Anaerobic Bacteria as Oncopathic Agents for Pancreatic Cancer

描述(由申请人提供)：癌症治疗中的一个主要挑战是选择性和有效地将治疗剂输送到病变部位。实体肿瘤，如胰腺癌，在其核心往往有血运不良的区域，这些区域是低氧的，不容易通过血管输送化疗药物。然而，缺氧提供了厌氧细菌定植的可能性，可以导致肿瘤的破坏，其中一种厌氧细菌是产气荚膜梭菌(Clostridium Perringens，CP)。我们已经删除了其编码磷脂酶c(Cp/plc-)的α毒素基因，并表明该突变株不再能引起小鼠气性坏疽。我们还表明，这种缺失突变体的休眠孢子，当静脉注射到携带胰腺癌原位模型的小鼠身上时，能够在具有肿瘤治疗作用的缺氧肿瘤区域萌发和增殖。然而，CP/plc-确实对氧气有残留的耐受性，并在有氧组织中保持有限的生长能力，当静脉注射高剂量时，会导致动物的显著毒性。超氧化物歧化酶(SOD)是一个主要的耐氧性基因，在CP/plc-中也被敲除。在荷瘤小鼠中，这种基因敲除菌株(Cp/plc-/sod-)的最大耐受量比Cp/plc-高一个对数。为了进一步提高安全性，我们假设sod基因敲除菌株中其他主要耐氧基因的顺序敲除将产生一种具有最大肿瘤选择性和最低毒性的耐氧菌株(oiCp/plc-)。然而，肿瘤内细菌的复制被具有杀菌作用的宿主炎性细胞的快速积累所抑制，这限制了肿瘤反应的程度。为了增强肿瘤治疗的效力，我们假设通过构建表达来自自然赋予这些特性的异源微生物的炎症抑制基因的重组体，可以显著提高oiCp/plc-的瘤内复制及其抗肿瘤效果。这些新的重组菌株将在血管稀少的肿瘤中具有最大的肿瘤选择性和肿瘤治疗能力。最后，大多数实体肿瘤还包含相对充血和充氧的区域，因此对厌氧菌治疗无效。然而，这些血管和氧合区域容易受到全身化疗药物的影响。因此，我们假设，针对血运不良的肿瘤的缺氧核心的厌氧菌治疗将补充以血运区域为目标的全身化疗，从而显著增强肿瘤的破坏和延长生存期。拟议研究的成功可能导致重组CP孢子作为一类新型治疗剂的开发，这种新型治疗剂可以在未来对晚期胰腺癌和其他血管较差的肿瘤患者进行系统和安全的治疗，与化疗相结合，可能会比目前的标准治疗标准--全身化疗--带来更好的治疗结果。公共卫生相关性：尽管胰腺癌在美国男性和女性新增癌症病例中分别只占第10位和第11位，但它是美国男性和女性癌症死亡的第4位主要原因。据估计，仅在美国，2007年就有33,370人死亡，37,170人新增病例。目前可用的治疗方法包括化疗和放射治疗，这对胰腺癌并不是特别有效。厌氧菌可以针对肿瘤中的缺氧核心，这将是化疗的补充，因此联合治疗的结果将优于单独化疗。

DESCRIPTION (provided by applicant): A major challenge in cancer therapy has been the selective and efficient delivery of therapeutic agents to the lesions. Solid tumors such as pancreatic cancer often have poorly-vascularized regions at their cores that are hypoxic and are not readily accessible to the vascular delivery of chemotherapeutic drugs. Hypoxia, however, offers the potential for anaerobic bacterial colonization that can lead to tumor destruction, and one such anaerobic bacterium is Clostridium perfringens (Cp). We have deleted its alpha toxin gene that encodes phospholipase c (Cp/plc-), and showed that the mutant strain can no longer cause gas gangrene in mice. We have also showed that the dormant spores of this deletion mutant, when administered intravenously in mice bearing an orthotopic model of pancreatic cancer, were capable of germination and proliferation in the hypoxic tumor regions with oncopathic effects. However, Cp/plc- does have residual tolerance to oxygen and retains limited capabilities for growth in oxygenated tissues, leading to significant toxicities in animals when administered intravenously at high doses. Superoxide dismutase (sod) is a major oxygen tolerance gene that has also been knocked out in Cp/plc-. The maximum tolerated dose of this knock-out strain (Cp/plc-/sod-) was elevated by one-log over that of Cp/plc- in tumor-bearing mice. To further improve safety, we hypothesize that sequential knock-out of the other major oxygen tolerance genes in the sod knock-out strain will produce an oxygen-intolerant strain (oiCp/plc-) with maximal tumor selectivity and minimal toxicity. However, intratumoral bacteria replication was inhibited by a rapid accumulation of host inflammatory cells that are bactericidal, which limited the extent of tumor response. To enhance oncopathic potency, we hypothesize that intratumoral replication of oiCp/plc- and its antitumor efficacy can be substantially elevated by constructing recombinants that express inflammation suppressive genes from heterologous microbes endowed naturally with such properties. These novel recombinant strains will have maximal tumor selectivity and oncopathic potency in poorly-vascularized tumors. Finally, most solid tumors also contain regions that are relatively well-vascularized and oxygenated, and hence refractory to the anaerobic bacteria treatment. These vascularized and oxygenated regions however, are susceptible to systemically administered chemotherapeutic drugs. Thus we hypothesize that anaerobic bacteria treatment that targets the hypoxic cores of the poorly- vascularized tumors will be complementary to systemic chemotherapy that targets the vascularized regions, leading to substantially enhanced tumor destruction and survival prolongation. Successful conduct of the proposed studies may lead to the development of recombinant Cp spores as a novel class of therapeutic agents that can be administered systemically and safely to patients with advanced pancreatic cancer and other poorly-vascular tumors in the future and which, in combination with chemotherapy, may lead to improved treatment outcome than systemic chemotherapy alone, which is the standard of care at present. PUBLIC HEALTH RELEVANCE: Although pancreatic cancer is only the 10th and 11th leading cause of new cancer cases in U.S. men and women, respectively, it is the 4th leading cause of cancer deaths for both U.S. men and women. 33,370 deaths and 37,170 new cases are estimated for 2007 in the U.S. alone. Currently available treatment modalities include chemotherapy and radiation therapy, which are not particularly effective for pancreatic cancer. Anaerobic bacteria can target the hypoxic cores in tumors that will be complementary to chemotherapy, such that the outcome of combination treatment will be superior to chemotherapy alone.