AFP-BASED PRIME-BOOST IMMUNOTHERAPY OF HCC

描述(申请人提供)：具体目的：肝细胞癌是全球癌症死亡的主要原因，目前还没有有效的系统疗法来治疗这种疾病。此前，我们进行了甲胎蛋白(AFP)可被人和小鼠免疫系统识别的新观察。在小鼠肿瘤模型中，自身抗原小鼠甲胎蛋白可以作为肿瘤排斥抗原。我们已经进行了广泛的CD8T细胞表位定位，并鉴定了几个免疫优势和劣势AFP衍生肽。在一项初步的临床试验中，我们用这四个免疫优势肽在佐剂中乳化，免疫AFP+/HLA-A*0201+肝癌患者。这些患者能够通过增加外周血中AFP特异性T细胞的频率来进行免疫反应。我们还在小鼠模型中测试了DNA原始腺病毒Boost基因免疫策略，发现它可以接近AFP基因工程树突状细胞的免疫激活水平。在这项应用中，我们建议在I/II期临床试验中测试这种基因免疫策略，并检查免疫反应和机制。我们提出了一项具有两个特定目标的临床遗传免疫治疗研究。 具体目的1：AFP Prime-Boost疫苗产生的免疫应答：我们将在佐剂环境下用编码AFP和GM-CSF的质粒免疫AFP+/HLA-A*0201+肝癌患者，然后用编码AFP的腺病毒(ADV)加强免疫。为此，我们将利用最先进的免疫学检测方法来检测外周血中的反应。通过用1.8kb的完整基因免疫，我们将激发广泛的反应，包括针对免疫优势表位和亚优势表位的CD8+T细胞，以及AFP特异性的CD4+T细胞。我们将分析这些细胞的频率、细胞因子的产生、亲和力和增殖情况。这将不仅表明患者是否已成功接种，而且还允许在人类受试者中对这一遗传免疫策略进行彻底的免疫学评估。特异性目的2：质粒腺病毒促进遗传免疫治疗的免疫学机制：为了阐明注射质粒DNA和注射ADV后在受试者中产生T细胞反应的机制，我们将分析注射AFP载体的活检组织，并对照注射生理盐水的皮内部位以及引流和对侧淋巴结。我们假设免疫抗原(AFP)以蛋白质的形式交叉递送到疫苗仓库的专业抗原提呈细胞(APC)，然后APC运输到引流淋巴结与T细胞相互作用。我们将通过分析前哨淋巴结引流和评估树突状细胞的载体序列及其刺激AFP特异性T细胞反应的能力来检验这一假设。 这些拟议的研究将使我们能够确定针对自身肿瘤抗原的质粒腺病毒增强的免疫学影响，并确定抗原呈递的机制。

DESCRIPTION (provided by applicant): Specific Aims: Hepatocellular cancer (HCC) is a major cause of cancer death globally, and there are no effective systemic therapies for this disease. Previously, we made the novel observation that alpha fetoprotein (AFP) can be recognized by human and murine immune systems. In a murine tumor model, the self-antigen murine AFP can serve as a tumor rejection antigen. We have performed extensive CD8 T cell epitope mapping and identified several immunodominant and subdominant AFP-derived peptides. In a pilot clinical trial, we immunized AFP+/HLA-A* 0201+ HCC patients with the four immunodominant peptides emulsified in adjuvant. These patients were able to respond immunologically by increasing the frequency of AFP-specific T cells in the peripheral blood. We have also tested a DNA prime-adenovirus boost genetic immunization strategy in murine models and found it can approach the levels of immune activation of AFP genetically engineered dendritic cells. In this application, we propose to test this genetic immunization strategy in a phase I/II clinical trial and examine the immunological responses and mechanism. We propose a clinical genetic immunotherapy study with two specific aims. Specific Aim 1: Immune Responses Generated By An AFP Prime-Boost Vaccine: We will immunize AFP+/HLA-A*0201+ HCC patients in the adjuvant setting with plasmids encoding AFP and GM-CSF then boost with an adenovirus (AdV) encoding AFP. In this aim, we will utilize state-of-the-art immunological assays to detect responses in peripheral blood. By immunizing with the entire 1.8 kb gene, we will stimulate a broad response encompassing CD8+ T cells specific for immunodominant and subdominant epitopes, as well as AFP-specific CD4+ T cells. We will analyze these cells with respect to their frequency, cytokine production, avidity and proliferation. This will indicate not only whether patients have been successfully immunized, but also allow for a thorough immunological assessment of this genetic immunization strategy in human subjects. Specific Aim 2: Immunological Mechanisms Governing Plasmid Prime/Adenovirus Boost Genetic lmmunotherapy: To elucidate the mechanism by which injection of plasmid DNA followed by injection of AdV generates T cell responses in human subjects, we will analyze biopsies of AFP vector-injected and control saline injected intradermal sites as well as draining and contralateral lymph nodes. We hypothesize that the immunizing antigen (AFP) is cross-presented as protein to professional antigen presenting cells (APC) at the vaccine depot which then traffic to draining lymph nodes to interact with T cells. We will test this hypothesis by analyzing draining sentinel lymph nodes and evaluating dendritic cells for vector sequences and their ability to stimulate AFP-specific T cell responses. These proposed studies will permit us to determine the immunological impact of a plasmid prime-adenoviral boost directed towards a self tumor antigen and to define mechanisms of antigen presentation.