Antibody Phage DisplayMeiling Xiong20180629ContentsContentsIntroduction of Ab phage Display TechnologyAb Formats for Phage DisplayAb Libraries ConstructionPhage Ab Selection Methods & StrategiesPhage Ab Screening ApplicationsIn vitro Affinity MaturationExpression & Purification of Phage Ab FragmentsIntroduction of Phage Display TechnologyTheFfbacteriophagestructureIntroduction of Phage Display TechnologyTheschemeofphagemidvectorIG region:intergenicregion,usuallycontainsthepackingsequenceandreplicationoriginofminusandplusstrandsMolecular tag:tofacilitatelibraryscreeningandforproteinanalysisRestriction enzyme recognition sites:usefulforDNArecombinationandgenemanipulation;multiplecloningsites(MCS)Coat protein:PIII(largerprotein,lessthan5copies,)PVIII(morethan5copies,decreasedlength)Amber codon TAG:supEstrains(glutamicacidcodon),non-suppressorstrains(stopcodon)Protease cleavage sitePromoterSignal peptides:phageproteintranslocation,crucialfordisplaylevelSelective marker:forselectionofinfectedhostcellsIntroduction of Phage Display TechnologyNonlyticfilamentousphageisthemostoftenusedforphagedisplay,primarilytheM13andFdstrains.Proteinstobeselectedareinfusedtoallfivecoatproteins,withpIIIandpVIIImostcommonlyused.pIIIproteinisessentialforinfectionofbacteriaHelperphage:wild-typepIIIhelperphageandspecialhelperphageAntigenimmobilizedonmagneticbeads,polystryrenesurfaces,oroncolumns,orisusedinsolutionasbiotinylatedantigenandlatercapturedbyimmobilizedstreptavidinAdvantages of Phage Display for Recombinant Antibody SelectionMoreefficientlythanthroughconventionalhybridomasystem.Cheapertoproducerecombinantantibodiesusingbacteria,ratherthanmammaliancellline.Easiertomaintainandgrowbacterialculturesforrecombinantantibodyproduction.Bypassimmunizationinantibodyselection.Bypasstheuseofanimalcellsforproductionofantibodies.Producingthecombinatoriallibrary(ideallywith108to109members)offunctionalantibodiestogeneratealargerrepertoireofantibodiesthanthoseavailablethroughconventionalhybridomatechnology.Easyisolationandexpressionoftheclonedgeneinabacterialhost.Excellentpotentialtofurtherimprovebindingpropertiesoftheselectedantibodybyproteinengineeringtechniques.Capableofgeneratingantibodiesagainstalmostanydesiredantigen,includinghighlyconservedorself-antigens,conformationalvariants,lowimmunogenicantigens,andalsotoxiccomponents,whichisnotpossiblebyinvivoimmunizationofanimals.Anumberofstartingmaterial:proteins,peptides,haptens,celllines,tissueslides,orvirusparticlesAntibody FormatsThemostcommonlyusedformat:single-chain variable fragment (scFv)SimplicityofcloningprocessFastandeasylibrarygenerationAhighdisplayrate(smallproteinsize25kDa)LessstablethanFabfragmentsTendtoformdimers(canbereducedwithlinkermorethan20aminoacids)Antibody Formats FabThelightchain(VL-CL)andtheFd-domain(VH-CH1)oftheheavychainofanantibody.Duringbacterialexpression,thesetwochainsaresynthesized separately,andsecretedintotheperiplasmwheretheyfoldtoformheterodimers.FabexhibithigherstabilitythanscFvsPossessbetterPKandPDqualitiesthanscFvsEasiertoconvertintofull-lengthantibodiesClinicalapplications:abciximab,lucentis,cimzia.Antibody Formats SingledomainantibodyVHH:VHdomainofcamelidantibody,heavychainsonly,IgNAR(newantigenreceptor):sharkantibody,heavychainsonly,UniqueCDRsAffibodiesAnticalinsDARPinsAvimersAffimersMonobodiesvNARAntibody Formats MultivalentfragmentsMiniantibodiesarescFvsorFabsconnectedviaaflexiblelinkertoself-associatingstructuressuchashelixbundlesorleucinezippers.DiabodiesarenoncovalentdimersofscFvs,whichspontaneouslyformdependingonthelinkerlengthbetweenVHandVL.AnotherformofdiabodiesistwoscFvsconnectedwithashortlinker.Fab-AiscreatedbygeneticfusionoftheFabFdgenewiththealkalinephosphatase(PhoA)geneandcoexpressingthelightchaingene.scFv-FcarescFvsdimerizedbytheFcdomain.Immune libraries:first,immunizeananimalwithanantigenandisolatethemRNAfromBlymphocytes(forimmunizedanimals)orperipheralbloodBcells(forimmunizeddonors).ThemRNAisthenreversetranscribedintocDNA,andthevariableregionsofexpressedantibodiesareamplifiedviaPCRandclonedintoaphagedisplayvector.Advantages:MaturedinvivoImmunelibrariescanbegeneratedfromanyanimalandevenhumans:mouse,human,chicken,rabbit,camelAnyspeciesthathavebeenimmunized,infected,orexposedtoanantigen.Usefulinanalyzingnaturalhumoralresponses,forexample,inpatientswithautoimmunedisease,viralinfection,neoplasticdiseases,etc.Antibody LibrariesNave natural libraries: universalantibodylibrariesgeneratedfromB-cellsofnonimmunizeddonorsandeliminatetheneedtoconstructnewlibrariesforeachantigen.loweraffinitiesthanthosegeneratedduringinvivoaffinitymaturation.tofindgoodantibodiesagainstdiverseantigens,theselibrariesneedtobeverylarge.Advantages:Absolutefreedominantigenchoice,includingself,nonimmunogenic,andtoxicAgsSeveralantibodiesselectedbyphagedisplayfromhumannavelibrarieshavealreadybeenapprovedasdrugs,suchasraxibacumab,ramucirumab,necitumumab,orbelimumab.Antibody LibrariesNave Semisynthetic libraries: NavesemisyntheticlibrariesareusuallylibrariesthathavebeenisolatedfromnonimmunehostsandwhereoneorseveralCDRs were exchanged with synthetic peptides or were randomly mutated.Thisapproachisawaytoachievehighdiversitywithoutrequiringalargenumberofdonorsandcangeneratespecificitiesnotnormallyincludedinnaturalrepertoires.Advantages:LowimmunogenicityinhostssinceonlyafewoftheCDRsareartificialTheselibrariescancovertheentirerepertoireofgermlinesAntibody LibrariesNave Synthetic librariesAdvantages:Theprincipleadvantageofnavesyntheticlibrariesoversemisyntheticlibrariesisthatthebiophysicalparametersandcodonusageoftheframeworkregioncanbeoptimizedforexpressibilityandstability.AdvancedDNAsynthesismethodssuchasTRIM,slonomics,orchip-basedDNAphotolithographyoffertheabilitytopreciselydefinethefrequencyofeachaminoacidateachpositionwithoptimizedcodons.CDRscanbeofhigherdiversity,differentincompositionthanbiologicallyoccurringCDRs,henceofferingapotentiallylargerparatopespace.Havebeenusedtogeneratetherapeuticantibodies,aswellasantibodiesforresearchanddiagnosticapplications.Antibody LibrariesStandard Fab Library ConstructionConstruction of Large Nave Fab LibraryAnefficientcloningmethod,inwhich restriction fragments instead of PCR products wereused.VHfragmentsareisolatedbydigestionofplamidDNApurifiedfromtheprimaryrepertoires,andclonedintotheacceptorphagemidvectorcontainingthelight-chain(LC)repertoires.Thisinnovationincreasesthesizeofthelibrariesdramatically.IgM-derived antibody repertoirewereused.scFv Library ConstructionToensurethatallfiveAbclassesarelikelytoberepresentedandincreasetheoverallsizeofthefinallibrary,random hexamers areemployedintheprimaryfirst-strandcDNAsynthesisfromPBLmRNA.ComponentVHandVLgenesegmentsareamplifiedinseparatePCRreactions,andinitiallyclonedintotwodifferentvectors,pCANTAB6andpCANTAB3his6(seeFig.1).ThelatterisusedforcloningtheVLrepertoirebecauseithasthe appropriate polylinker cloning sites forthedigestedVLfragments;theVHrepertoireisclonedintopCANTAB6.AshortlinkerfromanexistingscFviscloned(togetherwithanirrelevantor“dummy”VH)intotheVLrepertoire,upstreamoftheVLfragments.TheVHandlinker-VLrepertoiresarethenamplifiedfromtheirvectors,andthescFvconstructispreparedusingasimpletwo-fragmentPCRassemblyprocedure.ThisconstructisthenclonedintopCANTAB6tocreatethelargenavescFvlibraryPolyclonal antibody library constructionPolyclonal antibody libraries (PCALs)arestandardizedmixturesofantibodiesspecificforanantigenormulti-Agtargets.Theytargetmultipleepitopesonpoly-Ags,resultinginhigh-aviditybindingandefficienttriggeringofeffectorfunctions.PCALgenerationusuallyinvolvestherecoveryofVLandVHrepertoires,andtheir random pairing asFabsintoaphage-displayvector.Thelibraryispositively and negatively selected.SelectedVLVHgenepairsarethentransferred in mass to a mammalian expression vector.Theconstructsarethentransfectedintoamammalian cell line for expression.Phage Ab Selection Procedures and applicationsDiversity in Selection methodsImmobilizedAg:solidsupports,columns,BIAcoresensorchipsBiotinylatedAginsolutiontoavoidconformationalchangesProkaryoticormammaliancells,fluorescenceactivatedcellsorting,tissuesections,invivoselection,etc.ElutionAcidsolutions(HCl).Glycinebuffers;Basicsolutions,triethylaming;Chaotropicagents;Dithiothreitol;Enzymaticcleavage;CompetitionmethodsSelection of Abs for affinity or binding kineticsSelection on complex AgsSelection on cellsFinding new Ags with phage Ab librariesSelection for Ab stability and foldingIn vitro selection of antibodies for specific applicationsTissue panning for immunohistochemistry antibodies:antibodyselectionwithformalin-fixedparaffinembedded(FFPE)tissue.Sandwichpairselection,complex-specificantibodies,anddrugmonitoring:Drug monitoring:variousforms(freeantibodydrug,antibody-targetcomplex,orboth)ofantibodytherapeuticscanbeeasilytrackedandquantifiedinPKassays,usinganti-idiotypeantibodiesComplex-specific antibodies:guidedselectionmethodSandwich pair selectionSite-specific antibody conjugation usingmethodssuchasgeneticfusion(enzyme,orfluorescentprotein).Hapten-specific antibody selectionIsolation of anti-hapten specific antibody fragments from combinatorial librariesHaptentargetswithmolecularweightbelow1000DaltonTheyshouldbeconjugatedtoasuitableimmunogeniccarrierproteinforpresentationToavoidtheselectionofantibodiesspecificforthecarrierproteinorthelinker,wecanuseamethodthatutilizestwodifferenthaptenconjugatesforalternativeroundsofselection.Thelibrarycanbeimmunizedornave.Thenavelibraryshouldbelargebutimmunizedlibraryshouldbeconstructseparately.Competitive DeselectionAntigensfromaparticularpathogencanbeofvariableimmunogenicity,withtheantigenthatstimulatesthestrongestresponsebeingtheimmunodominantone.Toobtainantibodiesagainsttheepitopeofinterest,apreadsorption panning isused.ThisfacilitatesthemolecularcloningofMabfragmentsagainstnon-immunodominantAgdeterminants.ThephagelibraryisfirstpreabsorbedontheAgofinteresttoremovephagethatreactwiththeimmunodominantepitope.TheunboundphagearethenincubatedasecondtimewithAgandelutedandamplifiedaccordingtonormalprotocols.Epitope-masking StrategyCapture-lift Screening procedureCapture-sandwich ELISAStronglyeffectivetoselectAbsagainstAgsfromcrudepreparations.Absagainstconformation-sensitiveAgscanbeselected.MAbsagainstavarietyofAgepitopescanbeisolatedfromasinglelibrary.BothpAbandmAbcanbeusedascaptureAbs.Proximity-Guided SelectionItinvolvestheuseofcatalyzed reporter enzyme deposition (CARD),whichisamethodofsignalamplification.CARD usesHRP-conjugatedsecondaryantibody,biotin tyraminetobiotinylatephageparticlesthatbindaroundthesiteoftheHRPactivity.Thesephagecanberecoveredonstreptavidin-coatedmagneticbeads.ThisselectionstrategycanbesuedtoisolatephageAbagainstcellsurfacemarkers,andotherantigens,suchaspurifiedAgs,cellextracts,membranepreparations.Magnetic sorting for selection of antibodies to cell-surface antigensForselectionofantibodiestargetingcell-surfaceantigensAcompetitivecell-panningapproachisused,inwhichtargetcells(positivecells)areprecoatedwithmagneticbeads,andmixedwithanexcessofunmodifiedAg-negative cells.ThismethodismoreefficientthanjustseveralroundsofnegativeselectiononAg-negativecells.Phage Ab screening applicationsScreening for affinity or kinetics of bindingScreening for bioactivity/function:receptorblockingortriggering(dimerization),virusorcytokineneutralizationSelection for a particular function:Abwithagonistorantagonistactivityforagivenreceptor,fordrugdiscovery;Abthatdimerizesreceptors;Abinternalizationforgenetransfer;Abselectionforcellsurvivalorkilling;Combining phage display with other procedures suchasselectionusingamammalianhostcellorothercellsystems.High-throughput selection and screeningScreening for affinity or kinetics of bindingDependingontheintendedapplication,thebindingofamoleculetoitstargetisdesiredtobelong-livedorshort-lived.BIAcoretechnologyIn vitro affinity maturationMethodstogeneratemutations:Error-prone PCRDegenerate oligonucleotidesMutagenic strains of bacteria: mutD5-FITChain/CDR shufflingSite-directedmutagenesis,atrestrictedpositionsintheCDRregionRandommutagenesis,mutationsareintroducedintotheentireVregionTargetingrandommutationstohotspotsinantibodyvariabledomainsforaffinityimprovementExpression and purification of Abs in different cell linesCytoplasmicinclusionbodiesorexpressedascorrectlyfoldedAbSeveralwaystoenhancetheproportionofcorrectlyfoldedAbsandreduce aggregationPurification: His-taggedprotein,IMAC,affinitychromatographyExpressionofAntibodyfragmentsin Pichia pastorisExpressionofVHHAntibodyFragmentsinSaccharomycescerevisiaeIntrabodiesExpressionofscFvsandscFvFusionProteinsinEukaryoticCellsExpressionofAntibodyFabFragmentsandWholeImmunoglobulininMammalianCellsThank you!