Targeted tumoricidal 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。以前其他人的实验利用厌氧微生物进行癌症治疗。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。由于厌氧细菌不能在活的肿瘤组织中生长，因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（Nature Reviews Cancer 5,796 -806, 2005; 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。该菌株是兼性厌氧菌鼠伤寒沙门氏菌的一种营养不良但毒性很强的变种，称为A1，可在缺氧或常氧条件下生长。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。我们通过用GFP标记它们并成像它们靶向用RFP标记的肿瘤的能力来快速选择这种细菌。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。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。我们现在已经证明，当用A1治疗人类前列腺和乳腺肿瘤小鼠模型时，生存率显著提高。本应用的目的是扩大由营养不良鼠伤寒沙门氏菌靶向的肿瘤类型，并确定宿主免疫系统是否增强了细菌对肿瘤的杀伤。具体目的如下：(1)选择更多的鼠伤寒沙门氏菌多氨基酸营养缺陷菌，将肿瘤杀伤选择性扩大到更多的肿瘤类型，包括患者肿瘤模型；(2)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进一步开发用于最终的临床应用。缺失突变体将被开发出来，以减少营养不良细胞恢复为野生型的可能性。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。细菌治疗转移性癌症的描述。人们正在开发一种基因改变的细菌，这种细菌只在肿瘤中生长，并能摧毁肿瘤。细菌和肿瘤都被设计成发出不同颜色的荧光，这样就可以在使用的小鼠模型外部看到细菌对肿瘤的靶向。未来的肿瘤杀伤细菌的人体试验可以在第一阶段和第二阶段授权期完成后进行。

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 by others employed anaerobic microorganisms for cancer therapy. Target specificity appeared largely due to the anaerobic requirements met principally in necrotic tumor areas. The resulting tumor killing was at best limited since anaerobic bacteria could not grow in viable tumor tissue. 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 (Nature Reviews Cancer 5, 796-806, 2005; 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 variant of the facultative anaerobe Salmonella typhimurium, termed A1 that can grow under hypoxic or normoxic conditions. 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. We rapidly selected this bacteria by labeling them with GFP and imaging their ability to target tumor labeled with RFP. This remarkable selectivity apparently reflects the imposed nutritional requirements that are apparently met only in the cancer cell milieu. S. typhimurium A1 is a double amino acid auxotroph that requires Leu and Arg. We have now demonstrated that human prostate and breast tumor mouse models have highly significant survival increases when treated with A1. The goal of this application is to expand the tumor types targeted by auxotrophic S. typhimurium and to determine if the host immune system enhances tumor kill by the bacteria. The specific aims are as follows: (1) Select additional multiple amino-acid auxotrophs of S. typhimurium to expand tumor-killing selectivity to additional tumor types, including patient tumor models; (2) 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 for eventual clinical application. Deletion mutants will be developed to reduce the probability of reversion of auxotrophs to wild-type. 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. Bacterial therapy for metastatic cancer is described. Genetically-altered bacteria that grow only in tumors and destroy them are being developed. Both bacteria and tumors are engineered to fluoresce different colors such that the targeting of the bacteria to the tumors can be visualized external to the mouse models being used. Future human trials of the tumor-killing bacteria can be held after the Phase I and Phase II grant periods are completed.