Anaerobic Bacteria as Oncopathic Agents for Pancreatic Cancer

描述（由申请人提供）：癌症治疗的一个主要挑战是选择性和有效地将治疗药物输送到病变。实体肿瘤，如胰腺癌，通常在其核心有血管化不良的区域，这些区域缺氧，不易进入化疗药物的血管输送。然而，缺氧提供了可能导致肿瘤破坏的厌氧细菌定植的可能性，其中一种厌氧细菌是产气荚膜梭菌（Cp）。我们删除了其编码磷脂酶c的α毒素基因（Cp/plc-），并表明突变菌株不再引起小鼠气性坏疽。我们还发现，将这种缺失突变体的休眠孢子静脉注射到具有原位胰腺癌模型的小鼠中，能够在缺氧肿瘤区域萌发和增殖，具有致瘤作用。然而，Cp/plc-确实对氧有残余耐受性，并且在含氧组织中保持有限的生长能力，当高剂量静脉注射给药时，导致动物显着毒性。超氧化物歧化酶（sod）是一个主要的氧耐受性基因，在Cp/plc-中也被敲除。该敲除菌株（Cp/plc-/sod-）在荷瘤小鼠中的最大耐受剂量比Cp/plc-提高了1倍。为了进一步提高安全性，我们假设依次敲除sod敲除菌株中的其他主要氧耐受性基因将产生具有最大肿瘤选择性和最小毒性的氧耐受性菌株（oiCp/plc-）。然而，肿瘤内细菌的复制被宿主炎症细胞的快速积累所抑制，这限制了肿瘤反应的程度。为了增强肿瘤毒性，我们假设通过构建表达天然具有炎症抑制基因的异源微生物的重组体，可以显著提高肿瘤内oiCp/plc-的复制及其抗肿瘤功效。这些新的重组菌株在缺乏血管化的肿瘤中具有最大的肿瘤选择性和肿瘤病理效力。最后，大多数实体瘤还包含相对血管化和氧合良好的区域，因此对厌氧细菌治疗难以耐受。然而，这些血管化和含氧区域易受全身化疗药物的影响。因此，我们假设针对血管化不良肿瘤缺氧核心的厌氧细菌治疗将与针对血管化区域的全身化疗相辅相成，从而大大增强肿瘤破坏和延长生存期。这些研究的成功进行可能会导致重组Cp孢子作为一种新型的治疗药物的发展，它可以在未来系统安全地给药于晚期胰腺癌和其他血管不良肿瘤患者，并且与化疗联合使用，可能会导致比单独全身化疗更好的治疗结果，这是目前的治疗标准。公共卫生相关性：尽管胰腺癌仅是美国男性和女性新发癌症病例的第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，2007年仅在美国就有33,370人死亡，37,170例新病例。目前可用的治疗方式包括化疗和放疗，但对胰腺癌并不是特别有效。厌氧菌可以靶向肿瘤内的缺氧核心，作为化疗的补充，因此联合治疗的效果将优于单独化疗。

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.