Development and Testing of Recombinant Clostridia for Cancer Therapy

描述(由申请人提供)：这项STTR申请是为了开发和测试一种肿瘤特异性抗癌药物输送的新策略，并最终用于临床。这项名为梭状芽胞杆菌导向的酶前药物治疗或CDEPT的技术利用了这样一个事实，即梭状芽胞杆菌属的某些非致病细菌只在缺乏氧气(缺氧)的情况下生长，在实体肿瘤特有的缺氧和坏死区定居。这些细菌在这些地区的生长可以通过改造细菌来产生一种酶，这种酶通常不存在于人类身上，可以将无毒的“前药”转化为有毒的药物。跟随静脉注射。重组梭状芽胞杆菌的孢子注射、孢子萌发和前药物活化酶的产生仅发生在肿瘤中。由于酶只存在于肿瘤中，前药仅在肿瘤中转化为有毒药物，因此选择性地将药物的细胞毒活性靶向肿瘤。我们将使用一种已经被证明(在其非工程状态下)在人类患者中具有良好耐受性的产孢梭菌，以及一种新的抗癌药物PR-104，该药物最近进入临床测试，当它被大肠杆菌硝基还原酶(NTR)酶(我们已经改造产孢杆菌表达的酶)代谢时对细胞具有高度毒性。我们已经在啮齿动物肿瘤模型中证明了表达NTR和PR-104的产孢子球菌合用的有效性。我们在这项STTR中的总体目标是将SGM Biotech的专业知识与斯坦福大学的Brown实验室相结合，SGM Biotech在过去15年里一直在GMP条件下生产生孢杆菌孢子，布朗实验室为CDEPT策略对生孢杆菌进行了基因工程，以开发用于最终临床测试的微生物。我们有三个具体的目标来实现这一目标：1)将密码子优化的梭状芽胞杆菌NTR基因整合到产孢链霉菌(Stanford University)的基因组中。2)在斯坦福大学(SGM Biotech)进行初步的安全性和有效性研究，并在斯坦福大学(Stanford University)进行初步的安全性和有效性研究；3)用荷瘤小鼠(Stanford University)检测GMP产生的重组生孢杆菌在荷瘤小鼠(Stanford University)中的表达和有效性(与PR-104一起)。如果该项目成功，将能够在临床上测试一种细胞毒性化疗药物对患者实体肿瘤的特定输送，从而增强抗肿瘤活性，并将正常组织毒性降至最低。 与公共卫生相关：这项STTR提案提出了一种独特的策略，可以将特定化疗药物的有效杀伤浓度靶向实体肿瘤。我们提出的方法防止了对整个动物的间接毒性影响，但允许在肿瘤中实现药物的杀伤浓度，因为我们只在肿瘤中直接激活药物。

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.