RNA-based Immunotherapy Targeting Antigens Unique to Brain Tumor Stem Cells

描述(由申请人提供)：在多形性胶质母细胞瘤(GBM)中，已发现具有独特的肿瘤再生能力的细胞亚群。这些脑肿瘤干细胞(BTSC)可以被神经干细胞标记物CD133分离出来，人们普遍认为它们是导致对传统疗法产生抵抗的细胞。一种专门清除这些细胞的有效方法可能会减少强化和非特异性常规治疗的需要，并降低肿瘤复发的风险。EGFRvIII是在BTSC上发现的一种肿瘤特异性突变。我们已经成功地使用了一种多肽疫苗来靶向EGFRvIII，这种疫苗可以快速转化为正在进行的第三阶段试验。然而，EGFRvIII的表达是异质性的，而EGFRvIII阴性肿瘤的复发表明BTSC可以依赖于其他致癌途径。虽然我们的数据表明，在BTSC中靶向肿瘤特异性突变可能是重要的，但很少有像EGFRvIII这样高度保守的肿瘤特异性突变被识别出来，抗原定义的疫苗方法最终将受到限制。树突状细胞(DC)负载扩增的总肿瘤RNA是一种诱导细胞和体液抗肿瘤免疫反应的创新策略。虽然CD133()BTSC是GBM的少数亚群，不能可靠地分离或繁殖足够的数量作为人类疫苗接种的抗原源，但我们已经能够从仅500个分选的CD133()肿瘤细胞中重复扩增RNA内容，以生成足以用于临床规模DC疫苗的RNA文库。为了将免疫反应集中在BTSC优先或唯一表达的抗原上，并限制针对在正常细胞中表达的共同抗原的自身免疫反应的可能性，我们将评估通过基于全长cDNA亲和力的消减杂交或利用DNA错配结合蛋白MutS分离包含肿瘤特异性突变的cDNA的创新策略来丰富在BTSC中优先或唯一表达的抗原的方法。这些不同的制剂将在近交系转基因小鼠恶性星形细胞瘤模型中进行不同的毒性和疗效评估，在该模型中，已鉴定出具有BTSC特性的CD133()肿瘤细胞亚群，并发现了CD8()和CD4()表位。如果看到疗效，毒性最小的策略将在我们现有的临床试验平台的背景下转化为第一阶段研究。公共卫生相关性：恶性原发脑瘤是儿童最常见的死亡原因，成人死亡人数超过黑色素瘤，目前是美国目前提供的每一质量调整的生命年所提供的最昂贵的药物治疗。一组被称为脑肿瘤干细胞的恶性原发脑肿瘤细胞(BTSCs)具有独特的再生肿瘤和抵抗传统治疗的能力。在这项建议中，我们将观察BTSCs优先或唯一表达的靶向抗原是否会增强免疫治疗的疗效并降低毒性。

DESCRIPTION (provided by applicant): A subset of cells in glioblastoma multiforme (GBM) has been identified that enjoy a unique capacity to regenerate tumors. These brain tumor stem cells (BTSC) can be segregated by the neural stem cell marker, CD133, and are widely believed to be the cells responsible for resistance to conventional therapies. An effective means of specifically eliminating these cells may reduce the need for intensive and non-specific conventional therapy and lower the risk of tumor recurrence. EGFRvIII is a tumor-specific mutation found on BTSC. We have successfully targeted EGFRvIII using a peptide vaccine that allowed rapid translation to an ongoing Phase III trial. EGFRvIII expression is heterogeneous, however, and the recurrence of EGFRvIII-negative tumors suggests that BTSC can rely on other oncogenic pathways. While our data suggests that targeting tumor-specific mutations in BTSC may be important, few highly-conserved tumor-specific mutations like EGFRvIII will be identified and antigen defined vaccine approaches will ultimately be limited. Dendritic cells (DCs) loaded with amplified total tumor RNA is an innovative strategy to induce cellular and humoral antitumor immune responses. Although CD133(+) BTSC are a minority subpopulation of GBM that cannot be reliably isolated or propagated in sufficient quantities to serve as an antigen source for human vaccination protocols, we have been able to reproducibly amplify the RNA content from as few as 500 sorted CD133(+) tumor cells to generate RNA libraries sufficient for clinical scale DC-based vaccination. In order to focus the immunologic response on antigens preferentially or uniquely expressed within BTSC and limit the potential for autoimmune reactivity against shared antigens expressed in normal cells, we will evaluate approaches to enrich for antigens preferentially or uniquely expressed in BTSC by using full length cDNA affinity based substractive hybridization or an innovative strategy that leverages the ability of the DNA mismatch binding protein, MutS, to isolate cDNAs that contain tumor-specific mutations. These various preparations will be evaluated for differential toxicity and efficacy in an inbred transgenic murine malignant astrocytoma model, in which a subpopulation of CD133(+) tumor cells with BTSC qualities have been identified and CD8(+) and CD4(+) epitopes have been found. If efficacy is seen, the least toxic strategy will be translated into a Phase I study within the context of our existing clinical trial platform. PUBLIC HEALTH RELEVANCE: Treatment for malignant primary brain tumors, which are the most common cause of death among children and account for more deaths in adults than melanoma, currently represents the most expensive medical therapy per quality-adjusted life-year saved currently provided in the United States. A subset of malignant primary brain tumor cells (BTSCs), called brain tumor stem cells, enjoy a unique capacity to regenerate tumors and to resist conventional therapies. In this proposal we will see if targeting antigens preferentially or uniquely expressed by BTSCs will enhance the efficacy and reduce toxicity of immunotherapy.