Listeria Immunotherapy for Pancreatic and Ovarian Cancer

描述(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，是一个主要的未得到满足的医疗需求。也就是说：(1)胰腺癌患者的5年生存率不到5%，美国每年32,000新发病例接近年死亡人数；(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，估计美国每年有14,000人死亡。这项SBIR II期提案的总体目标是继续开发我们的基于单核细胞增生性李斯特氏菌(Lm)的免疫疗法，针对胰腺癌和卵巢癌患者的Mesothelin和密码子12突变的K-ras抗原。自从SBIR第一阶段拨款提交以来，已经发生了两项重大进展：(1)我们产生(并记录)了一种减毒活疫苗平台菌株，其ActA和InIB毒力决定因素的缺失程度超过了1000倍，但仍保留了与野生型LM几乎没有区别的免疫原性；(2)我们的合作者伊丽莎白·贾菲博士证明，来自胰腺癌患者的以间硫蛋白为靶标的T细胞可以靶向并摧毁胰腺癌细胞，患者的T细胞应答与长期无病生存相关。在第一阶段的资助和继续工作下，我们开发了表达和分泌肿瘤抗原的专有和独特的方法。通过利用密码子优化和来自不同革兰氏阳性细菌的信号肽，我们构建了能够有效合成和分泌间硫蛋白的LM疫苗。重叠的多肽文库被用来在原型疫苗免疫的小鼠中定位人间皮蛋白和LM(LLO)特异性的细胞反应。我们扩大了LLO多肽文库的使用范围，以测量人类的LM细胞免疫。此外，我们还证明了LM疫苗可以有效地将肿瘤抗原输送到人树突状细胞中，用于MHC-I类的体外呈递--这是第二阶段中提出的研究的关键概念验证。通过这一应用，我们建议：(1)基于同基因和HLA-A2转基因小鼠的免疫原性以及已建立的HLA-A2、A3和A24限制性人类间皮蛋白特异性T细胞株的激活，选择用于临床试验的LM-Mesothelin/ras疫苗株；(2)建立临床监测方法来评估接种患者中间皮蛋白和K-ras特异性的T细胞应答；(3)建立选定的LM间皮内毒素/ras疫苗的制造和加工方法；以及(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卓有成效的合作历史的团队，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拥有有据可查的专业知识。

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.