AntibodyPhageDisplayMeilingXiong20180629ContentsIntroductionofAbphageDisplayTechnologyAbFormatsforPhageDisplayAbLibrariesConstructionPhageAbSelectionMethods&StrategiesPhageAbScreeningApplicationsInvitroAffinityMaturationExpression&PurificationofPhageAbFragmentsIntroductionofPhageDisplayTechnologyTheFfbacteriophagestructureIntroductionofPhageDisplayTechnologyTheschemeofphagemidvectorIGregion:intergenicregion,usuallycontainsthepackingsequenceandreplicationoriginofminusandplusstrandsMoleculartag:tofacilitatelibraryscreeningandforproteinanalysisRestrictionenzymerecognitionsites:usefulforDNArecombinationandgenemanipulation;multiplecloningsites(MCS)Coatprotein:PIII(largerprotein,lessthan5copies,)PVIII(morethan5copies,decreasedlength)AmbercodonTAG:supEstrains(glutamicacidcodon),non-suppressorstrains(stopcodon)ProteasecleavagesitePromoterSignalpeptides:phageproteintranslocation,crucialfordisplaylevelSelectivemarker:forselectionofinfectedhostcellsIntroductionofPhageDisplayTechnologyNonlyticfilamentousphageisthemostoftenusedforphagedisplay,primarilytheM13andFdstrains.Proteinstobeselectedareinfusedtoallfivecoatproteins,withpIIIandpVIIImostcommonlyused.pIIIproteinisessentialforinfectionofbacteriaHelperphage:wild-typepIIIhelperphageandspecialhelperphageAntigenimmobilizedonmagneticbeads,polystryrenesurfaces,oroncolumns,orisusedinsolutionasbiotinylatedantigenandlatercapturedbyimmobilizedstreptavidinAdvantagesofPhageDisplayforRecombinantAntibodySelectionMoreefficientlythanthroughconventionalhybridomasystem.Cheapertoproducerecombinantantibodiesusingbacteria,ratherthanmammaliancellline.Easiertomaintainandgrowbacterialculturesforrecombinantantibodyproduction.Bypassimmunizationinantibodyselection.Bypasstheuseofanimalcellsforproductionofantibodies.Producingthecombinatoriallibrary(ideallywith108to109members)offunctionalantibodiestogeneratealargerrepertoireofantibodiesthanthoseavailablethroughconventionalhybridomatechnology.Easyisolationandexpressionoftheclonedgeneinabacterialhost.Excellentpotentialtofurtherimprovebindingpropertiesoftheselectedantibodybyproteinengineeringtechniques.Capableofgeneratingantibodiesagainstalmostanydesiredantigen,includinghighlyconservedorself-antigens,conformationalvariants,lowimmunogenicantigens,andalsotoxiccomponents,whichisnotpossiblebyinvivoimmunizationofanimals.Anumberofstartingmaterial:proteins,peptides,haptens,celllines,tissueslides,orvirusparticlesAntibodyFormatsThemostcommonlyusedformat:single-chainvariablefragment(scFv)SimplicityofcloningprocessFastandeasylibrarygenerationAhighdisplayrate(smallproteinsize~25kDa)LessstablethanFabfragmentsTendtoformdimers(canbereducedwithlinkermorethan20aminoacids)AntibodyFormatsFabThelightchain(VL-CL)andtheFd-domain(VH-CH1)oftheheavychainofanantibody.Duringbacterialexpression,thesetwochainsaresynthesizedseparately,andsecretedintotheperiplasmwheretheyfoldtoformheterodimers.FabexhibithigherstabilitythanscFvsPossessbetterPKandPDqualitiesthanscFvsEasiertoconvertintofull-lengthantibodiesClinicalapplications:abciximab,lucentis,cimzia.AntibodyFormatsSingledomainantibodyVHH:VHdomainofcamelidantibody,heavychainsonly,IgNAR(newantigenreceptor):sharkantibody,heavychainsonly,UniqueCDRsAffibodiesAnticalinsDARPinsAvimersAffimersMo...