Annexin A2 as a Novel Enhancer of Tumor-Associated Antigen Processing

描述(申请人提供)：原发恶性脑瘤预后不佳，四分之一的患者在诊断后存活5年以上(1)。尤其是胶质母细胞瘤细胞侵袭性很强，常常从肿瘤切除腔向能言善辩的脑组织迁移几厘米(2)。由于治疗不充分，高级别胶质瘤需要替代的细胞特异性治疗，如免疫疗法。到目前为止，一些神经胶质瘤的免疫治疗试验已经显示出有希望的结果(3-6)，但我们缺乏关于如何重复地安装抗肿瘤免疫反应的知识。1,000多项癌症疫苗试验已经并将继续将肿瘤细胞培养作为抗原源。氧对培养的肿瘤细胞免疫原性的影响知之甚少，直到我们最近发现，在5%O2中培养可以增加：(1)胶质瘤相关抗原的表达(参考文献8)；以及(2)胶质瘤裂解物的佐剂性(7，8)。这些发现导致了蛋白质的鉴定 膜联蛋白A2(以下简称A2)，一种新型的免疫佐剂，富含在5%O2中生长的胶质瘤细胞中。我假设A2是一种损伤相关的分子模式，并确定它是一种有效的Toll样受体2(TLR2)配体。A2‘S TLR2活性，除了在接受5%O2裂解疫苗的小鼠中强大的CD8T细胞反应外，导致了A2增加肿瘤抗原交叉呈递的假设。体外实验数据表明，A2促进树突状细胞(DC)的交叉提呈，DC能够启动抗肿瘤的CD8T细胞；然而，确切的机制尚不清楚。为了验证这一假设，这项建议将分为两个不同的具体目标：(1)研究Annexin A2‘S对抗原摄取、传递和蛋白降解的影响：a.测量标记抗原和肿瘤裂解物在DC中的摄取；b.利用DC的显微镜确定抗原的亚细胞位置；以及c.定量DC中的蛋白酶体活性和组成。(2)确定A2是否增强T细胞受体信号、肿瘤反应性CD8 T细胞的扩张，并延长胶质瘤荷瘤小鼠的生存时间：a.量化胶质瘤反应性CD8 T细胞中T细胞受体的相对信号强度；b.测量内源性胶质瘤反应性CD8 T细胞的扩张；c.确定A2/胶质瘤多肽疫苗是否延长胶质瘤荷瘤小鼠的生存时间，以及生存是否与体外CD8 T细胞反应相关。完成这些研究，基础科学癌症和免疫学培训，以及专注于癌症治疗的医学院培训，将为我的职业生涯做好准备，成为一名拥有脑瘤免疫疗法研究项目的内科科学家。

DESCRIPTION (provided by applicant): Primary malignant brain tumors bear dismal prognoses, with one quarter of patients surviving beyond five years of diagnosis (1). Glioblastoma cells in particular are fiercely invasive, often migrating several centimeters from the tumor resection cavity into eloquent brain (2). Due to inadequate treatment, high-grade gliomas require alternate, cell-specific treatments such as immunotherapy. Thus far several immunotherapy trials for glioma have shown promising results (3-6), but we lack knowledge on how to reproducibly mount an anti-tumor immune response. Over 1,000 cancer vaccine trials have and continue to involve the culture of tumor cells as the antigen source. Little was known on the influence of oxygen on the immunogenicity of cultured tumor cells until our recent findings that culture in 5% O2 increases: (1) glioma-associated antigen expression (Ref 8); and (2) adjuvanticity of glioma lysates (7, 8). These findings led to the identification of the protein annexin A2 ("A2" hereafter), a novel immune adjuvant enriched in glioma cells grown in 5% O2. I hypothesized that A2 is a damage-associated molecular pattern and determined that it is a potent toll-like receptor 2 (TLR2) ligand. A2's TLR2 activity, in addition to the powerful CD8 T cell responses in mice given lysate vaccines from 5% O2, led to the hypothesis that A2 increases cross presentation of tumor antigens. In vitro data indicate that A2 boosts cross presentation on dendritic cells (DCs), which are capable of priming anti-tumor CD8 T cells; however, the precise mechanisms are unclear. To test this hypothesis, this proposal will be divided into two separate specific aims: (1) Investigate annexin A2's effect on antigen uptake, routing, and proteolysis: a. Measure uptake of labeled antigens and tumor lysate in DCs; b. Determine the subcellular location of antigen using microscopy of DCs; and c. Quantify proteasome activity and composition in DCs. (2) Determine if A2 potentiates T cell receptor signaling, expansion of tumor-reactive CD8 T cells, and extends survival of glioma-bearing mice: a. Quantify relative T cell receptor signal strength in glioma-reactive CD8 T cells;b. Measure expansion of endogenous glioma-reactive CD8 T cells c. Determine if A2/glioma peptide vaccination extends survival of glioma-bearing mice, and if survival correlates with ex vivo CD8 T cell response. Completion of these studies, basic-science cancer and immunology training, and medical school training focused on cancer therapy will prepare me for a career as a physician-scientist with a research program in brain tumor immunotherapy.