腫瘤與免疫-腫瘤免疫治療課件

腫瘤免疫治療

Tumorimmunotherapy概述主動(dòng)免疫療法被動(dòng)免疫療法

腫瘤免疫治療概述1PaulEhrlich’smagicbulletconcept:100yearsofprogress,NatureReviews/CancerVol.8,2008概述PaulEhrlich’smagicbulletco2NATUREREVIEW/CANCER,2008,Vol.8:299-307NATUREREVIEW/CANCER,2008,Vol3

腫瘤免疫治療分為主動(dòng)免疫療法和被動(dòng)免疫療法兩大類。前者著重激發(fā)機(jī)體抗腫瘤免疫應(yīng)答能力；后者向宿主轉(zhuǎn)移有抗腫瘤活性的治療因子或細(xì)胞，抑制腫瘤生長。腫瘤免疫治療分為主動(dòng)免疫療法和被動(dòng)免疫療法兩大類。前4

腫瘤免疫療法的分類及常用的生物制劑

分類 治療因子或細(xì)胞

主動(dòng)免疫療法

非特異性

BCG、CpGODN、HSP、IFN-

等

特異性

減毒或滅活的瘤苗、修飾的瘤苗、

腫瘤抗原肽疫苗和基因重組產(chǎn)物

被動(dòng)免疫療法

非特異性

細(xì)胞因子誘導(dǎo)的殺傷細(xì)胞（CIK）

淋巴因子激活的殺傷細(xì)胞（LAK） DC介導(dǎo)的免疫治療

特異性

腫瘤浸潤淋巴細(xì)胞（TIL)

抗體（Antibody）

免疫偶聯(lián)物（Immuno-conjugates）

嵌合抗原受體（CAT）

BCG:BacilleCalmette-Guerin,CpGODN:CpGOligodeoxynucleotide,HSP:heatshockprotein,CIK:cytokineinducingkillercell,LAK:lymphokineactivatedkillercell,TIL:tumor-infiltrationlymphocytes,CAT：chimericantigenreceptor 腫瘤免疫療法的分類及常用的生物制劑5ApprovedimmunetherapiesforcancerApprovedimmunetherapiesfor6Antibodytherapyofcancer.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.Antibodytherapyofcancer.NA7一、非特異性主動(dòng)免疫療法二、特異性主動(dòng)免疫療法主動(dòng)免疫治療

(ActiveImmunotherapy)一、非特異性主動(dòng)免疫療法主動(dòng)免疫治療8

非特異性地激發(fā)機(jī)體的免疫系統(tǒng)，增強(qiáng)抗腫瘤免疫應(yīng)答能力，而達(dá)到殺傷腫瘤細(xì)胞。1.免疫因子

①胸腺素(Thymosin)，轉(zhuǎn)移因子(Transterfactor)免疫核糖核酸(ImmuneRNA)②細(xì)胞因子:IFN-α,IFN-β,IFN-γ,IL-2,TNF-α等2.微生物制劑

卡介苗(BCG)，CpG寡聚脫氧核苷酸(CpGODN)3.化學(xué)合成藥物

左旋咪唑(Levamisole，LMS)

4.中藥制劑(滋陰、補(bǔ)氣、補(bǔ)血)

人參、黃芪等(主要為多糖成分)

一、非特異性主動(dòng)免疫療法非特異性地激發(fā)機(jī)體的免疫系統(tǒng)，增強(qiáng)抗腫瘤免疫應(yīng)一、非9

卡介苗制劑

牛型結(jié)核桿菌或其細(xì)胞壁成分或細(xì)胞骨架成分，BCG中的有效成分是胞壁酰二肽(muramyldipeptide，MDP)。作用機(jī)制

直接活化Mφ，刺激Mφ

表達(dá)IL-6、IL-1,增強(qiáng)IFN-

對(duì)Mφ的刺激作用。

刺激特異性TDTH的產(chǎn)生，間接活化Mφ。

刺激NK細(xì)胞增殖。

非特異性刺激單核巨噬細(xì)胞。

促進(jìn)IL-2和IL-4對(duì)B細(xì)胞的作用。臨床應(yīng)用

黑色素瘤：瘤灶內(nèi)直接注射

淺表性膀胱癌：膀胱內(nèi)滴注 卡介苗10卡介苗的臨床應(yīng)用（黑色素瘤）

皮膚轉(zhuǎn)移灶內(nèi)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，提示局部注射可導(dǎo)致全身性抗腫瘤免疫的建立?？偣舶?69名患者的16項(xiàng)使用這一方法的研究都獲得不同程度的療效，完全緩解率最高達(dá)90%(范圍7-90%)，部分緩解率范圍為5-50%。在一項(xiàng)結(jié)合放療的試驗(yàn)中，74%患者獲得完全緩解，另外5%患者獲得部分緩解。

BCG瘤內(nèi)注射療法可提高患者生存率。不用BCG治療的皮膚癌復(fù)發(fā)患者，存活時(shí)間的中值為13.3月，而接受BCG瘤內(nèi)注射患者的5年生存率達(dá)27%。有相當(dāng)一部分原發(fā)性黑色素瘤患者經(jīng)皮內(nèi)注射BCG治療后，長期存活?？ń槊绲呐R床應(yīng)用（淺表性膀胱癌）BCG膀胱內(nèi)滴注可以消除肉眼可見的淺表性膀胱癌，并能預(yù)防復(fù)發(fā)。膀胱內(nèi)注射BCG用于預(yù)防膀胱癌復(fù)發(fā)，可以顯著延遲疾病的進(jìn)展，延長保留膀胱的時(shí)間，提高總存活率。5項(xiàng)追蹤期范圍為12-60月的隨機(jī)研究發(fā)現(xiàn)，具有高度復(fù)發(fā)危險(xiǎn)的患者，用膀胱內(nèi)注射BCG治療者，70%保持無瘤；而用反復(fù)尿道內(nèi)切除者，僅有31%的患者保持無瘤。報(bào)道實(shí)例卡介苗的臨床應(yīng)用（黑色素瘤）皮膚轉(zhuǎn)移灶內(nèi)BCG11CpG寡聚脫氧核苷酸CpG寡聚脫氧核苷酸12腫瘤與免疫-腫瘤免疫治療課件13ActivationofinnateandadaptiveimmunitybyTLR9activation.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.Activationofinnateandadapt14腫瘤與免疫-腫瘤免疫治療課件15腫瘤與免疫-腫瘤免疫治療課件16細(xì)胞因子Functionofcytokinesinhostdefense細(xì)胞因子Functionofcytokinesinh17①上調(diào)免疫細(xì)胞的表面分子和受體的表達(dá)；②促進(jìn)DC細(xì)胞的成熟,增強(qiáng)T細(xì)胞的增殖、分化和CTL的活

化和效應(yīng)功能，刺激B細(xì)胞產(chǎn)生抗體，提高NK細(xì)胞活性，激發(fā)巨噬細(xì)胞等產(chǎn)生抗腫瘤免疫應(yīng)答；③促進(jìn)免疫效應(yīng)細(xì)胞釋放淋巴毒素和效應(yīng)分子殺傷腫瘤；④促進(jìn)腫瘤細(xì)胞表達(dá)MHC分子，增強(qiáng)腫瘤細(xì)胞的免疫原性和對(duì)效應(yīng)細(xì)胞的敏感性；⑤某些細(xì)胞因子具有直接破壞腫瘤細(xì)胞和促使其發(fā)生凋亡的作用，如TNF。細(xì)胞因子殺傷腫瘤細(xì)胞的效應(yīng)機(jī)制①上調(diào)免疫細(xì)胞的表面分子和受體的表達(dá)；細(xì)胞因子殺傷腫瘤細(xì)胞18腫瘤與免疫-腫瘤免疫治療課件19二、特異性主動(dòng)免疫療法1.腫瘤疫苗2.抗獨(dú)特型抗體作為疫苗

二、特異性主動(dòng)免疫療法20腫瘤疫苗使用腫瘤疫苗的原理腫瘤疫苗分類腫瘤疫苗治療腫瘤的可能影響因素腫瘤疫苗使用腫瘤疫苗的原理21使用腫瘤疫苗的原理腫瘤細(xì)胞表達(dá)腫瘤抗原并被機(jī)體免疫系統(tǒng)所識(shí)別；腫瘤疫苗刺激主動(dòng)特異性抗腫瘤應(yīng)答免疫，激發(fā)或加強(qiáng)宿主免疫以消滅腫瘤；腫瘤患者中許多已知的和未知的因素（腫瘤免疫原性）造成了免疫應(yīng)答的失敗。使用腫瘤疫苗的原理腫瘤細(xì)胞表達(dá)腫瘤抗原并被機(jī)體免疫系統(tǒng)所識(shí)別22腫瘤與免疫-腫瘤免疫治療課件23腫瘤疫苗分類＊根據(jù)應(yīng)用目的分類①預(yù)防性腫瘤疫苗②治療性腫瘤疫苗＊根據(jù)腫瘤疫苗中腫瘤抗原的存在形式分類①細(xì)胞疫苗滅活腫瘤細(xì)胞、DC、DC/腫瘤融合細(xì)胞②腫瘤抗原疫苗

腫瘤細(xì)胞裂解物、腫瘤抗原、抗原肽③基因工程疫苗

腫瘤抗原、細(xì)胞因子或協(xié)同刺激分子等

(基因重組產(chǎn)物)

基因轉(zhuǎn)染DC、腫瘤細(xì)胞等④DNA疫苗腫瘤抗原基因＋質(zhì)粒腫瘤疫苗分類＊根據(jù)應(yīng)用目的分類24腫瘤細(xì)胞疫苗DC疫苗DC/腫瘤融合細(xì)胞①細(xì)胞疫苗腫瘤細(xì)胞疫苗①細(xì)胞疫苗25

腫瘤細(xì)胞疫苗腫瘤細(xì)胞疫苗26DC疫苗特征DC疫苗特征27制備和應(yīng)用制備和應(yīng)用28DC/腫瘤融合細(xì)胞

適用于大多數(shù)腫瘤抗原還未鑒定的腫瘤。DC/腫瘤融合細(xì)胞29腫瘤抗原的來源和應(yīng)用

來源 舉例癌基因產(chǎn)物ras12密碼子突變:胰腺癌

BCR/abl重排產(chǎn)物:CML靜止基因產(chǎn)物 MAGE家族:黑色素瘤，乳腺癌病毒基因產(chǎn)物 EBV:Burkett淋巴瘤，鼻咽癌

HPV:宮頸癌

HBV:肝細(xì)胞癌組織特異性蛋白(分化抗原)酪氨酸酶:黑色素瘤突變的抑癌基因產(chǎn)物P53:多種腫瘤抗獨(dú)特型抗體(Anti-Id-Ab)TCRId:T細(xì)胞淋巴瘤②腫瘤抗原疫苗腫瘤抗原的來源和應(yīng)用來源 30mRNAMAGE-1蛋白309氨基酸HLA-Cw16HLA-A1161169EADPTGHSYSAYGEPRKL

開放閱讀框230238MAGE-1基因X染色體q28區(qū)

外顯子3

外顯子2

外顯子1MAGE-1.Cw16肽段

MAGE1基因定位于X染色體q28區(qū)，mRNA轉(zhuǎn)錄表達(dá)的MAGE-1蛋白為309氨基酸，由HLA-A1和HLA-Cw16提呈的抗原肽位于161～169和230～238區(qū)域氨基酸序列。MAGE-1基因、編碼蛋白和抗原肽mRNAMAGE-1蛋白HLA-Cw16HLA-A116131黑色素瘤特異性CTL識(shí)別的黑色素細(xì)胞分化抗原肽分化抗原 抗原肽結(jié)構(gòu) 肽位置 遞呈分子酪氨酸酶

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黑色素瘤特異性CTL識(shí)別的黑色素細(xì)胞分化抗原肽分化抗原 抗原32制備和應(yīng)用制備和應(yīng)用33腫瘤與免疫-腫瘤免疫治療課件34Aneffectivevaccineagainsthumanpapillomavirus(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.Aneffectivevaccineagainsth35PLGbased3Dpolymerscaffoldvaccinetorecruitandprogramimmunecellstogenerateantitumorimmunity.(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.CurrentOpinioninImmunology2013,25:238–245PLGbased3Dpolymerscaffold36③基因工程疫苗修飾的腫瘤疫苗協(xié)同刺激分子、細(xì)胞因子基因修飾的腫瘤細(xì)胞重組病毒疫苗用已知肽的cDNA序列與滅活病毒重組產(chǎn)生的疫苗，可同時(shí)轉(zhuǎn)入MHC、B7等基因。③基因工程疫苗修飾的腫瘤疫苗37Enhancementoftumorcellimmunogenicitybytransfectionofcostimulatorandcytokinegenes.TumorcellsthatdonotadequatelystimulateTcellsontransplantationintoananimalwillnotberejectedandwillthereforegrowintotumors.Vaccinationwithtumorcellstransfectedwithgenesencodingcostimulatorsorcytokines,suchasIL-2,canleadtoenhancedactivationofTcells.Thisapproachofusingtransfectedtumorcellsasvaccineshasworkedinmousemodels,butclinicaltrialshavenotyetbeensuccessful.Tumorvaccines.Twotypesoftumorvaccinesthathaveshownefficacyinclinicaltrialsandanimalmodelsareillustrated.Autologousdendriticcellsarepreparedfrompatients’ownperipheralbloodcells.Thedendriticcellsareeitherpulsedwithrecombinantproteinortransfectedwithageneconstructthatexpressestheprotein.Theconstructmayalsoexpresscostimulatorymolecules(notshown).Enhancementoftumorcellimmu38腫瘤與免疫-腫瘤免疫治療課件39UseofDNAvaccinesraisesbothhumoralandcellularimmunity④DNA疫苗UseofDNAvaccinesraisesbot40

腫瘤DNA疫苗由腫瘤抗原基因與質(zhì)粒重組后形成，其中腫瘤抗原基因隨治療的腫瘤的類型及選用的腫瘤抗原不同而不同，質(zhì)粒則是作為腫瘤抗原基因的載體。腫瘤DNA疫苗中常用的質(zhì)粒有pSV2、pRSV、pcDNA3.1和pC1等，這些載體一般都設(shè)計(jì)有真核基因表達(dá)調(diào)控序列（如增強(qiáng)子和啟動(dòng)子等）、供目的基因插入的多克隆位點(diǎn)、轉(zhuǎn)錄終止序列以及可使質(zhì)粒在大腸桿菌中保持并多拷貝復(fù)制的序列ColEI等。

腫瘤DNA疫苗的免疫接種有多種途徑，包括直接注射、基因槍免疫和電穿孔免疫等。不同免疫途徑的免疫機(jī)制不同，所誘導(dǎo)的免疫保護(hù)力強(qiáng)弱和維持時(shí)間也不盡相同。肌肉注射是最早采用的免疫途徑，研究最充分，效果也較好。腫瘤DNA疫苗的臨床應(yīng)用目前報(bào)道較少，僅有少量進(jìn)入I/II期臨床試驗(yàn)，如gp100和蛋白酪氨酸激酶用于治療黑色素瘤、CEA疫苗用于治療結(jié)直腸癌、PSA和PSMA用于治療前列腺癌、HPV-E6，E7用于治療子宮頸癌以及NY-ESO-1用于治療非小細(xì)胞性肺癌等。腫瘤DNA疫苗由腫瘤抗原基因與質(zhì)粒重組后形成41AppropriateutilizationandregulationofDCsinvaccinedesigninduceamuchmorepotentCTLantitumorimmuneresponse.(a)Tumorantigen-loadingtechniquesactivateDCsexvivo.(b)TargeteddrugsfacilitatethecaptureoftumorantigensbyDCsandtheexpressionofcostimulatorymoleculesandMHC-IIinvivo.(c)StimulatoryadjuvantsinducematurationofDCsandenhancetheactivationofCTLs.Appropriateutilizationandre42腫瘤疫苗治療腫瘤的可能影響因素腫瘤抗原選擇MHCI、II類分子和共刺激分子的缺陷或降低誘導(dǎo)T細(xì)胞CTLA-4的表達(dá)CD4+CD25+Foxp3+調(diào)節(jié)T細(xì)胞的抑制作用TH1/TH2細(xì)胞不平衡免疫程序優(yōu)化組合(1)抗原和佐劑的劑量及途徑應(yīng)用恰當(dāng)(2)減輕腫瘤負(fù)荷(3)免疫活性細(xì)胞含量和功能檢測(cè)(4)抑制性DC、Treg細(xì)胞、MSC和抑制性巨噬細(xì)胞等含量分析(5)腫瘤治療的干預(yù)手段聯(lián)合應(yīng)用腫瘤疫苗治療腫瘤的可能影響因素腫瘤抗原選擇43Schematicillustrationofhumantelomerasereversetranscriptase(hTERT)antigenicpeptidespresentedbyMHCclassIcomplexonthetumorcellsurface.MultipleantigenicpeptidesderivedfromhTERTintumorcellarerecognizedbyimmunecells.ThepeptidesaregeneratedbyproteasomedegradationofhTERTprotein.ThehTERTpeptidesaretransportedbythetransporterassociatedwithantigenprocessingintotheERwheretheyareloadedontoMHCclassImoleculesandtransportedthroughtheGolgiapparatusontothecellsurface.CD8+cytotoxicTlymphocytewithspecificreceptorsrecognizeandattackthetumorcellbytheengagementoftheTcellreceptorwithanMHCclassI-hTERTpeptidecomplex.BiochimicaetBiophysicaActaxxx(2009)

Telomeraseincancerimmunotherapy(review)Schematicillustration44Modelofhumantelomerasereversetranscriptase(hTERT)antigenstimulationofDCs,CD4+Tcells,andexpansionofCD8+Tcellsinvivo.(1)TelomerasehTERTantigentransferandprocessinginAPC,(2)antigenpresentationfromAPCortumorcellstoTcells,(3)interactionbetweenCD4+andCD8+Tcells,(4)ExpansionofCD8+CTLsthathavesufficienttelomeres,(5)CTLswithshorttelomeresundergocellsenescenceandapoptosis,and(6)CD8+cytotoxiclymphocytesattackthetumorsexpressingtelomerasehTERTantigen.Modelofhumantelomera45Hypotheticalmodesofactionofdominantandcrypticimmuneepitopesinepitope-specificTcellclonalevasionorreactivation.(A)DominantepitopetoMHCclassIformsastableantigen-MHCclassIcomplexthatengageswithitsspecificTCRatsuchhighaffinitythatdeathoftheTcellcloneoccurs.(B)AnintermediateepitopewithvariableaviditytoMHCclassIformsantigen-MHCcomplexesthatinteractwithitsspecificTCRdiscontinuouslyunderparticularconditionsresultingineithereliminationoractivationoftheTcellclone.(C)CrypticepitopewithlowaffinitytoMHCclassIformsanunstableantigen-MHCclassIcomplexthathasapoorinteractionwithitsspecificTCR,resultinginthesurvivalandcontinuouspresenceoftheTcellclonespecifictothecrypticepitope.(D)Position1tyrosinesubstitution(P1Y)orposition9valinesubstitution(P9V)inthecrypticepitopeenhancesthecrypticepitopeinteractionwithMHCclassIandtherebyestablishesanoptimalcomplextointeractwithitsspecificTCR,resultinginactivationandexpansionoftheTcellclone.ActivatedTcellclonesbindtheirspecificantigenepitope-MHCclassIcomplexontumorcellstoexertkilleffectsonthetumorcells.BiochimicaetBiophysicaActaxxx(2009)Telomeraseincancerimmunotherapy(review)Hypotheticalmodesofactiono46AdvantagesandDisadvantagesofDifferentVaccinationStrategiesAdvantagesandDisadvantageso47Waystoimprovetheclinicaloutcomeofpeptide-basedvaccinesWaystoimprovetheclinicalo48Therepertoireofhumantumor-associatedepitopes-identificationandselectionofantigensandtheirapplicationinclinicaltrials.CurrentOpinioninImmunology2013,25:277–283Therepertoireofhumantumor-49腫瘤與免疫-腫瘤免疫治療課件50激活質(zhì)控癌癥患者

DC

激發(fā)性DC抗原靶向引入途徑劑量頻率成熟耐受性DCDC亞群DC疫苗有效治療腫瘤的臨床和免疫學(xué)指標(biāo)進(jìn)行質(zhì)量控制和標(biāo)準(zhǔn)化運(yùn)用激活質(zhì)癌癥患者DC激發(fā)性D51AbAg免疫系統(tǒng)感受淋巴細(xì)胞克隆容積的閾值TCR/BCR儲(chǔ)備庫:51013~11018

多樣性淋巴細(xì)胞抗原受體多樣性的體內(nèi)儲(chǔ)備及其對(duì)抗原激發(fā)的感知和應(yīng)答免疫網(wǎng)絡(luò)學(xué)說

帶有不同獨(dú)特型抗原受體的淋巴細(xì)胞克隆，因數(shù)量未能超過免疫系統(tǒng)的感知閾值而在體內(nèi)長期存在。圖中以不同的幾何圖形代表各種細(xì)胞克隆的受體（TCR或BCR）?？乖t三角）一旦到來，選擇出帶有相應(yīng)受體的淋巴細(xì)胞克隆使之發(fā)生增殖并跨越閾值，成為Ab1（紫紅色），后者再誘導(dǎo)產(chǎn)生Ab2（藍(lán)色和綠色），引發(fā)網(wǎng)絡(luò)式的連鎖反應(yīng)。注意Ab2作為抗原分子，其表位結(jié)構(gòu)與抗原分子相似。1974年NielsJerne提出抗體分子上的獨(dú)特型和抗獨(dú)特型相互識(shí)別而形成免疫網(wǎng)絡(luò)（免疫網(wǎng)絡(luò)學(xué)說）?？躬?dú)特型抗體作為疫苗AbAg免疫系統(tǒng)感受淋巴細(xì)胞克隆容積的閾值TCR/BCR儲(chǔ)52Preparationandapplicationofanti-idiotypeantibodyPreparationandapplicationof53一、過繼性免疫療法二、抗體導(dǎo)向療法三、其他相關(guān)免疫療法被動(dòng)免疫治療

(PassiveImmunotherapy)一、過繼性免疫療法被動(dòng)免疫治療54一、過繼性免疫療法

過繼性免疫療法(Adoptiveimmunotherapy)是指把自身或異體的具有抗腫瘤活性的免疫細(xì)胞轉(zhuǎn)輸?shù)矫庖吖δ艿拖碌哪[瘤患者，在體內(nèi)發(fā)揮抗腫瘤作用，以此達(dá)到治療腫瘤的目的。1.淋巴因子激活的殺傷細(xì)胞(LAK)2.腫瘤浸潤性淋巴細(xì)胞(TIL)一、過繼性免疫療法55

小鼠脾臟細(xì)胞或人外周血淋巴細(xì)胞在體外培養(yǎng)中，經(jīng)高濃度的細(xì)胞因子（IL-2）誘導(dǎo)后發(fā)生擴(kuò)增，產(chǎn)生一類能非特異性殺傷自身和異體腫瘤細(xì)胞的效應(yīng)細(xì)胞，稱為淋巴因子激活的殺傷細(xì)胞（lymphokineactivatedkillercell）,簡(jiǎn)稱LAK細(xì)胞。

淋巴因子激活的殺傷細(xì)胞

LAK細(xì)胞的特征

是一群異質(zhì)性的細(xì)胞群，主要來源于外周血淋巴細(xì)胞，其表型既可是CD3＋細(xì)胞，也可是CD3－細(xì)胞，往往具有NK細(xì)胞樣標(biāo)記（CD16和CD56)，其殺傷腫瘤細(xì)胞不需要抗原致敏，亦無MHC約束性。

IL-2/LAK細(xì)胞抗腫瘤機(jī)制LAK細(xì)胞對(duì)腫瘤細(xì)胞的非特異性直接殺傷；依賴腫瘤免疫原性的特異性抗瘤機(jī)制①對(duì)具有免疫原性腫瘤有效;②有長期療效，說明特異性免疫記憶的存在。小鼠脾臟細(xì)胞或人外周血淋巴細(xì)胞在體外培養(yǎng)中，經(jīng)高濃度的56CancertherapiestargetingNKcells.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|APRIL2012CancertherapiestargetingNK57LAK細(xì)胞的制備和應(yīng)用腫瘤患者

全血細(xì)胞（淋巴細(xì)胞）加IL-2，培養(yǎng)、擴(kuò)增過繼性輸注抗腫瘤淋巴細(xì)胞加IL-2培養(yǎng)、擴(kuò)增的淋巴細(xì)胞TreatmentofmelanomawithLAKcellsandIL-2BeforeAfterExperimentaldemonstrationoftumor-destroyingactivityofLAKcellsplusIL-2.SpleencellsorLAKcells,inthepresenceorabsenceofrecombinantIL-2,wereinfusedintomicewithpulmonarysarcoma.Theanimalswereevaluated13dayslaterforthenumberofpulmonarysarcomametastases.LAK細(xì)胞的制備和應(yīng)用腫瘤患者全血細(xì)胞（淋巴細(xì)胞）過繼性輸58

IL-2/LAK細(xì)胞對(duì)晚期腫瘤患者的療效腫瘤類別患者數(shù)CR人數(shù)PR人數(shù)CR+PR(%)腎細(xì)胞癌7281735黑色素瘤484621結(jié)腸癌301313非何杰金淋巴瘤71357乳腺瘤1000肺癌5000肉瘤6000其它9000總計(jì)178142924IL-2/LAK細(xì)胞對(duì)晚期腫瘤患者的療效59IL-2/LAK細(xì)胞的毒副作用

單獨(dú)使用LAK細(xì)胞幾乎不產(chǎn)生毒性作用，但是大劑量IL-2/LAK細(xì)胞可對(duì)全身各種器官、組織產(chǎn)生廣泛毒副作用。IL-2引起的毒副作用產(chǎn)生機(jī)制：重要器官中淋巴樣細(xì)胞的浸潤；IL-2引起的血管通透性增高，導(dǎo)致液體滯留和間質(zhì)水腫,損

傷器官功能；IL-2促進(jìn)IFN-γ等細(xì)胞因子的釋放，后者也能產(chǎn)生負(fù)作用。Vascularleaksyndrome(VLS)causedbyIL-2therapyIL-2TcellCD44endothelialcellsIL-2/LAK細(xì)胞的毒副作用單獨(dú)使用LAK細(xì)60

浸潤于實(shí)體瘤內(nèi)和周圍淋巴結(jié)中，往往已被腫瘤抗原致敏而具有特異性抗腫瘤作用的一類細(xì)胞，被稱為腫瘤浸潤性淋巴細(xì)胞(tumor-infiltrationlymphocytes,TIL)。TIL的特征

是一群異質(zhì)性細(xì)胞，主要由T淋巴細(xì)胞組成，其次是NK細(xì)胞和B細(xì)胞，大多數(shù)表達(dá)IL-2R、HLA-DR和粘附分子。TIL比LAK細(xì)胞具有更佳的增殖活性，對(duì)腫瘤細(xì)胞的殺傷特異性強(qiáng)、效率高。腫瘤浸潤性淋巴細(xì)胞

浸潤于實(shí)體瘤內(nèi)和周圍淋巴結(jié)中，往往已被腫瘤抗原致敏61TIL細(xì)胞的制備和應(yīng)用腫瘤患者腫瘤切除

TIL加IL-2反復(fù)培養(yǎng)ELISA測(cè)刺激后IFN-

水平用IL-2及抗CD3單抗克隆擴(kuò)增TIL過繼性輸注抗腫瘤淋巴細(xì)胞陽性克隆治療前以化療作非髓系淋巴細(xì)胞清除TIL過繼性免疫治療通常與IL-2聯(lián)合使用。同時(shí)使用TNF-α和IFN-α等細(xì)胞因子以提高TIL的療效。采用動(dòng)脈內(nèi)注射的方法提高腫瘤局部的TIL濃度。小劑量環(huán)磷酰胺和大劑量TIL能促進(jìn)TIL歸巢。TIL細(xì)胞的制備和應(yīng)用腫瘤患者腫瘤切除TIL加62BeforeAfterTreatmentofMelanomaswithTIL+IL-2Left:CompleteregressionofalargelivermetastasisfromkidneycancerinapatienttreatedTILwithIL-2.Right:Regressionisongoingsevenyearslater.Rosenberg(2001)Nature,411;381-4BeforeAfterTreatmentofMelano63二、抗體導(dǎo)向療法二、抗體導(dǎo)向療法64NatureReviews/CancerVol.15,2015NatureReviews/CancerVol.15,65NatureReviews/CancerVol.15,20151.非偶聯(lián)(單純)抗體2.抗體偶聯(lián)物(免疫偶聯(lián)物)NatureReviews/CancerVol.15,66Antibodytherapyofcancer.NATUREREVIEWS.CANCERVOLUME12APRIL2012AntibodyconstructsandpotentialusesinoncologyAntibodytherapyofcancer.NA67非偶聯(lián)(單純)抗體抗體的類型抗體療法的腫瘤抗原的基本條件抗體作用機(jī)制抗體的制備和應(yīng)用抗體療法特點(diǎn)和影響因素抗體的臨床應(yīng)用前景非偶聯(lián)(單純)抗體抗體的類型68抗體的類型鼠源性抗體(mouseantibody)多抗(Polyclonalantibody,PAb)鼠單抗(Monoclonalantibody,MAb)單鏈抗體(Singlechainantibody)

抗Id抗體(Anti-Idiotypicantibody)

基因工程抗體(Gene-engineeringantibody)

嵌合抗體(Chimericantibody)

重構(gòu)抗體(Reshapedantibody)

雙特異性抗體(Bispecificantibody)人源化抗體(Humanizedantibody)抗體的類型69抗體療法的腫瘤抗原的基本條件

靶抗原必須在腫瘤細(xì)胞表面表達(dá);靶抗原在細(xì)胞表面穩(wěn)定表達(dá),不存在分泌型；在腫瘤細(xì)胞表面的密度高;理想的靶抗原應(yīng)只在腫瘤細(xì)胞中表達(dá),或在正常組織中的分布范圍極其狹窄;在腫瘤的生長中起關(guān)鍵的作用。抗體療法的腫瘤抗原的基本條件靶抗原必須在腫瘤細(xì)胞表面表達(dá);70抗體作用機(jī)制CDC,ADCC,調(diào)理作用。阻斷和干擾作用：①抗生長因子受體抗體抑制腫瘤細(xì)胞生長；②抗T細(xì)胞活化的抑制分子，增強(qiáng)T細(xì)胞效應(yīng)功能?？笽d單抗：抗Id治療可分為被動(dòng)療法和主動(dòng)療法兩種。Id疫苗可能激發(fā)多克隆應(yīng)答；抗Id單抗可能通過ADCC殺傷腫瘤細(xì)胞?？贵w作用機(jī)制CDC,ADCC,調(diào)理作用。71NatureReviews/CancerVol.15,2015NatureReviews/CancerVol.15,72

抗體與腫瘤抗原結(jié)合后，發(fā)揮有效的阻遏作用，可阻斷其生物學(xué)活性，抑制腫瘤細(xì)胞增殖?？贵w與腫瘤抗原結(jié)合后，發(fā)揮有效的阻遏作用，可阻斷其生73腫瘤與免疫-腫瘤免疫治療課件74Immunoregulatoryreceptorsandtheirligands.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.CurrentOpinioninImmunology2013,25:214–221Immunoregulatoryreceptorsand75AntibodiestargetingmoleculesexpressedbyimmunecellsAntibodiestargetingmolecules76腫瘤與免疫-腫瘤免疫治療課件77DrugsinClinicalDevelopmentthatBlockPD-1

orPD-L1DrugsinClinicalDevelopment78A.引入一部分有待清除的Ab1，在體內(nèi)大量誘導(dǎo)Ab2，由Ab2發(fā)揮負(fù)向調(diào)節(jié)作用，抑制體內(nèi)原有的Ab1，削弱機(jī)體對(duì)抗原的特異性應(yīng)答。B.藉助抗原內(nèi)影像直接制備Ab1/Ab3,增強(qiáng)機(jī)體對(duì)抗原的特異性應(yīng)答。AgAb2

增強(qiáng)Ab1Ab1Ab2BAAb3/Ab1Ab1Ab2

Ab2

削弱

Ab1A.引入一部分有待清除的Ab1，在體內(nèi)大量誘導(dǎo)Ab2，由A79Mechanismsoftumourcellkillingbyantibodies.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.CANCERVOLUME12APRIL2012Mechanismsoftumourcellkill80抗體的制備和應(yīng)用多克隆抗體單克隆抗體抗Id單抗基因工程抗體人源化抗體

抗體的制備和應(yīng)用多克隆抗體81

Theconventionalpolyclonalantiserumproducedinresponsetoacomplexantigencontainsamixtureofmonoclonalantibodies,eachspecificforoneofthefourepitopesshownontheantigen(inset).Incontrast,amonoclonalantibody,whichisderivedfromasingleplasmacell,isspecificforoneepitopeonacomplexantigen.Theoutlineofthebasicmethodforobtainingamonoclonalantibodyisillustratedhere.多克隆抗體制備Theconventionalpolyclo82TheprocedureforproducingmonoclonalantibodiesspecificforagivenantigendevelopedbyG.KohlerandC.Milstein.Spleencells(HGPRT+andIg+)fromanantigen-primedmousearefusedwithmousemyelomacells(HGPRT-

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

AminopterinblocksDNAsynthesisbythedenovopathway.Itactsasananalogofdihydrofolicacidandbindswithahighaffinitytodihydrofolatereductase,inhibitingpurinesynthesis.Inthepresenceofaminopterin,cellsmustusethesalv