Antigen-specific T-cell activation, application to vaccines for Cancer and AIDS

抗原特异性 T 细胞激活，在癌症和艾滋病疫苗中的应用

• 批准号: 6433339
• 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/6433339
• 关键词: AIDS therapy; AIDS vaccines; HIV envelope protein gp160; MHC class I antigen; T cell receptor; cellular immunity; clinical research; clinical trial phase I; cytotoxic T lymphocyte; drug design /synthesis /production; epitope mapping; human subject; human therapy evaluation; immunotherapy; interleukin 2; laboratory mouse; leukocyte activation /transformation; neoplasm /cancer immunotherapy; neoplasm /cancer vaccine; neutralizing antibody; 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 improve on the peptide vaccine constructs. We have now 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. We are applying this ?epitope enhancement? approach to conserved helper epitopes presented by human class II 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. 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 one mechanism involving the ability of high avidity CTL to kill cells earlier in virus infection before viral progeny are produced. 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. Finally, 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 observed an asymmetry between mucosal and systemic immune responses in that systemic immunization induced only systemic CTL whereas mucosal immunization induced both mucosal and systemic CTL. This observation led us to develop an approach to overcome the problem of preexisting poxvirus immunity from smallpox vaccination in order to use recombinant vaccinia vector vaccines by taking advantage of the naivete of the mucosal immune system after systemic immunization to still be able to immunize with recombinant vaccinia vaccines through the mucosal route in vaccinia-immune animals. 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 also demonstrated striking protection of HLA-A2.1-transgenic mice from challenge with a recombinant vaccinia virus expressng HCV core protein by immunization with a DNA vaccine expressing HCV core, and showed that the protection was CD8-T cell dependent and correlated with HLA-A2.1-restricted CTL. Further, we found that T cell help against the hypervariable region 1 of HCV envelope was critical for induction of human antibodies to this region, believed to be a neutralizing epitope. 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, and we are exploring the cytokine mechanisms involved using receptor knock-out mice. 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 also induced murine CTL against fusion proteins from chromosomal translocations in pediatric tumors, alveolar rhabdomyosarcoma and Ewings sarcoma. We also identified novel epitopes spanning these fusion protein junctions in these sarcomas that could bind to several human HLA molecules, HLA-A1, A3, B7 and B27. 29 patients have been 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 have been seen. A trial of translocation fusion peptide immunization of patients with alveolar rhabdomyosarcoma and Ewings 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. A phase II trial of autologous dendritic cells pulsed with mutant ras peptides corresponding to the patients tumor in colon cancer patients with HLA-A2.1 that can present these ras peptides has opened and we have treated 3 patients to date. (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，以改进肽疫苗构建体。现在，我们已经显示了原理证明，我们可以修改HIV的辅助辅助表位，以使其更加免疫原性，并且在引起CTL中耦合到CTL表位时更有效。我们正在应用这种表位增强？人类II类HLA分子以及人类HLA-A2.1提出的丙型肝炎病毒（HCV）表位的保守辅助表位的方法（见下文）。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种细胞因子都提供三重协同作用，用于用肽疫苗诱导CTL，用于诱导干扰素 - 伽马并保护体内病毒攻击。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SCID小鼠中表达HIV GP160的重组疫苗病毒相比，对HIV-1包膜肽特异性的高亲和力CTL比对同一肽-MHC复合物的低接种CTL更为有效，并且对同一肽-MHC复合物进行了低的ctl，并且涉及一种涉及高流行病的能力以早些时候杀死Virus的能力。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。最后，我们首次表明，可以通过CD8 CTL介导防止病毒的粘膜传播的无抗体介导，但要求CTL存在于传播的粘膜部位，而全身CTL则不够。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。我们观察到粘膜和全身免疫反应之间的不对称性，因为该全身免疫仅诱导全身性CTL，而粘膜免疫诱导的粘膜和全身性CTL诱导。这一观察结果使我们开发了一种方法，以克服天花疫苗接种毒病毒免疫的问题，以便通过利用全身免疫后的粘膜免疫系统的天真疫苗来使用重组疫苗疫苗疫苗，以便通过MIMUCINE疫苗接种疫苗，以使其能够通过MIMUCINE疫苗接种。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，我们可以增加HCV核心蛋白表位的免疫原性，由最常见的人类I类HLA分子HLA-A2.1提出，均用于HLA-A2.1-A2.1- Transgenic小鼠体内的HLA-A2.1- Transgenic小鼠。这是增强的？表位被掺入疫苗中。我们还证明了HLA-A2.1- TRENGENIC小鼠免受挑战的惊人保护，并通过用表达HCV核心的DNA疫苗进行免疫，表达HCV核心蛋白，并表明该保护是CD8-T细胞依赖性并与HLA-A2.1-A2.1-A2.1种子限制的CTL相关的。此外，我们发现针对HCV包膜高变量区域1的T细胞有助于对该区域的人类抗体诱导至关重要，这被认为是中和表位。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细胞的耗竭允许通过CD8细胞完全消除肿瘤，我们正在探索使用受体敲除小鼠涉及的细胞因子机制。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。我们还通过小儿肿瘤，肺泡横纹肌肉瘤和ewings肉瘤中的染色体易位诱导鼠CTL诱导融合蛋白。我们还鉴定了这些肉瘤中这些融合蛋白连接的新型表位，这些肉瘤可能与几种人HLA分子HLA-A1，A3，B7和B27结合。 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进行了治疗，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eWings肉瘤患者的转运融合肽免疫试验正在进行中。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。一项与突变体RAS肽脉动的自体树突状细胞的II期试验，该肽对应于HLA-A2.1结肠癌患者的肿瘤患者，可以呈现这些RAS肽的肿瘤患者，迄今为止已经打开了3名患者。 （50％与艾滋病有关）