Imageable tumor-targeting bacteria

描述（由申请人提供）：本申请的目的是开发可成像的肿瘤靶向细菌，其可以治愈肿瘤而不会对宿主进行性感染。以前的实验使用厌氧微生物进行癌症治疗，但使用了一种明显不同的方法。天然无毒或减毒微生物主要用作治疗基因的递送载体。靶特异性主要是由于坏死肿瘤区域主要满足厌氧要求。由此产生的肿瘤杀伤充其量是有限的。因此，更有效的靶向是必要的，特别是在活的肿瘤组织中。为了这个目标，我们最近开发了全身成像系统，其使得能够可视化表达绿色荧光蛋白（GFP）和红色荧光蛋白（RFP）的肿瘤和细菌（Lancet Oncology 3，546-556，2002; Proc. Natl. Acad. Sci. USA 98，9814-9818，2001）。在成像技术的帮助下，我们已经开发了一种独特的肿瘤靶向鼠伤寒沙门氏菌菌株（Proc. Natl. Acad. Sci USA 102，755-760，2005）。该菌株是一种营养缺陷型但完全有毒的沙门氏菌。称为A1的鼠伤寒沙门氏菌变体。A1营养缺陷型菌株选择性地生长并破坏存活的以及坏死的恶性组织，但对正常组织几乎没有影响。即使在免疫缺陷的裸鼠中，A1细菌最终也会从正常组织中消失。这种显著的选择性显然反映了明显仅在癌细胞环境中满足的强加的营养需求。我们使用荧光荷瘤活小鼠试验快速选择了这种细菌。S.鼠伤寒A1是需要Leu和Arg的双氨基酸营养缺陷型。在活的动物模型中选择治疗性细菌将是极其昂贵、耗时且极其繁琐的，除了在该应用中使用的全身成像系统所提供的实时成像。具体目标如下：（1）筛选出附加的多氨基酸营养缺陷型菌株。鼠伤寒沙门氏菌，以提高肿瘤杀伤的选择性，并降低可能的宿主毒力在小鼠肿瘤模型;（2）建立依赖特定的营养缺陷型菌株的S。鼠伤寒沙门氏菌对肿瘤类型的影响，包括患者-肿瘤小鼠模型;和（3）通过比较所选S.裸鼠和免疫活性小鼠肿瘤模型中的鼠伤寒沙门氏菌营养缺陷型。在第二阶段的资助中，有效的抗肿瘤细菌菌株将进一步使用更大的动物进行开发，以最终用于临床应用。确定肿瘤靶向S.鼠伤寒营养缺陷型菌株和化疗剂以及放射学也将在II期申请中进行测试。此外，沙门氏菌氨基酸肿瘤靶向营养缺陷型菌株将被修饰以分泌细胞因子，以增强肿瘤的细菌杀伤，并在II期刺激宿主抗肿瘤免疫反应。

DESCRIPTION (provided by applicant): The goal of this application is to develop imageable tumor-targeting bacteria that can cure tumors without progressive infection of the host. Previous experiments employed anaerobic microorganisms for cancer therapy but used a markedly different approach. Naturally nonvirulent or attenuated microbes served primarily as delivery vehicles for therapeutic genes. Target specificity appeared largely due to the anaerobic requirements met principally in necrotic tumor areas. The resulting tumor killing was at best limited. Therefore, more effective targeting is necessary, especially in the viable tumor tissue. Toward this goal, we have recently developed whole-body imaging systems that enable the visualization of green fluorescent protein (GFP)- and red fluorescent protein (RFP)-expressing tumors and bacteria (Lancet Oncology 3, 546-556, 2002; Proc. Natl. Acad. Sci. USA 98, 9814-9818, 2001). With the help of the imaging technology, we have developed a unique tumor-targeting Salmonella typhimurium strain (Proc. Natl. Acad. Sci USA 102, 755-760, 2005). This strain is an auxotrophic but fully virulent S. typhimurium variant termed A1. The A1 auxotrophic strain selectively grows in and destroys viable as well as necrotic malignant tissue but has little effect on normal tissue. The A1 bacteria eventually disappears from normal tissue even in immunodeficient nude mice. This remarkable selectivity apparently reflects the imposed nutritional requirements that are apparently met only in the cancer cell milieu. We rapidly selected this bacteria using assays of fluorescent tumor-bearing live mice. S. typhimurium A1 is a double amino acid auxotroph that requires Leu and Arg. Selecting therapeutic bacteria in live animal models would be prohibitively expensive, time-consuming, and extremely tedious, save for the real-time imaging afforded by the whole- body imaging systems to be used in this application. The specific aims are as follows: (1) Select additional multiple amino-acid auxotrophs of S. typhimurium to enhance tumor-killing selectivity and reduce possible host virulence in mouse tumor models; (2) Establish dependence of particular auxotrophic strains of S. typhimurium on tumor type, including patient-tumor mouse models; and (3) Determine possible significance of host immunological status by comparing antitumor efficacy of selected S. typhimurium auxotrophs in nude-mouse and immunocompetent-mouse tumor models. In the Phase II grant, the effective antitumor bacterial strains will be further developed using larger animals for eventual clinical application. Determination of possible synergy of tumor targeting S. typhimurium auxotrophic strains and chemotherapeutic agents as well as radiology will also be tested in the Phase II application. In addition, Salmonella amino acid tumor-targeting auxotroph strains will be modified to secrete cytokines to enhance the bacterial killing of the tumor with stimulation of host antitumor immune reactions in Phase II.