04-Antibodies

Antibody(Ab)andImmunoglobulin(Ig),EmilvonBehring,1901,antitoxins,GeoregesKohlerandCesarMilstein,1984,monoclonalantibody,SusumuTonegama,1987,structureofIggene,GeraldEdelmanandRodneyPorter,1972,structureofantibody,PaulEhrlich,1908,productionofantibody,NobelPrizewinners,EmilvonBehring(1845-1917),EmilvonBehring,1901,antitoxins,Section1Introduction,Antibody(Ab)GlobulinproducedbyplasmacellsinresponsetoantigenandbindsspecificallytoparticularAg.AbisgenerallydefinedintermsoftheirspecificbindingtotheimmunizingAg,plasmacells,Bcells,Immunoglobulins(Ig)TheIgsareglobulinwhichfunctionasAbsorsimilartoAbsinchemicalstructure.,SecretedIg(sIg):Ab-mediatedhumoralimmunityMembraneIg(mIg):BCR,plasmacells,Bcells,StructuresofsecretedandmembraneIgs,AllantibodiesareimmunoglobulinsNotallofIghavethefunctionsofAb,e.g.PatientswithmultiplemyelomahaveveryhighlevelsofIg,butnotAb(toirrelevantthings),TherelationshipbetweenAbitsroleinserumremainsunclear.IgDisprimarilyfoundonnaiveBcellsurfaceswhereitfunctionsasareceptorforantigen.3.MembraneIgDdisappearinactivatedormemoryBcell,ImmatureBcell,MatureBcell,ActivatedBcell,5.IgE,IgEistheleastcommonserumIg.ItbindsverytightlytoFcreceptors(FcR)onbasophilsandmastcellsevenbeforeinteractingwithantigen.Involvedinallergicreactions(hypersensitivityI)ImportantphysiologicalfunctionsAnti-parasiticinfection:clearparasites(e.g.worms)byeosinophils.,IgE-DirectedEosinophilAttack,ImmunoglobulinIsotypesAreDistributedToDifferentPartsOfTheBodyIgMBloodIgGTissuesIgAMucosaIgE-Surfaces,Section6Productionofantibodies,1)Polyclonalantibody2)Monoclonalantibody3)Geneticengineeringantibody,1.Polyclonalantibody,Polyclonalantibodies(pAbs)areantibodiesthataresecretedbydifferentBcelllineageswithinthebody.Theyareacollectionofimmunoglobulinmoleculesthatreactagainstaspecificantigen,eachidentifyingadifferentepitope.Advantages:easily,inexpensive,quicklytoproducealargeamountofpolyclonalantibody.Disadvantages:acomplexmixtureofantibodiesdirectedagainstdifferentepitopesandthatdifferintheiraffinityfortheantigen.Instability,variation,Manyepitopes,DifferentBCR,mice,HeterogeneousPcAb,Adjuvant+,2.Monoclonalantibody,Monoclonalantibody(mAb)：anantibodyproducedbysingleBcellcloneorgeneticallyhomogeneouspopulationoffusedhybridcells,thatis,hybridoma.HybridcellsareproducedbyfusingBcellsfromtheimmunizedanimalwithmyelomacells.Unlikepolyclonalantibodies,monoclonalarehomogenouswithdefinedspecificitytooneepitope.,CesarMilsteinNobelPrizein1984,GeorgesKohlerNobelPrizein1984,MonoclonalAntibodyApplications,DiagnosticTestsDiagnosticImagingMolecularmissileincancertreatmentImmunotoxins,mouse-derivedantibodiesreducedtheireffectivenessasdiagnosticandtherapeuticagentsandraisedconcernsovertheriskoftreatment-associatedanaphylaxis“Humanization”ofthemouse-derivedMAbshasbeenthemostwidelyusedstrategytoreducetheirimmunogenicityfortherapeuticpurposesgenescodingforantibodyproductioncanbeclonedintotransfectionvectorstoproducerecombinantantibodies.,3)Geneticengineeringantibody,Frontier,3)Geneticengineeringantibody,ChimericAbMouseVgene+humanCgenehumanizedAb/CDR-graftedAbMouseCDRgene+humanframeworkregion,Frontier,Section7ThegeneticstructureofIganditsexpression,10differentconstantregions,Millionsofdifferentvariableregions,Antigens:Somanymany,ThequestionofGOD（GenerationofDiversity）,Side-chaintheoryofantibodyformation,PaulEhrlich(18541915),surfacereceptorsboundbylockAgstimulatedreceptors,TheoriesoftheGenerationofDiversity(G.O.D.),Templatemodel-Antigenservesastemplatearoundwhichantibodyisfolded.Germlinemodel-onegeneforeachantibody.Problems:needlotsofgenes,allotypes(i.e.constantregionpolymorphismsbehavelikesimpleMendeliangenes).Somaticmutationmodel-onegenethatgetsindependentlymutatedinindividualcells.Problem:mutatormustspareC-region,Theclonalselectiontheory,Eachlymphocytebearsasingletypeofreceptorwithauniquespecificity.(pre-existing)Receptoroccupationisrequiredforcellactivation.Thedifferentiatedeffectorcellsderivedfromanactivatedlymphocytewillbearreceptorsofidenticalspecificityastheparentalcell.ThoselymphocytesbearingreceptorsforselfmoleculeswillbeClonaldeletedatanearlydevelopmentstage.(tolerance),Important,Validatedbymonoclonalantibodytechnology,Conclusion,HowisthespecificityofAbgenerated？pre-existingspecificityOneBcellclone,oneAb,HowisthediversityofAbgenerated？Onegene,oneAbOnegene,multipleAbs,Tobecontinued,Arelativelysmallnumberofgenesegmentscanbecombinedindifferentwaystogenerateextensivediversity.,1.Organizationofimmunoglobulingerm-linegenesegments,Arelativelysmallnumberofgenesegmentscanbecombinedindifferentwaystogenerateextensivediversity.,Humanantibodygenes,Hchain:encodedbyV、D、J、Cgenesegments。Cgene：5-C-C-C3-C1-C1-C2-C4-C-C2-3L(/)chain:encodedbyV、J、Cgenesegments。,2.ImmunoglobulingenerearrangementDuringearlyB-celldifferentiationinthebonemarrow(BM),thevariable(V),diversity(D),andjoining(J)genesegmentsoftheimmunoglobulin(Ig)genesarerearrangedinanorderedfashiontogeneratetheprimaryIg,Important,Aim:V(D)Jrecombinationachievestheassemblyofnewexonsthatencodetheportionoftheantigenreceptorthatbindsantigen.Process:Functionalheavyandlightchaingenesareassembledfromseparatepiecesinthegenome.LightchaingenesareassembledfromoneofseveralVregiongenesandoneofseveralJsegments.HeavychaingenesareassembledfromoneofseveralVregiongenes,oneofseveralDsegments,andoneofseveralJsegments.TheJsegmentsareseparatedfromtheconstantregioncodingregionsbyanintron.Components:Recombinationsignalsequences(RSS)marktheendsofVgenes,DsegmentsandJsegmentsthataretargetsforrearrangement.Recombinationreactionsonlyoccuratpairsofsignalsequencesofdifferenttypes.V(D)JrecombinationisdonebytheRAG1andRAG2proteins.TheRAGcomplex(consistingofRAG1andRAG2,alongwithHMGB1)isonlyexpressedinpre-Bcellsandpre-Tcells.Co-transfectionofRAG1andRAG2genesintofibroblastsresultsinimmunoglobulingenerearrangement.Italsorepresentsthe“Achillesheal”oftheB-celllineage,occasionallyleadingtomalignanttransformationofthesecells,Figure2-18,V(D)JrecombinationisinitiatedbytheRecombinaseActivatorGeneProducts,RAG1andRAG2(foundonlyindevelopinglymphocytes),Unrearrangmentgeneswereexcisedbyloop-outfashion,Figure2-19part1of3,Imprecisionofcodingjoinspromotesgenerationofdiversity,Figure2-19part2of3,TdT=Terminaldeoxynucleotidyltransferase(Lymphocytespecific)canaddnucleotidesrandomlytothesinglestrandedendsexposedbynickingofthehairpin,Untemplated!,Figure2-19part3of3,JunctionalDiversity,Nucleotidescanbeaddedtothejunctionbyterminaldeoxynucleotidyl-transferase(Tdt),GenerationofdiversityofIg,NaveBcells(pre-antigenselection):DiversityofVDJgenesegmentsingermlineDNA.RandomrearrangmentofVDJgenes.PairingdiversitybetweenHandlightchain.JunctionaldiversityAntigen-experiencedBcells(post-antigenstimulation)Somatichypermutation,Important,GerminalCenter,Somatichypermutation,Followingantigenactivation,thevariableregionsofimmunoglobulinheavyandlightchainsarefurtherdiversifiedbysomatichypermutation.SomatichypermutationinvolvestheintroductionofnontemplatedpointmutationsintoVregionsofrapidlyproliferatingB-cellsinthegerminalcentersofLymphoidfollicles.Antigen-drivensomatichypermutationofvariableimmunoglobulingenescanresultinanincreaseinbindingaffinityoftheB-cellreceptorforitscognateligand.,AsB-cellswithhigheraffinityimmunoglobulinscanmoresuccessfullycompeteforlimitedamountsofantigenpresent,anincreaseintheaverageaffinityoftheantibodiesproducedduringanimmuneresponseisobserved.Thisincreaseintheaverageaffinityofimmunoglobulinsisknownasaffinitymaturation.,Somatichypermutation,ThepotentialdiversitygeneratedbyV(D)Jrecombinationisvirtuallyinfinite.,Totallymphocytesinahuman1012,Genomesize3X109bp,thesolerecipientoftheNobelPrizeforPhysiologyorMedicinein1987,forhisdiscoveryofthegeneticmechanismthatproducesantibodydiversity,SusumuTonegawa,3.allelicexclusionandisotypeexclusion,allelicexclusion:onlyonealleleof