肿瘤与免疫-肿瘤免疫治疗课件

肿瘤免疫治疗

Tumorimmunotherapy概述主动免疫疗法被动免疫疗法

PaulEhrlich’smagicbulletconcept:100yearsofprogress,NatureReviews/CancerVol.8,2008概述NATUREREVIEW/CANCER,2008,Vol.8:299-307

肿瘤免疫治疗分为主动免疫疗法和被动免疫疗法两大类。前者着重激发机体抗肿瘤免疫应答能力；后者向宿主转移有抗肿瘤活性的治疗因子或细胞，抑制肿瘤生长。

肿瘤免疫疗法的分类及常用的生物制剂

分类 治疗因子或细胞

主动免疫疗法

非特异性

BCG、CpGODN、HSP、IFN-等

特异性

减毒或灭活的瘤苗、修饰的瘤苗、

肿瘤抗原肽疫苗和基因重组产物

被动免疫疗法

非特异性

细胞因子诱导的杀伤细胞（CIK）

淋巴因子激活的杀伤细胞（LAK） DC介导的免疫治疗

特异性

肿瘤浸润淋巴细胞（TIL)

抗体（Antibody）

免疫偶联物（Immuno-conjugates）

嵌合抗原受体（CAT）

BCG:BacilleCalmette-Guerin,CpGODN:CpGOligodeoxynucleotide,HSP:heatshockprotein,CIK:cytokineinducingkillercell,LAK:lymphokineactivatedkillercell,TIL:tumor-infiltrationlymphocytes,CAT：chimericantigenreceptorApprovedimmunetherapiesforcancerAntibodytherapyofcancer.NATUREREVIEWS.CANCERVOLUME12APRIL2012ADCC,antibody-dependentcellularcytotoxicity;CDC,complement-dependentcytotoxicity;CLL,chroniclymphocyticleukaemia;CTLA4,cytotoxicTlymphocyte-associatedantigen4;EGFR,epidermalgrowthfactorreceptor;FDA,USFoodandDrugAdministration;IgG,immunoglobulinG;INFa;interferon-a;NHL,non-Hodgkin’slymphoma;NSCLC,non-small-celllungcancer;SCCHN,squamouscellcarcinomaoftheheadandneck;VEGF,vascularendothelialgrowthfactor.*BasedoninformationfromtheEuropeanMedicinesAgency.‡NotrecommendedforpatientswithcolorectalcancerwhosetumoursexpressmutatedKRAS.一、非特异性主动免疫疗法二、特异性主动免疫疗法主动免疫治疗

(ActiveImmunotherapy)

非特异性地激发机体的免疫系统，增强抗肿瘤免疫应答能力，而达到杀伤肿瘤细胞。1.免疫因子

①胸腺素(Thymosin)，转移因子(Transterfactor)免疫核糖核酸(ImmuneRNA)②细胞因子:IFN-α,IFN-β,IFN-γ,IL-2,TNF-α等2.微生物制剂

卡介苗(BCG)，CpG寡聚脱氧核苷酸(CpGODN)3.化学合成药物

左旋咪唑(Levamisole，LMS)

4.中药制剂(滋阴、补气、补血)

人参、黄芪等(主要为多糖成分)

一、非特异性主动免疫疗法

卡介苗制剂

牛型结核杆菌或其细胞壁成分或细胞骨架成分，BCG中的有效成分是胞壁酰二肽(muramyldipeptide，MDP)。作用机制

直接活化Mφ，刺激Mφ

表达IL-6、IL-1,增强IFN-

对Mφ的刺激作用。

刺激特异性TDTH的产生，间接活化Mφ。

刺激NK细胞增殖。

非特异性刺激单核巨噬细胞。

促进IL-2和IL-4对B细胞的作用。临床应用

黑色素瘤：瘤灶内直接注射

浅表性膀胱癌：膀胱内滴注卡介苗的临床应用（黑色素瘤）

皮肤转移灶内BCG直接注射可使60%左右接受注射的病灶消退，而且有15%未接受注射的病灶也消退，提示局部注射可导致全身性抗肿瘤免疫的建立。总共包括269名患者的16项使用这一方法的研究都获得不同程度的疗效，完全缓解率最高达90%(范围7-90%)，部分缓解率范围为5-50%。在一项结合放疗的试验中，74%患者获得完全缓解，另外5%患者获得部分缓解。

BCG瘤内注射疗法可提高患者生存率。不用BCG治疗的皮肤癌复发患者，存活时间的中值为13.3月，而接受BCG瘤内注射患者的5年生存率达27%。有相当一部分原发性黑色素瘤患者经皮内注射BCG治疗后，长期存活。卡介苗的临床应用（浅表性膀胱癌）BCG膀胱内滴注可以消除肉眼可见的浅表性膀胱癌，并能预防复发。膀胱内注射BCG用于预防膀胱癌复发，可以显著延迟疾病的进展，延长保留膀胱的时间，提高总存活率。5项追踪期范围为12-60月的随机研究发现，具有高度复发危险的患者，用膀胱内注射BCG治疗者，70%保持无瘤；而用反复尿道内切除者，仅有31%的患者保持无瘤。报道实例CpG寡聚脱氧核苷酸ActivationofinnateandadaptiveimmunitybyTLR9activation.Amonghumanimmunecells,onlyBcellsandpDCsconstitutivelyexpressTLR9.ThesecellsendocytoseDNAintoanendosomalcompartmentwhereitbindstoTLR9,formingasignalingcomplex.IftheDNAcontainsunmethylatedCpGmotifs,TLR9isstimulated,andthecellbecomesactivated.InpDCs,thisresultsintypeIIFNsecretion,whichactivatesNKcells,monocytes,andotherAPCs,andinthepDCmaturationintoamoreeffectiveAPCabletoactivatenaiveTcells.Opposingtheseimmuneboostingeffects,pDCsactivatedthroughTLR9alsomediateimmune-suppressiveeffectsthroughcounterregulatoryfactorssuchasindoleamine2,3-dioxygenase(35,36)andthegenerationofTregs.InBcells,TLR9stimulationresultsinthesecretionofproinflammatorycytokines,suchasIL-6,andinthereleaseofimmuneregulatorycytokinesthatmightlimittheintensityoftheinflammatoryresponse,suchasIL-10.TLR9activationofBcellsconfersagreatlyincreasedsensitivitytoantigenstimulationandenhancestheirdifferentiationintoantibody-secretingplasmacells.Onbalance,theseimmuneeffectsofCpGDNAgenerallypromotestrongTh1CD4+andCD8+Tcellresponses.However,theconcurrentactivationofcounterregulatorypathwayssuchastheinductionofTregslimitTLR9-inducedimmuneactivation,offeringapotentialforenhancingthetherapeuticefficacyofTLR9agonistsbycoadministrationofantagonistsofoneormoreoftheseinhibitorypathways.细胞因子Functionofcytokinesinhostdefense①上调免疫细胞的表面分子和受体的表达；②促进DC细胞的成熟,增强T细胞的增殖、分化和CTL的活

化和效应功能，刺激B细胞产生抗体，提高NK细胞活性，激发巨噬细胞等产生抗肿瘤免疫应答；③促进免疫效应细胞释放淋巴毒素和效应分子杀伤肿瘤；④促进肿瘤细胞表达MHC分子，增强肿瘤细胞的免疫原性和对效应细胞的敏感性；⑤某些细胞因子具有直接破坏肿瘤细胞和促使其发生凋亡的作用，如TNF。细胞因子杀伤肿瘤细胞的效应机制二、特异性主动免疫疗法1.肿瘤疫苗2.抗独特型抗体作为疫苗

肿瘤疫苗使用肿瘤疫苗的原理肿瘤疫苗分类肿瘤疫苗治疗肿瘤的可能影响因素使用肿瘤疫苗的原理肿瘤细胞表达肿瘤抗原并被机体免疫系统所识别；肿瘤疫苗刺激主动特异性抗肿瘤应答免疫，激发或加强宿主免疫以消灭肿瘤；肿瘤患者中许多已知的和未知的因素（肿瘤免疫原性）造成了免疫应答的失败。肿瘤疫苗分类＊根据应用目的分类①预防性肿瘤疫苗②治疗性肿瘤疫苗＊根据肿瘤疫苗中肿瘤抗原的存在形式分类①细胞疫苗灭活肿瘤细胞、DC、DC/肿瘤融合细胞②肿瘤抗原疫苗

肿瘤细胞裂解物、肿瘤抗原、抗原肽③基因工程疫苗

肿瘤抗原、细胞因子或协同刺激分子等

(基因重组产物)

基因转染DC、肿瘤细胞等④DNA疫苗肿瘤抗原基因＋质粒肿瘤细胞疫苗DC疫苗DC/肿瘤融合细胞①细胞疫苗

肿瘤细胞疫苗DC疫苗特征制备和应用DC/肿瘤融合细胞

适用于大多数肿瘤抗原还未鉴定的肿瘤。肿瘤抗原的来源和应用

来源 举例癌基因产物ras12密码子突变:胰腺癌

BCR/abl重排产物:CML静止基因产物 MAGE家族:黑色素瘤，乳腺癌病毒基因产物 EBV:Burkett淋巴瘤，鼻咽癌

HPV:宫颈癌

HBV:肝细胞癌组织特异性蛋白(分化抗原)酪氨酸酶:黑色素瘤突变的抑癌基因产物P53:多种肿瘤抗独特型抗体(Anti-Id-Ab)TCRId:T细胞淋巴瘤②肿瘤抗原疫苗mRNAMAGE-1蛋白309氨基酸HLA-Cw16HLA-A1161169EADPTGHSYSAYGEPRKL

开放阅读框230238MAGE-1基因X染色体q28区

外显子3

外显子2

外显子1MAGE-1.Cw16肽段

MAGE1基因定位于X染色体q28区，mRNA转录表达的MAGE-1蛋白为309氨基酸，由HLA-A1和HLA-Cw16提呈的抗原肽位于161～169和230～238区域氨基酸序列。MAGE-1基因、编码蛋白和抗原肽黑色素瘤特异性CTL识别的黑色素细胞分化抗原肽分化抗原 抗原肽结构 肽位置 递呈分子酪氨酸酶

MLLAVLYCL

1-9

HLA-A2

YMNGTMSQV

369-377

HLA-A2

AFLPWHRLF(L)

－

HLA-A24

SEIWRDIDF

192-200

HLA-B44Pmel17/gp100

KTWGQYWQV

154-162

HLA-A2

ITDQVQGSV

209-217

HLA-A2

YLEPGPVTA

280-288

HLA-A2

LLGDTATLRL

457-466

HLA-A2

VLYRYGSFSV

476-485

HLA-A2Melan-AMART-1

(E)AAGIGILTV

26(7)-35

HLA-A2

ILTVILGVL

32-40

HLA-A2gp75TRP1

－

－

HLA-A31制备和应用Aneffectivevaccineagainsthumanpapillomavirus(HPV)inducesantibodiesthatprotectagainstHPVinfection.Serotype16ofHPV(HPV-16)ishighlyassociatedwiththedevelopmentofcervicalcancer.Inaclinicaltrial,755healthyuninfectedwomenwereimmunizedwithavaccinegeneratedfromhighlypurifiednoninfectious'virus-likeparticles'(VLP)consistingofthecapsidproteinL1ofHPV-16andformulatedwithanalumadjuvant(inthiscasealuminumhydroxyphosphatesulfate).Incomparisonwiththeverylowtitersofantibodyinplacebo-treateduninfectedwomen(greenline),orwomenpreviouslyinfectedwithHPVthatreceivedplacebo(blueline),thewomentreatedwiththevirus-likeparticlevaccine(redline)developedhightitersofantibodyagainsttheL1capsidprotein.NoneoftheseimmunizedwomensubsequentlybecameinfectedbyHPV-16.Ananti-HPVvaccinemarketedasGardasilisnowavailableandrecommendedforuseingirlsandyoungwomenasaprotectionfromcervicalcancercausedbyHPVserotypes6,11,16,and18.PLGbased3Dpolymerscaffoldvaccinetorecruitandprogramimmunecellstogenerateantitumorimmunity.(a)Overallschematicofthevaccine.1.SEMimageofaPLG3Dscaffold;scalebar:1000mm.(b)NumberofCD11c+DCsisolatedfromthescaffoldonday14postimplantationinresponseto0,400ng,3000ngand7000ngofGM-CSF.(c)ThenumberofCD11c+CCR7+DCsisolatedfromthescaffoldloadedwithPEI-ODNcontrol,10mgPEI-CpG-ODN,400and3000ngGM-CSFand400and3000ngGM-CSFincombinationwith10mgPEI-CpG-ODNatday7afterimplantation.(d)Acomparisonofthesurvivalofmicefollowingprophylactic(left)andtherapeutic(right)vaccination.Intheprophylacticvaccination,micewerevaccinatedwithblankPLGscaffolds(Blank),antigen+100mgCpG-ODN(Lys+100CpG),antigen+3000ngGM-CSF+10mgCpG-ODN(Lys+3000GM+10CpG),antigen+3000ngGM-CSF+100mgCpG-ODN(Lys+3000GM+100CpG),orirradiated,GM-CSF-transducedB16-F10cells(cellbased).Micewerechallenged(day0ongraphs)with105B16-F10melanomatumorcellsandmonitoredfortheonsetoftumoroccurrence.Inthetherapeuticvaccination,micewereinoculatedwith5X105B16-F10cellsandallowedtodevelopfor9daysandtreatedwithblankPLGmatrices(Blank),PLGmatricesloadedwith3000ngofGM-CSFand100mgofCpG-ODN(GM+CpG).Micewerealsotreatedonce(Vax,1;atday9),ortwice(Vax,2;atdays9and19)withPLGmatricesincorporatingGM-CSF,CpG-ODN,andtumorlysates(Vax).Micewerealsotreatedwith5X105

irradiated,GM-CSF-transducedB16-F10cells.Panels(c)and(dleft)reproducedwithpermission[19],Copyright2009,NaturePublishingGroup.Panels(b)and(dright)reproducedwithpermission[20],Copyright2009,TheAmericanAssociationfortheAdvancementofScience.Materialsbasedtumorimmunotherapyvaccines.CurrentOpinioninImmunology2013,25:238–245③基因工程疫苗修饰的肿瘤疫苗协同刺激分子、细胞因子基因修饰的肿瘤细胞重组病毒疫苗用已知肽的cDNA序列与灭活病毒重组产生的疫苗，可同时转入MHC、B7等基因。Enhancementoftumorcellimmunogenicitybytransfectionofcostimulatorandcytokinegenes.TumorcellsthatdonotadequatelystimulateTcellsontransplantationintoananimalwillnotberejectedandwillthereforegrowintotumors.Vaccinationwithtumorcellstransfectedwithgenesencodingcostimulatorsorcytokines,suchasIL-2,canleadtoenhancedactivationofTcells.Thisapproachofusingtransfectedtumorcellsasvaccineshasworkedinmousemodels,butclinicaltrialshavenotyetbeensuccessful.Tumorvaccines.Twotypesoftumorvaccinesthathaveshownefficacyinclinicaltrialsandanimalmodelsareillustrated.Autologousdendriticcellsarepreparedfrompatients’ownperipheralbloodcells.Thedendriticcellsareeitherpulsedwithrecombinantproteinortransfectedwithageneconstructthatexpressestheprotein.Theconstructmayalsoexpresscostimulatorymolecules(notshown).UseofDNAvaccinesraisesbothhumoralandcellularimmunity④DNA疫苗

肿瘤DNA疫苗由肿瘤抗原基因与质粒重组后形成，其中肿瘤抗原基因随治疗的肿瘤的类型及选用的肿瘤抗原不同而不同，质粒则是作为肿瘤抗原基因的载体。肿瘤DNA疫苗中常用的质粒有pSV2、pRSV、pcDNA3.1和pC1等，这些载体一般都设计有真核基因表达调控序列（如增强子和启动子等）、供目的基因插入的多克隆位点、转录终止序列以及可使质粒在大肠杆菌中保持并多拷贝复制的序列ColEI等。

肿瘤DNA疫苗的免疫接种有多种途径，包括直接注射、基因枪免疫和电穿孔免疫等。不同免疫途径的免疫机制不同，所诱导的免疫保护力强弱和维持时间也不尽相同。肌肉注射是最早采用的免疫途径，研究最充分，效果也较好。肿瘤DNA疫苗的临床应用目前报道较少，仅有少量进入I/II期临床试验，如gp100和蛋白酪氨酸激酶用于治疗黑色素瘤、CEA疫苗用于治疗结直肠癌、PSA和PSMA用于治疗前列腺癌、HPV-E6，E7用于治疗子宫颈癌以及NY-ESO-1用于治疗非小细胞性肺癌等。AppropriateutilizationandregulationofDCsinvaccinedesigninduceamuchmorepotentCTLantitumorimmuneresponse.(a)Tumorantigen-loadingtechniquesactivateDCsexvivo.(b)TargeteddrugsfacilitatethecaptureoftumorantigensbyDCsandtheexpressionofcostimulatorymoleculesandMHC-IIinvivo.(c)StimulatoryadjuvantsinducematurationofDCsandenhancetheactivationofCTLs.肿瘤疫苗治疗肿瘤的可能影响因素肿瘤抗原选择MHCI、II类分子和共刺激分子的缺陷或降低诱导T细胞CTLA-4的表达CD4+CD25+Foxp3+调节T细胞的抑制作用TH1/TH2细胞不平衡免疫程序优化组合(1)抗原和佐剂的剂量及途径应用恰当(2)减轻肿瘤负荷(3)免疫活性细胞含量和功能检测(4)抑制性DC、Treg细胞、MSC和抑制性巨噬细胞等含量分析(5)肿瘤治疗的干预手段联合应用Schematicillustrationofhumantelomerasereversetranscriptase(hTERT)antigenicpeptidespresentedbyMHCclassIcomplexonthetumorcellsurface.MultipleantigenicpeptidesderivedfromhTERTintumorcellarerecognizedbyimmunecells.ThepeptidesaregeneratedbyproteasomedegradationofhTERTprotein.ThehTERTpeptidesaretransportedbythetransporterassociatedwithantigenprocessingintotheERwheretheyareloadedontoMHCclassImoleculesandtransportedthroughtheGolgiapparatusontothecellsurface.CD8+cytotoxicTlymphocytewithspecificreceptorsrecognizeandattackthetumorcellbytheengagementoftheTcellreceptorwithanMHCclassI-hTERTpeptidecomplex.BiochimicaetBiophysicaActaxxx(2009)

Telomeraseincancerimmunotherapy(review)Modelofhumantelomerasereversetranscriptase(hTERT)antigenstimulationofDCs,CD4+Tcells,andexpansionofCD8+Tcellsinvivo.(1)TelomerasehTERTantigentransferandprocessinginAPC,(2)antigenpresentationfromAPCortumorcellstoTcells,(3)interactionbetweenCD4+andCD8+Tcells,(4)ExpansionofCD8+CTLsthathavesufficienttelomeres,(5)CTLswithshorttelomeresundergocellsenescenceandapoptosis,and(6)CD8+cytotoxiclymphocytesattackthetumorsexpressingtelomerasehTERTantigen.Hypotheticalmodesofactionofdominantandcrypticimmuneepitopesinepitope-specificTcellclonalevasionorreactivation.(A)DominantepitopetoMHCclassIformsastableantigen-MHCclassIcomplexthatengageswithitsspecificTCRatsuchhighaffinitythatdeathoftheTcellcloneoccurs.(B)AnintermediateepitopewithvariableaviditytoMHCclassIformsantigen-MHCcomplexesthatinteractwithitsspecificTCRdiscontinuouslyunderparticularconditionsresultingineithereliminationoractivationoftheTcellclone.(C)CrypticepitopewithlowaffinitytoMHCclassIformsanunstableantigen-MHCclassIcomplexthathasapoorinteractionwithitsspecificTCR,resultinginthesurvivalandcontinuouspresenceoftheTcellclonespecifictothecrypticepitope.(D)Position1tyrosinesubstitution(P1Y)orposition9valinesubstitution(P9V)inthecrypticepitopeenhancesthecrypticepitopeinteractionwithMHCclassIandtherebyestablishesanoptimalcomplextointeractwithitsspecificTCR,resultinginactivationandexpansionoftheTcellclone.ActivatedTcellclonesbindtheirspecificantigenepitope-MHCclassIcomplexontumorcellstoexertkilleffectsonthetumorcells.BiochimicaetBiophysicaActaxxx(2009)Telomeraseincancerimmunotherapy(review)AdvantagesandDisadvantagesofDifferentVaccinationStrategiesWaystoimprovetheclinicaloutcomeofpeptide-basedvaccinesTherepertoireofhumantumor-associatedepitopes-identificationandselectionofantigensandtheirapplicationinclinicaltrials.CurrentOpinioninImmunology2013,25:277–283激活质控癌症患者

DC

激发性DC抗原靶向引入途径剂量频率成熟耐受性DCDC亚群DC疫苗有效治疗肿瘤的临床和免疫学指标进行质量控制和标准化运用AbAg免疫系统感受淋巴细胞克隆容积的阈值TCR/BCR储备库:51013~11018

多样性淋巴细胞抗原受体多样性的体内储备及其对抗原激发的感知和应答免疫网络学说

带有不同独特型抗原受体的淋巴细胞克隆，因数量未能超过免疫系统的感知阈值而在体内长期存在。图中以不同的几何图形代表各种细胞克隆的受体（TCR或BCR）。抗原（红三角）一旦到来，选择出带有相应受体的淋巴细胞克隆使之发生增殖并跨越阈值，成为Ab1（紫红色），后者再诱导产生Ab2（蓝色和绿色），引发网络式的连锁反应。注意Ab2作为抗原分子，其表位结构与抗原分子相似。1974年NielsJerne提出抗体分子上的独特型和抗独特型相互识别而形成免疫网络（免疫网络学说）。抗独特型抗体作为疫苗Preparationandapplicationofanti-idiotypeantibody一、过继性免疫疗法二、抗体导向疗法三、其他相关免疫疗法被动免疫治疗

(PassiveImmunotherapy)一、过继性免疫疗法

过继性免疫疗法(Adoptiveimmunotherapy)是指把自身或异体的具有抗肿瘤活性的免疫细胞转输到免疫功能低下的肿瘤患者，在体内发挥抗肿瘤作用，以此达到治疗肿瘤的目的。1.淋巴因子激活的杀伤细胞(LAK)2.肿瘤浸润性淋巴细胞(TIL)

小鼠脾脏细胞或人外周血淋巴细胞在体外培养中，经高浓度的细胞因子（IL-2）诱导后发生扩增，产生一类能非特异性杀伤自身和异体肿瘤细胞的效应细胞，称为淋巴因子激活的杀伤细胞（lymphokineactivatedkillercell）,简称LAK细胞。

淋巴因子激活的杀伤细胞

LAK细胞的特征

是一群异质性的细胞群，主要来源于外周血淋巴细胞，其表型既可是CD3＋细胞，也可是CD3－细胞，往往具有NK细胞样标记（CD16和CD56)，其杀伤肿瘤细胞不需要抗原致敏，亦无MHC约束性。

IL-2/LAK细胞抗肿瘤机制LAK细胞对肿瘤细胞的非特异性直接杀伤；依赖肿瘤免疫原性的特异性抗瘤机制①对具有免疫原性肿瘤有效;②有长期疗效，说明特异性免疫记忆的存在。CancertherapiestargetingNKcells.a.Followinghaplo-identicalorMHC-matchedhaematopoieticstemcelltransplantation(HSCT),naturalkiller(NK)cellsofdonororigindevelopinthepatientwithcancer.b.Alternatively,NKcellpopulationscanbeisolatedfromhealthydonorsandactivatedand/orexpandedinvitrobeforeinfusionintothepatientwithcancer.Inbothcases(allogeneicHSCTandNKcellinfusion),theaimistopromotetheantitumourfunctionofdonorNKcellsinthepatient.Indeed,afractionofdonorNKcellswillbenotbeinhibitedbytheMHCclassImoleculesofthepatient,asthekillercellimmunoglobulin-likereceptors(KIRs)expressedbythedonorNKcellswillnotinteractwiththeMHCclassImoleculesofthepatient,andthispromotestumourcellelimination.Incontrasttocancercells,mosthealthycellsofthepatientwillnotactivatedonorNKcells,astheylackasufficientcell-surfacedensityofactivatingligandsforthedonorNKcells.c.AnalternativeapproachistoboostendogenousNKcellactivitybytreatingpatientswithmonoclonalantibodiesspecificforNKcell-expressedinhibitoryreceptors.Theseantibodiesaredesignedtoenhancetheantitumouractivityofthepatient’sownNKcellswithoutinducingautoimmunity.TargetingnaturalkillercellsandnaturalkillerTcellsincancer.NATUREREVIEWS|IMMUNOLOGYVOLUME12|APRIL2012LAK细胞的制备和应用肿瘤患者

全血细胞（淋巴细胞）加IL-2，培养、扩增过继性输注抗肿瘤淋巴细胞加IL-2培养、扩增的淋巴细胞TreatmentofmelanomawithLAKcellsandIL-2BeforeAfterExperimentaldemonstrationoftumor-destroyingactivityofLAKcellsplusIL-2.SpleencellsorLAKcells,inthepresenceorabsenceofrecombinantIL-2,wereinfusedintomicewithpulmonarysarcoma.Theanimalswereevaluated13dayslaterforthenumberofpulmonarysarcomametastases.

IL-2/LAK细胞对晚期肿瘤患者的疗效肿瘤类别患者数CR人数PR人数CR+PR(%)肾细胞癌7281735黑色素瘤484621结肠癌301313非何杰金淋巴瘤71357乳腺瘤1000肺癌5000肉瘤6000其它9000总计178142924IL-2/LAK细胞的毒副作用

单独使用LAK细胞几乎不产生毒性作用，但是大剂量IL-2/LAK细胞可对全身各种器官、组织产生广泛毒副作用。IL-2引起的毒副作用产生机制：重要器官中淋巴样细胞的浸润；IL-2引起的血管通透性增高，导致液体滞留和间质水肿,损

伤器官功能；IL-2促进IFN-γ等细胞因子的释放，后者也能产生负作用。Vascularleaksyndrome(VLS)causedbyIL-2therapyIL-2TcellCD44endothelialcells

浸润于实体瘤内和周围淋巴结中，往往已被肿瘤抗原致敏而具有特异性抗肿瘤作用的一类细胞，被称为肿瘤浸润性淋巴细胞(tumor-infiltrationlymphocytes,TIL)。TIL的特征

是一群异质性细胞，主要由T淋巴细胞组成，其次是NK细胞和B细胞，大多数表达IL-2R、HLA-DR和粘附分子。TIL比LAK细胞具有更佳的增殖活性，对肿瘤细胞的杀伤特异性强、效率高。肿瘤浸润性淋巴细胞

TIL细胞的制备和应用肿瘤患者肿瘤切除

TIL加IL-2反复培养ELISA测刺激后IFN-水平用IL-2及抗CD3单抗克隆扩增TIL过继性输注抗肿瘤淋巴细胞阳性克隆治疗前以化疗作非髓系淋巴细胞清除TIL过继性免疫治疗通常与IL-2联合使用。同时使用TNF-α和IFN-α等细胞因子以提高TIL的疗效。采用动脉内注射的方法提高肿瘤局部的TIL浓度。小剂量环磷酰胺和大剂量TIL能促进TIL归巢。BeforeAfterTreatmentofMelanomaswithTIL+IL-2Left:CompleteregressionofalargelivermetastasisfromkidneycancerinapatienttreatedTILwithIL-2.Right:Regressionisongoingsevenyearslater.Rosenberg(2001)Nature,411;381-4二、抗体导向疗法NatureReviews/CancerVol.15,2015NatureReviews/CancerVol.15,20151.非偶联(单纯)抗体2.抗体偶联物(免疫偶联物)Antibodytherapyofcancer.NATUREREVIEWS.CANCERVOLUME12APRIL2012Antibodyconstructsandpotentialusesinoncology非偶联(单纯)抗体抗体的类型抗体疗法的肿瘤抗原的基本条件抗体作用机制抗体的制备和应用抗体疗法特点和影响因素抗体的临床应用前景抗体的类型鼠源性抗体(mouseantibody)多抗(Polyclonalantibody,PAb)鼠单抗(Monoclonalantibody,MAb)单链抗体(Singlechainantibody)

抗Id抗体(Anti-Idiotypicantibody)

基因工程抗体(Gene-engineeringantibody)

嵌合抗体(Chimericantibody)

重构抗体(Reshapedantibody)

双特异性抗体(Bispecificantibody)人源化抗体(Humanizedantibody)抗体疗法的肿瘤抗原的基本条件

靶抗原必须在肿瘤细胞表面表达;靶抗原在细胞表面稳定表达,不存在分泌型；在肿瘤细胞表面的密度高;理想的靶抗原应只在肿瘤细胞中表达,或在正常组织中的分布范围极其狭窄;在肿瘤的生长中起关键的作用。抗体作用机制CDC,ADCC,调理作用。阻断和干扰作用：①抗生长因子受体抗体抑制肿瘤细胞生长；②抗T细胞活化的抑制分子，增强T细胞效应功能。抗Id单抗：抗Id治疗可分为被动疗法和主动疗法两种。Id疫苗可能激发多克隆应答；抗Id单抗可能通过ADCC杀伤肿瘤细胞。NatureReviews/CancerVol.15,2015

抗体与肿瘤抗原结合后，发挥有效的阻遏作用，可阻断其生物学活性，抑制肿瘤细胞增殖。Immunoregulatoryreceptorsandtheirligands.TcellactivationreliesontheTcellreceptor(TCR)recognizingitscognateantigeninthecontextofMHCmoleculesfromanantigenpresentingcell(APC)oranAPC-likecell(tumorcell).Interactionbetweenco-stimulatorymoleculesCD80,andCD86andCD28iscrucialforappropriateTcellactivation.ImmunoregulatoryreceptorssuchasCTLA-4andPD-1aretofinetuneTcellactivation.Highlevelsofmultipleimmunoregulatoryreceptors(LAG-3,2B4,CD160,KLRG1,Tim-3,CTLA-4,andCD57)ortheirligandsarefoundinthetumormicroenvironment.PotentandlastingimmunoregulatorysignalingresultsinreducedTcellfunctionandtolerance.Tcellanergy,exhaustion,senescence,andstemnessinthetumormicroenvironment.CurrentOpinioninImmunology2013,25:214–221AntibodiestargetingmoleculesexpressedbyimmunecellsDrugsinClinicalDevelopmentthatBlockPD-1

orPD-L1A.引入一部分有待清除的Ab1，在体内大量诱导Ab2，由Ab2发挥负向调节作用，抑制体内原有的Ab1，削弱机体对抗原的特异性应答。B.藉助抗原内影像直接制备Ab1/Ab3,增强机体对抗原的特异性应答。AgAb2增强Ab1Ab1Ab2BAAb3/Ab1Ab1Ab2Ab2削弱

Ab1Mechanismsoftumourcellkillingbyantibodies.a|Directtumourcellkillingcanbeelicitedbyreceptoragonistactivity,suchasanantibodybindingtoatumourcellsurfacereceptorandactivatingit,leadingtoapoptosis(representedbythemitochondrion).Itcanalsobemediatedbyreceptorantagonistactivity,suchasanantibodybindingtoacellsurfacereceptorandblockingdimerization,kinaseactivationanddownstreamsignalling,leadingtoreducedproliferationandapoptosis.Anantibodybindingtoanenzymecanleadtoneutralization,signallingabrogationandcelldeath,andconjugatedantibodiescanbeusedtodeliverapayload(suchasadrug,toxin,smallinterferingRNAorradioisotope)toatumourcell.b|Immune-mediatedtumourcellkillingcanbecarriedoutbytheinductionofphagocytosis;complementactivation;antibody-dependentcellularcytotoxicity(ADCC);geneticallymodifiedTcellsbeingtargetedtothetumourbysingle-chainvariablefragment(scFv);Tcellsbeingactivatedbyantibody-mediatedcross-presentationofantigentodendriticcells;andinhibitionofTcellinhibitoryreceptors,suchascytotoxicTlymphocyte-associatedantigen4(CTLA4).c|Vascularandstromalcellablationcanbeinducedbyvasculaturereceptorantagonismorligandtrapping(notshown);stromalcellinhibition;deliveryofatoxintostromalcells;anddeliveryofatoxintothevasculature.MAC,membraneattackcomplex;MHC,majorhistocompatibilitycomplex;NK,naturalkiller.Antibodytherapyofcancer.NATUREREVIEWS.CANCERVOLUME12APRIL2012抗体的制备和应用多克隆抗体单克隆抗体抗Id单抗基因工程抗体人源化抗体

Theconventionalpolyclonalantiserumproducedinresponsetoacomplexantigencontainsamixtureofmonoclonalantibodies,eachspecificforoneofthefourepitopesshownontheantigen(inset).Incontrast,amonoclonalantibody,whichisderivedfromasingleplasmacell,isspecificforoneepitopeonacomplexantigen.Theoutlineofthebasicmethodforobtainingamonoclonalantibodyisillustratedhere.多克隆抗体制备TheprocedureforproducingmonoclonalantibodiesspecificforagivenantigendevelopedbyG.KohlerandC.Milstein.Spleencells(HGPRT+andIg+)fromanantigen-primedmousearefusedwithmousemyelomacells(HGPRT-

andIg-).ThespleencellprovidesthenecessaryenzymesforgrowthonHATmedium,whilethemyelomacellprovidesimmortal-growthproperties.Unfusedmyelomacellsormyeloma/myelomafusionsfailtogrowbecauseoftheirlackofHGPRT.Unfusedspleencellshavelimitedgrowthcapabilitiesinvitroandwilldiewithinafewdays.

AminopterinblocksDNAsynthesisbythedenovopathway.Itactsasananalogofdihydrofolicacidandbindswithahighaffinitytodihydrofolatereductase,inhibitingpurinesynthesis.Inthepresenceofaminopterin,cellsmustusethesalvagepathwaytoproduceDNA.单克隆抗体制备一种抗原决定簇或表位能刺激一种B细胞克隆。而识别单一抗原表位的B细胞克隆产生的同源抗体，称为单克隆抗体（monoclonalantibody,mAb）。mAb结构高度均一，其抗原结合部位和同种型相同，具有纯度高、特异性强等特点。TreatmentofB-celllymphomawithmonoclonalantibodyspecificforidiotypicdeterminantsonthecancercells抗Id单抗制备和应用

ChimericandhybridmonoclonalantibodiesengineeredbyrecombinantDNAtechnology.(a)Chimericmouse-humanmonoclonalantibodycontainingtheVHandVLdomainsofamousemonoclonalantibody(blue)andCLandCHdomainsofahumanmonoclonalantibody(gray).(b)AchimericmonoclonalantibodycontainingonlytheCDRsofamousemonoclonalantibody(bluebands)graftedwithintheframeworkregionsofahumanmonoclonalantibodyiscalleda“humanized”monoclonalantibody.(c)AchimericmonoclonalantibodyinwhichtheterminalFcdomainisreplacedbytoxinchains(white).(d)AheteroconjugateinwhichonehalfofthemouseantibodymoleculeisspecificforatumorantigenandtheotherhalfisspecificfortheCD3/T-cellreceptorcomplex.基因工程抗体

Productionofchimericmouse-humanmonoclonalantibodies.Chimericmouse-humanheavy-andlight-chainexpressionvectorsareproduced.ThesevectorsaretransfectedintoAbmyelomacells.Cultureinampicillinmediumselectsfortransfectedmyelomacellsthatsecretethechimericantibody.a.嵌合抗体b.重构抗体GeneralprocedureforproducinggenelibrariesencodingFabfragments.Inthisprocedure,isolatedmRNAthatencodesheavyandlightchainsisamplifiedbythepolymerasechainreaction(PCR)andclonedinvectors.Randomcombinationsofheavy-andlight-chaingenesgenerateanenormousnumberofheavy-lightconstructsencodingFabfragmentswithdifferentantigenicspecificity.c.双特异性抗体BsAb是一种基因工程抗体，其2个Fab的抗原特异性是不同的。其中的一个是肿瘤抗原特异性的，另一个则是针对效应细胞上的某一种分子的，后者与效应细胞上相应分子的结合，激活效应细胞，发挥杀伤肿瘤的作用。HER2/neu和FcgRIIIBsAb（2B1）治疗卵巢癌。人源化抗体

(Humanizedantibody)

Humanizedantibody

Ageneticallyengineeredantibodyinwhichtheminimummousepartfromamurineantibodyistransplantedontoahumanantibody;generallyhumanizedantibodiesare5-10%mouseand90-95%human.Humanizedantibodiesweredevelopedt