Anaerobic Bacteria as Therapeutic Agents for Metastatic Cancer

描述(申请人提供)：随着肿瘤体积的增长，许多肿瘤，包括胰腺癌和结肠癌，形成血管贫乏和缺氧(缺氧)区域，限制了治疗药物的进入，并限制了目前使用的抗癌方法的疗效。然而，缺氧的存在也提供了厌氧细菌定植的可能性，这可能导致肿瘤的破坏。产气荚膜梭菌就是这样一种厌氧细菌，它含有一个编码磷脂酶C(Plc)的主要毒素基因。本实验室已构建了一株缺失plc的CP(CP/plc-)，经静脉给药后，可在小鼠实体瘤中定植并诱导大量坏死。不幸的是，CP/plc-保留了一些对氧气的耐受性，使其能够在正常组织中以较低的速度生长，并且有效剂量的CP/plc-治疗荷瘤小鼠也显示出全身毒性。我们假设，CP/plc-可以通过基因修饰来显著降低其耐氧性，从而创造出能够在不对正常组织产生毒性的情况下导致肿瘤破坏的亚株。我们建议在一株表达荧光素酶的CP/plc-/Luc+(CP/plc-/sod-/Luc+)菌株中删除超氧化物歧化酶(Sod)基因。免疫功能正常的小鼠患有预先确定的同基因结直肠癌和胰腺癌，将通过静脉注射CP/plc-/sod-来治疗。剂量反应曲线和在最大耐受剂量下的长期存活将被确定。细菌的生物分布和肿瘤内生长将通过使用荧光素的重复无创全身成像来确定为时间的函数。治疗动物的肿瘤反应将通过组织学检查进行评估。毒性终点将包括血细胞计数、血液化学、血清促炎细胞因子水平和主要器官病史。如果有必要，可以从CP/plc-/sod-中删除额外的耐氧性基因，以进一步降低毒性。我们推测，这些耐氧突变细菌亚株可以安全地应用于荷瘤动物，它们将选择性地定位、萌发和生长，并摧毁缺氧区的肿瘤。应用厌氧细菌载体是一种很有前途的策略，可以开发一种有效的治疗剂，可以系统地给药来治疗有缺氧的播散性实体瘤，并可能在未来导致转移性结直肠癌和/或胰腺癌患者的临床翻译研究。

DESCRIPTION (provided by applicant): As they grow in volume many tumors, including pancreatic and colon cancers, construct poorly vascularized and oxygen-deficient (hypoxic) areas that restrict the access by therapeutic agents and limit the efficacy of currently used anti-cancer modalities. However, the presence of hypoxia also offers the potential for anaerobic bacterial colonization that can lead to tumor destruction. One such anerobic bacterium is Clostridium perfringens (Cp), which contains a major toxin gene that encodes phospholipase C (plc). A plc-deleted strain of Cp (Cp/plc-) has been constructed in our laboratory and shown, after intravenous administration, to colonize and induce massive necrosis in solid tumors in mice. Unfortunately Cp/plc- retains some tolerance to oxygen that enables it to grow in normal tissues at a much reduced rate, and tumor-bearing mice treated with Cp/plc- at the effective doses also exhibited systemic toxicity. We hypothesize that Cp/plc- can be genetically modified to reduce substantially its oxygen tolerance, thereby creating sub-strains that will lead to tumor destruction without toxicity to normal tissues. We propose to delete the superoxide dismutase (sod) gene in a luciferase-expressing strain of Cp/plc-/LUC+ (Cp/plc-/sod-/LUC+). Immune- competent mice with pre-established syngeneic colorectal and pancreatic cancers in the liver will be treated by intravenous administration of Cp/plc-/sod-. Dose response curve and long-term survival at the maximal tolerated dose will be determined. Bacterial biodistribution and intratumoral growth will be determined as a function of time by repeated non-invasive whole-body imaging using luciferrin. Tumor responses in the treated animals will be evaluated by histological examination. Toxicity endpoints will include CBC, blood chemistries, serum proinflammatory cytokine levels and history of major organs. If necessary, additional oxygen tolerance genes can be deleted from Cp/plc-/sod- to further reduce toxicity. We hypothesize that these oxygen-intolerant mutant bacterial sub-strains can be safely applied in tumor-bearing animals, which will selectively localize to, germinate and grow in, and destroy tumors in hypoxic regions. Application of anaerobic bacterial-based vectors is a promising strategy for the development of an effective therapeutic agent that can be administered systemically to treat disseminated solid tumors with hypoxia, and may lead to clinical translational studies in patients with metastatic colorectal and/or pancreatic cancers in the future.