Display of full length IgG antibodies on the surface of E. coli for the construction of antibody libraries.

Dilkaute C, Weckenbrock W, Jose J

Abstract

Thenumberofantibodiesusedfortherapeuticpurposeisincreasingcontinuously.Forthediscoveryofnewantibodiesdirectedagainstacertainantigen,inmostcases,phagedisplay1isused.Nevertheless,therearesomedisadvantagesusingthismethod,suchasthepossiblediscriminationofthemostpotentbindersduringthebiopanningprocessduetomildelutionconditions.Furthermore,phagedisplayisnotcompatiblewithflowcytometryandproteinsdisplayedarelimitedinsize.2TocircumventthesedrawbacksantibodiesweredisplayedonE.coliusingtheAIDA-Iautotransporter.3Thepresentationofantibodiesandinparticularantibodylibrariesenablesthescreeningfornewvariantsagainstpre-givenepitopesusingflowcytometrywithoutlosingthehighlypotentbinders.Furthermore,thedisplayedproteinsarenotrestrictedinsize.Thescreeningcanoccurdirectlywithfulllengthantibodies,whicharebeneficialfortherapeuticapplicationsinmostcases.Forproofofprinciple,theantibodyT84.664whichisdirectedagainstthecarcinoembryonicantigen(CEA)wasinvestigated.TheautotransporterfusionproteinsofitslightanditsheavychainwereproducedsimultaneouslyinasingleE.colicell.Toprovesurfaceexposureaproteaseaccessibilitytestwasperformed.Assemblyoftheheavyandthelightchainwasshownviaco-immunoprecipitationofbothchainsandthefunctionalityoftheantibodywasconfirmedwithaflowcytometrybasedantigenbindingassay.TheresultsconfirmedthepresenceofheavyandlightchainsatthebacterialsurfaceaswellastheirinteractiontoformafunctionalfulllengthIgGantibodybindingtheantigenCEA.ThedescribedtechniqueofdisplayingantibodiesonE.colicellswasusedtodisplayanantibodylibraryatthebacterialsurface.Therefore,thecomplementaritydeterminingregions3(CDR3)oftheheavyandthelightchainwererandomizedseparatelyusingrandomizedoligonucleotidesinalinearamplificationreaction.Afterco-expressionofthesemutatedantibodychains,theresultingcombinatoriallibrarycanbeusedforscreeningwithnewantigensfromrelevanttargetse.g.fromcancer.