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AntibodyPhageDisplay MeilingXiong20180629 Contents IntroductionofAbphageDisplayTechnologyAbFormatsforPhageDisplayAbLibrariesConstructionPhageAbSelectionMethods StrategiesPhageAbScreeningApplicationsInvitroAffinityMaturationExpression PurificationofPhageAbFragments IntroductionofPhageDisplayTechnology TheFfbacteriophagestructure IntroductionofPhageDisplayTechnology Theschemeofphagemidvector IGregion intergenicregion usuallycontainsthepackingsequenceandreplicationoriginofminusandplusstrandsMoleculartag tofacilitatelibraryscreeningandforproteinanalysisRestrictionenzymerecognitionsites usefulforDNArecombinationandgenemanipulation multiplecloningsites 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 smallproteinsize 25kDa 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 camel Anyspeciesthathavebeenimmunized infected orexposedtoanantigen Usefulinanalyzingnaturalhumoralresponses forexample inpatientswithautoimmunedisease viralinfection neoplasticdiseases etc AntibodyLibraries Na venaturallibraries universalantibodylibrariesgeneratedfromB cellsofnonimmunizeddonorsandeliminatetheneedtoconstructnewlibrariesforeachantigen loweraffinitiesthanthosegeneratedduringinvivoaffinitymaturation tofindgoodantibodiesagainstdiverseantigens theselibrariesneedtobeverylarge Advantages Absolutefreedominantigenchoice includingself nonimmunogenic andtoxicAgsSeveralantibodiesselectedbyphagedisplayfromhumanna velibrarieshavealreadybeenapprovedasdrugs suchasraxibacumab ramucirumab necitumumab orbelimumab AntibodyLibraries Na veSemisyntheticlibraries Na vesemisyntheticlibrariesareusuallylibrariesthathavebeenisolatedfromnonimmunehostsandwhereoneorseveralCDRswereexchangedwithsyntheticpeptidesorwererandomlymutated Thisapproachisawaytoachievehighdiversitywithoutrequiringalargenumberofdonorsandcangeneratespecificitiesnotnormallyincludedinnaturalrepertoires Advantages LowimmunogenicityinhostssinceonlyafewoftheCDRsareartificialTheselibrariescancovertheentirerepertoireofgermlines AntibodyLibraries Na veSyntheticlibrariesAdvantages Theprincipleadvantageofna vesyntheticlibrariesoversemisyntheticlibrariesisthatthebiophysicalparametersandcodonusageoftheframeworkregioncanbeoptimizedforexpressibilityandstability AdvancedDNAsynthesismethodssuchasTRIM slonomics orchip basedDNAphotolithographyoffertheabilitytopreciselydefinethefrequencyofeachaminoacidateachpositionwithoptimizedcodons CDRscanbeofhigherdiversity differentincompositionthanbiologicallyoccurringCDRs henceofferingapotentiallylargerparatopespace Havebeenusedtogeneratetherapeuticantibodies aswellasantibodiesforresearchanddiagnosticapplications AntibodyLibraries StandardFabLibraryConstruction ConstructionofLargeNa veFabLibrary 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 ThisconstructisthenclonedintopCANTAB6tocreatethelargena vescFvlibrary Polyclonalantibodylibraryconstruction Polyclonalantibodylibraries PCALs arestandardizedmixturesofantibodiesspecificforanantigenormulti Agtargets Theytargetmultipleepitopesonpoly Ags resultinginhigh aviditybindingandefficienttriggeringofeffectorfunctions PCALgenerationusuallyinvolvestherecoveryofVLandVHrepertoires andtheirrandompairingasFabsintoaphage displayvector Thelibraryispositivelyandnegativelyselected SelectedVL VHgenepairsarethentransferredinmasstoamammalianexpressionvector 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 Thelibrarycanbeimmunizedorna ve Thena velibraryshouldbelargebutimmunizedlibraryshouldbeconstructseparately 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 Methodst