Anaerobic Bacteria as Therapeutic Agents for Metastatic

描述（由申请人提供）：随着许多肿瘤（包括胰腺癌和结肠癌）的体积增长，它们会形成血管化不良和缺氧（缺氧）区域，这限制了治疗剂的进入，并限制了目前使用的抗癌方式的疗效。然而，缺氧的存在也提供了可能导致肿瘤破坏的厌氧菌定植的可能性。一种这样的厌氧细菌是产气荚膜梭菌（Cp），其含有编码磷脂酶C（plc）的主要毒素基因。在我们的实验室中构建了Cp的plc缺失菌株（Cp/plc-），并显示静脉给药后，在小鼠实体瘤中定殖并诱导大量坏死。不幸的是，Cp/plc-保留了一定的氧耐受性，使其能够以大大降低的速率在正常组织中生长，并且用有效剂量的Cp/plc-治疗的荷瘤小鼠也表现出全身毒性。我们假设Cp/plc-可以被遗传修饰以显著降低其氧耐受性，从而产生将导致肿瘤破坏而对正常组织没有毒性的亚株。我们建议删除的超氧化物歧化酶（sod）基因的表达菌株Cp/plc-/LUC+（Cp/plc-/sod-/LUC+）。将通过静脉内施用Cp/plc-/sod-来治疗在肝脏中具有预先建立的同基因结肠直肠癌和胰腺癌的免疫活性小鼠。将确定最大耐受剂量下的剂量反应曲线和长期生存期。细菌生物分布和肿瘤内生长将通过使用白藜芦醇的重复非侵入性全身成像来确定为时间的函数。将通过组织学检查评价给药动物的肿瘤缓解。毒性终点将包括CBC、血液化学、血清促炎细胞因子水平和主要器官病史。如有必要，可以从Cp/plc-/sod-中删除额外的耐氧基因，以进一步降低毒性。我们假设这些不耐氧突变细菌亚株可以安全地应用于荷瘤动物，其将选择性地定位于缺氧区域，在缺氧区域萌发和生长并破坏肿瘤。基于厌氧菌的载体的应用是一种有前途的策略，用于开发一种有效的治疗剂，该治疗剂可以全身给药以治疗伴有缺氧的播散性实体瘤，并且可能导致未来在转移性结直肠癌和/或胰腺癌患者中的临床转化研究。

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.