WithineachVdomain(VLandVH)therearethreenon-consecutive complementarity-determiningregions(CDRs)loopsorhypervariable regions,thatdirectlyinteractwithanantigenForthisreason,CDRsand adjointregionscontainthemostimportantinformationregardingantigenspecificityinagivenmoleculeThisknowledgehasledtothe developmentofoneofthemostpopularmethodsofantibody humanization,CDR-grafting,whichtakesparentalcomplementarity determiningregions(CDR)intohumanframework(FR)regions
Thus,reducingtheriskofthemoleculebeingrecognizedasforeignby thepatients’immunesystemParentalantibodyspecificityandaffinityare conservedthankstothepreservationofresiduesimplicatedinantigen binding
1HomologyModelingoftheMurineVariableRegion(homology modelingmethods)
2HumanAcceptorFrameworkSelection
3CDRGraftingandExpressionofReshapedAntibodies
4BindingAnalysisandFrameworkBack-mutations
Humanizedantibodiesconstitutethemajorityoftoday’sapproved therapeuticantibodiesHumanizationisimportantforreducingthe immunogenicityofmonoclonalantibodiesderivedfromxenogeneic sources(commonlyrodent)andforimprovingtheiractivationofthe humanimmunesystemSincethedevelopmentofthehybridoma technology,alargenumberofrodentmonoclonalantibodieswith specificityforantigensoftherapeuticinteresthavebeengeneratedand characterized
1FortheexpressionofantibodiesasfullIgGs,eukaryoticvectors thatprovidethelight-andheavy-chainconstantdomainsare availablefromAERESBiomedical,London,UK(SuperVector ExpressionSystem)andLonzaBiologics,NH,USA(pCON vectors)Forexpressionassingle-chainFvfragments(scFv), commonprokaryoticvectorsthatallowperiplasmicprotein expressioncanbeused
2Inthehomologyapproach,astructuraltemplate(s)ischosen basedonsequencehomologyandlengthConflflictsbetween aminoacidside-chainsarethenresolvedbyenergy minimizationThisapproachisespeciallyusefulfortheβ-sheet frameworkandCDRloopswithknowncanonicalstructures(see Subheading11)Incontrast,theconformationalsearch approachseekstobuildCDRconformationsabinitioby generatingalargenumberoftheoreticallypossible conformationsbyvaryingthedihedralanglesofthepeptide backboneThepredictedloopconformationisobtainedafter energeticevaluationoftheconformersThisisusefulforCDRs thatdonotbelongtoanyofthecanonicalclassesandthose thatarepoorlymodeledbyhomologyalone.
3V-BASEandIMGTareonlinedatabasesfeaturinghuman immunoglobulingermlinesequences
4TheFvexpressioncassettesareinitiallysynthesizedashalflengthinsertsandsequencedbeforethetwohalvesarestitched togetherThisallowstheearlydetec-154LotionofPCRartifacts, whicharelikelyatthenumerousprimerjunctions,andavoids thesubsequentneedtosequencealargenumberofclonesfor thecorrectfull-lengthcassettesequence
5Designoverhangingprimersthatarepartiallycomplementaryto the5′-and3′-endsofthemurineVLandVHgeneforthe introductionofadditionalsequences
6TheshufflflingofthemurineandreshapedVL/VHdomainsand subsequentanalysesofthehybridconstructswillreveal whetherthereshapedantibodyretainsthebindingaffifinityand specifificityofthemurineantibodyand,ifnot,theCDRgrafting ofwhichdomain(VL,VH,orboth)hasresultedinthechangein bindingcharacteristics
