Functional relevance of in vivo half antibody exchange of an IgG4 therapeutic antibody-drug

22 Pages·2017·5.08 MB·English

RESEARCHARTICLE
Functional relevance of in vivo half antibody
exchange of an IgG4 therapeutic antibody-
drug conjugate
PeterHerbener1*,KurtScho¨nfeld1,MartinKo¨nig1,MatthiasGermer1,Jude
M.Przyborski2¤,KatrinBerno¨ster3,Jo¨rgSchu¨ttrumpf1
1 CorporateResearch&Development,BiotestAG,Dreieich,Germany,2 Parasitology,FacultyofBiology,
PhilippsUniversityMarburg,Marburg,Germany,3 CorporateProject&PortfolioManagement,BiotestAG,
Dreieich,Germany
a1111111111
a1111111111
¤ Currentaddress:CentreforInfectiousDiseases,Parasitology,HeidelbergUniversityHospital,Heidelberg,
a1111111111 Germany
a1111111111 *[email protected]
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Abstract
Anincreasingnumberofmonoclonalantibodiesandderivativessuchasantibody-drugcon-
OPENACCESS
jugates(ADC)areoftheIgG1andIgG4isotypewithdistinctstructuralandfunctionalproper-
Citation:HerbenerP,Scho¨nfeldK,Ko¨nigM, ties.Incaseswhereantibody-mediatedcytotoxicityisnotdesired,IgG4isoftenused,asits
GermerM,PrzyborskiJM,Berno¨sterK,etal.
Fcregionisrelativelypooratinducingantibody-dependentcell-mediatedorcomplement-
(2018)Functionalrelevanceofinvivohalfantibody
exchangeofanIgG4therapeuticantibody-drug dependentcytotoxicity.IgG4ADCswithhighlycytotoxicdrugsagainstproliferatingtarget
conjugate.PLoSONE13(4):e0195823.https://doi. cellsbutwhichlackorhavediminishedantibodyeffectorfunctionsagainstquiescentcells
org/10.1371/journal.pone.0195823
mayhaveafavorablesafetyprofilecomparedtoIgG1.AnotheruniquepropertyoftheIgG4
Editor:HiroshiShiku,MieUniversityGraduate subclassisthecapabilitytoexchangehalfantibodiesinvivocreatingrandomlybispecific
SchoolofMedicine,JAPAN
antibodies.Toinvestigatethefunctionalpropertiesofprocess-derivedantibodyspecies,
Received:June27,2017 anddeterminetheinfluenceofshufflingonthetherapeuticefficacy,severalmodelantibod-
Accepted:March30,2018 iesonthebasisoftheanti-CD138antibody-drugconjugateBT062(Indatuximabravtansine)
weregenerated:(I)AwildtypenBT062,(II)astablenBT062comprisingmutationstopre-
Published:April19,2018
venthalf-antibodyexchange,(III)ahalfnBT062lackingcovalentbindingbetweentwo
Copyright:©2018Herbeneretal.Thisisanopen
heavychainsand(IV)astabilized,bispecificnBT062-natalizumabantibodywithasecond,
accessarticledistributedunderthetermsofthe
CreativeCommonsAttributionLicense,which monovalentspecificityagainstCD49d.AllnBT062modelvariantswerecapableofCD138-
permitsunrestricteduse,distribution,and specificbindingandantigen-mediatedinternalizationintocells.Furthermore,allnBT062
reproductioninanymedium,providedtheoriginal
modelsinhibitedtumorgrowthinvitroafterconjugationwiththemaytansinoidDM4.Thein
authorandsourcearecredited.
vivoeffectsofthedifferentmolecularvariantswereassessedintheMAXF1322xenograft
DataAvailabilityStatement:Allrelevantdataare
model.ThebispecificnBT062-natalizumab-DM4demonstratedtheleastefficacyandwas
withinthepaperanditsSupportingInformation
onlymoderatelyactiveevenwithouttheco-administrationofahumanIgGpreparation.Wild
files.Anyfurtherinformationcanberequestedto
theauthors. type,stableandhalfnBT062-DM4modelsdemonstratedgreatanti-tumoractivities.The
efficacyofwildtypeandhalfnBT062-DM4wasreducedinthepresenceofIgG,whilestable
Funding:BiotestAGprovidedallfundingforstudy
design,datacollectionandanalysis,and nBT062-DM4wasonlymarginallyinfluenced.Thesepre-clinicaldatademonstratethe
preparationofthemanuscriptincludingsalariesfor advantageofintroducinghalf-antibodyexchange-preventingmutationsintotherapeutic
authorsP.H.,K.S.,M.K.,M.G.,K.B.andJ.S.This
IgG4-basedantibodydrug-conjugates.
preclinicalresearchispartofadevelopment
programforBT062inmalignantdiseases.The
developmentcandidateiscurrentlytestedinphase
PLOSONE|https://doi.org/10.1371/journal.pone.0195823 April19,2018 1/22
InfluenceofIgG4halfantibodyexchangeonADCs
IIclinicaltrialsinmultiplemyeloma,breastand Introduction
bladdercancer.
Thechoicebetweentheimmunoglobulingamma(IgG)1,IgG2andIgG4isotypesduringthe
Competinginterests:DuringdatacollectionP.H.,
discoveryphaseofmonoclonalantibodiesinvolvescarefulconsiderationoftheintendedinvivo
K.S.,M.K.,M.G.,K.B.,andJ.S.wereemployedby
effect.Antibodytherapeuticscanbedifferentintheirmodeofaction.Mechanismsincluding
BiotestAG.P.H.,M.K.,M.G.,K.B.andJ.S.hold
neutralizingsolublemediators,bindingtocellsandkillingthem,andregulatingcellfunctions
sharesofBiotestAG.M.K.,M.G.andK.B.are
inventorsonBT062relatedpatents.Thispreclinical canbeoptimized.Theselectionofthevariableantigenbindingdomainincludescriteriasuchas
researchispartofadevelopmentprogramfor targetspecificity,affinity,andpharmacology.Thechoiceoftheconstantfragmentcrystallizable
BT062inmalignantdiseases.Thedevelopment (Fc)regionfocusesonwhetherspecificeffectorfunctionsarerequiredforbestefficacyorunde-
candidateiscurrentlytestedinphaseIIclinical
sirableforsafetyreasons,andadditionallyontheneedforasuitablehalf-lifeinthepatient.
trialsinmultiplemyeloma,breastandbladder
IncontrasttootherIgGs,IgG4antibodiesexhibitonlyweakFcγ-receptorandC1qbinding
cancer.ThisdoesnotalterouradherencetoPLOS
necessaryforantibody-dependentcell-mediatedcytotoxicity(ADCC)andcomplement-
ONEpoliciesonsharingdataandmaterials.
Provisionstoprotectintellectualpropertiesmight dependentcytotoxicity(CDC)[1,2].Inaddition,theyaretheonlyIgclasscapableofexchange
berequiredforsharingmaterials.J.M.P.isan halfantibodies(heavy(H)+light(L)chain)invivo,whichresultsinmonovalent,bispecific
academicEditoratPLOSONE.Theauthorsdeclare
antibodies[3,4].IgG1moleculesharboraCPPCmotifwithinthehingeregion.Thesetwopro-
nofurtherconflictofinterest.
linescreatesterichindrancebetweenthecysteinesthatIgG1halfantibodiesareonlyableto
forminterchaindisulfidebondswithasecondIgG1half-antibody.Onthecontrary,IgG4anti-
bodieshaveaCPSCmotifresultinginamoreflexiblehingeregionwithadecreaseddistance
betweenthetwocysteines,whichallowstheformationofeitherinter-orintrachaindisulfide
bonds(Fig1A)[5,6].R409isthecrucialresidueattheCH3-CH3interface,weakeningthe
non-covalentassociationbetweenthesedomainsandallowinghalfantibodyexchange[7,8].
BymutatingeitherS228PorR409KIgG4antibodiesarestabilized,whileintroductionof
P228SandK409RmutationsinIgG1antibodiesenablehalf-antibodyshuffling[7].
CD138(Syndecan1)isatransmembraneheparinsulfateproteoglycanandamemberofthe
syndecanproteoglycanfamily.Itisinvolvedinseveralcellularfunctions,includingcell-cell
adhesion,migration,signaling,cell-matrixinteractionsandproliferation.Thelatterismedi-
atedbygrowthfactorbindingsuchasfibroblastgrowthfactor2andtransforminggrowthfac-
torß1[9–13].CD138isexpressedonepithelialcellsanddifferenthematopoieticcells:While
antibodyproducingplasmacellsandBcellprecursorsexpressCD138,itisabsentoncirculat-
ingmatureBcellsandCD34-positivestemcells[11,14,15].However,duringmalignanthema-
topoiesisandseveralsolidtumorsincludingbreast,pancreas,bladder,lungandprostate
cancerexpressionofCD138ishighlyupregulated[11,16,17].
Indatuximabravtansine,alsoreferredtoasBT062,isanantibodydrugconjugatecomposed
oftheCD138-specificantibodynBT062(nakedBT062)chemicallylinkedtothetubulin-bind-
ingmaytansinoidDM4.nBT062isahinge-unmodifiedhumanIgG4chimerizedantibody
basedonthemurineB-B4precursor.B-B4wasshowntobindthelinearepitopebetweenresi-
dues90to93oftheCD138coreprotein[18,19].BT062iscurrentlyevaluatedinseveralclinical
trialsinmultiplemyeloma[20],breastandbladdercancer(EudraCTNo.:2013-003252-20).
Theproposedmodeofactionfordisulfidebond-basedantibody-maytansinoidconjugates
includesantigen-binding,internalizationbyendocytosisandreleaseofthetoxicagentwithin
thecellleadingtotargetcelldeathandbeneficialbystanderkillingofdirectlyneighboringcells
viaDM4diffusion[18].InproliferatingcellsDM4inhibitspropermicrotubuleassembly.
Thus,cellcyclearrestandapoptosisareinduced[21].Onlyfree,butnotantibody-conjugated
DM4isabletopassthroughplasmamembranes[22].Therefore,BT062mightexhibitnodam-
agetohealthytissuesandpossesslowsystemictoxicity.Gemtuzumabozogamicinandinotu-
zumabozogamicinaretwoFDAapprovedIgG4-basedADCs.Despitetheybothcontaina
hinge-regionstabilizingS228Pmutationtopreventhalfantibodyexchange[23],adirectinflu-
enceofthisinvivomechanismonthefunctionalityandefficacyonADCshasneverbeenstud-
iedindetailsofar.
PLOSONE|https://doi.org/10.1371/journal.pone.0195823 April19,2018 2/22
InfluenceofIgG4halfantibodyexchangeonADCs
Inthiscurrentstudyfourantibodyvariantswildtype(WT)nBT062,stablenBT062,half
nBT062,andbispecificnBT062-natalizumabweregenerated.Natalizumab(Biogen)isathera-
peuticIgG4antibodyapprovedforthetreatmentofmultiplessclerosisandCrohn’sdisease
andbindingtotheintegrinα4subunit(CD49d).Sinceitisunstabilizedandhalf-antibody
exchangehaspreviouslybeendemonstrated[24],itscomplementaritydeterminingregions
(CDR)wereusedtodesignabispecificmodelantibody.Thesefourantibodyvariantswere
usedtoinvestigatethefunctionalactivityofpotentiallyinvivoderivedIgG4antibodiesspecies
anddeterminetheinfluenceofIgG4shufflingontheefficacy.
Materialsandmethods
GenerationofCD138-specificantibodies
ThesequenceofWTnBT062wasusedasbasisforthegenerationofstablenBT062,half
nBT062andbispecificnBT062-natalizumab.Allmutationswereintroducedbyfulllength
Fig1.SchematichalfantibodyexchangeandrespectivenBT062modelantibodies.(A)OverviewofinvivoIgG4halfantibodyexchangemainlydrivenbythepotential
toformeitherinterchainorintrachaindisulfidebondsinthehingeregion.AS228Pmutationincreasesthestericaldistanceofthetwocysteinespreventingintrachain
disulfidebonds.(B)GeneratednBT062modelantibodies:Wildtype(WT)nBT062,stablenBT062,halfnBT062andbispecificnBT062-natalizumab.Respectivemutations
areindicated.
https://doi.org/10.1371/journal.pone.0195823.g001
PLOSONE|https://doi.org/10.1371/journal.pone.0195823 April19,2018 3/22
InfluenceofIgG4halfantibodyexchangeonADCs
DNAsynthesisatThermoFisherScientific,Germany.Thestabilizingaminoacidmutations
S228PandR409KwereintroducedtodesignthestablenBT062antibody[7].Additionallya
L235EmutationwasintroducedintothisvariantforreductionofFcγRbinding[25].Thecys-
teinesbuildingupthedisulfidebondsofthehingeregionwereexchangedbyserines(C226S
+C229S)tocreatethehalfnBT062.ForthegenerationofthebispecificnBT062-natalizumab
antibody,theavailableCDRandframeworkregionsofnatalizumabHandLchains[26]were
alignedtothestablenBT062sequencetodeterminetheexchangeoftherespectiveaminoacid
sections.Knobandholetechnologywasusedtoreceivecorrectheavychainpairingduringco-
expressionofthetwodifferentheavyandlightchains[27].ToensurecorrectHtoLchain
pairing,orthogonalantigen-bindingfragment(Fab)interfaceswereused[28]:ThenBT062
FabofthebispecificnBT062-natalizumabcomprisesD1RandQ38Dmutationsinthelight
chainandQ39KandK62Emutationsinthevariableregionoftheheavychain.Q38R,S176W
andL135YmutationswereintroducedtothelightchainofthenatalizumabFabofbispecific
nBT062-natalizumab,whileQ39Y,F174GandH172Amutationswereappliedtotheheavy
chain.FulllengthDNAsequencesofthemodelantibodiesweresynthesizedandclonedinto
pEFmammalianexpressionvectors.HeavyandlightchainsofmonovalentantibodiesWT
nBT062,stablenBT062andhalfnBT062wereclonedintoseparatevectorscontainingeithera
puromycinormethotrexate(MTX)resistance.Twoplasmidscarryingthesequencesofthebis-
pecificnBT062-natalizumabwereclonedasfollows:Heavyandlightchainsequencesofthe
nBT062partwerebothclonedintothepEFvectorcontainingapuromycinresistance,whereat
aT2Aelement[29]andafurincleavingsitewereintroducedbetweenheavyandlightchains
forfurtherseparation.Heavyandlightchainsofthenatalizumabpart,alsointerruptedbya
T2Aelementandfurinrecognitionsite,wereclonedintothepEFvectorcomprisingaMTX
resistance.TheT2Aelementprocessesheavyandlightchainsequencesonthetranslational
levelwhilefurinfurtherdeletespost-translationallyintroducedspacerresidues.
1x106FreeStyleCHO-Scells(LifeTechnology)wereco-transfectedwith5μgofeachplas-
midDNA(10μgintotalforco-expression)usingprogramFF-137onthe4Dnucleofector
system(Lonza).Forthegenerationofcellpoolsstablyexpressingtheantibodies,cellswerecul-
turedinMTX/PuromycincontainingFreeStyleCHOExpressionMedium(ThermoFisher
Scientific)in24wellplatesuntilpopulationsgrewupwheregenomeintegrationtookplace.
Forantibodyproduction,cellsweregrownin1Lshakingcultureat37˚C/5%CO /125rpm
2
untilviabilitydroppedbelow80%.Thecellswerecentrifugedandtheantibodiesinthesuper-
natantwerepurifiedon5mlMabSelectProteinAcolumns(GEHealthcare)usinganA¨kta
Avant150System(GEHealthcare).
Electrophoresisandwesternblotting
10μgofpurifiedWTnBT062,stablenBT062,halfnBT062,bispecificnBT062-natalizumab
andnatalizumabwereseparatedbysodiumdodecylsulfatepolyacrylamidegelelectrophoresis
(SDS-PAGE)usinga4–12%Bis-Trisgel(Novex)andMOPSSDSRunningbuffer.Samples
wereeitherseparatedunderreducing(25%NuPAGELDSSampleBuffer(ThermoFisher)+
10%NuPAGESampleReducingAgent(ThermoFisher),10minat70˚C)ornon-reducing
(25%NuPAGELDSSampleBuffer+25%of400mMN-ethylmaleimidesolution,30sat70˚C)
conditions.Gelswerefixedfor45minin37.2%methanol/7%aceticacidsolutionandstained
for3hinCoomassiebrilliantbluesolution.Destainingwasperformedovernightin25%
methanolsolution.
Forisoelectricfocusing(IEF),5μgofWTnBT062,stablenBT062,halfnBT062,bispecific
nBT062-natalizumabandnatalizumabweremixedwith50%NovexIEFSampleBufferpH
3–10(ThermoFisherScientific)andappliedtoaNovexpH3–10IEFgelinIEFAnodeand
PLOSONE|https://doi.org/10.1371/journal.pone.0195823 April19,2018 4/22
InfluenceofIgG4halfantibodyexchangeonADCs
Cathodebuffer(ThermoFisherScientific).Proteinswereseparatedinastepwiseprocess:100
Vfor60min,200Vfor60minand500Vfor30min.Gelswereeitherfixedfor30minin12%
TCA/3.5%sulfosalicylicacidandstainedwithCoomassieblueorsubsequentlyblottedontoa
PVDFmembrane.Themembranewasblockedovernightat4˚CinOdysseyBlockingbuffer
(Licor)andincubatedsimultaneouslywith1μg/mlanti-BT062-Biotin(Biotest)andanti-nata-
lizumab-HRP(Bio-Rad)for2hatroomtemperature.Bindingofanti-idotypicantibodieswas
visualizedafter2hofsimultaneousincubationwithanti-HRP-800CWandStreptavidin-
680RD(Licor)onLicorsOdysseyImager.
Cellcultivation
Humancelllines,NCI-H929(Cat.-No.CRL-9068)andJurkat(Cat.-No.TIB-152)were
obtainedfromtheAmericanTypeCultureCollection(ATCC)andmurineBaF3cells(Cat.-
No.ACC300)wereobtainedfromLeibniz-InstitutDSMZ(Germany).BaF3-hCD138cells
weregeneratedbylentiviraltransductionofBaF3cellswithhumanCD138atSIRIONBiotech
(Germany).Allcelllineswereculturedat37˚C,5%CO2and100%atmospherichumidity
inaThermoScientificIncubator.NCI-H929cellsweremaintainedinRPMI1640medium
(Lonza)supplementedwith10%fetalbovineserum(FBS,ThermoFisherScientific)and2
mML-glutamine(Lonza);Jurkatcellmediumwasadditionallysupplementedwith10mM
HEPESbuffer(ThermoFisherScientific)and1mMsodiumpyruvate(ThermoFisherScien-
tific).BaF3cellswereculturedinRPMI1640mediumsupplementedwith10%FBSand10ng/
mlmouseIL-3(R&DSystems).Cellswerepassagedtwotothreetimesperweekandhandled
asrecommendedbythesuppliers.
AntibodylabelingwithDylight488
FordirectdetectionofnBT062modelvariantsduringflowcytometricandfluorescencemicro-
scopicinternalizationexperiments,WTnBT062,stablenBT062,halfnBT062andbispecific
nBT062-natalizumabwerelabeledwithaDylight488(Dy488)fluorescentdye(ThermoFisher
Scientific)accordingtothemanufacturer’sinstructions.Inaddition,natalizumabwaslabeled
ascontrolantibody.Inbrief,40μlof0.67Mboratebufferwereaddedto500μlofeachanti-
bodysolutioninDPBS(2.2mg/ml).Eachresultingsolutionwastransferredintoavialcontain-
ingtheDy488dyepowder,mixedbyvortexingandthelabelingreactionwascarriedoutfor60
mininthedark.Unbounddyemoleculeswereremovedbythekit-containingspincolumns.
Antibodyconcentrationsandantibody-to-dyeratiosweredeterminedbyphotometricabsorp-
tionsmeasuredat280and493nmwavelengthusingthefollowingformulas:
(cid:16) (cid:17)
g A (cid:0) ðA (cid:3)CFÞ
Proteinconcentration ¼ 280 max (cid:3)DF(cid:3)m
l ε
protein
A (cid:3)DF
ratiodye:protein¼ max
ε (cid:3)c
Dye Protein
DF(dilutionfactor)=10
ε =210000forIgG
protein
m(specificmass)=150000Da
Amax=493
CF=0.147
ε =70.000
Dye
PLOSONE|https://doi.org/10.1371/journal.pone.0195823 April19,2018 5/22
InfluenceofIgG4halfantibodyexchangeonADCs
TheDy488labelingreactionresultsindye-to-antibodyratiosof1.92,2.17,1.82,and1.99
forWTnBT062,stablenBT062,halfnBT062andbispecificnBT062-natalizumab,respectively.
Flowcytometry
InordertoanalyzethespecificbindingcapacityofthefourdifferentnBT062variantsortheir
respectiveDM4-conjugatesaswellasnatalizumab,BaF3-hCD138(CD138+/CD49d-),Jurkat
(CD138-/CD49d+)orNCI-H929(CD138+/CD49d+)cellswereincubatedwith3-foldserial
antibodydilutions.Cellswereseededin96wellplates(150,000cells/well)andincubatedfor30
minat4˚Cin100μlantibodysolution(5x10-7–2.82x10-12molar).Cellswerewashedtwice
withPBS/3%FBSandincubatedwith10μg/mlofalexafluor647-labelledgoatanti-humansec-
ondaryantibody(ThermoFisher)for0.5hat4˚C.Subsequently,theantibodysolutionwas
discarded,cellswerewashedtwicewithPBS/3%FBSandanalyzedwithaFACSCantoIIflow
cytometer(BecktonDickinson).
TodeterminenBT062variantinternalization,NCI-H929cells(CD138+/CD49d+)were
seededin96wellplates(100,000cells/well).Cellswereincubatedwith15μg/mlofeither
Dylight-488(Dy488)labeledWTnBT062,stablenBT062,halfnBT062orbispecificnBT062-
natalizumabantibodies.Cellsurfacestainingwasanalyzedafter0.5hofantibodyincubation
at4˚C,whileinternalizationwasanalyzedafter24hincubationat37˚C.Topreventbispecific
nBT062-natalizumabfrombindingtoCD49d,natalizumabwasco-incubatedwitha50-times
excess.CellswereeitherincubatedwithTrypsinfor10minat37˚CtoremoveCD138includ-
ingboundedantibodiesornotandwashedtwicewithPBS/3%FBS.Analysiswasperformed
usingaFACSCantoIIflowcytometeranddataevaluationwasdonewithGraphPadPrism
6.1.
Fluorescencemicroscopy
TofurtherinvestigateCD138-mediatedinternalizationofnBT062variantantibodies,BaF3
(CD138-/CD49d-)andBaF3-hCD138(CD138+/CD49d-)cellswereseededin96wellplates
(150,000cells/wells)andincubatedfor0.5hat4˚Cwith10μg/mlofDy488labeledWT
nBT062,stablenBT062,halfnBT062orbispecificnBT062-natalizumabantibodiesornatalizu-
mabinPBS/3%FBS.Cellswerewashedonceandincubatedingrowthmediafor3hat37˚Cto
enableantibodyinternalization.Subsequently,extracellularsurfaceanchoredCD138including
non-internalizedantibodieswasremovedbyTrypsindigest(10min/37˚C).
Afterwashing,cellswerefixedin4%paraformaldehydeandpermeabilizedby0.02%Sapo-
nin(Sigma-Aldrich).Intracellularblockingwasperformedin1xBMB/PBS(BoehringerBlock-
ingAgent,Roche)for10minatRT.CellswerewashedtwiceinPBS/3%FBS.Co-stainingwith
lysosomal-associatedmembraneprotein1(LAMP1)wasperformedbypolyclonalanti-
LAMP1incubation(Abcam,ab24170,1:500in1xBMB/PBS)followedbytwowashingsteps
andstainingwithaCy3-labeledgoatanti-rabbitH+Lsecondaryantibody(JacksonImmunoR-
esearch,111-165-003,1:500in1xBMB/PBS).Forsamplefinalization,cellswerewashedtwice
inPBS/3%FBS,resuspendedinProlongGoldMountingMediumwithDAPI(ThermoFisher
Scientific)andtransferredontomicroscopyslides.Dataevaluationwasdoneonthefollowing
dayusinganOlympusIX53invertedfluorescencemicroscope(Olympus)andCellSensSoft-
ware(Olympus).
DM4conjugation
Tocreateanti-CD138ADCs,theconjugationtechnologieofImmunogen(USA)wasusedto
attachthemaytansinderivateDM4ontoWTnBT062,stablenBT062,halfnBT062aswellas
ontobispecificnBT062-natalizumab.Inafirststep,theantibodyformulationbufferwas
PLOSONE|https://doi.org/10.1371/journal.pone.0195823 April19,2018 6/22
InfluenceofIgG4halfantibodyexchangeonADCs
exchangedbyconjugationbuffer(50mMpotassiumphosphate(Merck),50mMsodiumchlo-
ride(Merck),2mMEDTA(Merck),pH6.5)usingaNAP-25column(GEHealthcare)and
proteinsolutionswereconcentratedto10mg/mlusingAmiconUltra30kDaMWCOcentri-
fugationfilters(Merck).TheN-succinimidyl-4-(2-pyridyldithio)butanoate(SPDB)Linker
(Immunogen,solvedin100%Ethanol)wasaddedina6.5-timesmolarquantitytotheanti-
body.Finallinkerconjugationreactionincludedanantibodyconcentrationof8mg/mland
5%Ethanol.Thereactionwasperformedfor4hatRTwhilestirring.SPDB-modifiedantibod-
ieswerefilteredandpurifiedbyNAP-25columnusingconjugationbuffer.Subsequently,
DM4(Immunogen,15mMinDimethylacetamid(DMA,Merck))wasaddedina7.1-times
molarquantitytotheantibody.DMAwasaddedtoafinalconcentrationof6%andthetarget
antibodyconcentrationwas5mg/ml.TheDM4conjugationreactionwascarriedoutfor16h
atRTduringstirring.ANAP-25columnwasusedforpurificationandbufferexchangetoanti-
bodyformulationbufferfollowedbya0.2μmfiltration.ThehalfnBT062wasconjugatedwith
identicalamountsofSPDBandDM4comparedtofulllengthantibodiesWTnBT062andsta-
blenBT062assumingHchaindimerizationofthismodelduringtheconjugationreaction.To
mimicthesituationofashuffledantibodyinvivo,thebispecificnBT062-natalizumabwascon-
jugatedwith50%ofSPDBand50%ofDM4.
DM4-to-antibodyratioandDM4distribution
ThedeterminationoftheDM4toantibodyratioisbasedonthemeasurementoftheoptical
density(OD)at252and280nm.Formulationbufferwasusedasblank.TheDM4toantibody
ratiowascalculatedusingthefollowingformulas:
ðOD252(cid:3)dilutionfactorÞ(cid:0) ð0:344(cid:3)OD280(cid:3)dilutionfactorÞ(cid:3)1000000
DM4½mM(cid:138)¼
24377
ð1000000(cid:3)OD280(cid:3)dilutionfactorÞ(cid:0) ð5180(cid:3)DM4½mM(cid:138)Þ
BT062variant ½mM(cid:138)¼
216060
DM4½mM(cid:138)
DM4toantibodyratio¼
BT062variant ½mM(cid:138)
Invitrocytotoxicity
ToinvestigatethecytotoxicityofthefourdifferentDM4-conjugatednBT062variants,1x104
CD138-expressingNCI-H929cellswereseededinto96wellplatesonthedaybeforetheexper-
imentandwereincubatedovernightat37˚C.Jurkatcells(CD138-/CD49d+)wereusedasneg-
ativecontrolcells.WTnBT062-DM4,stablenBT062-DM4,halfnBT062-DM4orbispecific
nBT062-natalizumab-DM4wereaddedtothecellswithfinalantibody-basedconcentrations
of4,1,0.4,0.2,0.1,0.06,0.04,0.02,0.01and0nM.Natalizumabwasusedascontrolwith50x
excess.Cellswereincubatedandafter5daystheviabilitywasdeterminedusingtheWST-1cell
proliferationassay(Roche)accordingtothemanufactures’instructions.Dataevaluationwas
doneusingGraphPadPrism6.1.
Xenograftmousestudy
AnimalexperimentswereperformedatOncotestGmbH(Freiburg,Germany).Experiments
wereapprovedbytheCommitteeontheEthicsofAnimalExperimentsoftheRegierungspra¨si-
diumFreiburg(Freiburg,Germany;permitnumber:G-13/13)andconductedaccordingtothe
guidelinesoftheGermanAnimalWelfareAct(Tierschutzgesetz).Researchstaffhasrelevant
PLOSONE|https://doi.org/10.1371/journal.pone.0195823 April19,2018 7/22
InfluenceofIgG4halfantibodyexchangeonADCs
educationandisFELASA-Bqualified.Duringsurgeryanimalswereanesthetizedbyinhalation
ofisoflurane.
HumanprimaryMAXF1322mammarycarcinoma(Oncotest)passagedindonormicewas
subcutaneouslyinoculatedas3-4mmfragmentsintotheflankofrecipientNMRInudemice.
Animalsbearingtumorsof50–250mm3(preferably80–200mm3)wererandomizedtothe
studygroups(5mice/group).WTnBT062-DM4,stablenBT062-DM4,halfnBT062-DM4and
bispecificnBT062-natlizumab-DM4wereadministeredi.v.asmonotherapyeitherusingan
antibody-baseddoseof4or2mg/kg/weekforthreeinjectionsintotal.Toinvestigatewhether
theefficacyofthemodelantibodieswasinfluencedbyendogenousIgG4,allmonotherapy
groupsreceivedadditionallya10%intravenousimmunoglobulinGpreparation(IVIg,Intra-
tect10%,BiotestAG).Intratect10%wasadministeredi.v.onehourbeforeanti-CD138anti-
bodiesatadoseof10ml/kg/week.PBSwasusedasvehiclecontrol.Mortalitywasassessed
daily;bodyweightandabsolutetumorvolumewasmeasuredtwice-weekly.Tumorvolume
wascalculatedas(AxB2)x0.5,whereAwasthelargestandBtheperpendiculartumordiame-
ter;tumorvolumewascalculatedrelativetoday0(Startoftreatment).Theexperimentwas
endedonday86afterstartoftreatmentorwhentumorsreachedavolumeof2000mm3,
whichevercamefirst.Accordingtoanimalwelfareregulations,thefollowingcriteriaforimme-
diateeuthanasiawereappliedtoindividualanimals,irrespectiveoftheexperimentalstatus:(I)
tumorvolume>2000mm3;(II)ulcerating,skin-penetratingtumor;(III)bodyweightloss>
30%onanyonemeasuringday;(IV)recorded,continuedbodyweightloss>20%formore
thantwodays;(V)recorded,rapiddecreaseinbodyweight>20%withintwodays;(VI)severe
impairmentofgeneralcondition(apathy,pain,markedlyreducedfeedandwaterintake,dys-
pnea,abnormalhabitusorbehavior).AnimalswereeuthanizedbyCO orcervicaldislocation.
2
EfficacyoftheADCswasassessedasmedianrelativetumorvolumeofthetestgroupcompared
tothecontrolgroup.CriteriaareshowninTable1.
Forgraphicaldemonstrationoftumorgrowthcurves,tumorvolumesofanimalsthatwere
terminatedduetotheirtumorloadwerecarriedforwardusingtheLast-Obersation-Carried-
Forwardmethodologyforaslongasthisincreasedthegroupmediantumorvolume.Toevalu-
atethedifferencesintimetotumorprogression,thecutoffofabsolutetumorvolumewasset
to2000mm2andKaplan-Meiersurvival-stylestatisticscombinedwiththelog-rankMantel-
Coxtestforpairwisecomparisonswereemployed[30].
Results
nBT062modelantibodiesarecapableofantigen-specificbinding
TostudytheIgG4-relatedhalf-antibodyexchangeanditspotentialeffectontheantibodies
functionalityandefficacy,wehavegeneratedfouranti-CD138antibodies:(I)WTnBT062,
Table1. Tumorcontrolefficacycriteria.
Classification T/C(cid:3)
Inactive (cid:21)65%
Borderline 50%–<65%
Moderate 25%–<50%
High 10%–<25%
Veryhigh 5%–<10%
Completeremission <5%
(cid:3)T/C=mediantumorvolumeintestgroup/mediantumorvolumeincontrolgroup
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InfluenceofIgG4halfantibodyexchangeonADCs
comprisesthebackboneofanunmodifiedhumanIgG4antibody.(II)StablenBT062wasgen-
eratedbyintroducingstabilizingchangeswithinthehingeandCH3domainsintotheWT
nBT062topreventhalfantibodyexchange.(III)Byexchangingthecysteinesatposition226
and229inthehingeregiontoserinesinWTnBT062,covalentdimerizationoftheheavy
chainswasinhibitedresultinginnBT062halfantibodies(halfnBT062).(IV)Tostudyeffects
ofbispecificantibodiesderivedbyhalfantibodyexchangewithendogenousIgG4,astabilized,
bispecificnBT062-natalizumabwasdesigned(Fig1B).AllvariantswereexpressedinFreeStyle
CHO-ScellsandpurifiedbyProteinAaffinitychromatography.Reducingandnon-reducing
SDS-PAGEanalysisdemonstratedexpressionofallgeneratedproteinsandtheirexpected
molecularweights(MW;Fig2A).WhileinpurifiedWTnBT062asmallfractionofhalfanti-
bodieswasdetectableatapprox.70kDaundernon-reducingconditions,nohalfantibodies
werepresentinstablenBT062andbispecificnBT062-natalizumabpreparations.C226Sand
C229SmutationinthehingeregionofhalfnBT062resultedinnofulllengthH2L2antibodies
atapprox.150kDa.Interestingly,non-reducing,non-denaturingsizeexclusionchromatogra-
phy(SEC)revealeddimerizationofhalfnBT062antibodiesassumingtheirconformationsta-
tusdependsonsurroundingconditions(S1Fig).ReducingSDS-PAGEdemonstratedheavy
andlightchainsofallfournBT062variantsatapprox.50kDaand25kDa,respectively.For
bispecificnBT062-natalizumab,twoheavychainbandswithminimaldifferentmolecular
weightswereobservedindicatingtheseparatenBT062andnatalizumabheavychain.Within
Fig2.AnalyticalcharacterizationofnBT062modelvariants.(A)Reducingandnon-reducingSDS-PAGEanalysisofindicatednBT062variants.L=lightchain,
H=heavychain,HL=halfantibody,H2=heavychaindimer,H2L2=intactantibody.Molecularweightandantibodyexplanationsarerepresentativeforreducingand
non-reducingconditions.(B)IndicatednBT062modelvariantswereseparatedbyisoelectricfocusingandblottedontoaPVDFmembrane.SelectiveBT062and
natalizumabspecificitiesweredetectedbyanti-idiotypicantibodiesvisualizedingreenandredrespectively.ThebispecificnBT062-natalizumabfractionisshowingtwo
natalizumabexclusivebands.
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InfluenceofIgG4halfantibodyexchangeonADCs
thestablenBT062preparationanadditionalbandatapprox.85kDawasdetected,indicatinga
H2fraction.Multicolorwesternblottingofisoelectricfocusingrevealedthatbispecific
nBT062-natalizumabdidnotcontainmonospecificnBT062,butaminimalfractionofmono-
specificnatalizumab(Fig2B).Sincethisphenomenonisknownfortheknobandholeengi-
neeringtechnology[31],natalizumabwaschosentocomprisethehole-heavychaintoprevent
influenceondatafrommonospecificnBT062withinthismodel.SECanalysisobtainedhigher
amountsofaggregatesforstablenBT062comparedtotheothervariants,whichmightbean
effectofanon-optimizedformulationbuffer(S1Fig).Evidenceforthepreventionofhalfanti-
bodyexchangebyS228PandR409KmutationsintroducedintostablenBT062andbispecific
nBT062-natalizumabwasdemonstratedbyaninvitroassay:ExposingstablenBT062andWT
nBT062inthepresenceofnatalizumabwith10mMreducedglutathioneandsubsequentre-
oxidationwithoxidizedglutathioneresultedinfractionofbispecificWTnBT062/natalizumab
antibodies,whilesuchantibodyshufflingwasnotobservedforstablenBT062(S2Fig).
Flowcytometricanalysiswasusedtocomparespecificantigenbindingofthe4different
nBT062antibodyvariants(Fig3,Table2).WhileWTnBT062,stablenBT062andhalfnBT062
antibodiesshowedsimilarbindingtoBaF3-hCD138(CD138+/CD49d-)cells,thebindingof
bispecificnBT062-natalizumabwasslightlyreduced.However,theaffinityofnBT062-natali-
zumabagainstCD138wasstillinthenanomolarrange(Table2).Additionally,thebispecific
nBT062-natalizumabantibodyexhibitedbindingofCD49d-expressing,CD138-Jurkatcells
similartonatalizumab(Fig3B),indicatingthatbothantigenswererecognizedbythismodel
antibody.
AllnBT062modelantibodiesareinternalizeduponCD138-specificbinding
CD138-mediatedantibodyuptakeintoCD138+cellswasinvestigatedbyflowcytometryand
fluorescencemicroscopy.
ForflowcytometricanalysisDy488labelednBT062variantswereaddedtoCD138+
NCI-H929cellsforeither0.5hor24h.Todistinguishbetweensurface-boundandinternal-
izedantibodies,cellsweretreatedwithorwithoutTrypsinbeforethemeasurement(Fig4A
and4B).Antibodyinternalizationpatternsincludinginitialsurfacebindingandinternalized
antibodieswerecomparableforWTnBT062,stablenBT062andhalfnBT062.Despitethe
excessofeachnBT062variantduringthe24hincubation,thedetectedinternalizedantibody
levelswereapprox.halfoftheinitialboundantibodylevels.Incontrasttothemonospecific
variants,bispecificnBT062-natalizumabdemonstratedareducedinitialsurfacestainingbuta
significantlyhigherfluorescenceintensityofinternalizedantibodies.Bindingandpotential
internalizationofbispecificnBT062-natalizumabduetoCD49dwassuccessfullyblockedby
anexcessofnatalizumab.OnepossibleexplanationmightbethelackofCD138cross-linking
potentiallyincreasingtheinternalizationprocessitself.Thismighttakeplaceundertheprem-
iseofkeepinginmindthatthehalfnBT062modelstillpossessesthepotentialtodimerizedue
tonon-covalentinteractionsbetweentheCH3domainsoftwoheavychains.
Tofurtherinvestigatethetypeofinternalization,epifluorescencemicroscopywasperformed
after3hoursofcellincubationwiththefourdifferentfluorescent-labelednBT062antibodies.
Co-localizationwithanantibodytargetingtheLysosomal-associatedmembraneprotein1
(LAMP-1)indicatedaCD138-mediateduptakebytheendosomal/lysosomalpathway(Fig4C).
TheDM4-conjugationisnotinfluencedbymutationsanddoesnot
interferewithantigenbinding
Togenerateantigen-specificcytotoxicvariantsofthedifferentnBT062models,theywerecon-
jugatedtoDM4.Thestrivendrug-to-antibodyratioforeachmodelvariantwasbasedon
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