Imageable tumor-targeting bacteria

描述（由申请人提供）：本申请的目标是开发可想象的肿瘤靶向细菌，该细菌可以在不感染宿主的情况下治愈肿瘤。以前的实验使用厌氧微生物治疗癌症，但使用了明显不同的方法。自然无毒或减毒的微生物主要作为治疗基因的传递载体。靶特异性的出现主要是由于主要在坏死肿瘤区域满足厌氧需求。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。为了实现这一目标，我们最近开发了能够可视化表达绿色荧光蛋白（GFP）和红色荧光蛋白（RFP）的肿瘤和细菌的全身成像系统（柳叶刀肿瘤学3,546-556,2002;Proc. Natl）。学会科学。美国，98,9814-9818,2001)。在成像技术的帮助下，我们开发了一种独特的肿瘤靶向鼠伤寒沙门氏菌菌株（Proc. Natl）。《科学通报》（英文版），2005年6月。该菌株是一种营养不良但完全致命的鼠伤寒沙门氏菌变种，称为A1。A1型营养不良菌株选择性地生长并破坏活的和坏死的恶性组织，但对正常组织几乎没有影响。即使在免疫缺陷的裸鼠体内，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强加的营养需求，而这种需求显然只有在癌细胞环境中才能得到满足。我们用荧光荷瘤活小鼠的实验快速选择了这种细菌。鼠伤寒沙门氏菌A1是一种双氨基酸营养不良菌，需要亮氨酸和精氨酸。在活体动物模型中选择治疗性细菌将是非常昂贵、耗时和极其繁琐的，除了在该应用中使用的全身成像系统所提供的实时成像。具体目的如下：(1)在小鼠肿瘤模型中，选择鼠伤寒沙门氏菌额外的多种氨基酸营养不良因子，增强肿瘤杀伤选择性，降低可能的宿主毒力；(2)建立鼠伤寒沙门氏菌特定营养不良菌株对肿瘤类型的依赖性，包括患者-肿瘤小鼠模型；(3)通过比较选定的鼠伤寒沙门氏菌在裸鼠和免疫功能小鼠肿瘤模型中的抗肿瘤效果，确定宿主免疫状态的可能意义。在II期资助中，有效的抗肿瘤菌株将在更大的动物身上进一步开发，最终用于临床应用。确定肿瘤靶向鼠伤寒沙门氏菌营养不良菌株和化疗药物以及放射学的可能协同作用也将在II期应用中进行测试。此外，在II期，将对靶向肿瘤的氨基酸型营养不良沙门氏菌进行修饰，使其分泌细胞因子，通过刺激宿主抗肿瘤免疫反应来增强细菌对肿瘤的杀伤作用。

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.