Listeria Immunotherapy for Pancreatic and Ovarian Cancer

描述（由申请人提供）：胰腺癌和卵巢癌是最具侵袭性和致命性的恶性肿瘤之一，代表了一个主要的未满足的医疗需求。即：(1)胰腺癌患者的5年生存率不到5%，美国每年3.2万例新病例接近每年的死亡人数；(2)卵巢癌约占妇科恶性肿瘤的25%，但占妇科癌症死亡人数的50%以上，在美国每年估计有14,000人死亡。SBIR II期提案的总体目标是继续开发基于单核细胞增生李斯特菌（Lm）的免疫疗法，以靶向胰腺癌和卵巢癌患者的间皮素和密码子12突变活化K-ras抗原。自SBIR I期拨款提交以来，发生了两项重大进展：(1)我们生成（并记录）了一种减毒活Lm疫苗平台菌株，该菌株删除了ActA和InIB毒力决定因素，其强度大于减毒后的1000倍，但保留了与野生型Lm的免疫原性；(2)我们的合作者Elizabeth Jaffee博士已经证明来自胰腺癌患者的间皮素靶向t细胞可以靶向并破坏胰腺肿瘤细胞，并且患者的t细胞反应与长期无病生存相关。在第一阶段的资助和持续的工作下，我们开发了专有的和独特的肿瘤抗原表达和分泌方法。利用密码子优化和不同革兰氏+菌的信号肽，构建了高效合成和分泌间皮素的Lm疫苗。利用重叠肽文库绘制了原型疫苗免疫小鼠的人间皮素和Lm特异性细胞反应。我们扩展了LLO肽库的使用，以测量人类的Lm细胞免疫。此外，我们证明了Lm疫苗有效地将肿瘤抗原递送到人树突状细胞中，用于MHC I类体外呈递——这是II期研究的关键概念证明。为此，我们建议：(1)选择lm -间皮素/ras疫苗株进行临床试验，基于对同源和HLA-A2转基因小鼠的免疫原性，并激活已建立的HLA-A2-、A3-和a24限制性人间皮素特异性t细胞系；(2)建立临床监测方法，评估接种疫苗患者间皮素和k -ras特异性t细胞反应；(3)建立所选Lm间皮素/ras疫苗的生产和工艺方法；(4)进行使能ind的小鼠毒理学研究。这些活动中的每一项都处于实现I期临床试验的关键路径上。为了实现这些目标，我们组建了一个具有富有成效合作历史的团队，该团队由科学家和外部顾问组成，他们在革兰氏阳性细菌遗传学、小鼠和人类免疫学、新型癌症疫苗平台开发和转化医学方面具有专业知识。

DESCRIPTION (provided by applicant): Pancreatic and ovarian cancers are among the most aggressive and lethal malignancies, and represent a major unmet medical need. To wit: (1) pancreatic cancer patients have a 5-year survival rate of less than 5% and the 32,000 new cases per year in the U.S. is close to the number of annual deaths; and, (2) ovarian cancer represents about 25% of gynecologic malignancies but accounts for over 50% of gynecologic cancer deaths, an estimated 14,000 per annum in the U.S. The overall goal of this SBIR phase II proposal is to continue development of our Listeria monocytogenes (Lm)-based immunotherapy to target Mesothelin and codon 12-mutated activated K-ras antigens in patients with pancreatic and ovarian cancers. Two significant developments have occurred since the SBIR Phase I grant was submitted: (1) We generated (and documented) a live-attenuated Lm vaccine platform strain deleted of ActA and InIB virulence determinants that is greater that 1000-fold attenuated, yet retains immunogenicity that is indistinguishable from wild-type Lm; and, (2) Mesothelin-targeted T-cells from pancreatic cancer patients have been shown by our collaborator, Dr. Elizabeth Jaffee, to target and destroy pancreatic tumor cells, and patient T-cell responses correlate with long-term disease-free survival. Under Phase I funding and continued work, we developed proprietary and unique means for expression and secretion of tumor antigens. By utilizing both codon optimization and signal peptides from distinct Gram+ bacteria, we constructed Lm vaccines that efficiently synthesize and secrete Mesothelin. Overlapping peptide libraries were used to map human Mesothelin- and Lm (LLO)-specific cellular responses in mice immunized with prototype vaccines. We extended the use of the LLO peptide library to measure Lm cellular immunity in humans. Additionally, we demonstrated that Lm vaccines efficiently deliver tumor antigens into human dendritic cells for MHC class I presentation in vitro-a critical proof of concept for studies proposed in Phase II. With this application, we propose to: (1) select the Lm-Mesothelin/ras vaccine strain for clinical trials based on immunogenicity in syngeneic and HLA-A2 transgenic mice, and activation of established HLA-A2-, A3-, and A24-restricted human Mesothelin specific Tcell lines; (2) establish clinical monitoring methods to evaluate Mesothelin- and K-ras-specific T-cell responses in vaccinated patients; (3) establish manufacturing and process methods for the selected Lm Mesothelin/ras vaccine; and, (4) perform IND-enabling murine toxicology studies. Each of these activities is on the critical path to enabling a Phase I clinical trial. To accomplish these goals, we have assembled a team with a history of productive collaboration consisting of scientists and external consultants with documented expertise in Gram-positive bacterial genetics, murine and human immunology, novel cancer vaccine platform development, and translational medicine.