肿瘤疫苗的分子设计课件.ppt

1、Somatic mutations are not transmitted to progeny,but germinal mutations may be transmitted to some or all progenypro-oncogeneSuppressor gene诱因诱因Suppressor genepro-oncogeneImmune cells killed a tumor cell The Tumors As A grafted tissueImmune Surveillance(recognizing abnormal cell)Tumor escape mechani

2、smsICAM-1:intercellular adhesion molecule-1RCAS1:a growth inhibitory molecule,which react with T cell bearing their corresponding receptors and stop them in their trackes.1234567肿瘤分泌免疫抑制分子肿瘤分泌免疫抑制分子表表-1 免疫抑制性细胞因子对肿瘤浸润性淋巴细胞功能的作用免疫抑制性细胞因子对肿瘤浸润性淋巴细胞功能的作用 作用作用 TGF-IL-10 VEGFT细胞生长抑制细胞生长抑制 +-+CTL分化抑制分化抑制

3、+细胞因子产生抑制细胞因子产生抑制 +-T细胞无反应性诱导细胞无反应性诱导 +-细胞毒性作用细胞毒性作用下调下调 +-抗原呈递作用抑制抗原呈递作用抑制 +-Th1-Th2细胞平衡移至细胞平衡移至Th2细胞细胞 +-粘附粘附/协同协同刺激分子下调刺激分子下调 +-CTL介导裂解的抗性介导裂解的抗性 -+-可溶性抗原封闭抗体可溶性抗原封闭抗体下调下调TGF-,IL-6,IL-10(-)凋亡凋亡(1)对凋亡的抵抗性对凋亡的抵抗性 表达表达FasL,并通过并通过Fas-FasL相互作用而杀灭浸润相互作用而杀灭浸润的免疫效应细胞。的免疫效应细胞。a growth inhibitory molecule,

4、Detection of Fas ligand expression by immunofluorescence in a human lung carcinoma cell line.Fas ligand expression by a variety of tumor cell lines and primary tumor isolates.Expression of Fas ligand by tumor cells has been suggested as a tumor escape mechanism,how tumor cells kill activated cytotox

5、ic T cells and thus avoid an anti-tumor immune response.伴随蛋白伴随蛋白多肽转运蛋白1、MHC I 类抗原呈递类抗原呈递*缺乏协同刺激分子缺乏协同刺激分子IL-2 AbActive Non-specific BCG,Propionibacterium acnes,levamisole,cytokine genes,etc.Specific killed tumor cells or their extract,recombinant antigens,idiotype,co-stimulatory molecule genes,etc.P

6、assive Nonspecific LAK cells,cytokines Specific antibodies alone or coupled to drugs,pro-drug toxins or radioisotope;bispecific antibodies;T-cells Combined LAK cells and bispecific antibody *BCG:Bacillus Calmette Geurin is a bovine strain of Mycobacterium tuberculosis.Table 1.Immunotherapy of tumors

7、Table 1.Immunotherapy of tumorsType of BRM examplesbacterial product BCG,P.acnes,muramyl di -peptide,trehalose dimycolate synthetic molecules pyran,poly I:C,pyrimidines cytokines interferon-alpha,-beta,-gamma,IL-2,TNF Table 2.Non-specific active immunotherapy:Table 2.Non-specific active immunotherap

8、y:biological response modifiers(BRMs)biological response modifiers(BRMs)activate macrophages and NK cells(via cytokines)major effectinduce interferon productionactivate macrophages and NK cellsFigure1.Recurrent melanoma,unresponsive to radiotherapy,prior to immunotherapy with intralesional(病灶内的病灶内的)

9、injections of human monoclonal antibody to GM2 or GD2.GD3,a prominent ganglioside(神经节糖神经节糖苷苷)on the surface of melanoma cells.Figure 2.The same patient 2 years later following complete regression of all disease.2、天然抗、天然抗Gal抗体增强自体肿瘤免疫原性的作用抗体增强自体肿瘤免疫原性的作用半乳糖成分半乳糖成分:a-1,3-半乳糖苷半乳糖苷(Gala-1,3Gal)天然抗体天然抗体天

10、然抗体天然抗体激活补体激活补体半乳糖成分半乳糖成分:a-1,3-半乳糖苷半乳糖苷(Gala-1,3Gal)半乳糖成分半乳糖成分:a-1,3-半乳糖苷半乳糖苷(Gala-1,3Gal)肿瘤细胞膜肿瘤细胞膜 天然抗体天然抗体antibodycomplement CD16(Fc R III)CD11b/CD18 CD25CD28 CD32(Fc R II)CD35(CR1)CD64(Fc R I)CD71B7-2IL-2antibody antibodyrecognizing receptor on macrophagecomplementLactoferrinSurface receptor on

11、 macrophage半乳糖成分半乳糖成分:a-1,3-半乳糖苷半乳糖苷(Gala-1,3Gal)肿瘤细胞膜肿瘤细胞膜 天然抗体天然抗体 CD16 1、CML的细胞免疫治疗的细胞免疫治疗2、骨髓移植后疾病复发的细胞免疫治疗、骨髓移植后疾病复发的细胞免疫治疗Treatment o leukemias by autologous(自体自体)bone marrow rescue.Treatment of leukemia with allogeneic(同种异体同种异体)bone marrow transfer.Table 3.Cytokine therapy of tumorscytokinetu

12、mor type and resultantitumor mechanism(s)IFN-alpha,beta remission of hairy cell leukemia,weak effect on some carcinomasincreased expression of class I MHC,possible cytostatic anti-tumor effect,IFN-gamma remission of peritoneal carcinoma of ovary:ineffective systemically increased MHC antigens;macrop

13、hage,Tc and NK cell activationIL-2 remission in renal carcinoma and melanoma T-cell proliferation and activation,NK cells activationTNF-alpha can reduce malignant ascites macrophage and lymphocyte activationTumor celltransfection肿瘤基因治疗系指将外源的正常基肿瘤基因治疗系指将外源的正常基因导入人体因导入人体,达到直接或间接抑制达到直接或间接抑制或杀伤肿瘤细胞的目的。或

14、杀伤肿瘤细胞的目的。1、细胞因子基因治疗 TNF-基因治疗计划,从晚期黑色素细胞瘤患者瘤体内分离出TIL细胞,转染克隆有TNF-基因逆转录病毒载体,再回输给患者体内,期望TIL所表达分泌的TNF-能增强其杀瘤作用.IL-2、IL-4、IL-6、IL-7、TNF、GM-CSF和IFN等 2、导入MHC基因 通过基因转染可使肿瘤细胞MHC-1类抗原增加,从而降低小鼠肿瘤细胞的致瘤能力,肿瘤致瘤能力降低被认为是肿瘤细胞上肿瘤特异性多肽表达增多,从而引起体内CD8+T细胞对肿瘤的排斥。3、以抑癌基因作为目的基因的基因治疗方法 目的基因即弥补缺损基因及纠正异常基因的外源性正常基因序列,包括标志基因(ma

15、rker genes)、反义结构基因、免疫刺激性细胞因子基因、病毒酶类基因、药物抗性基因、组织相容性复合体基因等。这些基因无论整合染色体还是游离细胞浆中皆能稳定地随细胞分裂而复制。4、药物敏感性基因的选择及在肿瘤基因治疗中的作用 细菌和真菌能产生胞嘧啶脱氧氨酶(CD),CD能催化5-氟胞嘧啶(5-FCYT)转化为5-氟尿嘧啶(5-FU),后者为具有高度的细胞毒性的化疗药物,能抑制RNA和DNA的合成而致细胞死亡。CDHSV-tkCD/HSV-tkExpression of CD/HSV-tkTumor celltransfectionInjection of GCV(丙氧鸟苷丙氧鸟苷),5-氟

16、胞嘧啶氟胞嘧啶(5-FCYT)自杀基因疗法自杀基因疗法:单纯疱疹病毒胸苷激酶基因单纯疱疹病毒胸苷激酶基因(HSV-tk)(HSV-tk)细菌胞嘧啶脱氨酶基因细菌胞嘧啶脱氨酶基因(CD)(CD)几个特点几个特点:1通过主动免疫能够激发全身性的抗肿瘤效应通过主动免疫能够激发全身性的抗肿瘤效应,特别适用于多特别适用于多发病灶或有广泛转移的恶性肿瘤发病灶或有广泛转移的恶性肿瘤;2 主动免疫治疗通过调动机体自身的力量达到抗肿瘤作用主动免疫治疗通过调动机体自身的力量达到抗肿瘤作用,与放与放疗和化疗相比疗和化疗相比,副作用很小副作用很小;3 由于部分肿瘤表达的抗原是自身由于部分肿瘤表达的抗原是自身组织的正常

17、成分组织的正常成分,所以用该抗原进所以用该抗原进行主动免疫可能打破自身免疫耐受行主动免疫可能打破自身免疫耐受而导致自身免疫性疾病的发生。而导致自身免疫性疾病的发生。肿瘤组织肿瘤组织免疫免疫肿瘤肿瘤接种接种排斥肿瘤排斥肿瘤接种接种肿瘤生长肿瘤生长20世纪世纪20年代年代肿瘤特异性抗原肿瘤特异性抗原?建立建立基本一致的近交系小鼠基本一致的近交系小鼠肿瘤组织肿瘤组织免疫免疫肿瘤肿瘤接种接种排斥肿瘤排斥肿瘤接种接种肿瘤生长肿瘤生长MHC MHC MHC MHC MHC The specificity of immunity induced by tumors(同系小鼠同系小鼠)化学致癌剂甲基胆恩化学致

18、癌剂甲基胆恩(Methylchoanthrene,MCA)20世纪世纪50年代年代排斥肿瘤排斥肿瘤肿瘤不被排斥肿瘤不被排斥Tumor specific transplantation antigen TSTA(1)黑色素瘤抗原黑色素瘤抗原:特异性的黑色素瘤抗原特异性的黑色素瘤抗原(2)基因突变产生的肿瘤抗原基因突变产生的肿瘤抗原,包括癌基因如包括癌基因如ras、抑癌、抑癌基因如基因如p53等等p53,a tumor suppressor molecule基因突变产生的肿瘤抗原基因突变产生的肿瘤抗原Onco-fetal antigens(癌胚抗原癌胚抗原)Two major oncofetal

19、antigens:alpha-fetoprotein(AFP)(甲胎蛋白甲胎蛋白)-0-20 ng/ml.carcino-embryonic antigen(癌胚抗原癌胚抗原)(CEA)-2.5 ng/ml(抗原抗原)+特异性特异性单克隆抗体单克隆抗体(一抗一抗)+同位素标记的兔同位素标记的兔(羊羊)抗人抗体抗人抗体(二抗二抗)TTAGGG TTAGGG TTAGGG102-104 chromosomeTELTELTelomerase enzymeAAUCCChTRhTERT(EST/TRT)hTLP1Catalytic subunit,Reverse transcriptase activi

20、tySSL/GG 2000Telomerase catalytic subunit=universal tumor antigen?Cell linesTissueTissue of origin NormalTumorSkin0/58/8Breast0/822/22Lung0/318/18Cervix0/13/3Bladder0/13/3Prostata0/22/2Joint0/1Uterus0/1Connective1/1Adipose1/1Stomach1/1Pancreas3/3Ovary5/5Kidney8/8Colon7/7CNS3/3Blood9/9Tissue typeTumo

21、rAdj.tissueColon8/80/7Squamous carc.14/166/16Kidney(Wilms)6/62/6Breast18/202/20Prostate2/2Neuroblastoma5/5Brain6/8Lung4/4Lymph.leuk.14/16Lymphoma5/5NormalFetal testis2/2Adult testis1/1Fetal ovary2/2Ovarian follicle1/1Breast0/8Prostate0/8肿瘤相关移植抗原肿瘤相关移植抗原:病毒诱导的抗原病毒诱导的抗原(4)致瘤病毒的抗原成分致瘤病毒的抗原成分Tumor assoc

22、iated transplantation antigens(TATA)on viral tumorsA number of viruses cause different types of tumors HTLV-1 in leukemia(白血病白血病),hepatitis-B virus in hepatic carcinoma(肝癌肝癌),papilloma virus in cervical cancer(宫颈癌宫颈癌)1 肿瘤细胞疫苗肿瘤细胞疫苗2 多肽疫苗多肽疫苗3 表达肿瘤抗原的重组病毒和表达肿瘤抗原的重组病毒和DNA疫苗疫苗:4 APC为基础的肿瘤疫苗或称为为基础的肿瘤疫苗或

23、称为APC疫苗疫苗5 独特型与抗独特型疫苗独特型与抗独特型疫苗6 细胞因子基因转导的肿瘤疫苗细胞因子基因转导的肿瘤疫苗7 MHC和和B7分子转基因的肿瘤疫苗分子转基因的肿瘤疫苗Evidence for immunity against malignancy comes mostly from experimental tumors,although there is ample evidence for anti-tumor immune reactivity in humans.In experimental studies,animals can be immunized by admin

24、istering inactivated tumor cells or by removal of a primary tumor.Also,immunity can be transferred from an animal,in which a tumor has regressed,to a naive animal by injection of lymphocytes(T cells).All components of the immune system(nonspecific and specific;humoral and cellular)can affect the gro

25、wth and progression of a tumor.Immunity against tumors特异性信号和协同刺激特异性信号和协同刺激Tumor cellT cellImmunotherapy by transfection with costimulatory molecules.Immunotherapy by transfection with costimulatory molecules.The tumor can only simulate the resting T cell with the costimulatory help of B7-1 and 2 and

26、/or cytokines such as GM-CSF,-interferon and various interleukins,IK-2,-4 and-7,CTLA-4 blockade enhances immunogenicity.Alternatively,the T-cell can be stimulated directly by tumor antigens presented by dendriitic cells which can themselves be activated by cross-linking their surface CD40 with antib

27、ody.Once activated,the T cell with upregulated accessory molecules can now attack the original tumor lacking costimulators.GM-CSF基因转染基因转染Recruitment of dendritic cells into epidermis by injection of DNA encoding the cytokine GM-CSFP1P2P3P6P7P9P4 P5P8NH3COO A B D C E FCD8+T细胞所识细胞所识别的是别的是MHC-I类类分子呈递的分

28、子呈递的79肽肽Htlv-1 PeptideIdentify T cell epitopes in hTERT(human enzyme Telomere Reverse transcriptase)人的端粒反转录酶人的端粒反转录酶Look for”spontaneous”T cell responses against hTERT peptides in cancer patientsTake into account the extreme polymorphism(多肽性多肽性)of the HLA systemEARPALLTSRLRFIPK (hTERT RT site peptid

29、e p545)EARPALLTSR EARPALLTS EARPALLT ARPALLTSRL ARPALLTSR ARPALLTS RPALLTSRLR RPALLTSRL RPALLTSR PALLTSRLRF PALLTSRLR PALLTSRL ALLTSRLRFI ALLTSRLRF ALLTSRLR LLTSRLRFIP LLTSRLRFI LLTSRLRF LTSRLRFIPK LTSRLRFIP LTSRLRFI TSRLRFIPK TSRLRFIP已知蛋白序列已知蛋白序列,并有相应的并有相应的CTL克隆克隆,人工方法合成人工方法合成大量前后重叠的短肽大量前后重叠的短肽(810

30、),再用再用CTL进行筛选进行筛选hTERT分别免疫分别免疫CTLCTL筛选筛选T cell 表位表位(多肽疫苗多肽疫苗)DNA VaccinesPlasmidvaccineDNA VaccinesMCS肿瘤抗原基因肿瘤抗原基因Identification of using tumor-specific cytotoxic T cell ()clones derived from tumor lymphocyte culture.Identification of tumor-specific gene using tumor-specific cytotoxic T cell clones

31、derived from mixed tumor lymphocyte culture.克隆性抗原克隆性抗原具有独特性决定簇的膜表面免疫球蛋白分子具有独特性决定簇的膜表面免疫球蛋白分子淋巴细胞肿瘤淋巴细胞肿瘤特异性肿瘤抗原特异性肿瘤抗原抗独特型抗体抗独特型抗体被动免疫治疗被动免疫治疗肿瘤的消退肿瘤的消退Tumor cell:Complement-mediated lysisutilizing dendritic cells pulsed with idiotype from a B-cell lymphoma(patients chest)paracardiac mass(a)prevaccine(b)10 months after completion of three vaccine treatments.