Antigen-specific T-cell activation--cancer /AIDS vaccine

抗原特异性T细胞激活--癌症/艾滋病疫苗

• 批准号: 6558253
• 负责人: JAY A BERZOFSKY
• 金额: --
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6558253
• 关键词: AIDS therapy; AIDS vaccines; HIV envelope protein gp160; MHC class I antigen; Macaca mulatta; T cell receptor; biotechnology; cellular immunity; clinical trial phase I; cytotoxic T lymphocyte; drug design /synthesis /production; epitope mapping; human subject; human therapy evaluation; immunotherapy; interleukin 13; laboratory mouse; leukocyte activation /transformation; neoplasm /cancer immunotherapy; neoplasm /cancer vaccine; neutralizing antibody; patient oriented research; synthetic vaccines; vaccine development; virus infection mechanism

We studied mechanisms for T cell recognition of antigens in association with major histocompatibility complex (MHC)-encoded molecules, and applications to the design of synthetic vaccines for AIDS and cancer. We have been characterizing the helper and cytotoxic T lymphocyte (CTL) responses to HIV envelope and reverse transcriptase, mapping the key epitopes, and defining the role of individual residues in these epitopes to be able to modify the structures to make more potent immunogens as vaccines. We have made vaccine constructs in which clusters of helper epitopes are synthesized coupled to a peptide that is a CTL epitope presented promiscuously by multiple class I MHC molecules in the human and mouse as well as a neutralizing antibody epitope. These constructs can induce all three arms of the immune response, neutralizing antibodies, CTL, and Th1 helper cells. Results of the first arm of a phase I clinical trial with one of these peptides show ability to induce CTL, helper T cell responses, and neutralizing antibodies to HIV in at least a subset of human recipients. Meanwhile, we are developing new approaches in mouse models to develop second generation vaccine constructs. We have shown proof of principle that we can modify the sequence of a helper epitope of HIV to make it more immunogenic and also much more potent, when coupled to a CTL epitope, in eliciting CTL and protecting against viral infection. The enhanced helper epitopes elicit a stronger Th1 response and upregulate CD40L on the helper cells, which in turn induce more IL-12 production by dendritic cells, which then polarize the T helper cells to Th1. We are applying this "epitope enhancement" approach to conserved HIV helper and CTL epitopes from env, gag, and pol, presented by human class II and class I HLA molecules, as well as to hepatitis C virus (HCV) epitopes presented by human HLA-A2.1 (see below). We have discovered ways of increasing CTL, helper, and antibody responses and steering them toward desired phenotypes, such as Th1 or Th2 or particular antibody isotypes, by incorporating cytokines into the emulsion adjuvant with the antigen. We compared a panel of 8 cytokines for their effects on 8 types of immune response, and discovered a novel synergy between GM-CSF and IL-12 and between TNF and IL-12 in induction of CTL. We found that all 3 cytokines provide triple synergy for induction of CTL with a peptide vaccine, for induction of interferon-gamma, and for protection against viral challenge in vivo, which we show to be interferon-gamma dependent. The mechanism of this synergy appears to relate to the upregulation of antigen presenting function and cytokine receptors. We have shown that high avidity CTL specific for HIV-1 envelope peptide are much more effective at clearing a recombinant vaccinia virus expressing HIV gp160 from SCID mice than are low avidity CTL specific for the same peptide-MHC complex, and have worked out two complementary mechanisms involving the ability of high avidity CTL to kill cells earlier in virus infection before viral progeny are produced, and to lyse targets more quickly. However, we found that high avidity CTL are exquisitely sensitive to high dose antigen and will undergo programmed cell death, mediated by TNF and the TNF receptor II, but also requiring a permissive state involving a decrease in Bcl-2, IAP1, and TRAF2, and correlating with downmodulation of the T cell receptor. This effect may explain clonal exhaustion in viral infections. We have shown for the first time that protection against mucosal transmission of virus can be mediated by CD8 CTL without antibodies, but requires that the CTL be present at the mucosal site of transmission, whereas systemic CTL are not sufficient. The protection can be accomplished by intrarectal immunization with a peptide vaccine and increased by inclusion of IL-12 and GM-CSF with the vaccine. We found that endogenous IL-12 is less inhibited by the mucosal adjuvant LT(R192G) than by cholera toxin, and substituting this, the mucosal CTL response and protection are less dependent on exogenous IL-12. Using this mutant LT, we immunized MamuA*01-positive Rhesus macaques intrarectally with a similar peptide vaccine and induced CTL in the colon and mesenteric lymph nodes that have impacted the clearance of virus after intrarectal challenge with pathogenic SHIV-Ku. Intrarectal immunization was more effective than subcutaneous immunization with the same peptide vaccine at protecting against SHIV, in part because we found the induction of mucosal CTL provided for greater clearance from a major site of virus replication, the gut mucosa, which was seeding the bloodstream. With regard to cancer, we identified several CTL epitopes in proteins of hepatitis C virus (HCV), that causes liver cancer, using a novel approach, and have analyzed the role of each amino acid residue in order to modify one of the peptides to make a more potent vaccine. Using this epitope enhancement approach, we could increase the immunogenicity of an epitope of the HCV core protein, presented by the most common human class I HLA molecule, HLA-A2.1, both for HLA-A2.1-transgenic mice in vivo and for human T cells in vitro. This enhanced epitope is being incorporated into a vaccine. We are attempting to enhance other HCV core epitopes to incorporate into a DNA vaccine. We also developed a model of immunosurveillance of cancer in which tumors are rejected by CD8 T cells, but the rejection is incomplete in the presence of normal CD4 regulatory cells, and an escape variant of the tumor recurs. However, depletion of CD4 cells allows complete eradication of the tumor by CD8 cells. Using receptor knock-out mice, we found that the key regulatory cytokine inhibiting immunosurveillance against cancer was IL-13, acting through the IL-4 receptor/STAT6 pathway, although IL-4 itself was neither necessary nor sufficient. We discovered that the major source of IL-13 was NKT cells, and that absence of these in CD1-knockout mice prevented tumor recurrence in these mice. We have recently found that this regulatory pathway applies to other tumor models, and we are also determining the mechanism by which IL-13 indirectly inhibits CD8 T cell-mediated immunosurveillance when the CD8 T cells do not have IL-13 receptors. We are also developing clinical trial approaches to amplify immunotherapy of cancer by inhibiting IL-13. We developed peptide cancer vaccines inducing CTL immunity to mutant p53 expressed in cancer cells. We found that mutant p53 peptides, coated on dendritic cells, elicit CTL that kill tumor cells expressing the mutation and suppress established tumors in animals. Common mutations in ras peptides were found to enhance binding to HLA-A2.1, but also to influence antigen processing. We are applying epitope enhancement to the mutant ras peptides to make more effective vaccines. We also induced murine CTL against fusion proteins from chromosomal translocations in pediatric tumors, alveolar rhabdomyosarcoma and Ewing's sarcoma. We also identified novel epitopes spanning these fusion protein junctions in these sarcomas, synovial sarcoma, and others, that could bind to several human HLA molecules, HLA-A1, A3, B7 and B27, and were able to map a minimal epitope in synovial sarcoma presented by HLA-B7 and elicit human CTL that could kill human sarcoma tumor cells, proving that these fusion proteins are promising tumor antigens for cancer immunotherapy. 29 patients were treated in a phase I/II clinical trial of the mutant p53/ras peptide vaccine approach to treating cancer, and a large fraction have made CTL or cytokine responses, and no adverse effects were seen. A trial of translocation fusion peptide immunization of patients with alveolar rhabdomyosarcoma and Ewing's sarcoma is underway. We have also started a trial of immunization of cervical cancer patients with peptides from the E6 and E7 oncoproteins of human papillomavirus type 16 that bind to HLA-A2.1 in patients who express this HLA molecule. Many patients made CTL responses, and some had unexpectedly stable disease. A phase II trial of autologous dendritic cells pulsed with mutant ras peptides corresponding to the patient's tumor in colon cancer patients with HLA-A2.1 that can present these ras peptides is underway. This protocol and a new one that just opened to treat non-small cell lung cancer patients with autologous mutant p53 peptide-pulsed dendritic cells have been modified to mature the dendritic cells with CD40 ligand, which has recently been made available. This should greatly improve immunogenicity of the peptide-dendritic cell vaccine. The second patient treated had a CTL response and a mixed clinical response. (50% AIDS related)

我们研究了与主要组织相容性复合物（MHC）编码分子相关的T细胞识别的机制，并应用于艾滋病和癌症的合成疫苗设计。我们一直在表征助手和细胞毒性T淋巴细胞（CTL）对HIV包膜和逆转录酶的反应，映射关键表位，并确定各个残基在这些表位中的作用，以便能够修饰结构以使疫苗成为更多有效的免疫原。我们制造了疫苗构建体，其中将辅助表位的簇簇合成与肽偶联，该肽是人类和小鼠中多个I类MHC分子以及中和抗体表位的CTL表位。这些构建体可以诱导免疫反应的所有三个臂，中和抗体，CTL和Th1辅助细胞。 I期临床试验的第一臂的结果，其中一项肽表明在至少一部分人类受体中诱导CTL，辅助T细胞反应以及对HIV的中和抗体的能力。同时，我们正在开发小鼠模型中的新方法，以开发第二代疫苗构建体。我们已经显示了原理证明，即我们可以修改HIV的辅助辅助表位的序列，从而使其更加免疫原性，并且在耦合到CTL表位时，可以引起CTL并保护病毒感染。增强的辅助辅助表位会引起更强的Th1响应，并在辅助细胞上上调CD40L，进而诱导树突状细胞的更多IL-12产生，然后将T辅助细胞偏振至TH1。我们正在将这种“表位增强”方法应用于ENV，GAG和POL的保守HIV助手和CTL表位，由人类II类和I类HLA分子提出，以及乙型肝炎病毒（HCV）表位，由人类HLA-A2.1提出。我们发现了增加CTL，助手和抗体反应的方法，并将其转向所需的表型，例如Th1或Th2或特定的抗体同型，或通过将细胞因子掺入与抗原的乳液辅助剂中。我们比较了一组8个细胞因子对8种免疫反应的影响，并发现了GM-CSF和IL-12之间的新型协同作用以及TNF和IL-12之间的CTL诱导。我们发现，所有3种细胞因子都提供了三重协同作用，可用于用肽疫苗诱导CTL，用于诱导干扰素 - 伽玛以及在体内防止病毒攻击的保护，我们表明这是依赖性的γ-γ。该协同作用的机制似乎与抗原表现功能和细胞因子受体的上调有关。我们已经表明，与HIV-1包膜肽相比，对HIV-1包膜肽特异性的高流相关CTL在清除SCID小鼠中表达HIV HIV GP160的重组疫苗的病毒更有效，而不是针对同一肽-MHC复合物特异性的低ctl，并且已经杀死了较高的互补机制，以前杀死了两个互补机制。目标更快。但是，我们发现高剂量抗原的高潮CTL非常敏感，并将经历由TNF和TNF受体II介导的程序性细胞死亡，但也需要允许的状态，涉及Bcl-2，IAP1和TRAF2的降低，并与T细胞受体下降相关。这种作用可能解释了病毒感染的克隆疲劳。我们首次表明，CD8 CTL无抗体可以介导防止病毒的粘膜传播，但要求CTL存在于传播的粘膜部位，而全身CTL则不足。可以通过肽疫苗内直肠免疫来实现该保护，并通过将IL-12和GM-CSF纳入疫苗中增加。我们发现，内源性IL-12受粘膜辅助LT（R192G）的抑制作用要小于霍乱毒素，而取代此症状，粘膜CTL反应和保护较少依赖于外源IL-12。使用该突变型LT，我们对MAMUA*01阳性恒河猕猴在内部用相似的肽疫苗内部直立型猕猴，并在结肠和肠系膜淋巴结中诱导CTL，这些淋巴结在与致病性Shiv-ku的直肠挑战后影响了病毒的清除。直肠内免疫比皮下免疫更有效，在预防SHIV时具有相同的肽疫苗，部分原因是我们发现诱导粘膜CTL可从病毒复制的主要部位，肠粘膜的主要清除，这是肠粘膜，这是在血液中播种。 关于癌症，我们确定了丙型肝炎病毒（HCV）中引起肝癌的几个CTL表位，它们使用一种新方法，并分析了每个氨基酸残基的作用，以修改其中一种肽以制造更有效的疫苗。使用这种表位增强方法，我们可以提高由最常见的人类I类HLA分子HLA-A2.1提出的HCV核心蛋白表位的免疫原性，均用于HLA-A2.1- TRANSGENIC小鼠体内的HLA-A2.1-转基因小鼠。这种增强的表位被掺入疫苗中。我们正在尝试增强其他HCV核心表位以掺入DNA疫苗中。我们还开发了一种癌症免疫监视的模型，其中肿瘤被CD8 T细胞拒绝，但是在存在正常CD4调节细胞的情况下，排斥反应是不完整的，并且肿瘤的逃脱变体又出现了。但是，CD4细胞的耗竭允许CD8细胞完全消除肿瘤。使用受体敲除小鼠，我们发现抑制癌症免疫监测的关键调节细胞因子是IL-13，它通过IL-4受体/STAT6途径作用，尽管IL-4本身既不必需也​​不足够。我们发现IL-13的主要来源是NKT细胞，并且在CD1-敲除小鼠中缺乏这些细胞会阻止这些小鼠的肿瘤复发。我们最近发现，该调节途径适用于其他肿瘤模型，并且我们还确定IL-13间接抑制CD8 T细胞介导的免疫监视的机制，当CD8 T细胞没有IL-13受体时。我们还正在开发通过抑制IL-13来扩增癌症免疫疗法的临床试验方法。我们开发了诱导在癌细胞中表达的突变体p53的CTL免疫的肽癌疫苗。我们发现，突变体p53肽涂在树突状细胞上，引起CTL杀死表达突变的肿瘤细胞并抑制动物中已建立的肿瘤。发现RAS肽中的常见突变增强了与HLA-A2.1的结合，但也会影响抗原加工。我们正在将表位增强应用于突变的RAS肽以制造更有效的疫苗。我们还通过小儿肿瘤，肺泡横纹肌肉瘤和尤因的肉瘤中的染色体易位诱导了鼠CTL，以针对融合蛋白进行融合蛋白。我们还确定了这些肉瘤，滑膜肉瘤等的新型表位，可以与几个人类HLA分子，HLA-A1，A3，B7和B27结合结合，并能够绘制Hla-B7 sarcom carcon的最小表位，并能够杀死Hla-b7的carma，并能够杀死Hla-b7 ct的最小表位。蛋白质是有望用于癌症免疫疗法的肿瘤抗原。 29例患者在I/II期突变p53/RAS肽疫苗治疗癌症方法的临床试验中接受治疗，并且很大一部分已经产生了CTL或细胞因子反应，并且没有看到不良影响。肺泡横纹肌肉瘤和尤因肉瘤患者的转运融合肽免疫的试验正在进行中。我们还开始了对16型人乳头瘤病毒的E6和E7癌蛋白的宫颈癌患者免疫的试验，该试验与表达该HLA分子的患者中与HLA-A2.1结合。许多患者做出了CTL反应，有些患者出乎意料地稳定疾病。与突变体RAS肽脉动的自体树突状细胞的II期试验正在进行HLA-A2.1患者的肿瘤患者中，可以呈现这些RAS肽。该方案和一种刚刚开放的方案可以治疗自体突变体p53肽脉冲的树突状细胞的非小细胞肺癌患者，已修饰以用CD40配体成熟树突状细胞，最近可用。这应该大大改善肽树枝状细胞疫苗的免疫原性。接受治疗的第二名患者的CTL反应和混合的临床反应。 （50％与艾滋病有关）