基因工程抗体及其发展前景PPT课件

.,1,主要内容,第一节:基因工程抗体的基本原理：第二节:人源化抗体制备的主要方法：第三节:抗体靶向治疗的新的思考：第四节:工程抗体的未来发展与展望：,.,2,第一节：基因工程抗体的基本原理(antibodyengineering）,.,3,Antibodyhasahigherspecificityandaffinitytobindtotargetprotein.,药物(drug)：是一类分子（化学合成物，蛋白质，核酸等）可以干扰人体内细胞生命过程中一些重要的生理和病理活动或通路。,.,4,基因抗体：通过基因工程手段，保留抗体重轻链可变区中主要与抗原结合的CDR功能区,而形成的各种功能抗体,Complementary-determiningregions:CDR,.,5,基因工程抗体的分类,.,6,基因工程抗体的分类,2.小分子抗体（1）Fab片段（2）单链抗体（singlechainantibody，scFv）、双链抗体、三链抗体（3）微型抗体（minibody，两个scFv与抗体CH3区连接）（4）双特异性抗体（diabody）（5）其他形式抗体（细胞内抗体、催化抗体、免疫脂质体、最小结合单位等）,.,7,抗体药物在应用中存在的问题：,一般必须用小鼠骨髓瘤制备单抗，故所得鼠源性单抗，必须人源化，在临床上可减少异源性蛋白所引起的过敏反应和增加疗效。鼠源抗体人源化后，抗体效价明显降低，导致临床疗效降低。临床治疗需要大量的抗体(克级），故需要生物反应器制备抗体。由于抗体的产量和质量受到限制，而影响疗效。,问题:工程细胞系,大规模生产工艺技术,.,8,基因工程抗体的优点和缺点,优点：不受动物品系（species)和抗体类型（isotype)的限制。利用嵌合抗体，使鼠源抗体人源化，减少潜在的抗原表位，增强抗体的疗效。全人源化抗体，可以降低抗体的异源性和免疫源性，最大化提升抗体的的疗效。缺点：抗体的亲和力减弱，与完整抗体结构相比，功能明显降低。,.,9,目的：减小鼠源性成份，降低HAMS反应（humanagainstmousesyndrome)。易于大规模生产和应用于临床。保留抗体的抗原结合能力。基本原理：借助基因工程手段，将编码Ab的重轻链可变区基因重组到真核表达载体上并进行表达。,.,10,基因工程抗体的表达,原核细胞和酵母可以用于表达小分子抗体和抗体片段大部分完整抗体和双链抗体、微型抗体等需要在CHO等哺乳动物细胞中表达利用完整的动植物体通过转基因的方法表达外源蛋白：如利用转基因烟草生产抗狂犬病毒抗体,.,11,抗体工程的发展历程,抗体工程就是指利用分子生物学、基因工程等手段对抗体进行各种不同的改造并在原核、真核细胞中表达制备的工程技术。,马血清,破伤风,狂犬病,.,12,TheEvolutionofTherapeuticAntibodies,Murine,FullyHuman,LonbergN.NatBiotechnol.2005;23:1117-1125.TernantD,etal.ExpertOpinBiolTher.2005;5(Suppl1):S37-S47.YangX-D,etal.CritRevOncolHematol.2001;38:17-23.WeinerLM.JImmunother.2006;29:1-9.,.,嵌合抗体,人源化抗体,全人抗体,.,13,.,14,第二节：基因工程抗体制备的主要方法：,.,15,1.人鼠嵌合抗体（ChimericAntibodies）,原理：利用基因重组技术，把鼠抗体的重轻链可变区部分与人抗体重轻链恒定区的进行重组，减少鼠源结构，增加人源结构，而保持抗体与原抗原的特异性结合。缺点：鼠抗体部分亦能作为一种异种抗原，多次反复使用在人体产生抗体及过敏反应（HAMS反应，humanagainstmousesyndrome)。嵌合抗体可保持特异性结合和外源性抗原降低，但亲和力明显下降。,.,16,Chimericantibodies,1。获得鼠单抗重轻链可变区的基因片段。,2.基因片段插入含有人IgG重轻链恒定区的表达载体。,.,17,人鼠嵌合抗体的真核表达在CHO细胞（共转染模式和单载体转染模式）,.,18,Until2003,7，8437peoplehavebeeninfectedwithSARSoverthe32countries,inwhich813patientswerediedformdisease.Thediseaseincidenceisabout10.5%.Therearemanyunresolvedquestionsaboutdiseasepathogenesis,treatmentanddiagnosis.,.,19,2.humanizedantibodies,Humanizationorreshapingofmurineantibodiesisanattempttotransferthefullantigenspecificityandbindingavidityofmurinemonoclonalantibodiestoahumanantibodybygraftingthemurinecomplementaritydeterminingregions(CDRs)ontoahumanvariableregionframework。,保留鼠CDR的人源化Ab,65to90%,.,20,Mouse,Human,Humanised,hypervariable,framework,.,21,humanization-CDRgrafting,CDRresiduesfromvariableregionofamousemAbaretransferredtohumanantibodyframeworksthathavehighsequencehomologywiththemousecounterparts,.,22,HumanizedAntibodies,AllowsspecificityAllowseffectorfunctionsLessimmunogenic,.,23,humanization-advantage,MurinemAbshavebeenextensivelycharacterizedandhavedesirableantiviralpropertiesand/orsuperiorfullyhumanmAbsarehardtoobtain.Thisviableapproachistheclassicandsimpleststrategyofhumanizationamongmanyotheravailablestrategies,suchasgraftingspecificitydeterminingresidues17.ThefactthatnearlyhalfofallUSFoodandDrugAdministration(FDA)-approvedtherapeuticmAbsarehumanizedantibodiestestifiestotheirsafetyandtolerancebyhumans.,.,24,3。FullyhumanmAbs,1）。transgenicmice2）。Humanantibody-displaylibraries3）。TomakeHumanmAbfromhumanbeing,全人抗体100%,.,25,1）Transgenicmice,首先把小鼠编码Ig重轻链的基因剔除。制备表达人的Ig重轻链的转基因小鼠。上二种小鼠回交，获得只表达人Ig重轻链的基因的小鼠。当用抗原免疫后，小鼠可产生完全人源抗体。,.,26,CreationofFullyHumanmAbsFromTransgenicMice,Replacemurineimmunoglobulin(Ig)GgeneswithhumanIgGgenes,WeinerLM.JImmunother.2006;29:1-9.YangX-D,etal.CritRevOncolHematol.2001;38:17-23.,Mousestrainincapableofproducingmouseantibodies,HumanIgloci,Breeding,Breeding,Mouseembryonicstemcellscontaininghumanantibodygenes,Mousestrainproducinghumanandmouseantibodies,Mouseembryonicstemcellswithinactivatedmouseheavyandlightchainloci,MouseIgloci,Mousestrainproducingonlyhumanantibodies,.,27,Transgenicmouse-advantage,HavearepertoireoffullyhumanIgG1gaveresearchersanalternativeroutetohumanmAbsthroughactiveimmunization,withouttheprotocolrestrictionsassociatedwithhumanstudies.,.,28,Transgenicmouse-drawbacks,AstheimmuneresponseintransgenicmiceissometimeslessrobustthaninstrainsthatareusedtogeneratemousemAbs,moreimmunizationsorantibodyscreensareoftenrequired.Thesetransgenicmicecannotexactlyreproducethehumanantibodyresponsebecauseofrepertoiredifferencesandbecausethegeneticbackgroundofthemurinehostaffectsantigenprocessing,B-cellontologyandregulation(e.g.,tolerancemechanisms).Atpresent,thesetransgenicmiceareprotectedintellectualpropertyandthereforenotcommonlyavailable,.,29,2）噬菌体抗体库技术(Phageantibodylibrarytechnique),把人的Ig重轻链可变区基因片段展示在l噬菌体表面,组成抗体库。通过噬菌体把抗体的表型和基因型相偶联，易进行分子克隆和基因操作。抗体库的来源影响筛选结果（免疫和正常人）。高通量筛选与抗原结合的抗体，但亲和力低。,.,30,scFv-single-chainvariablefragments,.,31,Displaytechnologies-strategy,.,32,PhageDisplayLibrary-Antibodylibrary,-fromImmunologyToday(2000),21,371-378,.,33,FlowchartofPhageDisplayApplication,.,34,StartfromhumangenesNewspecificities?Lessimmunogenic?CanscreenalargerrepertoireLossofcombinatorialspecificitiesBypassimmunisationnaivelibraryLowaffinityAffinitymaturationinvitroExtrasteps,moreimmunogenic?EconomicalproductioninbacteriaOnlyfragments,PhageDisplay-advantages,.,35,PhageDisplay-drawback,UnnaturalpairingsoftheantibodyheavyandlightchaincomponentsThemethodofpresentationofantigenasbaitforpanningmayaffectthenumberanddistributionofavailableneutralizingepitopes.,.,36,Naveorimmunized,Keypoint,FunctionalAb,.,37,3）用人的骨髓瘤细胞直接制备全人抗体,关键点：建立好的人骨髓瘤细胞。稳定性高和融合率高。,(Humanantibodies2002,11:85-96),.,38,Mousemyeloma,Humanmyeloma,DoubleselectingmarkersG418andHAT,HumanLNBcells,.,39,Breastcancertissue,Breastcancercells,WeworkonHCV/E2mAbandRabbisvirusSproteinbyusingFMP2cells.,.,40,4.B细胞永生化技术：用EB病毒将人淋巴细胞永生化可产生分泌抗体的B细胞克隆。这一技术较为成熟，但是存在抗体分泌不稳定的缺点，限制了其应用。或直接分离分泌抗体的B细胞,用PCR获得重轻连,构建全人抗体.,.,41,Bcellimmortalization:EBVTransformation,Epstein-BarrvirusWhenEBVinfectsB-lymphocytesinvitro,lymphoblastoidcelllineseventuallyemergethatarecapableofindefinitegrowth.Thegrowthtransformationofthesecelllinesistheconsequenceofviralproteinexpression.,.,42,Bcellimmortalization,MemoryB-cellsareobtainedfromthebloodofanindividualrecoveringfromavirusinfection.Therebyobviatingtheneedforafusionsteptogeneratehybridcells,asinthehybridomatechnique.,.,43,MemoryB-cellimmortalization,MemoryBcellsisolatedfromPBMC,ImmortalizedbyEBVinthepresenceofaCpGoligodeoxynucleotideandirradiatedallogeneicPBMCs,.,44,Relatedpaper,MemoryB-cellsisolatedfromthebloodofindividualswhohaverecoveredfromSARS-CoVandH5N1avianinfluenzainfectionshavebeenimmortalizedusingthismethodtoproducenAbsagainsttheseviruses.Traggiai,E.etal.AnefficientmethodtomakehumanmonoclonalantibodiesfrommemoryBcells:potentneutralizationofSARScoronavirus.Nat.Med.10,871875(2004).Simmons,C.P.etal.ProphylacticandtherapeuticefficacyofhumanmonoclonalantibodiesagainstH5N1influenza.PLoSMed.4,178(2007).,.,45,SingleBcellRT-PCR,NatureBiotechnology,2008,26:886-887.,.,46,thestrategyofsinglecellRT-PCR,.,47,Thetimeframe-onemonth,.,48,各种全人抗体生产技术比较,.,49,Sourceofantibodiesonthemarket(accordingtoa“marketanalyst”),.,50,第三节：抗体靶向治疗的新思考,问题1：在临床上抗体用量大（几百毫克到克级），价格昂贵。在不发达国家难于承受。问题2：由于专利限制，中国难于进行metoo药仿制。解决方案：能否利用中和性抗体的靶向作用，确定抗体作用的靶点位子，用小分子替代，设计新药？,.,51,SinceaneutralizedmAbisvaluableindiseasetreatment,thetechniquesusedhereallowustoidentifycorefunctionalregionsofgivenreceptor/ligand.therefore,thereisapotentialtodesignrationalmimickingmoleculesbasedontheidentifiedregion.DefiningthefunctionalregionofligandorreceptorrecognizedbyneutralizingmAbwillbeessentialinidentifyingnoveldrugtargets.,Prospective,展望,.,52,抗体药物发展现状,FDA已批准上市的抗体药物SFDA(中国)已批准上市及临床研究的的抗体药物,.,53,.,54,.,55,第四节：工程抗体的未来发展与展望,1.单克隆抗体的市场需求,.,56,对20042011年全球治疗单抗市场的预测及分析,.,57,2.抗体药物的应用领域,抗器官移植排斥反应；肿瘤导向治疗；哮喘、银屑病、类风湿性关节炎、红斑狼疮、急性心梗、脓毒症、多发性硬化症及其他自身免疫性疾病；心脑血管疾病；感染性疾病；其他,.,58,四种抗体的临床应用概况,.,59,.,60,.,61,.,62,.,63,小分子抗体,分子量小，穿透性强，抗原性弱，表达容易，易于进行基因工程改造，可