AFP-BASED PRIME-BOOST IMMUNOTHERAPY OF HCC

描述（由申请方提供）：特定目的：肝细胞癌（HCC）是全球癌症死亡的主要原因，目前尚无有效的全身性治疗方法。以前，我们做了新的观察，甲胎蛋白（AFP）可以识别的人类和小鼠的免疫系统。在鼠肿瘤模型中，自身抗原鼠AFP可用作肿瘤排斥抗原。我们已经进行了广泛的CD 8 T细胞表位定位，并确定了几个免疫显性和亚显性AFP衍生肽。在初步临床试验中，我们用佐剂乳化的四种免疫显性肽免疫AFP+/HLA-A* 0201+ HCC患者。这些患者能够通过增加外周血中AFP特异性T细胞的频率来免疫应答。我们还在小鼠模型中测试了DNA引发-腺病毒加强遗传免疫策略，发现它可以接近AFP基因工程树突状细胞的免疫活化水平。在本申请中，我们建议在I/II期临床试验中测试这种基因免疫策略，并检查免疫应答和机制。我们提出了一个临床遗传免疫治疗研究有两个具体目标。 具体目标1：由AFP初免-加强疫苗产生的免疫应答：我们将在佐剂环境中用编码AFP和GM-CSF的质粒免疫AFP+/HLA-A*0201+ HCC患者，然后用编码AFP的腺病毒（AdV）加强。在这个目标中，我们将利用最先进的免疫学测定来检测外周血中的反应。通过用完整的1.8 kb基因免疫，我们将刺激广泛的应答，包括对免疫显性和次显性表位特异性的CD 8 + T细胞，以及AFP特异性的CD 4 + 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。这将不仅表明患者是否已成功免疫，而且还允许在人类受试者中对这种遗传免疫策略进行彻底的免疫学评估。具体目标二：控制质粒初免/腺病毒加强遗传免疫疗法的免疫机制：为了阐明注射质粒DNA然后注射AdV在人类受试者中产生T细胞应答的机制，我们将分析注射AFP载体和注射对照盐水的皮内部位以及引流和对侧淋巴结的活组织检查。我们假设免疫抗原（AFP）作为蛋白质交叉呈递给疫苗库中的专职抗原呈递细胞（APC），然后运输到引流淋巴结与T细胞相互作用。我们将通过分析引流前哨淋巴结和评估树突状细胞的载体序列及其刺激AFP特异性T细胞应答的能力来验证这一假设。 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引发-腺病毒加强免疫的影响，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.