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Peer-reviewed
ResearchArticle
Editor:KarenL.Mossman,McMasterUniversity,Canada
Received:January3,2013;Accepted:April8,2013;Published:May16,2013
Funding:Theauthorshavenosupportorfundingtoreport.
Competinginterests:DonglinLiu,Chien-HsingChang,EdmundA.Rossi,ThomasM.CardilloandDavidMGoldenbergareemployeesofbothImmunomedics,Inc.,andIBCPharmaceuticals,Inc.(awhollyownedsubsidiaryofImmunomedics,Inc.)Allauthorshavestock,and/orstockoptionswithImmunomedicsInc.;CHC,EARandDMGhavestockand/orstockoptionswithIBCPharmaceuticalsInc.Forthisstudyhumanizedantibodiesincludingveltuzumab(anti-CD20),hRS7(anti-Trop2),hMN-15(anti-CEACAM6),andhL243(anti-HLADR)wereprovidedbyImmunomedics,Inc.OneormoreUSand/orforeignpatentapplications(Title-TargetingIFN-lambda1withantibodiespotentlyenhancesbothanti-tumorandanti-viralactivities,Numbers-PCT/US13/23093and13/749,985)havebeenfiledonthesubjectmatterofthemanuscript;however,thisdoesnotaltertheauthors'adherencetoallthePLOSONEpoliciesonsharingdataandmaterials.
Humanizedantibodies,includingveltuzumab(anti-CD20),hRS7(anti-Trop2),hMN-15(anti-CEACAM6),hL243(anti-HLA-DR),andh225(anti-EGFR),andcontrolmodules,suchashRS7-Fab-AD2andhMN-15-IgG-AD2,wereprovidedbyImmunomedics,Inc.RecombinanthumanIFN-λ1(rhIFN-λ1),mouseanti-humanIFN-λ1,andFITCmouseanti-humanHLA-ABCwerepurchasedfromR&DSystems(Minneapolis,MN).PolyclonalrabbitantibodiesforSTAT1,STAT3,pY-STAT1,andpY-STAT3wereobtainedfromCellSignalingTechnology(Danvers,MA).Polyclonalrabbitanti-STAT2andanti-pY-STAT2antibodieswerefromSantaCruzBiotechnology(SantaCruz,CA)andMilliporeCorporation(Billerica,MA),respectively.
(A)ConstructionofpET-26b-AD2-IFN-λ1expressionplasmid.ThecassetteofAD2-hingelinker-IFN-λ1wassynthesizedandclonedintotheexpressionvectorpET-26b.AD2,ananchoringdomainofAkinaseanchorprotein;hingelinker,a15aminoacidlongflexiblelinkerpeptide.(B)ConstructionofpdHL2-Fab-DDD2expressionplasmids.TheseconstructswerederivedfromtheirparentalplasmidspdHL2-IgGwherethecodingsequencesfortheCH1–CH3domainswerereplacedwithaCH1-GS-DDD2cassette.VHandCH,variableandconstantdomainsoftheheavychain;VKandCK,variableandconstantdomainsofthekappachain;LDR,leadingregion;DDD2,thedimerizationanddockingdomainofhumanPKA-RIIprotein.(C)DNLconjugationofAD2-IFN-λ1andFab-DDD2.ThehRS7-Fab-DDD2,hMN15-Fab-DDD2,c225-Fab-DDD2,orhL243-Fab-DDD2module(I)wasdockedandlockedwithAD2-IFN-λ1module(II)toproduce2(Fab)-λ1,namely(E1)-λ1,(15)-λ1,(c225)-λ1,and(C2)-λ1,respectively(III).
Forcell-bindingassays,cellsweretrypsinizedbriefly,washed,re-suspendedin1%BSA-PBS,andincubatedwithhumanizedmAbs(10μg/ml)orseriallydilutedIFN-λ1-basedagents.BindingwasdetectedwitheitherFITC-labeledgoatanti-humanIgGorwithmouseanti-humanIFN-λ1followedbyFITC-labeledgoatanti-mouseIgG.Allincubationswere45minat4°Cwith1%BSA-PBSwashesbetweenincubations.BindingwasmeasuredbyflowcytometryusingFACSCalibur(BDBiosciences,SanJose,CA).
ChangesofMHC-IexpressionwereevaluatedbyflowcytometryonFACSCaliburfollowingtreatmentofcellswithindicatedagentsfor3days.CellswereprobedwithFITC-labeledmouseanti-humanHLA-ABC.FITC-labelednon-specificmouseIgG1kwasusedasanegativecontrol.
ME-180,SK-MES-1,andTE-11wereseededin96-wellplates(1,000cells/well)andheldat37°Covernightpriortoincubationwiththeindicatedagentsfor4days.ViablecellsweremeasuredusingaCellTiter96CellProliferationAssayagent(Promega,Madison,WI).
SDS-PAGEwasperformedunderreducingandnon-reducingconditionsusing4–20%Tris-Glycinegels(Lonza,Allendale,NJ).ToevaluatephosphorylationofSTATs,HepG2cells(5×105)weretreatedwithIFN-λ1agentsfor1hourpriortolysiswithPhosphoSafeextractionreagent(Novagen).CelllysateswereresolvedonSDS-PAGE,transferredtonitrocellulosemembranes,andprobedwithrabbitantibodiesagainsttotalSTAT1,STAT2,orSTAT3orthephosphotyrosine-specificantibodiespY-STAT1,pY-STAT2,orpY-STAT3.SignalsweredetectedwithHRP-goatanti-rabbitantibody.Ananti-β-actinantibodywasusedforassessingβ-actinastheloadingcontrol.
AnimalstudieswereapprovedbytheCenterforMolecularMedicineandImmunologyInstitutionalAnimalCareandUseCommittee,andperformedinaccordancewiththeAssociationforAssessmentandAccreditationofLaboratoryAnimalCare,U.S.DepartmentofAgriculture,andDepartmentofHealthandHumanServicesregulations.Fourgroupsof8-week-oldfemaleathymicnudemice(Taconic,Germantown,NY)wereinjectedsubcutaneouslywith2.4nmolof(E1)-λ1andbledat6,16,24,and48hours.Serumconcentrationsofintact(E1)-λ1weremeasuredusingenzyme-linkedimmunosorbentassay(ELISA).PharmacokineticparameterswerecalculatedusingWinNonLinNoncompartmentalAnalysisProgram(Version5.3,PharsightCorporation,St.Louis,MO).TocorroboratewiththeresultsofELISA,thebioactivityofIFN-λ1inselectiveserumsampleswasalsoevaluatedbyexvivoproliferationassayofME-180cells,using(E1)-λ1asastandard.
MaxiSorp96-wellplates(Nunc,Roskilde,Denmark)werecoatedovernightwithgoatanti-humanF(ab’)2antibodyandblockedwithPBScontaining2%bovineserumalbumin(BSA)for1h.Serumsamplesforpharmacokineticstudywereseriallydilutedandaddedtothecoatedplates.Thecaptured(E1)-λ1wasdetectedwithmouseanti-humanIFN-λ1,followedbyHRP-conjugatedgoatanti-mouseIgGFcantibodies.Pure(E1)-λ1wasincludedasastandardforquantifyingserumsamples.
Statisticalsignificance(P<0.05)wasdeterminedwithFtestsforallresultsusingthePrismGraphPadsoftwarepackage(AdvancedGraphicsSoftware,RanchoSantaFe,CA).
Cellswereincubatedat4°Cfor45mininthepresenceof200,40,8nmol/Lofindicatedagentspriortodetectionwithmouseanti-IFN-λ1mAbandFITC-labeledgoatanti-mouseIgG.MFI,meanfluorescenceintensity.N=3×10,000cells.Mean±SD,95%confidenceinterval.(A)Enhancedbindingof(E1)-λ1relativetoAD2-IFN-λ1toME-180cells.(B)Enhancedbindingof(15)-λ1relativetoAD2-IFN-λ1toHepG2cells.(C)Enhancedbindingof(C2)-λ1relativetoAD2-IFN-λ1toA375cells.
Indicatedcelllineswereculturedfor4daysinthepresenceofincreasingconcentrationsof(E1)-λ1(A-C)or(15)-λ1(D-F),andtherelativecellviabilitiesweremeasuredwithMTS.Thepercentageofthesignalobtainedfromuntreatedcellswasplottedversusthelogofthemolarconcentration.Dose–responsecurvesandEC50valuesweregeneratedusingPrismsoftware.Eachstudyalsoincludedparallelevaluationofconstitutivemodules(AD2-IFN-λ1,hRS7-Fab-DDD2,hMN15-Fab-DDD2)testedalone,orAD2-IFN-λ1andcontrolmodules(hRS7-Fab-AD2,hMN-15-IgG-AD2)testedincombination.
(A)Enhancedanti-HCVpotencyof(c225)-λ1inHuh-7cells.AHuh-7stablecelllinewithHCVgenotype1bCon1repliconexpressingfireflyluciferasewastreatedwithindicatedconcentrationsof(c225)-λ1,(C2)-λ1,orrhIFN-λ1agents.After3days,luciferaseactivitywasmeasuredandantiviraleffectsweredeterminedbypercentactivityreductionrelativetountreatedcells.DatawereanalyzedbyGraphPadPrismusingasigmoidalfit(variableslope).Sampleswereruntwiceindependentlyinduplicate.(B)Enhancedanti-EMCVpotencyof(15)-λ1inA549cells.A549cellswereincubatedwithserialdilutionsof(15)-λ1,(C2)-λ1,rhIFN-λ1,orhMN15-Fab-DDD2beforebeingchallengedwithEMCV.Avisualcytopathiceffect(CPE)determinationwasperformed,andthedatawereanalyzedbyGraphPadPrismusingasigmoidalfit(variableslope).Sampleswereruntwiceindependentlyinduplicate.Mean±SD,95%confidenceinterval.
(A)ImmunoblotanalysisofSTATphosphorylation.HepG2cellsweretreatedwithindicatedtestarticlesfor1h,andphosphorylatedSTAT1,2,and3weremeasuredwithloading30μgoftotalprotein/lane.TotalSTATsandβ-actinwereprobedtoverifyequalloading.(B)FlowcytometricanalysisofMHCclassIantigenexpression.HepG2cellswereculturedforthreedaysinthepresenceofincreasingconcentrationsof(15)-λ1,AD2-IFN-λ1,or15-Fab-DDD2andthenstainedwithFITClabeledmouseanti-humanHLA-ABCorisotopic-matchedcontrolAb.MFI,meanfluorescenceintensity.N=10,000cells.DatawereanalyzedbyFlowJosoftware(TreeStar,Ashland,OR).(C)RT-PCRanalysisofMxAmRNAexpression.HepG2cellsweretreatedwithIFN-λ1agentsfor24h,andthemRNAexpressionofMxAgenewasanalyzedin100ngoftotalRNA/sample.GAPDHcDNAwasamplifiedasaninternalcontrolforcDNAamounts.Dataarerepresentativeoftwoindependentexperiments.
(A)Meanserumconcentrationofintactmoleculesversustimeinmiceafterasingledose(2.4nmol/animal)of(E1)-λ1.ThepharmacokineticparametersarederivedfromnoncompartmentalanalysisofELISAdata.T1/2,halflife;MRT,meanresidencetime;Cl,clearance.N=3animals/timepoint.Mean±SD,95%confidenceinterval.(B)SpecificbioactivityofserumsamplesintheexvivoassayofME-180cellgrowth.Serumsweredilutedat1∶20astheinitialtestconcentration,andthenseriallydilutedat1∶10.(E1)-λ1wasusedasastandardcontrolwithtestconcentrationsfrom1nMto109nM.Cellsweretreatedfor4days,andviablecellsweremeasuredwithMTS.Dose–responsecurvesweregeneratedusingPrismsoftware.N=3animals/timepoint.Mean±SD,95%confidenceinterval.
Generationandcharacterizationof2(Fab)-λ1.(A)SDS-PAGEanalysisofrefoldedAD2-IFN-λ1module.M,Mrstandard;lanes1and3,BSA;lanes2and4,AD2-IFN-λ1;R,reducing;NR,nonreducing.(B)BioactivitycomparisonbetweenAD2-IFN-λ1andcommercialrhIFN-λ1.ME-180,TE-11,andSK-MES-1cellsweregrowninthepresenceofincreasingconcentrationsofAD2-IFN-λ1orrhIFN-λ1andtherelativeviablecelldensitiesweremeasuredwithMTS.Dose–responsecurvesweregeneratedusingPrismsoftware.(C-F)SDS-PAGEanalysisofpurified(E1)-λ1,(15)-λ1,(C225)-λ1,or(C2)-λ1andtheirconstituentsunderreducing(R)andnon-reducing(NR)conditions.ReducingconditionresolvedthreebandsrepresentingpolypeptidesforFab-DDD2-heavychain,kappalightchain,andAD2-IFN-λ1,andnon-reducingconditionresolvedamajorhigh-relativemobilitybandrepresentingthecovalentDNLstructure.Lanes:M,Mrstandards;1and4,AD2-IFN-λ1;2and5,hRS7-,hMN15-,C225-,orhL243-Fab-DDD2;3and6,(E1)-λ1,(15)-λ1,(c225)-λ1,or(C2)-λ1.
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ImmunoblotanalysisofSTATphosphorylation.Cellsweretreatedwithindicatedtestarticlesfor1h,andphosphorylatedSTAT-1,-2,and-3weremeasuredwithloading30μgoftotalprotein/lane.TotalSTATsandβ-actinwereprobedtoverifyequalloading.(A)ME-180cells;(B)A375cells.
InvitroproliferationsensitivityofcancercelllinestorhIFN-λ1.
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WethankRongxiuLi,MeiyuLoo,JohnKopinski,andDionYeldellfortheirexcellenttechnicalhelp,andHiroshiNakagawa(UniversityofPennsylvania)forprovidingTE-11cellline.
Conceivedanddesignedtheexperiments:CHCDLTMC.Performedtheexperiments:DL.Analyzedthedata:DLCHCEARDMGTMC.Wrotethepaper:DLCHCEARDMG.
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