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. ? ? ?
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