高级免疫学-抗体细胞因子

Antibody(Ab)IntroductionAntibody(Ab)

proteinproducedbyplasmacellsinresponsetoimmunizationandbindsspecificallytoparticularAg.

1854-1917Nationality:GermanyFields:PhysiologyandimmunologyKnown

forDiphtheriavaccineNobelPrizeinPhysiologyormedicine(1901)for“Antitoxinimmunity”theory

EmilvonBehring(1845-1917)1901,antitoxinsNobelPrizewinnerexotoxins1937年，Tiselius和Kabat创建了血清蛋白电泳技术，提出抗体就是

g球蛋白。Immunoglobulin1948RodneyPorter1917-1985NobelPrizein1972GeraldEdelman1929--NobelPrizein1972"fortheirdiscoveriesconcerningthechemicalstructureofantibodies"structureoftheAbHandLchainVandCRegionHingeregionDomainJchainandSPIgFragmentStructuresofAb四肽链二硫键“Y”1.HeavyChainandLightChain

heavychain(Hchain)50~75Kd450~550aaHchain(classes):

isotype:IgM,IgG,IgA,IgE,IgDIgG:IgG1~4IgA:IgA1,IgA2

StructureoftheAb1.HeavyChainandLightChain

lightchain(Lchain)

25Kd214aaclassesofLchain:

:1~42.variableregionandconstantregion

variableregion(Vregion)LightChain-VL(110aa)HeavyChain-VH(110aa)HVRorCDRFR

constantregion(Cregion)LightChain-CL(110aa)HeavyChain-CH(330-440aa):CH1,CH2,CH3,(CH4)StructuresofAb可变区和恒定区（VariableregionandConstantregion）VCdomain可变区和恒定区（VariableregionandConstantregion）高变区（hypervariableregion,HVR）/互补决定区（complementaritydeterminingregion,CDR）骨架区FrameworkregionsFR(frameworkregion)regionsbetweentheCDRsintheVregionarecalledtheFR.VLandVH:Agbindingsite铰链区（hingeregion）CH1与CH2之间（IgM和IgE无）flexibility:allowthetwoFabarmstobindepitopeJ链（Joiningchain）分泌片（secretorypiece,SP）

木瓜蛋白酶水解片段：2Fab段＋Fc段

胃蛋白酶水解片段：F(ab’)2段＋pFc’段ProteolyiticFragmentofanIgGmoleculeHeterogeneityofAb1.ClassificationofIgclassμγαεδsubclassIgG1~IgG4type

subtype12

342.DiversityofIg

inducedbyexogenousfactors3.SerotypesofIginducedbyendogenousfactorsg链e链m链d链a链轻链Lightchain：k链和l链l链：l1、2、3、4HeterogeneityofAbIgG1IgG4IgG3IgG2免疫球蛋白的血清型（一）同种型（二）同种异型（三）独特型isotypeIgCregionHumanantibodyRabbitantibodyallotype

(IgC

region)HumanantibodyHumanantibodyIdiotype(Id)(CDR)HumanantibodyHumanantibodyFunctionsofAbsFunctionsofAbsIgV

regionValency：结合抗原表位的个数。FunctionsofAbsAffinity=

attractiveandrepulsiveforcesAbAgHighAffinityAbAgLowAffinityAffinity（亲和力）StrengthofthereactionbetweenasingleantigenicdeterminantandasingleAbcombiningsiteAvidity（亲合力）TheoverallstrengthofbindingbetweenanAgwithmanydeterminantsandmultivalentAbsYKeq=104AffinityY106AvidityYYYYY1010AvidityreversalcombinationnoncovalentforcehydrogenbondelectrostaticattractionVanderWalsforces

hydrophobicbonddegreeofdissociationAg-AbaffinityEnvironmentalfactorsAsincreasingconcentrationsofAgareaddedtoaconstantamountofAb,theamountofICprecipitatedrisesandthenfalls.Theprecipitincurvegeneratedinthiswayhasthreezones.

Abexcesszone(prozone)equivalencezoneAgexcesszone(postzone)FactorsAffectingMeasurementofAg/AbReactionsAffinityAvidityAg:AbratioPhysicalformofAgAbexcessAgexcessEquivalence–Latticeformation(Visible)SmallcomplexSmallcomplexFunctionsofAbsFunctionofVregionBindingofantigensFunctionsofAbsFunctionofCregion1.activationcomplement:IgM,IgG1-3,IgA2.bindingtoFcreceptorofcells(1)opsonization:Aprocessbywhichphagocytosisisfacilitatedbythedepositionofopsonins(AborC3b)ontheantigen.

ActivatecomplementFunctionofIgOpsonizationofantibody（2）ADCC(Ab-dependentcell-mediatedcytotoxicity)AcytotoxicreactioninwhichFcreceptor-bearingkillercellsrecognizetargetcellsviaspecificantibodies.（3）IgEmediatestypeⅠhypersensitivityFunctionofimmunoglobulinsADCC

3.passagethroughtheplacenta(IgG)andmucosal(SIgA)穿过胎盘和黏膜ImmunityistransferredfrommothertofetusthroughplacentaltransferofIgG.CharacteristicandfunctionofDifferentclassofIgPage96Table5-2IgGimmunoglobulina)IgGisthemostimportantclassofIginserum-75%ofserumIghalf-lifeinserum:23daysb)Fixescomplement–IgG1-3canwhileIgG4cannotIgG3＞IgG1＞IgG2＞IgG4c)crossestheplacentaconfersahighdegreeofpassiveimmunitytothenewborn.d)BindingtocellsMacrophages,monocytes,PMNandsomelymphocytesBindingtoSPANeutralizationofToxinbyIgGneutralizationofviralactivity

IgMimmunoglobulina)Penatmerb)IgMisthefirstIgsynthesizedinlifec)IgMisagoodcomplementfixingIgd)IgMisalsoagoodagglutinatingIge)IgMismIgM(IgD)ofBcells:f)IgMisthefirstIgproduced

inprimaryimmuneresponse.IgAimmunoglobulinIgA:monomerandsecretarysIgAexistsinsecretionssuchastears,saliva,colostrum,mucusandhasimportantroleinlocal(mucosal)immunity.c)IgAcanbetransmittedfrommothertonewbornIgEimmunoglobulinIgEistheleastserumIg.b)InvolvedintypeⅠallergicreactionsc)IgEplaysaroleinparasitichelminthdiseasesIgDimmunoglobulinIgDisfoundinlowlevelsinserum;itsroleinserumuncertain.b)IgDisprimarilyfoundonBcellsurfaceswhereitfunctionsasareceptorforantigen.MembraneIgDdisappearinactvatedormemoryBcellPreparationofAbs1)Polyclonalantibody2)Monoclonalantibody3)Geneticengineeringantibodymonoclonalantibody(mAb)针对单一抗原表位的高度均一的特异性IgGeorgesKohlerandCesarMilstein,1975,discoveredmonoclonalantibody1984immunoglobulinMonoclonalantibodyproductionGeneticengineeringantibodyGeneticengineeringantibodyFabantibodyFvantibodySinglechainfragmentvariable,ScFvSingledomainantibodyMinimalrecognitionantibody,MRUbispecificantibodyGeneticengineeringantibodyRearrangementofIggenesandsynthesisofIgTheNobelPrizeinPhysiologyorMedicine1987"forhisdiscoveryofthegeneticprincipleforgenerationofantibodydiversity"SusumuTonegawa

ⅡRearrangementofIggenesandsynthesisofIgOrganizationofIggenelociThreeseparatelociencode,respectively,alltheIgheavychains,theIgκlightchain,andtheIgλlightchain.Eachlocusisonadifferentchromosome.RearrangementofVregionofIgmoleculeMechanismsofV(D)JRecombinationV(D)JRecombinase:Rag-1/Rag-2Recombinationsignalsequences(RSS)RSS=heptamer+spacer+nonamer12/23ruleLoopingoutandinversionConservedheptamer(7bp)andnonamer(9bp)sequences,separatedby12-or23-bpspacers,arelocatedadjacenttoVandJexons(forκandλloci)ortoV,D,andJexons(intheHchainlocus).TheV(D)Jrecombinaserecognizestheserecombinationsignalsequencesandbringstheexonstogether

ProcessofV(D)JrecombinationConsequencesofVDJrecombinationIggenerecombinationandexpressionAllelicexclusionandIsotypeexclusionIggenerecombinationandexpressionⅢGenerationofDiversityofIgDiversity

(repertoire)isthetotalofalltheAbspecificitiesthatanorganismiscapableofexpressing.History-Germlinetheory-SomaticmutationⅢGenerationofDiversityofIgCurrentconceptsCombinatorialdiversity

V-D-J-CorV-J-CH-LJunctionaldiversity

P-nucleotideN-regioninsertionSomatichypermutation

antigenstimulation

CombinatorialdiversityJunctionaldiversityJunctionaldiversitySomatichypermutationⅣApplicationofAbScientificresearchDiagnosisTherapyProphylaxisClinicaltherapyTheNobelPrizeinPhysiologyorMedicine1901"forhisworkonserumtherapy,especiallyitsapplicationagainstdiphtheria,bywhichhehasopenedanewroadinthedomainofmedicalscienceandtherebyplacedinthehandsofthephysicianavictoriousweaponagainstillnessanddeaths"EmilvonBehring(1845-1917)肿瘤靶向治疗的单克隆抗体商品名英文名靶点肿瘤类型赫赛汀HerceptinHER-2转移性乳腺癌阿瓦斯汀Avastin血管内皮生长因子非小细胞肺癌爱必妥ErbituxEGFR结直肠癌美罗华RituxanCD20淋巴瘤FDA批准的抗肿瘤单抗和进入临床试验的单抗Numberofnovelanticancerantibodiesateachphaseofclinicaldevelopment.CTLA-4、PD-1与配体结合可导致T细胞对肿瘤的杀伤减弱，T细胞功能耗竭

靶向负性免疫调节分子的抗体药物（靶向T细胞的负性共刺激分子）Targetimmunecheckpoint(CTLA-4，PD-1，PD-L1……)阻断共抑制分子信号能逆转T细胞的抑制状态激活抗肿瘤免疫Ipilimumab(Yervoy)CTLA-4:Down-regulatesT-cellactivationIpilimumab(Yervoy):FullyhumanmonoclonalantibodyBlocksCTLA-4receptorPotentiatesTcellactivationKorman,PeggsandAllison:Adv.InImmunol.2006;90:297-339Ipilimumab:MechanismofActionTcellTCRCTLA4APCMHCB7T-cellinhibitionTcellTCRCTLA4APCMHCB7T-cellactivationTcellTCRCTLA4APCMHCB7T-cellpotentiationIPILIMUMABblocksCTLA-4CD28CD28Anti-PD-1antibody

Nivolumab(BMS-936558)Safety&activityTopalianSLetal.NEJM2012;366:2443Docetaxelvsnivolumab:phase3Lambrolizumab(MK-3475)Anti-PD-L1antibodyBMS-936559Safety&activityBrahmerJRetal.NEJM2012;366:2455Medi-4736MPDL-3280AAnti-PD-1,PD-L12014年11月，在同一期Nature上发表了5篇文章和1篇述评，研究内容都是免疫抑制性分子的单抗(anti-PD-L1,anti-PD-1,anti-CTLA4,anti-CD40)在肿瘤免疫治疗中的作用Nivolumab和ipilimumab联合治疗黑色素瘤NEJM2013;369(2):122-33CTLA-4和PD-1单抗联合治疗1例恶性黑色素瘤NEnglJMed.2015休息一下！Cytokines，CKsCytokinesConceptCharacteristicsClassificationCytokinereceptorBiologicactivityIntroductiontocytokinesWhatarecytokines

？

Cytokinesaresmallproteinsthataresecretedbycellsandexerttheirbiologicalactivitythroughspecificcellsurfacereceptors.

IntroductiontocytokineIntroductiontocytokineDifferfromgrowthfactorsthatcytokines

involvedinhostdefensethatactonwhitebloodcells(leukocytes),

whereasgrowthfactorsactonothersomaticcelltypes.Differfromgrowthfactorsthatareproducedconstitutively,whilecytokineproductioniscarefullyregulated

IntroductiontocytokineNomenclaturemonokine,LymphokinecolonystimulatingfactorInterleukinInterferontumornecrosisfactorgrowthfactorchemokineIntroductiontoCKsGeneralpropertiesofCKs

1.Smallproteins(MW:approx.15-30KD);2.Extremelypotent,actingatpMorfM3.Theproductionistransientandtightlyregulated;4.Autocrine,paracrineorendocrine;5.Non-specificandnon-MHCrestriction；6.DiversityinactionsCytokinescanactinthreedifferentmannersAutocrine:IL-2CytokinebindstoreceptoroncellthatsecreteditParacrine:IL-12CytokinebindstoreceptorsonnearbycellsEndocrine:IL-1CytokinebindscellsindistantpartsofthebodyIntroductiontoCKs

CytokineActions

Pleiotropy:Actonmorethanonecelltype(IFN-α/β)Redundancy:Morethanonecytokinehavethesameaction(IFN-α/βandIFN-γ)IntroductiontoCKs

CytokineActions

Synergy:TwoormoreCKscooperatetoproduceaneffectthatisdifferentorgreaterthanthecombinedeffectofthetwocytokineswhenfunctioningseparately(IL-12andIL-18)Antagonism:Twoormorecytokinesworkagainsteachother(IL-4andIL-12)IntroductiontoCKs

CytokineActions

Cytokinecascade

Acytokinemayincrease(ordecrease)theproductionofanothercytokineIntroductiontocytokineTheCKsnetworkIthasbeenshownabovethattherearemanyCKswithmultiplefunctionsandapparentredundancyofactionformingacomplexcommunicationnetwork.Figure：cytokinesnetworkHowcannon-specificcytokinesactspecifically?OnlycellsexpressingreceptorsforspecificcytokinescanbeactivatedbythemManycytokineshaveveryshorthalf-livesOnlycellsincloseproximitywillbeactivatedHighconcentrationsofcytokinesareneededforactivationOnlycellsincloseproximitywillbeactivatedMayrequirecell-tocellcontactCategoriesofCKsInterleukin(IL)Interferon(IFN)Tumornecrosisfactor(TNF)Colonystimulatingfactor(CSF)ChemokineGrowthfactor(GF)

Cytokinereceptor(CKR)1.FivefamiliesIgsuperfamilyreceptorsClassIcytokinereceptorfamilyClassⅡcytokinereceptorfamilyClassⅢcytokinereceptorfamily(TNFreceptorfamily)ChemokinereceptorfamilyCKreceptorMulti-subunitreceptor

oneforcytokinebindinganotherforcytokinesignalingCommonreceptorsubunitThereissamereceptorsubunitforcytokinesignalingamongthedifferentcytokinereceptors.e.g.IL-2Rγ

CKReceptorDavidPhillipVetter(September21,1971–February22,1984)wasaboyfromShenandoah,Texas,UnitedStateswhosufferedfromararegeneticdiseasenowknownasseverecombinedimmunedeficiencysyndrome(SCIDS).SignalTransductionbycytokinereceptorsCytokinereceptorsondifferentcelltypestriggerdifferenteventsinterleukin,ILIL1～37IL-2（Tcellgrowthfactor）IL-4（Th2type）IL-6（Bcellgrowthfactor,Th2type）IL-8（chemokine）IL-10（Th2type）IL-11（stimulatorofplatelet）IL-12（Th1type）IL-2TheschematicstructureoftheIL-2receptorsubunitsIL-4IL-12interferon,IFNIFNsmediatetheearlyinnateimmuneresponse;groupstypeIIFN:IFN-αandIFN-β.Themajorsourceisleukocyte,fibroblastsandvirusinfectedcells；typeIIIFN:IFN-γ.IFN-γismainlyproducedbyactivatedTcellsandNKcells.InterferonActionViralreplicationstimulatestheinfectedhostcelltoproduceinterferon.InterferoninducesuninfectedcellstoproduceantiviralproteinsthatpreventtranslationofviralmRNAdegradeviralnucleicacidViralreplicationisblockedinuninfectedcellsTumornecrosisfactor,TNFTNF:causethenecrosisoftumorsTNF-αandTNF-β.TNF-αwasproducedbyLPS-stimulatedmononuclearphagocytesandactivtedTcells；TNF-β:alsotermedaslymphotoxin(LT)，andthemajorsourceisactivatedTcells.Colony-stimulatingfactor,CSFStimulatingthedifferentiationandexpansionofbonemarrowprogenitor;beassayedbytheirabilitytostimulatetheformationofcellclonesinculture.IncludingIL-3,CSF(G-CSF,M-CSF,GM-CSF),SCF,EPO,TPO,etc.ChemokineChemokinesarealargefamilyofstructurallyhomologousCKsthatstimulatemovementandregulatethemigrationofleukocytesfromthebloodtotissues,includingabout50differentchemokines.subfamily:CXC,CC,C,CX3C,basedonstructuralcharacteristic(cysteineresidues).ChemokineIL-8CXCneutrophilsMCP-1CCmonocyteLymphotactinClymphocyteFractalkineCX3ClymphocyteGrowthfactor(GF)Promotingtheproliferationanddifferentiationofcells;IncludingTGF-

、EGF、VEGF、FGF、NGF、PDGF,etc.BiologicactionsofCKsMediateandregulateinnateimmunityMediateandregul