Development and Testing of Recombinant Clostridia for Cancer Therapy

描述（由申请人提供）：这个STTR申请是为了开发和测试最终临床使用的肿瘤特异性抗癌药物递送新策略。这项名为梭状芽孢杆菌定向酶前药物治疗（CDEPT）的技术，利用了梭状芽孢杆菌属的某些非致病性细菌在缺氧（缺氧）条件下生长的事实，这些细菌在实体瘤特有的缺氧和坏死区域定植。这些细菌在这些区域的生长可以通过改造细菌产生一种酶来利用，这种酶通常不存在于人类中，它将无毒的“前药”转化为有毒的药物。在静脉注射重组梭菌孢子后，孢子的萌发和前药激活酶的产生只发生在肿瘤中。由于酶只存在于肿瘤中，因此前药只在肿瘤中转化为毒性药物，从而选择性地靶向药物对肿瘤的细胞毒性活性。我们将使用一种梭状芽孢杆菌（C. sporogenes），它已被证明（在其非工程状态下）在人类患者中具有良好的耐受性，以及一种新的抗癌药物PR-104，它最近已进入临床测试，当它被大肠杆菌硝基还原酶（NTR）酶代谢时对细胞具有高度毒性（我们已经设计了C. sporogenes表达的酶）。我们已经在啮齿类动物肿瘤模型中证明了我们的表达NTR的孢子孢杆菌与PR-104结合的有效性。我们这次STTR的总体目标是将SGM生物技术公司的专业知识与斯坦福大学布朗实验室的专业知识结合起来，SGM生物技术公司在过去的15年里一直在GMP条件下生产孢子孢杆菌孢子，斯坦福大学布朗实验室对孢子孢杆菌进行了基因工程改造，用于CDEPT策略，开发出用于最终临床试验的生物。为了实现这一目标，我们有三个具体的目标：1)将Clostridial密码子优化的大肠杆菌NTR基因整合到C. sporogenes （Stanford University）的基因组中。2)在斯坦福大学（SGM Biotech）用整合的e.c oli NTR基因生产出足够数量的重组孢子菌，进行初步的安全性和有效性研究。3)在斯坦福大学（Stanford University）用PR-104检测GMP生产的重组孢子菌在荷瘤小鼠（Stanford University）的表达和有效性。该项目如果成功，将使细胞毒性化疗药物特异性递送到实体肿瘤患者的临床试验成为可能，从而增强抗肿瘤活性并将正常组织毒性降至最低。

DESCRIPTION (provided by applicant): This STTR application is to develop and test for eventual clinical use a new strategy for tumor specific anticancer drug delivery. The technology, Clostridia-directed enzyme prodrug therapy, or CDEPT, exploits the fact that certain non-pathogenic bacteria of the Clostridia genus, which only grow in the absence of oxygen (hypoxia), colonize the hypoxic and necrotic areas that are unique to solid tumors. Growth of these bacteria in these areas can be exploited by engineering the bacteria to produce an enzyme, not normally present in humans that will convert a non-toxic "prodrug" into a toxic drug. Following i.v. injection of spores of the recombinant clostridia, germination of the spores and production of the prodrug activating enzyme occurs solely in the tumors. Since the enzyme is only in the tumor, the prodrug is converted to the toxic drug only in the tumor, hence targeting the cytotoxic activity of the drug selectively to the tumor. We will use a Clostridial species, C. sporogenes, that has been shown (in its non-engineered state) to be well tolerated in human patients, and a new anticancer drug, PR-104, which has recently entered clinical testing and which is highly toxic to cells when it is metabolized by the E. coli nitroreductase (NTR) enzyme (the enzyme that we have engineered C. sporogenes to express). We have demonstrated the efficacy of the combination of our C. sporogenes expressing NTR with PR-104 in rodent tumor models. Our overall goal in this STTR is to combine the expertise of SGM Biotech, which has manufactured C. sporogenes spores under GMP conditions for the past 15 years, with the Brown laboratory at Stanford University, which has genetically engineered C. sporogenes for the CDEPT strategy, to develop the organism for eventual clinical testing. We have three specific aims to achieve this goal: 1) Integrate the Clostridial codon optimized E. coli NTR gene into the genome of C. sporogenes (Stanford University). 2) Produce the recombinant C. sporogenes with the integrated E. coli NTR gene in sufficient quantities to conduct the initial safety and efficacy studies at Stanford University (SGM Biotech), and 3) Test the expression and efficacy (with PR-104) of the GMP produced recombinant C. sporogenes with tumor bearing mice (Stanford University). This project, if successful, will enable clinical testing of the specific delivery of a cytotoxic chemotherapeutic agent to solid tumors in patients thereby enhancing antitumor activity and minimizing normal tissue toxicity. PUBLIC HEALTH RELEVANCE: This STTR proposal presents a unique strategy for targeting effective killing concentrations of a specific chemotherapeutic agent to solid tumors. The approach we are proposing prevents indirect toxic effects on the animal as a whole, but allows for killing concentrations of the drug to be achieved specifically in the tumor, as we are directly activating the drug only in the tumors.