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

描述（由申请人提供）：多形性胶质母细胞瘤（GBM）中的一个细胞亚群已被确定具有独特的肿瘤再生能力。这些脑肿瘤干细胞（BTSC）可以通过神经干细胞标记物CD133分离出来，并且被广泛认为是对常规治疗产生耐药性的细胞。特异性清除这些细胞的有效手段可能减少对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上发现的肿瘤特异性突变。我们已经成功地使用一种肽疫苗靶向EGFRvIII，这种疫苗可以快速转化为正在进行的III期试验。然而，EGFRvIII的表达是异质性的，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靶向BTSC中的肿瘤特异性突变可能很重要，但很少有高度保守的肿瘤特异性突变，如EGFRvIII，将被鉴定出来，抗原定义的疫苗方法最终将受到限制。负载扩增的肿瘤总RNA的树突状细胞（dc）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的一个少数亚群，不能可靠地分离或繁殖足够数量的抗原，作为人类疫苗接种方案的抗原来源，但我们已经能够从500个分类的CD133（+）肿瘤细胞中重复扩增RNA含量，以产生足够的RNA文库，用于临床规模的dc疫苗接种。为了将免疫应答集中在BTSC中优先或唯一表达的抗原上，并限制对正常细胞中表达的共享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亲和的减法杂交或利用DNA错配结合蛋白MutS的能力的创新策略来富集BTSC中优先或唯一表达的抗原的方法。来分离含有肿瘤特异性突变的cdna。这些不同的制剂将在一个近亲繁殖的转基因小鼠恶性星形细胞瘤模型中评估其不同的毒性和疗效，其中已鉴定出具有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I期研究。公共卫生相关性：恶性原发性脑肿瘤是儿童中最常见的死亡原因，成人死亡人数超过黑色素瘤，目前是美国目前提供的每质量调整生命年节省的最昂贵的药物治疗。恶性原发性脑肿瘤细胞（BTSCs）的一个子集，称为脑肿瘤干细胞，具有肿瘤再生和抵抗常规治疗的独特能力。在这一提议中，我们将看到是否靶向抗原优先或唯一表达的BTSCs将提高疗效和降低免疫治疗的毒性。

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.