肿瘤免疫研课程班

肿瘤免疫Tumor Immunity,肿瘤抗原机体抗肿瘤的免疫机制肿瘤的免疫逃逸机制肿瘤的免疫治疗,Model of sequential genetic alterations leading to metastatic colon cancer. Each of the stages indicated at the bottom is morphologically distinct, allowing researchers to determine the sequence of genetic alterations.,多次打击模型(Multihit Model),一、肿瘤特异性抗原二、肿瘤相关性抗原,肿瘤抗原 (Tumor Antigens),化学和物理致癌因素诱发的肿瘤抗原病毒诱发的肿瘤抗原 癌基因和突变型抑癌基因表达的肿瘤抗原 正常静止基因表达的肿瘤抗原,一、肿瘤特异性抗原,Mice were induced to produce tumors by the injection of a chemical carcinogen (methyl-cholanthrene). Tumor cells from these mice were then injected subcutaneously into genetically identical mice. Later, the growing tumor were removed surgically. Mice challenged with the same tumor were able to reject it, but those challenged with a different tumor (induced with the same carcinogen) were not. The ability to reject the tumor could be transferred with lymphoid cells.,化学和物理(理化)致癌因素诱发的肿瘤抗原,Experimental induction of immunity against tumor cells induced by polyoma virus(PV),病毒诱发的肿瘤抗原,癌基因和突变型抑癌基因表达的肿瘤抗原,在不同致癌因素和特定条件下，原癌基因可被异常激活，抑癌基因发生突变，导致正常细胞癌变，并能检测到异常表达产物或相应的编码蛋白。这类蛋白在胞内经处理分解为不同小肽，通过MHC I类分子递呈可作为肿瘤特异性抗原肽被T细胞所识别，激活CTL反应。,Wild-type p53 is required to restrain cell growth. Its activity may be lost by deletion of both wild-type alleles or by a dominant mutation in one allele.,突变的抑癌基因编码蛋白,Activation of a growth factor receptor involves ligand binding, dimerization, and autophosphorylation, A truncated oncogenic receptor that lacks the ligand-binding region is constitutively active because it is not repressed by the N-terminal domain.,癌基因异常激活蛋白,Pathways that rely on Ras could function by controlling either GNRF or GAP. Oncogenic Ras mutants are refractory to control, because Ras remains in the active form.,Translocations between chromosome 22 and chromosome 9 generate Philadephia chromosomes that synthesize bcl-abl fusion transcripts that are responsible for two types of leukemia.,机体中某些染色体易位形成新的癌基因，所编码蛋白由于融合点上出现新的氨基酸顺序和形成新的空间构象，仅在恶性细胞中表达，并与细胞的恶性转化密切相关, 是T细胞识别的特异性肿瘤抗原。,染色体易位产生的融合蛋白,Alteration Function of protein Tumor typePoint mutationERB B2 Growth factor receptor Breast carcinomaFMS CSF-1 receptor AML, myelodysplasiaRas GTP-binding protein Carcinomas and othersp53 Tumor suppressor cell cycle control Many including bladder, colon, lungRB1 Tumor suppressor cell cycle control Retinoblastoma, osteosarcoma Pancreatic carcinomaChromosomal translocationBCR-ABL Tyrosine kinase CML, ALLEZA-PRL Transcription factor Pre-B cell ALL H4-RET Growth factor receptor/ tyrosine kinase Thyroid carcinomaTPR-MET Growth factor receptor/ tyrosine kinase Gastric carcinoma LMYC-RLF Transcription factor Small cell lung carcinomaNPM/ALK Tyrosine kinase LymphomaDeletion mutations ERB-B Growth factor receptor Glimas,Genetic alterations in human tumors producing new protein sequences,正常静止基因表达的肿瘤抗原,肿瘤细胞中某些被T细胞所识别的抗原往往由正常状态下的静止基因(silent genes)所表达，除人的正常睾丸细胞外，这些基因一般只在恶性细胞中被激活而呈异常高表达,又被称为C-T抗原(cancer-testis antigen)；其中,已知MAGE家族至少有14个成员(MAGE-1MAGE-14)，它们之间具有高度同源性(8090)；MAGE家族成员表达特征：在不同肿瘤中有不同程度的表达，多个成员也可在同一肿瘤中表达。同一肿瘤中可测出多种静止基因的表达。 这类基因的编码蛋白经胞质溶胶途径处理成小肽，通过MHC I类分子递呈于肿瘤细胞表面，被CD8+T细胞所识别。,Tumour antigens recognized by T lymphocytes: at the core of cancer immunotherapy. NATURE REVIEWS | CANCER VOLUME 14 | FEBRUARY 2014,Immunogenicity of NY-ESO-1, MAGE-A1, MAGE-A3, and SSX-2CT antigen mRNA Expression frequency Frequency of serum T-cell epitopes antibody in cancer patients CD8 CD4NY-ESO-1 34% Melanoma 9% Melanoma A2 DR4 25% Ovarian Cancer 12% Ovarian Cancer A31 16% Lung Cancer 4% Lung Cancer Cw3 24% Breast Cancer 8% Breast Cancer Cw6MAGE-A1 16% Melanoma 1% Melanoma A1 B35 Cw2 DR13 28% Ovarian Cancer 3% Ovarian Cancer A3 B7 Cw3 DR15 49% Lung Cancer 4% Lung Cancer A24 B53 Cw16 18% Breast Cancer 0 Breast Cancer A28MAGE-A3 36% Melanoma 2% Melanoma A1 B35 DR4 17% Ovarian Cancer 0 Ovarian Cancer A2 B37 DR7 47% Lung Cancer 0 Lung Cancer A2 B40 DR11 11% Breast Cancer 0 Breast Cancer A24 B44 DR13 A24 B52SSX-2 35% Melanoma 1% Melanoma A2 Unknown 0 Ovarian Cancer 0 Ovarian Cancer 17% Lung Cancer 0 Lung Cancer 7% Breast Cancer 0 Breast Cancer,Immunological Reviews 2002; Vol 188: 2232,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161169和230238区域氨基酸序列。,MAGE-1基因、编码蛋白和抗原肽,肿瘤相关性抗原胚胎性抗原分化抗原癌基因高表达的抗原 异常表达的糖脂/糖蛋白抗原,Elevation of AFP and CEA in serum of patients with various diseases,胚胎性抗原 细胞发生恶性转化时，胚胎抗原编码基因可被激活呈异常表达，出现在细胞质、膜表面或分泌在血流中，其蛋白含量与细胞的恶性程度往往呈正相关。 此类抗原一般难以激发机体产生抗体，但发现某些抗原可经胞质溶胶途径处理成抗原肽由MHC I类分子递呈于细胞膜表面，被T细胞识别。,分化抗原,分化抗原是细胞在分化成熟不同阶段出现的抗原，不同来源、不同分化阶段的细胞可表达不同的分化抗原。这些抗原在多种黑色素瘤细胞呈异常高表达、结构高度同源，即很少显示个体差异。异常表达的分化抗原可经胞内途径处理成为抗原肽，通过MHCI或II类分子递呈于细胞表面，被CD8+或CD4+ T细胞所识别。,某些肿瘤细胞癌基因表达产物未发现突变特征仅有表达量的差别。这些过度表达蛋白中某些肽经MHCI类分子递呈于细胞表面可被机体CD8+T细胞所识别，同时在某些患者体内可测出相应的抗体。,癌基因高表达的抗原,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MLLAVLYCL1-9HLA-A2 YMNGTMSQV369-377HLA-A2 AFLPWHRLF(L)HLA-A24 SEIWRDIDF192-200HLA-B44Pmel 17/gp100KTWGQYWQV154-162HLA-A2 ITDQVQGSV209-217HLA-A2 YLEPGPVTA280-288HLA-A2 LLGDTATLRL457-466HLA-A2 VLYRYGSFSV476-485HLA-A2Melan-AMART-1(E)AAGIGILTV26(7)-35HLA-A2 ILTVILGVL32-40HLA-A2gp75TRP1HLA-A31,Overexpression of oncogene-encoded proteins in human tumors,Protein Normal function Tumor known to express high levels of the proteinCyclin D1 Regulator of the G1-S Breast cancer (approx. 20%), Colon cancer (20%) transition via dependent kinase Thyroid cancer, Liver cancer (11-13%) Cyclin E Regulator of the G1-S transition Breast cancer (90%) via bind to cyclin-dependent kinaseMdm2 Nuclear phosphoprotein. Sarcomas (30-36%), Leukaemia (between 42-73%) Inhibits the function of p53 breast cancer cell line, Gliomas (10-15%) by interaction with the proteinReceptor tyrosine kinasesEGF receptor Receptor for the epidermal Breast cancer (30-40%), Lung cancer (80%), Gliomas, growth factor Bladder cancer (70%), Renal cancer (73%), Head and neck cancer (50%) or more, dependent on type)Erb B2 Receptor belonging to the same Breast cancer (26%), Ovarian cancer (20-30%), Stomach (Her-2/neu ) family as the EGFR receptor. Cancer (4%) Non-small-cell lung cancer (up to 30%). Ligand not clearly identified.Nuclear oncogenesC-myc Transcription factor Breast cancer (6-57%), Small-cell lung (20-30%), Cervical cancer (30%), Testicular cancer, Colon cancer and Head and neck cancer.Proteins involved in the regulation of apoptosisBcl-2 inhibits apoptosis Non-Hodgkins lymphoma (40-80%)WT p53 Tumor suppressor protein, Gliomas (astrocytomas) (60-80%) transcription factor can induce Head and neck cancer (34%), Sarcoma (17%), apoptosis in response to DNA damage. Acute myeloid leukaemia (69%),异常表达的糖脂/糖蛋白抗原,某些肿瘤细胞可出现膜结构改变，表达过量或结构异常的糖脂和糖蛋白，其中包括神经节苷脂、血型抗原和粘蛋白等。表面结构改变和异常有助于肿瘤的侵袭和转移。 这类异常的糖脂和糖蛋白可诱发B细胞产生抗体和激发CTL反应。应用相应单抗检测其含量，可为肿瘤的诊断和预后判断提供参考。,机体抗肿瘤的免疫机制Mechanism of Immune Response to Tumor,一、体液免疫抗肿瘤作用二、细胞免疫抗肿瘤作用,免疫系统识别肿瘤细胞表达抗原产生免疫应答，激活效应细胞和释放一系列效应分子，攻击和清除肿瘤细胞。,二、细胞免疫抗肿瘤作用,1.T细胞CD4+T细胞与CD8+T细胞3.NK细胞4.巨噬细胞,CD4+T细胞,Tumor,Cathepsins(blue) LFA-1(green) Talin(red),CD8+T细胞,杀伤肿瘤细胞过程分为效靶细胞结合，攻击杀伤和靶细胞裂解。显示对肿瘤细胞效应功能具有高度的特异性和有效性。,NK细胞,特点 NK细胞是一类对多种靶细胞自发性细胞毒活性的淋巴细胞谱系的特殊亚群，不表达T、B细胞特有表面标志物(TCR、BCR、CD4和CD8等)，人类NK细胞表达CD16和CD56等分化抗原，占外周淋巴细胞的10-15%。,杀伤靶细胞的机制 抗体依赖的细胞介导的细胞毒作用 受体介导杀伤作用,受体介导杀伤作用,受体介导的NK细胞激活及其对肿瘤细胞的杀伤机制 MHC I类分子是KIR配体，相互作用产生抑制性信号，可抑制NK细胞激活。当肿瘤细胞I类分子表达下降或缺陷，缺乏炎症信号，抑制NK细胞激活，发挥杀伤效应。,抗体依赖的细胞介导的细胞毒作用(ADCC),巨噬细胞,巨噬细胞参与非特异性免疫和特异性免疫，杀伤肿瘤细胞机制主要为：吞噬和杀伤作用；介导炎症反应；释放细胞因子参与免疫调节；加工递呈抗原、启动免疫应答；参与ADCC释放效应分子杀伤靶细胞。,一、肿瘤细胞免疫原性低下二、免疫增强三、效应细胞的功能异常四、肿瘤微环境中的免疫抑制细胞和相关分子,肿瘤的免疫逃逸机制Mechanism of Tumor Evasion of the Immune System,一、肿瘤细胞免疫原性低下,1.抗原表达不稳定性2.MHC I类分子表达异常3.肿瘤细胞抗原加工处理缺陷4.缺乏共刺激信号,Low immunogenicity and antigenic modulation,遗传不稳定性和抗原表达异质性抗原调变,抗原表达不稳定性,Nature Rev. Cancer Vol.15, 473-483(2015),MHC I类分子表达异常,肿瘤细胞表面MHC类分子表达下降或丢失2微球蛋白表达异常,肿瘤细胞异常的HLA表型,A.正常细胞HLA I类分子表型(举例)。肿瘤细胞异常HLA I类分子表型：B.全部丢失，包括HLA-A、B、C；C.HLA一条单元型丢失，即HLA-A、B、三位点均丢失一半等位基因；D.某一位点丢失(此处为HLA-B或A位点);E.某一等位基因丢失(此处为HLA-B44)。,参见表18-5,某些病毒在肿瘤细胞内可抑制蛋白酶体活性，阻断TAP转运，干扰MHC分子合成及其与抗原肽的结合等，从而影响MHC分子-抗原肽复合物在肿瘤细胞膜上的表达和CD8 CTL对肿瘤细胞的识别。,肿瘤细胞抗原加工处理途径的缺陷,多种病毒干扰肿瘤细胞抗原加工处理途径,参见表18-6,缺乏共刺激信号,协同刺激信号（B7分子）与T细胞增殖、活化,（A）肿瘤细胞缺乏B7分子，不能有效激活T细胞；（B）转染B7基因的肿瘤细胞表达B7分子后可激活CTL显示对肿瘤细胞的细胞毒活性。,二、免疫增强 在实验中发现，给荷瘤动物输入抗肿瘤免疫血清可促进肿瘤细胞的生长，称之为免疫增强。（削弱机体的抗肿瘤能力，从而有利于肿瘤细胞逃避效应细胞的识别和攻击）。现认为，免疫增强是由于血清中存在封闭因子(blocking factor)，后者遮盖了肿瘤细胞表面的抗原决定簇。肿瘤的免疫增强不仅与封闭因子有关，也可能涉及某些淋巴细胞。在过继性免疫治疗中发现，某些致敏淋巴细胞过继性注入带瘤宿主，对肿瘤细胞出现刺激而非抑制其生长。这些淋巴细胞有可能属于抑制性细胞。,三、效应细胞的功能异常,1.T细胞信号转导途径缺陷2.细胞因子产生异常引起Th1/Th2细胞漂移,Abnormal and deficient of TCR-mediated signal transduction,T细胞信号转导途径缺陷,参见表18-7,Two subsets of CD4+ T cells each regulation by producing cytokines,细胞因子产生异常引起Th1/Th2细胞漂移,髓样抑制性细胞M2巨噬细胞调节性T细胞肿瘤细胞分泌免疫抑制因子肿瘤微环境中存在多种蛋白酶和相关分子,四、肿瘤微环境中的免疫抑制细胞和相关分子,在肿瘤微环境中VEGF等因子诱导作用下，髓样抑制细胞（MSC）通过分泌精氨酸酶、ROS和IL-10，下调效应T细胞的杀伤活性, 抑制DC细胞成熟。IL-10也可抑制巨噬细胞分泌IL-12，从而降低NK细胞的活性。,髓样抑制细胞发挥免疫抑制作用的可能机制,髓样抑制性细胞,巨噬细胞,TGF-, IL-6, PGE2, COX-2,M2巨噬细胞,肿瘤细胞以及肿瘤浸润的巨噬细胞分泌CCL22及H铁蛋白，招募初始的CCR4+Treg细胞在肿瘤部位积聚，Treg细胞与耐受性DC细胞相互作用下，抑制效应T细胞的功能。,Treg细胞的诱导和免疫抑制作用,调节性T细胞,肿瘤细胞产生的免疫抑制因子和表达蛋白对效应细胞的作用,-,-,肿瘤细胞分泌免疫抑制因子,FasL and the possible fates of antitumor CTLs,肿瘤微环境中存在多种蛋白酶和相关分子,肿瘤的免疫治疗（Tumor immunotherapy）,一、肿瘤的主动免疫治疗二、肿瘤的被动免疫治疗,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。,1.非特异性主动免疫疗法2.特异性主动免疫疗法,肿瘤的主动免疫治疗,非特异性刺激因子 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的目的。目前常用的有卡介苗(BCG)、短小棒状杆菌(PV)、左旋咪唑(Levamisole，LMS)和寡聚脱氧核苷酸(CpG)等。,非特异性主动免疫疗法,细胞因子,特异性主动免疫疗法,处理的瘤细胞作为疫苗,肿瘤抗原(肽)和人工合成肽抗原作为疫苗,Enhancement of tumor cell immunogenicity by transfection of costimulator and cytokine genes. Tumor cells that do not adequately stimulate T cells on transplantation into an animal will not be rejected and will therefore grow into tumors. Vaccination with tumor cells transfected with genes encoding costimulators or cytokines, such as IL-2, can lead to enhanced activation of T cells. This approach of using transfected tumor cells as vaccines has worked in mouse models, but clinical trials have not yet been successful.,Tumor vaccines. Two types of tumor vaccines that have shown efficacy in clinical trials and animal models are illustrated. Autologous dendritic cells are prepared from patients own peripheral blood cells. The dendritic cells are either pulsed with recombinant protein or transfected with a gene construct that expresses the protein. The construct may also express costimulatory molecules (not shown).,基因工程疫苗,Use of DNA vaccines raises both humoral and cellular immunity,DNA疫苗,DC治疗性疫苗,DC是专职抗原提呈细胞，负责对抗原进行加工处理后提呈给T细胞，诱导T细胞的活化和增殖，激发有效的免疫应答。由目前基于DC 的治疗性肿瘤疫苗，主要分为两种，一是DC体外分离培养、扩增鉴定后，荷载特定抗原回输到患者体内，二是诱导DC在体内增殖、成熟，有效摄取特定肿瘤抗原提呈并激活T细胞。二者的共同目的都是最大程度活化肿瘤抗原特异性CD4 T和CD8 T细胞，发挥抗肿瘤效应。目前认为，单纯DC疫苗虽有疗效但仍须改进，需要寻找更为合适的肿瘤抗原、更有效地促进DC增殖活化的方法或者与其他方法联合使用。需在整体设计实施合理，以保证在促进DC抗肿瘤效应，同时注意检测负向调节方面的变化。,Appropriate utilization and regulation of DCs in vaccine design induce a much more potent CTL antitumor immune response. (a) Tumor antigen-loading techniques activate DCs ex vivo. (b) Targeted drugs facilitate the capture of tumor antigens by DCs and the expression of costimulatory molecules and MHC-II in vivo. (c) Stimulatory adjuvants induce maturation of DCs and enhance the activation of CTLs.,An effective vaccine against human papilloma virus (HPV) induces antibodies that protect against HPV infection. Serotype 16 of HPV (HPV-16) is highly associated with the development of cervical cancer. In a clinical trial, 755 healthy uninfected women were immunized with a vaccine generated from highly purified noninfectious virus-like particles (VLP) consisting of the capsid protein L 1 of HPV-16 and formulated with an alum adjuvant (in this case aluminum hydroxyphosphate sulfate). In comparison with the very low titers of antibody in placebo-treated uninfected women (green line), or women previously infected with HPV that received placebo (blue line), the women treated with the virus-like particle vaccine (red line) developed high titers of antibody against the L 1 capsid protein. None of these immunized women subsequently became infected by HPV-16. An anti-HPV vaccine marketed as Gardasil is now available and recommended for use in girls and young women as a protection from cervical cancer caused by HPV serotypes 6, 11, 16, and 18.,肿瘤的被动免疫治疗 1.过继性免疫疗法 2.抗体导向疗法,过继性免疫疗法 过继性免疫疗法是指把自身或异体的具有抗肿瘤活性的免疫血清或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。淋巴因子激活的杀伤细胞（LAK） 肿瘤浸润性淋巴细胞（TIL）,LAK细胞的制备和应用,Before,After,Treatment of melanoma with LAK cells and IL-2,淋巴因子激活的杀伤细胞,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（主要为IL-2）诱导后发生扩增，产生一类能非特异性地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（lymphokine activated killer cell）, 简称LAK细胞。 LAK细胞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。,TIL细胞的制备和应用,Before,After,Treatment of Melanomas with TIL + IL-2,肿瘤浸润性淋巴细胞,（tumor-infiltration lymphocytes, TIL）,嵌合抗原受体(CAT),Examples of tumor antigens that have been targeted by monoclonal antibodies in therapeutic trials.,抗体导向疗法,人源化抗体,A制备抗肿瘤的抗体与效应分子（毒蛋白、化疗药物或放射性同位素）偶联物，利用抗体特异性识别肿瘤抗原作用，使效应分子有效地到达肿瘤部位，选择性杀伤肿瘤细胞。B. 双特异性抗体中包含着两种不同识别特异性抗原的Fab段，通过特异结合肿瘤抗原同时结合不同效应细胞和分子，达到有效杀伤肿瘤的作用。,抗体偶联物/双特异性抗体的抗肿瘤作用机制,双特异性抗体,抗体偶联物,Mechanisms of tumour cell killing by antibodies. a | Direct tumour cell killing can be elicited by receptor agonist activity, such as an antibody binding to a tumour cell surface receptor and activating it, leading to apoptosis (represented by the mitochondrion). It can also be mediated by receptor antagonist activity, such as an antibody binding to a cell surface receptor and blocking dimerization, kinase activation and downstream signalling, leading to reduced proliferation and apoptosis. An antibody binding to an enzyme can lead to neutralization, signalling abrogation and cell death, and conjugated antibodies can be used to deliver a payload (such as a drug, toxin, small interfering RNA or radioisotope) to a tumour cell. b | Immune-mediated tumour cell killing can be carried out by the induction of phagocytosis; complement activation; antibody-dependent cellular cytotoxicity (ADCC); genetically modified T cells being targeted to the tumour by single-chain variable fragment (scFv); T cells being activated by antibody-mediated cross-presentation of antigen to dendritic cells; and inhibition of T cell inhibitory receptors, such as cytotoxic T lymphocyte-associated antigen 4 (CTLA4). c | Vascular and stromal cell ablation can be induced by vasculature receptor antagonism or ligand trapping (not shown); stromal cell inhibition; delivery of a toxin to stromal cells; and delivery of a toxin to the vasculature. MAC, membrane attack complex; MHC, major histocompatibility complex; NK, natural killer.,Antibody therapy of cancer. NATURE REVIEWS.CANCER VOLUME 12 APRIL 2012,Strategies to inactivate and/or deplete CD4+CD25+ Treg cells in cancer.Based on the currently available evidence in favour of Treg-mediated suppression of anti-tumor T-cell responses, several strategies to inactivate and/or deplete Tregs have been evaluated in pre-clinical models of cancer. Chemotherapeutic drugs such as CY and fludarabine interfere with Treg functions. Ab directed against Ag assigned to Treg cells (CD25, GITR, CTLA-4, CD4) deplete and/or functionally inhibit Tregs, thus contributing to therapeutic efficacy of concomitantly administered chemotherapy and/or anti-tumour vaccines. Finally, inhibition of IDO, an enzyme involved in tryptophan catabolism and Treg induction and whose expression might be high in cancer-bearing hosts, is a novel and promising avenue to break tolerance and restore effective anti-tumor responses.,Targeted agents may antagonize immunosuppression in the tumour microenvironment. Multiple factors within tumours promote immune tolerance and curb the anti-tumour immune response. Tumour cells secrete