抗体噬菌体展示技术ppt课件

.,AntibodyPhageDisplay,MeilingXiong20180629,.,Contents,IntroductionofAbphageDisplayTechnologyAbFormatsforPhageDisplayAbLibrariesConstructionPhageAbSelectionMethodsmultiplecloningsites(MCS)Coatprotein:PIII(largerprotein,lessthan5copies,)PVIII(morethan5copies,decreasedlength)AmbercodonTAG:supEstrains(glutamicacidcodon),non-suppressorstrains(stopcodon)ProteasecleavagesitePromoterSignalpeptides:phageproteintranslocation,crucialfordisplaylevelSelectivemarker:forselectionofinfectedhostcells,.,IntroductionofPhageDisplayTechnology,Nonlyticfilamentousphageisthemostoftenusedforphagedisplay,primarilytheM13andFdstrains.Proteinstobeselectedareinfusedtoallfivecoatproteins,withpIIIandpVIIImostcommonlyused.pIIIproteinisessentialforinfectionofbacteriaHelperphage:wild-typepIIIhelperphageandspecialhelperphageAntigenimmobilizedonmagneticbeads,polystryrenesurfaces,oroncolumns,orisusedinsolutionasbiotinylatedantigenandlatercapturedbyimmobilizedstreptavidin,.,AdvantagesofPhageDisplayforRecombinantAntibodySelection,Moreefficientlythanthroughconventionalhybridomasystem.Cheapertoproducerecombinantantibodiesusingbacteria,ratherthanmammaliancellline.Easiertomaintainandgrowbacterialculturesforrecombinantantibodyproduction.Bypassimmunizationinantibodyselection.Bypasstheuseofanimalcellsforproductionofantibodies.Producingthecombinatoriallibrary(ideallywith108to109members)offunctionalantibodiestogeneratealargerrepertoireofantibodiesthanthoseavailablethroughconventionalhybridomatechnology.Easyisolationandexpressionoftheclonedgeneinabacterialhost.Excellentpotentialtofurtherimprovebindingpropertiesoftheselectedantibodybyproteinengineeringtechniques.Capableofgeneratingantibodiesagainstalmostanydesiredantigen,includinghighlyconservedorself-antigens,conformationalvariants,lowimmunogenicantigens,andalsotoxiccomponents,whichisnotpossiblebyinvivoimmunizationofanimals.Anumberofstartingmaterial:proteins,peptides,haptens,celllines,tissueslides,orvirusparticles,.,AntibodyFormats,Themostcommonlyusedformat:single-chainvariablefragment(scFv)SimplicityofcloningprocessFastandeasylibrarygenerationAhighdisplayrate(smallproteinsize25kDa)LessstablethanFabfragmentsTendtoformdimers(canbereducedwithlinkermorethan20aminoacids),.,AntibodyFormats,FabThelightchain(VL-CL)andtheFd-domain(VH-CH1)oftheheavychainofanantibody.Duringbacterialexpression,thesetwochainsaresynthesizedseparately,andsecretedintotheperiplasmwheretheyfoldtoformheterodimers.FabexhibithigherstabilitythanscFvsPossessbetterPKandPDqualitiesthanscFvsEasiertoconvertintofull-lengthantibodiesClinicalapplications:abciximab,lucentis,cimzia.,.,AntibodyFormats,SingledomainantibodyVHH:VHdomainofcamelidantibody,heavychainsonly,IgNAR(newantigenreceptor):sharkantibody,heavychainsonly,UniqueCDRsAffibodiesAnticalinsDARPinsAvimersAffimersMonobodies,.,AntibodyFormats,MultivalentfragmentsMiniantibodiesarescFvsorFabsconnectedviaaflexiblelinkertoself-associatingstructuressuchashelixbundlesorleucinezippers.DiabodiesarenoncovalentdimersofscFvs,whichspontaneouslyformdependingonthelinkerlengthbetweenVHandVL.AnotherformofdiabodiesistwoscFvsconnectedwithashortlinker.Fab-AiscreatedbygeneticfusionoftheFabFdgenewiththealkalinephosphatase(PhoA)geneandcoexpressingthelightchaingene.scFv-FcarescFvsdimerizedbytheFcdomain.,.,Immunelibraries:first,immunizeananimalwithanantigenandisolatethemRNAfromBlymphocytes(forimmunizedanimals)orperipheralbloodBcells(forimmunizeddonors).ThemRNAisthenreversetranscribedintocDNA,andthevariableregionsofexpressedantibodiesareamplifiedviaPCRandclonedintoaphagedisplayvector.Advantages:MaturedinvivoImmunelibrariescanbegeneratedfromanyanimalandevenhumans:mouse,human,chicken,rabbit,camelAnyspeciesthathavebeenimmunized,infected,orexposedtoanantigen.Usefulinanalyzingnaturalhumoralresponses,forexample,inpatientswithautoimmunedisease,viralinfection,neoplasticdiseases,etc.,AntibodyLibraries,.,Navenaturallibraries:universalantibodylibrariesgeneratedfromB-cellsofnonimmunizeddonorsandeliminatetheneedtoconstructnewlibrariesforeachantigen.loweraffinitiesthanthosegeneratedduringinvivoaffinitymaturation.tofindgoodantibodiesagainstdiverseantigens,theselibrariesneedtobeverylarge.Advantages:Absolutefreedominantigenchoice,includingself,nonimmunogenic,andtoxicAgsSeveralantibodiesselectedbyphagedisplayfromhumannavelibrarieshavealreadybeenapprovedasdrugs,suchasraxibacumab,ramucirumab,necitumumab,orbelimumab.,AntibodyLibraries,.,NaveSemisyntheticlibraries:NavesemisyntheticlibrariesareusuallylibrariesthathavebeenisolatedfromnonimmunehostsandwhereoneorseveralCDRswereexchangedwithsyntheticpeptidesorwererandomlymutated.Thisapproachisawaytoachievehighdiversitywithoutrequiringalargenumberofdonorsandcangeneratespecificitiesnotnormallyincludedinnaturalrepertoires.Advantages:LowimmunogenicityinhostssinceonlyafewoftheCDRsareartificialTheselibrariescancovertheentirerepertoireofgermlines,AntibodyLibraries,.,NaveSyntheticlibrariesAdvantages:Theprincipleadvantageofnavesyntheticlibrariesoversemisyntheticlibrariesisthatthebiophysicalparametersandcodonusageoftheframeworkregioncanbeoptimizedforexpressibilityandstability.AdvancedDNAsynthesismethodssuchasTRIM,slonomics,orchip-basedDNAphotolithographyoffertheabilitytopreciselydefinethefrequencyofeachaminoacidateachpositionwithoptimizedcodons.CDRscanbeofhigherdiversity,differentincompositionthanbiologicallyoccurringCDRs,henceofferingapotentiallylargerparatopespace.Havebeenusedtogeneratetherapeuticantibodies,aswellasantibodiesforresearchanddiagnosticapplications.,AntibodyLibraries,.,StandardFabLibraryConstruction,ConstructionofLargeNaveFabLibrary,Anefficientcloningmethod,inwhichrestrictionfragmentsinsteadofPCRproductswereused.VHfragmentsareisolatedbydigestionofplamidDNApurifiedfromtheprimaryrepertoires,andclonedintotheacceptorphagemidvectorcontainingthelight-chain(LC)repertoires.Thisinnovationincreasesthesizeofthelibrariesdramatically.IgM-derivedantibodyrepertoirewereused.,.,scFvLibraryConstruction,ToensurethatallfiveAbclassesarelikelytoberepresentedandincreasetheoverallsizeofthefinallibrary,randomhexamersareemployedintheprimaryfirst-strandcDNAsynthesisfromPBLmRNA.ComponentVHandVLgenesegmentsareamplifiedinseparatePCRreactions,andinitiallyclonedintotwodifferentvectors,pCANTAB6andpCANTAB3his6(seeFig.1).ThelatterisusedforcloningtheVLrepertoirebecauseithastheappropriatepolylinkercloningsitesforthedigestedVLfragments;theVHrepertoireisclonedintopCANTAB6.AshortlinkerfromanexistingscFviscloned(togetherwithanirrelevantor“dummy”VH)intotheVLrepertoire,upstreamoftheVLfragments.TheVHandlinker-VLrepertoiresarethenamplifiedfromtheirvectors,andthescFvconstructispreparedusingasimpletwo-fragmentPCRassemblyprocedure.ThisconstructisthenclonedintopCANTAB6tocreatethelargenavescFvlibrary,.,Polyclonalantibodylibraryconstruction,Polyclonalantibodylibraries(PCALs)arestandardizedmixturesofantibodiesspecificforanantigenormulti-Agtargets.Theytargetmultipleepitopesonpoly-Ags,resultinginhigh-aviditybindingandefficienttriggeringofeffectorfunctions.PCALgenerationusuallyinvolvestherecoveryofVLandVHrepertoires,andtheirrandompairingasFabsintoaphage-displayvector.Thelibraryispositivelyandnegativelyselected.SelectedVLVHgenepairsarethentransferredinmasstoamammalianexpressionvector.Theconstructsarethentransfectedintoamammaliancelllineforexpression.,.,PhageAbSelectionProceduresandapplications,DiversityinSelectionmethodsImmobilizedAg:solidsupports,columns,BIAcoresensorchipsBiotinylatedAginsolutiontoavoidconformationalchangesProkaryoticormammaliancells,fluorescenceactivatedcellsorting,tissuesections,invivoselection,etc.ElutionAcidsolutions(HCl).Glycinebuffers;Basicsolutions,triethylaming;Chaotropicagents;Dithiothreitol;Enzymaticcleavage;CompetitionmethodsSelectionofAbsforaffinityorbindingkineticsSelectiononcomplexAgsSelectiononcellsFindingnewAgswithphageAblibrariesSelectionforAbstabilityandfolding,.,Invitroselectionofantibodiesforspecificapplications,Tissuepanningforimmunohistochemistryantibodies:antibodyselectionwithformalin-fixedparaffinembedded(FFPE)tissue.Sandwichpairselection,complex-specificantibodies,anddrugmonitoring:Drugmonitoring:variousforms(freeantibodydrug,antibody-targetcomplex,orboth)ofantibodytherapeuticscanbeeasilytrackedandquantifiedinPKassays,usinganti-idiotypeantibodiesComplex-specificantibodies:guidedselectionmethodSandwichpairselectionSite-specificantibodyconjugationusingmethodssuchasgeneticfusion(enzyme,orfluorescentprotein).,.,Hapten-specificantibodyselection,Isolationofanti-haptenspecificantibodyfragmentsfromcombinatoriallibrariesHaptentargetswithmolecularweightbelow1000DaltonTheyshouldbeconjugatedtoasuitableimmunogeniccarrierproteinforpresentationToavoidtheselectionofantibodiesspecificforthecarrierproteinorthelinker,wecanuseamethodthatutilizestwodifferenthaptenconjugatesforalternativeroundsofselection.Thelibrarycanbeimmunizedornave.Thenavelibraryshouldbelargebutimmunizedlibraryshouldbeconstructseparately.,.,CompetitiveDeselection,Antigensfromaparticularpathogencanbeofvariableimmunogenicity,withtheantigenthatstimulatesthestrongestresponsebeingtheimmunodominantone.Toobtainantibodiesagainsttheepitopeofinterest,apreadsorptionpanningisused.ThisfacilitatesthemolecularcloningofMabfragmentsagainstnon-immunodominantAgdeterminants.ThephagelibraryisfirstpreabsorbedontheAgofinteresttoremovephagethatreactwiththeimmunodominantepitope.TheunboundphagearethenincubatedasecondtimewithAgandelutedandamplifiedaccordingtonormalprotocols.,.,Epitope-maskingStrategy,.,Capture-liftScreeningprocedure,.,Capture-sandwichELISA,StronglyeffectivetoselectAbsagainstAgsfromcrudepreparations.Absagainstconformation-sensitiveAgscanbeselected.MAbsagainstavarietyofAgepitopescanbeisolatedfromasinglelibrary.BothpAbandmAbcanbeusedascaptureAbs.,.,Proximity-GuidedSelection,Itinvolvestheuseofcatalyzedreporterenzymedeposition(CARD),whichisamethodofsignalamplification.CARDusesHRP-conjugatedsecondaryantibody,biotintyraminetobiotinylatephageparticlesthatbindaroundthesiteoftheHRPactivity.Thesephagecanberecoveredonstreptavidin-coatedmagneticbeads.ThisselectionstrategycanbesuedtoisolatephageAbagainstcellsurfacemarkers,andotherantigens,suchaspurifiedAgs,cellextracts,membranepreparations.,.,Magneticsortingforselectionofantibodiestocell-surfaceantigens,Forselectionofantibodiestargetingcell-surfaceantigensAcompetitivecell-panningapproachisused,inwhichtargetcells(positivecells)areprecoatedwithmagneticbeads,andmixedwithanexcessofunmodifiedAg-negativecells.ThismethodismoreefficientthanjustseveralroundsofnegativeselectiononAg-negativecells.,.,PhageAbscreeningapplications,ScreeningforaffinityorkineticsofbindingScreeningforbioactivity/function:receptorblockingortriggering(dimerization),virusorcytokineneutralizationSelectionforaparticularfunction:Abwithagonistorantagonistactivityforagivenreceptor,fordrugdiscovery;Abthatdimerizesreceptors;Abinternalizationforgenetransfer;Abselectionforcellsurvivalorkilling;Combiningphagedisplaywithotherproceduressuchasselectionusingamammalianhostcellorothercellsystems.High-throughputselectionandscreening,.,Screeningforaffinityorkineticsofbinding,Dependingontheintendedapplication,thebindingofamoleculetoitstargetisdesiredtobelong-livedorshort-lived.BIAcoretechnology,.,Invitroaffinitymaturation,Methodstogeneratemu