SBIR-STTR Award

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

We have developed an effective bacterial cancer therapy strategy by targeting viable tumor tissue by using Salmonella typhimurium auxotrophs that we have generated which grow in viable as well as necrotic areas of tumors. However, the auxotrophy severely restricts growth of these bacteria in normal tissue enabling them to be used for cancer treatment. The S. typhimurium A1-R mutant, which is auxotrophic for leu-arg and has high antitumor virulence was developed in our laboratory. In vitro, A1-R infects tumor cells and causes nuclear destruction. A1-R was initially used to treat metastatic human prostate and breast tumors that had been orthotopically implanted in nude mice. Forty percent of treated mice were cured completely and survived as long as non-tumor-bearing mice. A1-R administered i.v. to the nude mice with primary osteosarcoma and lung metastasis was highly effective, especially against metastasis. A1-R was also targeted to both axillary lymph and popliteal lymph node metastasis of human pancreatic cancer and fibrosarcoma, respectively, as well as lung metastasis of the fibrosarcoma in nude mice. The bacteria were delivered via a lymphatic channel to target the lymph-node metastases and systemically via the tail vein to target the lung metastasis. The metastases were cured without the need of chemotherapy or any other treatment. A1-R was administered intratumorally to nude mice with an orthotopically-transplanted human pancreatic tumor. The primary pancreatic cancer regressed without additional chemotherapy or any other treatment. A1-R also was effective against pancreatic cancer liver metastasis when administered intrasplenically to nude mice. A1-R has also shown to be effective for spinal glioma in orthotopic mouse models. The approach described here, where bacterial monotherapy effectively treats primary and metastatic tumors, is a significant improvement over previous bacterial tumor-therapy strategies that require combination with toxic chemotherapy. The present application will develop bacterial therapy of cancer with S. typhimurium A1-R in immunocompetent mice as a bridge to the clinic. The specific aims of the present application are (1) Determine the maximum tolerated dose (MTD) of S. typhimurium A1-R in tumor-bearing C57 immunocompetent mice. (2) Determine the optimal dose, route and schedule of S. typhimurium A1-R in tumor-bearing immunocompetent mice. Exploitation of the tumor-killing capability of Salmonella has great potential for a new paradigm of cancer therapy.

Public Health Relevance:
We have developed a bacterial cancer therapy strategy using Salmonella typhimurium variants. These bacteria kill cancer cells but do not grow in normal tissue. Our S. typhimurium A1-R mutant has antitumor virulence against a number of cancer cell lines in nude mouse models. In vitro, A1-R infects tumor cells and can cause rapid nuclear fragmentation and apoptosis. A1-R was found to be effective against metastatic human prostate, breast, and pancreatic cancer cell lines as well as osteosarcoma, fibrosarcoma and glioma cell lines in clinically-relevant mouse models. Treated mice were often cured completely and survived as long as non- tumor-bearing mice. The approach described here, where bacterial monotherapy effectively treats primary and metastatic tumors, is a significant improvement over previous bacterial tumor-therapy strategies that require combination with toxic chemotherapy. The present application will develop bacterial therapy of cancer in immunocompetent mouse models as a bridge to clinical trials. Exploitation of the tumor-killing capability of Salmonella has great potential for a new paradigm of cancer therapy.

Thesaurus Terms:
Apoptosis; Apoptosis Pathway; Area; Armpit; Athymic Nude Mouse; Axilla; Axillary; Bacteria; Bone Sarcoma; Breast; Breast Neoplasms; Breast Tumors; Cancer Treatment; Cancer Cell Line; Cell Death, Programmed; Clinic; Clinical Trials; Clinical Trials, Unspecified; Dose; Generalized Growth; Genital System, Male, Prostate; Glial Cell Tumors; Glial Neoplasm; Glial Tumor; Glioma; Growth; Hepatic Neoplasm Secondary; Hepatic Metastasis; Human; Human Prostate; Human Prostate Gland; Human, General; Immunocompetent; Implant; In Vitro; Killings; L-Leucyl-L-Arginine; Laboratories; Leu-Arg; Liver Metastasis; Liver Secondaries; Liver Secondary Cancer; Lung; Lymph; Lymph Node Proper; Lymphatic; Malignant Cell; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; Malignant Pancreatic Neoplasm; Malignant Neoplasm Of Liver, Specified As Secondary; Malignant Neoplasm Of Pancreas; Mammals, Mice; Mammary Cancer; Mammary Neoplasms; Man (Taxonomy); Man, Modern; Maximal Tolerated Dose; Maximally Tolerated Dose; Maximum Tolerated Dose; Metastasis; Metastasize; Metastatic Neoplasm; Metastatic Neoplasm To The Liver; Metastatic Tumor; Metastatic Tumor To The Liver; Metastatic Malignant Neoplasm To Liver; Metastatic Neoplasm Of Liver; Mice; Mice, Athymic; Mice, Nude; Murine; Mus; Necrosis; Necrotic; Neoplasm Metastasis; Neoplasms Of Neuroglia; Neuroglial Neoplasm; Neuroglial Tumor; Normal Tissue; Normal Tissue Morphology; Nuclear; Nude Mice; Osseous Sarcoma; Pancreas Cancer; Pancreas Neoplasms; Pancreatic Cancer; Pancreatic Tumor; Prostate; Prostate Gland; Prostatic Gland; Respiratory System, Lung; Reticuloendothelial System, Lymph; Reticuloendothelial System, Lymph Node; Route; S. Typhimurium; S.Typhimurium; Sched; Safety; Salmonella; Salmonella Typhimurium; Sarcoma, Osteogenic; Schedule; Secondary Neoplasm; Secondary Tumor; Secondary Malignancy Of Liver; Secondary Malignant Neoplasm Of Liver; Skeletal Sarcoma; Spinal; Tail; Tissue Growth; Transplantation; Tumor Cell; Tumor Cell Migration; Tumor Tissue; Tumor Of The Pancreas; Tumors Of Neuroglia; Underarm; Variant; Variation; Veins; Virulence; Anticancer Therapy; Auxotrophy; Cancer Cell; Cancer Metastasis; Cancer Therapy; Chemotherapy; Clinical Investigation; Clinical Relevance; Clinically Relevant; Fibrosarcoma; Glioma Cell Line; Leucyl-Arginine; Leucylarginine; Liver Metastases; Lymph Gland; Lymph Nodes; Lymphatic Fluid; Mammary Tumor; Metastatic Cancer To Liver; Mouse Model; Mutant; Neoplastic Cell; Ontogeny; Osteochondrosarcoma; Osteoid Sarcoma; Osteosarcoma; Pancreatic Neoplasm; Public Health Relevance; Pulmonary; Transplant; Tumor