Anaerobic Bacteria as Oncopathic Agents for Pancreatic Cancer

描述（由申请人提供）：癌症治疗的主要挑战是选择性和有效地将治疗剂递送至病变。诸如胰腺癌的实体瘤通常在其核心处具有血管化不良的区域，这些区域是缺氧的并且不容易接近化疗药物的血管递送。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，并且一种这样的厌氧细菌是产气荚膜梭菌（Cp）。我们已经删除了编码磷脂酶C（Cp/plc-）的α毒素基因，并表明突变株不再引起小鼠气性坏疽。我们还表明，这种缺失突变体的休眠孢子，当静脉注射给药的小鼠携带原位模型的胰腺癌，能够发芽和增殖的缺氧肿瘤区域与肿瘤病变的影响。然而，Cp/plc-对氧具有残余耐受性，并且在含氧组织中保留有限的生长能力，当以高剂量静脉内给药时，导致动物中的显著毒性。超氧化物歧化酶（SOD）是一个主要的耐氧基因，也已在Cp/PLC-中被敲除。在荷瘤小鼠中，该敲除菌株（Cp/plc-/sod-）的最大耐受剂量比Cp/plc-的最大耐受剂量升高一个对数。为了进一步提高安全性，我们假设顺序敲除sod敲除菌株中的其他主要耐氧基因将产生具有最大肿瘤选择性和最小毒性的氧不耐受菌株（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。为了增强肿瘤致病效力，我们假设oiCp/plc-的肿瘤内复制及其抗肿瘤效力可以通过构建表达来自天然赋予此类性质的异源微生物的炎症抑制基因的重组体来显著提高。这些新的重组菌株将在血管化不良的肿瘤中具有最大的肿瘤选择性和致瘤效力。最后，大多数实体瘤还含有相对良好血管化和氧合的区域，因此对厌氧菌治疗无效。然而，这些血管化和氧合区域对全身施用的化疗药物敏感。因此，我们假设靶向血管化不良肿瘤的缺氧核心的厌氧菌治疗将与靶向血管化区域的全身化疗互补，导致实质上增强的肿瘤破坏和存活延长。拟议研究的成功实施可能导致重组Cp孢子作为一类新型治疗药物的开发，该药物可在未来安全地全身给药于晚期胰腺癌和其他血管不良肿瘤患者，与化疗联合使用，可能导致治疗结局比单独全身化疗（目前的标准治疗）改善。公共卫生相关性：虽然胰腺癌分别是美国男性和女性新发癌症病例的第10和第11大原因，但它是美国男性和女性癌症死亡的第4大原因。仅在美国，2007年估计就有33,370例死亡和37,170例新病例。目前可用的治疗方式包括化疗和放射治疗，这对胰腺癌不是特别有效。厌氧细菌可以靶向肿瘤中的缺氧核心，这将是化疗的补充，因此联合治疗的结果将上级单独化疗。

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.