibody againsthuman C5(1.1g/mL in0.05Mcarbonatebuffer).Standardsand serumsampleswere addedinduplicates,diluted1:100inPBScontaining0.2%Tween 20andincubatedatroomtemperaturefor60min.Samplesfrom thepatientsreceivingeculizumabneededfurtherdilution,upto 10times,inordertoobtainabsorbancevaluesattheoptimumof thestandardcurve.HRP-conjugatedmouseanti-humanIgG4anti- bodywasdiluted1:2500inPBScontaining0.2%Tween20,added totheplateandincubatedatroomtemperaturefor60min.The plateswereautomaticallywashedthreetimesaftereachincubation (Hydrospeed,Tecan TradingAG, Männedorf, Switzerland)using PBScontaining0.1%Tween20.Assubstrate,H2O2wasusedata finalconcentrationof2.4×10−3%,in0.15Msodiumacetatebuffer, pH4.0,containing180mg/LABTS.Thewellswerefilledwith100L inallsteps,exceptforthewashingssteps(250L).Opticalden- sity(OD)wasdeterminedat405nm(DynexMRXereader,DYNEX Technologies,Inc,Chantilly,VA,USA)whenthehigheststandard hadreachedanODofapproximately1.0(10–15min).
ValidityandspecificityoftheassaywasverifiedusingC5defi- cient serum reconstituted withpurified C5.The reliability was investigatedby calculating inter-andintra assayvariation. The sensitivityandaccuracywasexaminedbyspikingnormalhuman serumaswellasthepatientserumsampleswereeculizumab(1, 10,30and100g/mL)and/orC5protein(100g/mL).Theconcen- trationoftheE-C5complexesisgivenaseculizumab-equivalent amounting/mL.
Immunofixation
Theanti-kappa antibody for immunofixationelectrophoresis wasobtainedfrom Sebia (Evry Cedex,France). Immunofixation electrophoresisofserumsamples,eculizumab,freeC5andE-C5 complexesinPBSwasperformedusingaHYDRASYSFocusingelec- trophoresisandimmunofixationsystemfromSebia(EvryCedex, France). Eculizumab-C5 complexes were generated in vitro by mixing eculizumab and purified C5 at different molar ratios.
Immunofixationwasperformedusing12Lanti-kappaantibody andHydragel4IFkits(Sebia).Afterelectrophoresis,thegelswere stainedwithCoomassiebrilliantblueandair-dried.Thegelswere photographedusingaChemiDocXRS+gelimaging systemwith ImageLabsoftwarefromBioRad(Hercules,CA).
Enzyme-immunoassayforFcRnbinding
Nunc MaxiSorp® microtiter wells were coated withtitrated amounts of eculizumab, rituximab (anti-CD20, Roche, Basel, Switzerland)orinfliximab(anti-tumornecrosisfactor␣,Janssen Biologics, Leiden, The Netherlands) diluted to 4g/mL in PBS. Following incubation overnight at 4◦C, the plates were blocked with 4% skimmed milk/PBS/Tween20 (S/PBS/T) for 2h atroomtemperatureandwashedfourtimesinPBS/T.Recombi- nantglutathione-S-transferase(GST)-taggedsolublehumanFcRn
(shFcRn-GST)(Andersenet al.,2008)wasdilutedto2g/mLin S/PBS/T andincubated for1hpriorto washingas above.Bind- ing was detectedby a HRP-conjugated goat anti-GSTantibody (GEHealthcare,Oslo,Norway).Platesweredevelopedbyadding 3,3,5,5-Tetramethylbenzidinesubstrate(Calbiochem,SanDiego, CA)andthereactionwasterminatedbyadditionof100L1M HCl.Absorbancewasmeasuredat450nmusingaSunriseTECAN spectrophotometer(TecanTradingAG,Männedorf,Switzerland).
Statistics
Student’st-test wasusedtocomparethecomplement activ- ityineculizumab-treatedpatientsandtheirnewbornswiththe respectivecontrols.
Ethics
Thestudywasapprovedbytheregionalethicalcommittees.
Writteninformedconsentwasobtainedfromthepatientsandcon- trols.
Results
Anovelassayforquantificationofeculizumab-C5(E-C5) complexesinhumanserum
Assaydesign,reliabilityandvalidity
TheE-C5assaywasdesigned asa double-antibody enzyme- immunoassay using an anti-C5 antibody for capture and an anti-IgG4antibodyfordetection.AnE-C5standardwasproduced byadding eculizumabtoa normal humanserumpool, andthe standard curve was designed by two-fold dilution starting at 100g/mL(Fig.1A).TheamountsofE-C5aregivenineculizumab- equivalents, i.e. 1g/mL E-C5 corresponds to 1g eculizumab addedto1mLofserum.Thelowerdetectionlimit(definedasback- ground+2SD)wasapproximately1g/mL(Fig.1A).Theintra-and inter-assaycoefficients ofvariationwere8.9% and4.9%,respec- tively.
TheassaydetectedE-C5inhumanserumwithhighspecificity asdemonstratedusingserumfromageneticC5-deficientindivid- ual(Lappegardetal.,2009)withandwithoutadditionofpurified C5(Fig.1B).Nosignalwasdetecteduponadditionofupto100g eculizumab/mLtoC5-deficientserum,whereasE-C5wasdetected inadose-dependentmannerafterreconstitutionofthedeficient serumwithC5.Duringspikingexperimentsusingnormalhuman sera,theamountsofE-C5detectedcorrespondedexactlytothe exogenously added 1, 10 and 30geculizumab permL serum (Fig.1C).Thisindicatesthattherewasnointerferenceintheassay.
E-C5complexesinhumanserumsamples
Serumsampleswereadded inoptimaldilutions(seeSection
“Methods”)andtheresultswerecorrectedforthedilutiontogive theE-C5concentrationinundilutedserum (Fig.1D). Using this assay,133g/mLand115g/mLofE-C5weredetectedinserum fromtwoeculizumabtreatedmothers(P1andP2).Incontrast,only 8.1g/mLand7.8g/mLweredetectedintheirnewborns,corre- spondingto6–7%ofthatinthemothers’sera.Incontrolsera(n=6 foradultsandnewborns)andintheserumofanon-eculizumab- treatedPNHpatient(P3)andhernewborn,E-C5wasnotdetected (Fig.1D).
Effectofeculizumabonfunctionalcomplementactivity Assayforfunctionalcomplementactivity
Serum samples from the eculizumab-treated PNH mothers (P1 and P2)and theirnewborns, as well asfrom the controls,
Fig.1. Assayforquantificationofeculizumab-C5(E-C5)complexes.(A)Thestandard wasmadebyaddingeculizumab(0–100g/mL)toanormalhumanserumpool.
Thisstandardshowedadose-dependentcurvereadasopticaldensity(OD)andwas usedasstandardcurvefortheassay.TheamountofE-C5complexesisindicatedin eculicumabequivalents,i.e.correspondingtotheamounting/mLofeculizumab addedpermLofserum.Brokenlinesindicatelowerdetectionlimit.(B)Specificityof theassaywasexaminedusingC5-deficientserum(C5D,closedcircles).Eculizumab wasaddedtoC5DwithoutanyincreaseinOD.Incontrast,adoseresponsecurve wasobtainedwithformationofE-C5afterreconstitutionofC5DwithpurifiedC5 (C5D+C5,opencircle).(C)Normalhumanserumwasspikedwith1,20and30g eculizumabpermLandmeasuredintheE-C5assay.Theobtainedvaluescorre- spondedcloselytothoseexpected.(D)TheconcentrationofE-C5-complexeswas 133and115g/mLinP1andP2serum(closedcircles),whereasthenewborns’
correspondingserumcontained8.1and7.8g/mL,respectively(closedtriangles).
Adult(opencircles)andnewborn(opentriangles)controlsera(n=6ofeach)didnot containdetectableE-C5complexes,aswasthecaseforP3(PNHwithouteculizumab, openasterisk)andhernewborn(closedasterisk).Brokenlineindicatelowerdetec- tionlimit.
wereexaminedintheTotalComplementFunctionalScreenELISA (Wieslab®),whichissuperiortothetraditionalCH50basedonred celllysis withrespect tosensitivityand reproducibility(Seelen et al.,2005).The assayspecificallydetects complement activity of thethree initialpathwaysusingterminal assembly ofC5b-9 asreadout.Thus,completeinhibitionofC5wouldabolishactiv- ityof theterminal pathwayirrespectiveof whichpathwaythat is initially activated, whereas the initialpathway activationup toand including C3is intact.We here usedthe classicalpath- way(CP)and alternativepathway (AP)assays for evaluationof their suitability for evaluation of eculizumab. Serum from P1 and P2 had nodetectable activityin CPand AP (Fig.2A), con- sistentwithcomplete inhibition ofC5.Thesixhealthy controls and P3 showed normal complement activity(significant differ- encebetween P1 and P2 versuscontrols: p<0.0001 for CPand p=0.018forAP).SerumfromthenewbornsofP1andP2showed completely normal complement activity (Fig. 2B), with nosig- nificantdifferencefromthecontrols(p=0.54forCPandp=0.30 forAP).
Fig.2.Complementfunctionalactivity.(A)Serumcomplementactivitywasmea- suredinP1andP2atdelivery(closedcircles),insixhealthycontrols(opencircles)as wellasinP3atdelivery(PNHwithouteculizumab,openasterisk)intheclassical(CP) andalternative(AP)pathways.Consistentwithfullinhibitoryeffectontheterminal pathwaybyeculizumab,therewasnoactivityintheP1andP2sera.Horizontallines indicateloverreferencevalues.(B)Newbornshavelowercomplementactivityand greaterrangethanadults(cfr.scaleony-axis)andreferencerangesforthefunc- tionalassayisnotestablished.ThenewbornsofP1andP2showedhighactivityin bothpathways(closedtriangles),consistentwithafullyactivecomplementsystem.
Controlsincludehealthynormalnewborns(opentriangels)andnewbornfromP3 (PNHwithouteculizumab,closedasterisk).(C)EffectofaddingC5inincreasingcon- centrationsonserumfunctionalactivityoftheterminalpathwaymeasuredbythe classicalpathwayassayfromanormaladultserum(opencircles)andserumfrom P1.(D)SameexperimentasdescribedinpanelC,usingserumfromanormalhealthy newborn(opentriangles)andserumfromthenewbornofP1(closedtriangles).
EffectofadditionofC5totheserumonfunctionalcomplement activity
TheeffectofaddingincreasingamountsofpurifiedC5tothe serumwasstudiedusingtheCPassay.Theaveragenormalserum concentrationinadultsis75g/mL.Additionof1mg/mLofC5to serumfromP1restoredfunctionalcomplementactivity(Fig.2C), whereasnoadditionalcomplementactivitywasobservedinserum fromhernewborn(Fig.2D),suggestingefficientsuppressionofC5 andthepresenceoffreeeculizumab inthemother,butnofree eculizumabavailableforC5suppressioninhernewborn.
Effectofeculizumabonfunctionalcomplementactivityintitrated sera
Titrationofserainthefunctionalassay
Since the functional complement activity examined in the experimentspresentedinFig.2wasmeasuredasend-pointata fixeddilutionoftheserum,weinvestigatedtheeffectofeculizumab onthecomplement activityover a long dilution range(Fig.3).
By usingboth CP(Fig.3A)and AP (Fig. 3B) theterminal path- wayactivationwascompletelyabolishedinP1andP2regardless ofthedilution,whereascomplementactivityinthehealthycon- trolsdecreasedinaccordancewithtitration.Notably,thenewborns fromP1andP2 showedasimilardecreaseinterminalpathway
Fig.3. Complementactivityintitratedsera.(AandB)SerumfromP1andP2at delivery(pregnantwomenwitheculizumab,closedcircles)andserumfromtwo healthycontrols(opencircles)weretitratedinthefunctionalcomplementassay.(C andD)SerumfromthenewbornsofP1andP2(closedtriangles)andserafromtwo healthynewbornsweretitratedinthefunctionalcomplementassay.CP,classical pathway;AP,alternativepathway.
activityastheircontrolsasmeasuredbytheCP(Fig.3C)andAP (Fig.3D).Collectively,thesedatademonstratethatthenewborns fromeculizumabtreatedmothershadcompletelynormalcomple- mentactivityindependentofserumdilution.
DetectionofE-C5inserumfromP1andhernewbornwithand withoutsubstitutionwitheculizumaborC5
InordertocontrolforsufficienttherapeuticC5inhibitioninthe eculizumab-treatedmotherandfreeC5inhernewborn,thepres- enceofE-C5wasmeasuredinserumfromP1andhernewborn beforeandafteradditionofeculizumaborC5(Fig.4).Consistently, theE-C5concentrationin P1wasunaffectedbytheadditionof eculizumaborC5(Fig.4A).AdditionofeculizumabtotheP1new- bornserumsubstantiallyincreasedtheE-C5levels,consistentwith bindingofeculizumabtofreeC5(Fig.4B).
Visualizationofeculizumab,C5andE-C5inserumsamplesfrom P1andhernewbornbyimmunofixation
DetectionofpurifiedC5,eculizumabandE-C5byagarosegel electrophoresisandimmunofixation
PurifiedE-C5wasidentifiedbyproteinstaining(Fig.4D,lane 1)andcomparedtoIgGK)-specificimmunofixationusingdifferent molarratiosbetweenC5andeculizumab(Fig.4C,lanes2–6).The anti-kappaantibodyspecificallydetectsfreeeculizumab(IgG2/4K) andE-C5.WhenE-C5wasformedinthepresenceofanexcessof freeeculizumab,thelatterappearsinalowermolecularweight band(Fig.4C,lanes2–4),whichcorrespondstothebandobserved
Fig.4.EffectofaddingeculizumabandC5totheseraanddetectionofE-C5in mother’sserumusingimmunofixationelectrophoresis.(A)E-C5complexeswere measuredinP1serum(PBS)andinP1serumtowhichwasaddedeculizumab(E) orpurifiedC5(C5)(meanwithrangeofn=3).(B)Sameexperimentasdescribed inpanelA,butusingserumfromthenewbornofP1(meanwithrangeofn=3).
(C)Lanes1and7representproteinstainingwithCoomassiebrilliantblue(CB)in electrophoresis,identifyingthelinesforpurifiedE-C5andpurifiedC5,respectively.
Lanes2–6and8–12representimmunofixationbasedonanantibodyreactingwith thekappa-chain(eculizumabhaskappalightchains).Lane12identifieseculizumab alone.Lanes2–6showsEC-C5complexesmadeinvitroandtitratedfromtheleft (eculizumabinexcess)totheright(C5inexcess).Lane8:SerumfromP1.Lane 9:SerumfromP1’snewborn.Lane10:normaladultserum.Lane11:normalnew- bornserum.Solidhorizontalline:indicatingpositionofC5.Brokenhorizontalline:
indicatingpositionofeculizumab.E-C5complexesappearbetweenthelines.
afteradditionofeculizumabalone(lane12).PurifiedfreeC5was identifiedbyproteinstaining(Fig.4C,lane7).
DetectionofE-C5complexesinserumsamplesfromP1andher newborn
Theimmunofixationrevealedadistinctbandintheserumfrom P1(Fig.4C,lane8),correspondingtoE-C5,whilenosuchbandwas observedinserumfromhernewborn(lane9).Also,serumsamples frombothahealthyadultcontrolandahealthynewborncontrol didnotcontaindetectableE-C5(Fig.4C,lanes11and12).
BindingofFcRntoeculizumab
ThetherapeuticeffectofIgGantibodiesisdependentontheir longserumhalf-life,whichisregulatedbyFcRn-mediatedrecycling inendothelialandhematopoieticcells(Montoyoetal.,2009;Wani etal., 2006).Specifically,IgG bindsFcRn withinacidified endo- somesthat aresubsequently recycledtothecellsurface where exposuretotheneutral pHoftheblood triggers releaseof IgG (Ward,2009;Roopenian andAkilesh,2007).Thus, FcRnrescues IgGfromintracellulardegradationandextendsitshalf-life.Inaddi- tion,FcRnprovidespassiveimmunitytothefetusbytranscytosing maternally-derived IgG across the placenta(Firan et al., 2001;
Mathiesenetal.,2013).ToaddresswhetherthehumanizedIgG2/4K eculizumabbindshumanFcRnwiththesamestrengthashuman IgG1,weperformedanELISAwherepHdependentbindingtoFcRn
Fig.5. EffectofpHoneculizumabbindingtoFcRn.ELISAbindingresponsesof titratedamountsofeculizumab,infliximabandrituximabtoGST-taggedhFcRnat pH6.0(A)andpH7.4(B).Thedatarepresentsthemeanofduplicates.
wascomparedwiththatofrituximab(chimericIgG1K)andinflix- imab(chimericIgG1K).Titratedamountsoftheantibodieswere coatedinwellsfollowedbyadditionofGST-taggedshFcRnateither pH6.0(Fig.5A)orpH7.4(Fig.5B).Eculizumabwasshowntobind thereceptor equallywellasrituximaband infliximab,in a pH- dependentmanner,withstrongbindingatpH6.0andnobindingat neutralpH.Thus,theabsenceofeculizumabinfetalplasmacannot beexplainedbyreducedbindingtoFcRn.
Discussion
Wehaveheredescribed acomprehensivesetofexperiments documentingthereliabilityandvalidityofanovelassayfordetec- tionofcomplexesbetweeneculizumabandC5.Usingthisassay, aswellasfunctionalcomplementactivityassaysandimmunofix- ation, we reveal that there is very littleeculizumab as wellas E-C5intheserumofnewbornsofpregnantwomentreatedwith eculizumab.Consequently,thenewbornshavea fullyfunctional complementsystem.Theimportanceofthedataisemphasizedby theincreasingnumberofpregnantwomenwhoarecandidatesfor eculizumabtreatment.PNH isthediseasewheremostpregnant patientshavebeentreatedwitheculizumab(deGuibertetal.,2011;
Kellyetal.,2010;Danilovetal.,2010;Marascaetal.,2010),but therearealsoreportsforaHUSandpreeclampsia(Ardissinoetal., 2013;Canigraletal.,2014;Burwick,2012).Otherconditions,in whichpregnancyisanoption,willlikelycomeupinthefutureas candidatesforcomplementinhibition,liketheanti-phospholipid syndrome(Erkanetal.,2014).Thesafetyoftreatmentandeffecton thenewbornwill,however,beamainconcern.Despitealimited numberofpatientsavailableforthepresentstudy,robustmeth- odswereestablishedandusedtodocumentthattreatmentwith eculizumabduringpregnancywouldbeofminorrisktothenew- born.Thiscouldbeindividuallyevaluatedinthefutureusingthe setofanalysespresentedhere.
TheamountofE-C5,complement blockageand complement activityinthenewbornhavetoourknowledgenotpreviouslybeen described. In one review onseven pregnant patients receiving eculizumabduringpregnancy,eculizumabwasquantifiedincord bloodfromfournewborns(Kellyetal.,2010).Intwo,eculizumab was undetectableand in two (twins,cesarean section at week 35 ofgestation) low levelswerefound, which accordingtothe authors, “were withinthe backgroundlevels for theassay and insufficienttoblockcomplement”.Thepregnantwomenhadther- apeuticlevelsofeculizumabintheirserum.Traditionalhemolytic assayswereusedforevaluationofthecomplementactivity.These assays do not have the same sensitivity and reliability as the novelassaysrecentlyusedtoevaluateaHUSpatientstreatedwith
eculizumab(Cugnoet al.,2014;Noriset al.,2014).Using these robustELISA-basedassaysforcomplementactivityandthenovel E-C5assaydevelopedinthepresentstudy,weconsistentlyshow thateculizumabintherapeuticdosesinthepregnantwomendid notaffectthecomplementsystemoftheirnewborn.
TheE-C5assaywedescribewasdocumentedtobehighlyspe- cific and sensitive. Thus, the reactivity was nil in serum from a genetic C5-deficient individual, even when eculizumab was addedtohighconcentrations.AdditionofpurifiedC5totheC5- deficientserumrestoredadose-dependentincreaseinE-C5.The sensitivitylimitinserumwasfoundtobebelow1g/mLadded eculizumab,whichequalslessthan1%oftheamountdetectedin thepatientstreatedwitheculizumab.TheE-C5levelsinthenew- borns,7–8g/mL,weredetectedthreetiterstepsabovethelower detectionlimit,andwerethushighlyreliable.
EculizumabisahumanizedIgG2/4Khybridantibodyaimedat minimizingtheeffectorfunctionsmediatedbycomplementacti- vationandFc␥receptorengagement,aspreviouslydemonstrated (Lauetal.,2013).WhilethebindingactivitytowardtheclassicalFc␥ receptorsarelackingorlow,eculizumabbindsthenon-classicalFc receptorFcRninastrictlypHdependentmanner,similartothat oftwocommerciallyavailablehumanIgG1Kantibodies,infliximab andrituximab.ThisisasexpectedsincebindingtoFcRnisdepend- entontwohistidineresidues(His310andHis435)attheCH2-CH3 elbowregion,whichareconservedinallsubclassesexceptIgG3, whichhasArg435(Ward,2009;RoopenianandAkilesh,2007).FcRn isahomeostaticregulatorofIgG,andhenceresponsibleforthe 3-weeklonghalf-lifeofendogenousIgG.However,theserumhalf- lifeoftherapeuticIgGantibodiesvaries,andpreviousstudieshave shownthehalf-lifeofeculizumab,infliximabandrituximabtobe 11.3,9.5and22days,respectively,thelattertwobeingchimeric IgG1Kantibodiesthatcrosstheplacentaandarefoundintheserum ofthenewborn(Maleketal.,1996;Kane,2009;Regazzietal.,2005;
Klinketal.,2008;McKeage,2011).Asweheredemonstratethat thesethreeantibodiesbindFcRnwithequalbindingstrength,it explainstheirprolongedhalf-life,butnotwhytheyshowdiffer- entserumpersistenceingeneralandpossiblydifferentplacental transferand/orfetal serumpersistence. Thedifferencesmaybe relatedtotheamountsoftherapeuticantibodygivenaswellasthe concentration,biophysicalpropertiesandbiodistributionoftheir respectiveantigens.Furthermore,itcouldbeduetodifferencesin howtheantibodiesaretransportedandsortedbyFcRnwhenbound totheircognateantigens.Astheantigensforeculizumabandinflix- imabarebothsolublewithonlyoneantibodybindingsite,these complexesshouldnotcross-bindclassicalFc␥receptors,whichis aprerequisiteforremovalbyphagocytosis.
The four human IgG subclasses are all actively transported acrosstheplacenta,andwhereasIgG1,IgG3andIgG4aredetected inthefetusinamountssimilartothosefoundinthemother,IgG2 isdetectedinloweramounts(Palmeiraetal.,2012;Hashiraetal., 2000).ItisgenerallyacceptedthatFcRnplaysakeyroleinshutt- lingofIgGtothefetusassubstitutionoftheaminoacidsthatare directlyinvolvedinFcRn,butnotclassicalFc␥receptorbinding, abolishestransplacentaltransportinexvivoperfusionmodelsys- tems(Palmeiraet al.,2012;Firan etal.,2001;Mathiesen etal., 2013).However,thereasonwhyIgG2isfoundinloweramounts isstillnotknown.Inthisstudy,wedemonstratethatverysmall amountsof theIgG2/4chimeric eculizumabcan bedetectedin thenewborn.Still,eculizumabmaywellbetransportedacrossthe placenta,andtheobservedlowsteadystatelevelduetotherela- tiveratesoftransportandclearance.Thisneedstobeaddressedin futurestudies.
Furthermore,theE-C5detectedintheserumofthenewborn couldbeformedeitherafterpassageofeculizumab,orcouldbethe resultofE-C5passage.WeexaminedE-C5concentrationandcom- plementactivityintheserumfromP1indetail,alsoafteraddition
ofpurifiedC5.NormalphysiologicalC5concentrationinadultsis 75g/mL.Sincefullcomplementactivitywasobtainedonlyafter additionofatleast1mg/mLC5,eculizumabwaspresentinexcessin theserumofthemother,consistentwiththesamplebeingobtained sixdaysafteraneculizumabinfusion.Thus,ourdatashowthatboth freeeculizumabaswellasE-C5ispresentinmother,andavailable fortransporttothenewborn.
In conclusion, our data indicate no adverse effects of the eculizumabtreatmentofthepregnantwomenonthecomplement activityofhernewborn.Thus,ourstudysuggeststhateculizumab shouldberegardedassafewithrespecttothedefensesystemofthe newborn.Wesuggestthatthecombinationofmethodsdescribed inthepresentstudyisparticularlysuitableforfurtherinvestigation ofpregnantpatientstreatedwitheculizumabandtheirnewborns.
Conflictofinterest
Noneoftheauthorshaveanyconflictofinteresttodeclare.
Acknowledgements
BrithSveiaKvarvisgreatlyacknowledgedforexcellenttechnical assistance.
Financialsupport:T.E.M.wassupportedbyTheResearchCoun- cil ofNorway (projectno.204874/F20),TheNorwegianCouncil onCardiovascularDisease,TheNorthernNorwayRegionalHealth Authority (Project no. SFP926-10), The Southern and Eastern NorwayRegionalHealthAuthority(Projectno.2012060),TheOdd FellowFoundation,andtheEuropeanCommunity’sSeventhFrame- workProgrammeundergrantagreementno.602699(DIREKT).I.S.
andJ.T.A.weresupportedinpartbytheResearchCouncilofNorway throughitsCentersofExcellencefundingscheme(projectnumber 179573).J.T.A.wassupportedbytheResearchCouncilofNorway (Grantnos.230526/F20and179573/V40)andtheSouth-Eastern NorwayRegionalHealthAuthority(Grantno.39375).
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