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1、 AntibodyPhageDisplay MeilingXiong20180629 Contents IntroductionofAbphageDisplayTechnologyAbFormatsforPhageDisplayAbLibrariesConstructionPhageAbSelectionMethods StrategiesPhageAbScreeningApplicationsInvitroAffinityMaturationExpression PurificationofPhageAbFragments IntroductionofPhageDisplayTechnolo
2、gy TheFfbacteriophagestructure IntroductionofPhageDisplayTechnology Theschemeofphagemidvector IGregion intergenicregion usuallycontainsthepackingsequenceandreplicationoriginofminusandplusstrandsMoleculartag tofacilitatelibraryscreeningandforproteinanalysisRestrictionenzymerecognitionsites usefulforD
3、NArecombinationandgenemanipulation multiplecloningsites MCS Coatprotein PIII largerprotein lessthan5copies PVIII morethan5copies decreasedlength AmbercodonTAG supEstrains glutamicacidcodon non suppressorstrains stopcodon ProteasecleavagesitePromoterSignalpeptides phageproteintranslocation crucialfor
4、displaylevelSelectivemarker forselectionofinfectedhostcells IntroductionofPhageDisplayTechnology Nonlyticfilamentousphageisthemostoftenusedforphagedisplay primarilytheM13andFdstrains Proteinstobeselectedareinfusedtoallfivecoatproteins withpIIIandpVIIImostcommonlyused pIIIproteinisessentialforinfecti
5、onofbacteriaHelperphage wild typepIIIhelperphageandspecialhelperphageAntigenimmobilizedonmagneticbeads polystryrenesurfaces oroncolumns orisusedinsolutionasbiotinylatedantigenandlatercapturedbyimmobilizedstreptavidin AdvantagesofPhageDisplayforRecombinantAntibodySelection Moreefficientlythanthroughc
6、onventionalhybridomasystem Cheapertoproducerecombinantantibodiesusingbacteria ratherthanmammaliancellline Easiertomaintainandgrowbacterialculturesforrecombinantantibodyproduction Bypassimmunizationinantibodyselection Bypasstheuseofanimalcellsforproductionofantibodies Producingthecombinatoriallibrary
7、 ideallywith108to109members offunctionalantibodiestogeneratealargerrepertoireofantibodiesthanthoseavailablethroughconventionalhybridomatechnology Easyisolationandexpressionoftheclonedgeneinabacterialhost Excellentpotentialtofurtherimprovebindingpropertiesoftheselectedantibodybyproteinengineeringtech
8、niques Capableofgeneratingantibodiesagainstalmostanydesiredantigen includinghighlyconservedorself antigens conformationalvariants lowimmunogenicantigens andalsotoxiccomponents whichisnotpossiblebyinvivoimmunizationofanimals Anumberofstartingmaterial proteins peptides haptens celllines tissueslides o
9、rvirusparticles AntibodyFormats Themostcommonlyusedformat single chainvariablefragment scFv SimplicityofcloningprocessFastandeasylibrarygenerationAhighdisplayrate smallproteinsize 25kDa LessstablethanFabfragmentsTendtoformdimers canbereducedwithlinkermorethan20aminoacids AntibodyFormats FabThelightc
10、hain VL CL andtheFd domain VH CH1 oftheheavychainofanantibody Duringbacterialexpression thesetwochainsaresynthesizedseparately andsecretedintotheperiplasmwheretheyfoldtoformheterodimers FabexhibithigherstabilitythanscFvsPossessbetterPKandPDqualitiesthanscFvsEasiertoconvertintofull lengthantibodiesCl
11、inicalapplications abciximab lucentis cimzia AntibodyFormats SingledomainantibodyVHH VHdomainofcamelidantibody heavychainsonly IgNAR newantigenreceptor sharkantibody heavychainsonly UniqueCDRsAffibodiesAnticalinsDARPinsAvimersAffimersMonobodies AntibodyFormats MultivalentfragmentsMiniantibodiesaresc
12、FvsorFabsconnectedviaaflexiblelinkertoself associatingstructuressuchashelixbundlesorleucinezippers DiabodiesarenoncovalentdimersofscFvs whichspontaneouslyformdependingonthelinkerlengthbetweenVHandVL AnotherformofdiabodiesistwoscFvsconnectedwithashortlinker Fab AiscreatedbygeneticfusionoftheFabFdgene
13、withthealkalinephosphatase PhoA geneandcoexpressingthelightchaingene scFv FcarescFvsdimerizedbytheFcdomain Immunelibraries first immunizeananimalwithanantigenandisolatethemRNAfromBlymphocytes forimmunizedanimals orperipheralbloodBcells forimmunizeddonors ThemRNAisthenreversetranscribedintocDNA andth
14、evariableregionsofexpressedantibodiesareamplifiedviaPCRandclonedintoaphagedisplayvector Advantages MaturedinvivoImmunelibrariescanbegeneratedfromanyanimalandevenhumans mouse human chicken rabbit camel Anyspeciesthathavebeenimmunized infected orexposedtoanantigen Usefulinanalyzingnaturalhumoralrespon
15、ses forexample inpatientswithautoimmunedisease viralinfection neoplasticdiseases etc AntibodyLibraries Na venaturallibraries universalantibodylibrariesgeneratedfromB cellsofnonimmunizeddonorsandeliminatetheneedtoconstructnewlibrariesforeachantigen loweraffinitiesthanthosegeneratedduringinvivoaffinit
16、ymaturation tofindgoodantibodiesagainstdiverseantigens theselibrariesneedtobeverylarge Advantages Absolutefreedominantigenchoice includingself nonimmunogenic andtoxicAgsSeveralantibodiesselectedbyphagedisplayfromhumanna velibrarieshavealreadybeenapprovedasdrugs suchasraxibacumab ramucirumab necitumumab orbelimumab AntibodyLibraries Na veSemisyntheticlibraries Na vesemisyntheticlibrariesareusuallylibrariesthathavebeenisolatedfromnonimmunehostsandwhereoneorseveralCDRswereexchangedwithsyntheticpept
