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1、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 Fragments
2、Introduction of Phage Display TechnologyTheFfbacteriophagestructureIntroduction of Phage Display TechnologyTheschemeofphagemidvectorIG region:intergenicregion,usuallycontainsthepackingsequenceandreplicationoriginofminusandplusstrandsMolecular tag:tofacilitatelibraryscreeningandforproteinanalysisRest
3、riction enzyme recognition sites:usefulforDNArecombinationandgenemanipulation;multiplecloningsites(MCS)Coat protein:PIII(largerprotein,lessthan5copies,)PVIII(morethan5copies,decreasedlength)Amber codon TAG:supEstrains(glutamicacidcodon),non-suppressorstrains(stopcodon)Protease cleavage sitePromoterS
4、ignal peptides:phageproteintranslocation,crucialfordisplaylevelSelective marker:forselectionofinfectedhostcellsIntroduction of Phage Display TechnologyNonlyticfilamentousphageisthemostoftenusedforphagedisplay,primarilytheM13andFdstrains.Proteinstobeselectedareinfusedtoallfivecoatproteins,withpIIIand
5、pVIIImostcommonlyused.pIIIproteinisessentialforinfectionofbacteriaHelperphage:wild-typepIIIhelperphageandspecialhelperphageAntigenimmobilizedonmagneticbeads,polystryrenesurfaces,oroncolumns,orisusedinsolutionasbiotinylatedantigenandlatercapturedbyimmobilizedstreptavidinAdvantages of Phage Display fo
6、r Recombinant Antibody SelectionMoreefficientlythanthroughconventionalhybridomasystem.Cheapertoproducerecombinantantibodiesusingbacteria,ratherthanmammaliancellline.Easiertomaintainandgrowbacterialculturesforrecombinantantibodyproduction.Bypassimmunizationinantibodyselection.Bypasstheuseofanimalcell
7、sforproductionofantibodies.Producingthecombinatoriallibrary(ideallywith108to109members)offunctionalantibodiestogeneratealargerrepertoireofantibodiesthanthoseavailablethroughconventionalhybridomatechnology.Easyisolationandexpressionoftheclonedgeneinabacterialhost.Excellentpotentialtofurtherimprovebin
8、dingpropertiesoftheselectedantibodybyproteinengineeringtechniques.Capableofgeneratingantibodiesagainstalmostanydesiredantigen,includinghighlyconservedorself-antigens,conformationalvariants,lowimmunogenicantigens,andalsotoxiccomponents,whichisnotpossiblebyinvivoimmunizationofanimals.Anumberofstarting
9、material:proteins,peptides,haptens,celllines,tissueslides,orvirusparticlesAntibody FormatsThemostcommonlyusedformat:single-chain variable fragment (scFv)SimplicityofcloningprocessFastandeasylibrarygenerationAhighdisplayrate(smallproteinsize25kDa)LessstablethanFabfragmentsTendtoformdimers(canbereduce
10、dwithlinkermorethan20aminoacids)Antibody Formats FabThelightchain(VL-CL)andtheFd-domain(VH-CH1)oftheheavychainofanantibody.Duringbacterialexpression,thesetwochainsaresynthesized separately,andsecretedintotheperiplasmwheretheyfoldtoformheterodimers.FabexhibithigherstabilitythanscFvsPossessbetterPKand
11、PDqualitiesthanscFvsEasiertoconvertintofull-lengthantibodiesClinicalapplications:abciximab,lucentis,cimzia.Antibody Formats SingledomainantibodyVHH:VHdomainofcamelidantibody,heavychainsonly,IgNAR(newantigenreceptor):sharkantibody,heavychainsonly,UniqueCDRsAffibodiesAnticalinsDARPinsAvimersAffimersMo
12、nobodiesvNARAntibody Formats MultivalentfragmentsMiniantibodiesarescFvsorFabsconnectedviaaflexiblelinkertoself-associatingstructuressuchashelixbundlesorleucinezippers.DiabodiesarenoncovalentdimersofscFvs,whichspontaneouslyformdependingonthelinkerlengthbetweenVHandVL.AnotherformofdiabodiesistwoscFvsc
13、onnectedwithashortlinker.Fab-AiscreatedbygeneticfusionoftheFabFdgenewiththealkalinephosphatase(PhoA)geneandcoexpressingthelightchaingene.scFv-FcarescFvsdimerizedbytheFcdomain.Immune libraries:first,immunizeananimalwithanantigenandisolatethemRNAfromBlymphocytes(forimmunizedanimals)orperipheralbloodBc
14、ells(forimmunizeddonors).ThemRNAisthenreversetranscribedintocDNA,andthevariableregionsofexpressedantibodiesareamplifiedviaPCRandclonedintoaphagedisplayvector.Advantages:MaturedinvivoImmunelibrariescanbegeneratedfromanyanimalandevenhumans:mouse,human,chicken,rabbit,camelAnyspeciesthathavebeenimmunize
15、d,infected,orexposedtoanantigen.Usefulinanalyzingnaturalhumoralresponses,forexample,inpatientswithautoimmunedisease,viralinfection,neoplasticdiseases,etc.Antibody LibrariesNave natural libraries: universalantibodylibrariesgeneratedfromB-cellsofnonimmunizeddonorsandeliminatetheneedtoconstructnewlibra
16、riesforeachantigen.loweraffinitiesthanthosegeneratedduringinvivoaffinitymaturation.tofindgoodantibodiesagainstdiverseantigens,theselibrariesneedtobeverylarge.Advantages:Absolutefreedominantigenchoice,includingself,nonimmunogenic,andtoxicAgsSeveralantibodiesselectedbyphagedisplayfromhumannavelibrarie
17、shavealreadybeenapprovedasdrugs,suchasraxibacumab,ramucirumab,necitumumab,orbelimumab.Antibody LibrariesNave Semisynthetic libraries: NavesemisyntheticlibrariesareusuallylibrariesthathavebeenisolatedfromnonimmunehostsandwhereoneorseveralCDRs were exchanged with synthetic peptides or were randomly mu
18、tated.Thisapproachisawaytoachievehighdiversitywithoutrequiringalargenumberofdonorsandcangeneratespecificitiesnotnormallyincludedinnaturalrepertoires.Advantages:LowimmunogenicityinhostssinceonlyafewoftheCDRsareartificialTheselibrariescancovertheentirerepertoireofgermlinesAntibody LibrariesNave Synthe
19、tic librariesAdvantages:Theprincipleadvantageofnavesyntheticlibrariesoversemisyntheticlibrariesisthatthebiophysicalparametersandcodonusageoftheframeworkregioncanbeoptimizedforexpressibilityandstability.AdvancedDNAsynthesismethodssuchasTRIM,slonomics,orchip-basedDNAphotolithographyoffertheabilitytopr
20、eciselydefinethefrequencyofeachaminoacidateachpositionwithoptimizedcodons.CDRscanbeofhigherdiversity,differentincompositionthanbiologicallyoccurringCDRs,henceofferingapotentiallylargerparatopespace.Havebeenusedtogeneratetherapeuticantibodies,aswellasantibodiesforresearchanddiagnosticapplications.Ant
21、ibody LibrariesStandard Fab Library ConstructionConstruction of Large Nave Fab LibraryAnefficientcloningmethod,inwhich restriction fragments instead of PCR products wereused.VHfragmentsareisolatedbydigestionofplamidDNApurifiedfromtheprimaryrepertoires,andclonedintotheacceptorphagemidvectorcontaining
22、thelight-chain(LC)repertoires.Thisinnovationincreasesthesizeofthelibrariesdramatically.IgM-derived antibody repertoirewereused.scFv Library ConstructionToensurethatallfiveAbclassesarelikelytoberepresentedandincreasetheoverallsizeofthefinallibrary,random hexamers areemployedintheprimaryfirst-strandcD
23、NAsynthesisfromPBLmRNA.ComponentVHandVLgenesegmentsareamplifiedinseparatePCRreactions,andinitiallyclonedintotwodifferentvectors,pCANTAB6andpCANTAB3his6(seeFig.1).ThelatterisusedforcloningtheVLrepertoirebecauseithasthe appropriate polylinker cloning sites forthedigestedVLfragments;theVHrepertoireiscl
24、onedintopCANTAB6.AshortlinkerfromanexistingscFviscloned(togetherwithanirrelevantor“dummy”VH)intotheVLrepertoire,upstreamoftheVLfragments.TheVHandlinker-VLrepertoiresarethenamplifiedfromtheirvectors,andthescFvconstructispreparedusingasimpletwo-fragmentPCRassemblyprocedure.Thisconstructisthenclonedint
25、opCANTAB6tocreatethelargenavescFvlibraryPolyclonal antibody library constructionPolyclonal antibody libraries (PCALs)arestandardizedmixturesofantibodiesspecificforanantigenormulti-Agtargets.Theytargetmultipleepitopesonpoly-Ags,resultinginhigh-aviditybindingandefficienttriggeringofeffectorfunctions.P
26、CALgenerationusuallyinvolvestherecoveryofVLandVHrepertoires,andtheir random pairing asFabsintoaphage-displayvector.Thelibraryispositively and negatively selected.SelectedVLVHgenepairsarethentransferred in mass to a mammalian expression vector.Theconstructsarethentransfectedintoamammalian cell line f
27、or expression.Phage Ab Selection Procedures and applicationsDiversity in Selection methodsImmobilizedAg:solidsupports,columns,BIAcoresensorchipsBiotinylatedAginsolutiontoavoidconformationalchangesProkaryoticormammaliancells,fluorescenceactivatedcellsorting,tissuesections,invivoselection,etc.ElutionA
28、cidsolutions(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
29、 vitro selection of antibodies for specific applicationsTissue panning for immunohistochemistry antibodies:antibodyselectionwithformalin-fixedparaffinembedded(FFPE)tissue.Sandwichpairselection,complex-specificantibodies,anddrugmonitoring:Drug monitoring:variousforms(freeantibodydrug,antibody-targetc
30、omplex,orboth)ofantibodytherapeuticscanbeeasilytrackedandquantifiedinPKassays,usinganti-idiotypeantibodiesComplex-specific antibodies:guidedselectionmethodSandwich pair selectionSite-specific antibody conjugation usingmethodssuchasgeneticfusion(enzyme,orfluorescentprotein).Hapten-specific antibody s
31、electionIsolation of anti-hapten specific antibody fragments from combinatorial librariesHaptentargetswithmolecularweightbelow1000DaltonTheyshouldbeconjugatedtoasuitableimmunogeniccarrierproteinforpresentationToavoidtheselectionofantibodiesspecificforthecarrierproteinorthelinker,wecanuseamethodthatu
32、tilizestwodifferenthaptenconjugatesforalternativeroundsofselection.Thelibrarycanbeimmunizedornave.Thenavelibraryshouldbelargebutimmunizedlibraryshouldbeconstructseparately.Competitive DeselectionAntigensfromaparticularpathogencanbeofvariableimmunogenicity,withtheantigenthatstimulatesthestrongestresp
33、onsebeingtheimmunodominantone.Toobtainantibodiesagainsttheepitopeofinterest,apreadsorption panning isused.ThisfacilitatesthemolecularcloningofMabfragmentsagainstnon-immunodominantAgdeterminants.ThephagelibraryisfirstpreabsorbedontheAgofinteresttoremovephagethatreactwiththeimmunodominantepitope.Theun
34、boundphagearethenincubatedasecondtimewithAgandelutedandamplifiedaccordingtonormalprotocols.Epitope-masking StrategyCapture-lift Screening procedureCapture-sandwich ELISAStronglyeffectivetoselectAbsagainstAgsfromcrudepreparations.Absagainstconformation-sensitiveAgscanbeselected.MAbsagainstavarietyofA
35、gepitopescanbeisolatedfromasinglelibrary.BothpAbandmAbcanbeusedascaptureAbs.Proximity-Guided SelectionItinvolvestheuseofcatalyzed reporter enzyme deposition (CARD),whichisamethodofsignalamplification.CARD usesHRP-conjugatedsecondaryantibody,biotin tyraminetobiotinylatephageparticlesthatbindaroundthe
36、siteoftheHRPactivity.Thesephagecanberecoveredonstreptavidin-coatedmagneticbeads.ThisselectionstrategycanbesuedtoisolatephageAbagainstcellsurfacemarkers,andotherantigens,suchaspurifiedAgs,cellextracts,membranepreparations.Magnetic sorting for selection of antibodies to cell-surface antigensForselecti
37、onofantibodiestargetingcell-surfaceantigensAcompetitivecell-panningapproachisused,inwhichtargetcells(positivecells)areprecoatedwithmagneticbeads,andmixedwithanexcessofunmodifiedAg-negative cells.ThismethodismoreefficientthanjustseveralroundsofnegativeselectiononAg-negativecells.Phage Ab screening ap
38、plicationsScreening for affinity or kinetics of bindingScreening for bioactivity/function:receptorblockingortriggering(dimerization),virusorcytokineneutralizationSelection for a particular function:Abwithagonistorantagonistactivityforagivenreceptor,fordrugdiscovery;Abthatdimerizesreceptors;Abinterna
39、lizationforgenetransfer;Abselectionforcellsurvivalorkilling;Combining phage display with other procedures suchasselectionusingamammalianhostcellorothercellsystems.High-throughput selection and screeningScreening for affinity or kinetics of bindingDependingontheintendedapplication,thebindingofamolecu
40、letoitstargetisdesiredtobelong-livedorshort-lived.BIAcoretechnologyIn vitro affinity maturationMethodstogeneratemutations:Error-prone PCRDegenerate oligonucleotidesMutagenic strains of bacteria: mutD5-FITChain/CDR shufflingSite-directedmutagenesis,atrestrictedpositionsintheCDRregionRandommutagenesis
41、,mutationsareintroducedintotheentireVregionTargetingrandommutationstohotspotsinantibodyvariabledomainsforaffinityimprovementExpression and purification of Abs in different cell linesCytoplasmicinclusionbodiesorexpressedascorrectlyfoldedAbSeveralwaystoenhancetheproportionofcorrectlyfoldedAbsandreduce
42、 aggregationPurification: His-taggedprotein,IMAC,affinitychromatographyExpressionofAntibodyfragmentsin Pichia pastorisExpressionofVHHAntibodyFragmentsinSaccharomycescerevisiaeIntrabodiesExpressionofscFvsandscFvFusionProteinsinEukaryoticCellsExpressionofAntibodyFabFragmentsandWholeImmunoglobulininMammalianCellsThank you!
