WO2009111177A2 - Compositions and methods comprising genetically enhanced obligate and facultative anaerobic bacteria for oncopathic cancer therapy - Google Patents

Abstract

Provided are genetically enhanced anaerobic bacteria for the oncopathic therapy of cancers, including pancreatic and otheτ cancers that contain avascular, hypoxic regions. Within certain embodiments, genetically enhanced anaerobic bacteria contain one or more mutation(s). such as, for example, a deletion in one or more oxygen tolerance gene(s) such as a superoxide dismutase (sod), glutathione peroxidase (gpo), rubrerythrin (rbr), and/or an alcohol dehydrogenase family mernbei (ydaD) gene. Within further embodiments, genetically enhanced anaerobic bacteria contain one or more mutation(s), such as, for example, a deletion in one or more toxin gene(s) such as a phospholipase c (plc) gene. Within other embodiments, genetically enhanced anaerobic bacteria further express an inflammation suppressive gene such as, for example, a Staphylococcus aureus Panton-Valentine Leukocidin (PVL) gene and/or a Yersinia enterocolitica virulence factor (LcrV) gene. Genetically enhanced anaerobic bacteria disclosed herein, as exemplified by Clostridium, Bifidobacterium, and Salmonella species, are capable of selectively targeting hypoxic tumors.

Description

COMPOSITIONS AND METHODS COMPRISING GENETICALLY ENHANCED

OBLIGATE AND FACULTATIVE ANAEROBIC BACTERIA

FOR ONCOPATHIC CANCER THERAPY

CROSS REFERENCE TO RELATED APPLICATIONS The present application claims the benefit of U S Provisional Patent Application

Serial No 61/034,111, filed March 5, 2008, the contents of which are hereby incorporated by reference in their entirety

GOVERNMENT SUPPORT

The work disclosed in the present application was supported, in part, by an NIH grant (R21 -CA- 120017) The Federal Government may have rights in certain aspects of the presently disclosed invention

TECHNICAL FIELD

The present disclosure relates generally to genetically enhanced obligate and facultative anaerobic bacteπa for the oncopathic therapy of cancers, including pancreatic and other cancers that contain avascular, hypoxic regions Within certain embodiments, genetically enhanced anaerobic bacteπa contain one or more mutation(s), such as, for example, a deletion in one or more oxygen tolerance gene(s) such as a superoxide dismutase (sod), glutathione peroxidase (gpo), rubrerythπn (rbr), and/or an alcohol dehydrogenase family member (ydaD) gene Within further embodiments, genetically enhanced anaerobic bacteπa contain one or more mutation(s), in one or more toxin gene(s) such as, for example, a phosphohpase c (pic) gene Within other embodiments, genetically enhanced anaerobic bacteπa further express an inflammation suppressive gene such as, for example, a Staphylococcus aureus Panton- Valentine Leukocidin (PVL) gene and/or a Yersinia enterocolitica virulence factor (LcrV) gene Genetically enhanced anaerobic bactena disclosed herein, as exemplified by Clostridium, Bifidobacterium, and Salmonella species, are capable of selectively targeting hypoxic tumors

BACKGROUND

Pancreatic carcinoma is currently the fourth leading cause of cancer-related death in the USA Greenlee et al , CA Cancer J Clin 5J. 15-36, 2001, Jemal et al , CA Cancer J Ctø 54 8-29, 2004, and Lebedeva er a/ , Cancer Res 66 2403-13, 2006 There were >30,000 new cases diagnosed in the U S in 2005 Id About 10-15% of patients undergo complete resection and, of these, less than a fifth are alive at 5-year follow-up Sener et al , J Am Coll Surg 189 1-7,1999 Median survival time is 6 to 10 months for patients with locally advanced disease, and 3 to 6 months for those with metastatic disease Id and Evans et al , "Cancer Principles and Practice of Oncology" Ed 4, 1054-87 (DeVita et al , (eds), Philadelphia, PA, Lippincott, 1997)

Pancreatic cancer is a complex disorder in which multiple subsets of genes undergo genetic change, either activation or inactivation, during tumor development and progression Jaffee el al , Cancer Cell 2 25-8, 2002 and Xiong, Cancer Chemother Pharmacol 54 69-77, 2004 Common genetic modifications in pancreatic carcinomas include activation of the K- ras oncogene (85-95%), over-expression of specific growth factors and their associated receptors, and inactivation of the pl6/RBl (>90%), p53 (75%), DPC4 (55%), and BRCA2 tumor suppressor genes Jaffee et al , Cancer Cell 2 25-8, 2002 and Xiong, Cancer Chemother Pharmacol 54 69-77, 2004 These findings accentuate the complexity of this heterogenous cancer and may underlie the aggressiveness and inherent resistance of this neoplasm to conventional therapies Bardeesy and DePinho, Nat Rev Cancer 2 897-909, 2002

Conventional treatment options, including chemotherapy and radiation options, for advanced or metastatic pancreatic cancer are limited Casper, Eur J Cancer 29 171-2, 1993, Storniolo et al , Cancer 85 1261-8, 1999, and Stephens, Oncol Nurs Forum 25 87-93, 1998 The single-agent fluorouracil (FU) results in tumor response rates of 7% or less Casper, Eur J Cancer 29 171-2, 1993 and Storniolo et al , Cancer 85 1261-8, 1999 Combination chemotherapy with FU has, unfortunately, resulted in increased toxicity without an improvement in efficacy Stephens, Oncol Nurs Forum 25 87-93, 1998 Gemcitabine, which is a nucleoside analog that can be converted to nucleotides by cellular kinases and cytotoxic to cells undergoing DNA replication, is the current standard of treatment for advanced disease because of clinical benefit (symptom amelioration), provides only modest survival benefit when compared to fluorouracil treatment (5 65 vs 441 months) Burns et al j CIm Oncol 15 2403-2413, 1997

Since its approval for treatment of pancreatic cancer in 1997, the only new drug that has received FDA approval is erlotimb, a small molecule that inhibits the EGFR tyrosine kinase pathway, when given in combination with gemcitabine Moore et al , J CIm Oncol 25_ 1960-1966, 2007 Although the survival benefit of the combination was modest (median survival of 5 91 to 6 24 months and 1-year survival of 17% to 23%), these data suggest the potential therapeutic importance of combination treatment modalities that target different pathways in cancers such as pancreatic cancer Radiation therapy administered concurrently with chemotherapy has been tested in locally advanced pancreatic cancer External beam radiation therapy alone has a 72% local failure rate Roldan et al , Cancer 61 1110-1116, 1988 Studies of concurrent chemotherapy with radiation compared to radiation therapy alone have showed conflicting results, from superior overall survival (Moertel et al , Cancer, 48 1705-10, 1981) to no benefit in response rate or survival Cohen, InI J Radial Oncol Biol Phys 62 1345-50, 2005

Most tumors, including pancreatic cancers, contain large, poorly vascularized areas that limit the efficacy of radiation and chemotherapeutic drugs Kondoh et al , Cancer Sci 94 1021-8, 2003 A major obstacle in cancer therapy is the specific delivery of an anticancer agent not only to the primary but also to metastatic solid tumors As metastases grow, they also construct poorly vascularized, oxygen-deficient, hypoxic areas that decrease the efficiency of currently used anti-cancer modalities Hypoxic regions are characteristic of tumors in rodents and occur with high frequency in many types of human tumors, including colorectal and pancreatic primary tumors and metastases to the liver Moulder and Rockwell, Int J Radial Oncol Biol Phys K) 695-712, 1984 and Vaupel and Hockel in Tumor Oxygenation 219-232 {eds Vaupel, Kelleher, & Gunderoth, Gustav Fischer, Stuttgart, 1995) For many years, the importance of hypoxia in solid tumors was linked solely to the fact that hypoxic cells are intrinsically more resistant to treatment A markedly lower mtra- tumoral oxygen level is a primary problem because ionizing radiation and certain drugs require oxygen-derived free radicals to destroy target cells In addition, chemotherapeutic drug resistance can be caused by hypoxia-induced inhibition of cell cycle progression and proliferation, since a number of drugs specifically target highly proliferating cells Proliferation decreases as a result of decreasing oxygen levels, and it has been shown that drug toxicity falls off as a function of distance from blood vessels Furthermore, inefficient drug delivery to hypoxic regions is a fundamental problem Because the blood flow to hypoxic tumor cells is very low, drugs are inefficiently delivered and the drug concentration never reaches an effective level

Hypoxia is a tumor characteristic that is potentially exploitable using bio-reductive drugs or gene therapy Theys el al , Curr Gene Ther 3 207-21, 2003 and Pawelek et al , Lancet Oncol 4 548-56, 2003 The presence of severe hypoxia in solid tumors also offers the potential for specific anaerobic bacterial colonization and tumor destruction Although early attempts to exploit the tumor-specific nature of hypoxia were disappointing, more recent strategies appear promising Hypoxic cell cytotoxins, such as tirapazamine, have shown that hypoxia can be converted to a therapeutic advantage, while gene therapy or the use of engineered non-pathogenic live obligate anaerobes may offer an even more specific means of targeting hypoxic tumor cells Brown, Cancer Biology & Therapy X_ 453-8, 2002

Anaerobic bacteria are microbes that thrive in oxygen-deprived areas and their growth in oxygen-proficient areas is severely limited Anaerobic bacteπa include facultative anaerobic bactena and obligate anaerobic bacteria Facultative anaerobic bacteria make ATP by aerobic respiration if oxygen is present but are also capable of switching to fermentation in the absence of oxygen Facultative anaerobic bactena include, for example, the Salmonella, Staphylococci, Escherichia coll, Corynebacterium, and Listeria species In contrast, obligate anaerobic bacteπa are incapable of aerobic respiration Obligate anaerobic bactena include, for example, Bacteroides and Bifidobacterium (non-spore forming) and Clostridium (spore-forming) species

Compared with viral vectors or liposome delivery, bactenal vectors have unique advantages They can be engineered to carry more than one gene, they are easy to produce, they do not alter the genome of the recipient, and they can be eliminated by antibiotics once treatment is complete Sznol et al , J Clin Invest 105 1027-30, 2000 Since solid tumor cores are oxygen-deficient and the normal tissues in the body are oxygen-proficient, anaerobic bacteπa offer the potential for tumor-specific killing while spanng normal tissues Preferential replication in tumors offers great potential to amplify the therapeutic effect of the anaerobic microorganisms

Beneficial effects of bactenal infection on tumors have been observed since the 18th century, and hundreds of cases of spontaneous regression of many types of malignancies following bactenal infections had been recorded Cπtchley el al , Gene Therapy X X, 1224- 33, 2004 In the 1960s, anaerobic bactena were shown to target tumors and replicate within the hypoxic and necrotic regions of these tumors Clostridium was shown to be effective in causing tumor regression in rodent models but a subsequent clinical tπal failed to demonstrate any benefit that would outweigh the toxicities Engelbart and Gencke, Cancer Res 24 239-43, 1964 More recently, the use of anaerobic bacteπa for the treatment of tumors has been re-evaluated using attenuated strains of Salmonella and Clostridium Ryan et al , BioEssays 28 84-94, 2006, Pawelek et al , Cancer Res 57 4537-44, 1997, Theys et al , Cancer Gene Ther 8 294-7, 2001 , and Forbes, Nature Biotechnology 24 1484-85, 2006

A number of approaches to developing oncopathic bacteria, including Gram-negative Salmonella, have been described In contrast to obligate anaerobic bacteria such as Clostridia and Bifidobacteria, Salmonella are facultative anaerobic bacteria and have the potential to colonize oxygenated small metastatic lesions as well as large tumors with a hypoxic center A major problem for the full exploitation of Salmonella in cancer treatment is, however, that it can induce TNF-alpha mediated septic shock Recently, attenuated Salmonella typhimurium strains have been descnbed as anticancer agents Pawelek et al , Lancet Oncol 4 548-56, 2003 Bifidobacterium longum, a nonpathogenic Gram-positive anaerobic bacterium, has been shown to selectively germinate and grow in the hypoxic regions of solid tumors after intravenous injection Yazawa et al , Breast Cancer Res and Treatment 66 16S-70, 2001

Clostridium sp are obligate anaerobic, spore-forming, Gram-positive bacteria All require anaerobic conditions to grow but they vary in their oxygen tolerance and their biochemical profiles Germination of clostridial spores will only occur when they encounter the requisite anaerobic conditions In the past few years, several Clostridium species were studied for anti-tumor potential Heppner and Mose, Acta Neurocbir 42 123-5, 1978

Vogelstein and coworkers investigated Clostridium novyi for its capacity to grow within transplanted tumors Dang et al , Proc Natl Acad Sci USA 98 15155-60, 2001 A recombinant strain of C novyi, devoid of its lethal toxin gene, was engineered (C novyi- NT) and was shown to germinate within the avascular regions of tumors and destroy surrounding tumor cells in mice Spores were systemically administered to immune- competent mice bearing 16 melanomas, alone or in combination with chemotherapeutic agents Tumor colonization occurred, leading to significant tumor necrosis This so-called combined bacteriolytic therapy (COBALT) was, however, associated with high toxicity and caused the death of most animals within 1 to 3 days following treatment, in spite of the removal of the toxin gene The toxicity is thought to be due to bacterial toxin products released from the tumors or oxygen tolerance leading to spread of the microorganisms into normal tissues Very recently, the same group showed that treatment of mice bearing large tumors with C novyi-ΗT plus a single dose of liposomal doxorubicin led to eradication of the tumors Cheong et al , Science 314 1308-11, 2006 The bacteπal factor responsible for the enhanced drug release was identified as a previously unrecognized protein termed "liposomase."

Clostridia have also been genetically engineered to selectively deliver pro-drug- activating enzymes such as E. coli cytosine deaminase (Theys et al., Cancer Gene Ther. 8:294-7, 2001) and nitroreductase (Lemmon et al, Gene Ther. 4:791-6, 1997) that enhance treatment efficacy via a bystander effect.

Another anaerobic bacterial strain, Clostridium perfringens (Cp), has been investigated for its ability to selectively colonize and induce necrosis in solid pancreatic tumors in mice. Unfortunately, the wild type Cp strain retained certain levels of residual oxygen tolerance and can cause systemic toxicities in animals. Briolat and Reysset, J. Bacteriol. 184:2333-43, 2002; Geissmann et al, J. Bacteriology 181:7136-9, 1999 and Lehmann et al, J. Bacteriol. 178:7152-8, 1996. The major gene associated with oxygen tolerance in Clostridia perfringens is the superoxide dismutase (sod) gene.

Dormant spores of wild-type Clostridium perfringens (Cp) are capable of preferential germination and proliferation within the hypoxic cores of pancreatic cancer in mice. Oncopathic effects of Cp are, however, limited by host inflammatory responses, and Cp's residual tolerance to oxygen caused toxicities in animals. Thus, there remains a need in the art for improved anaerobic bacteria that are capable of colonizing and destroying hypoxic tumors while exhibiting reduced in vivo toxicities. SUMMARY

The present disclosure fulfills these and other related needs by providing genetically enhanced facultative and obligate anaerobic bacteria for the oncopathic therapy of cancers, including pancreatic cancers. Genetically enhanced anaerobic bacteria disclosed herein are capable of colonizing and destroying hypoxic tumors and are less toxic in vivo as compared to the corresponding anaerobic bacteria from which it derives.

Within certain embodiments, genetically enhanced anaerobic bacteria contain one or more mutation(s) in one or more gene(s) associated with oxygen tolerance. Exemplified herein are genetically enhanced anaerobic bacteria containing one or more mutation(s) is an oxygen tolerance gene selected from the group consisting of a superoxide dismutase (sod) gene, a glutathione peroxidase (gpo) gene, a rubrerythrin (rbr) gene, and an alcohol dehydrogenase family member (ydaD) gene.

Within other embodiments, genetically enhanced anaerobic bacteria express one or more inflammation suppressive gene to enhance anti-tumor efficacy. Exemplary inflammation suppressive genes employed in genetically enhanced anaerobic bactena presented herein are the Staphylococcus aureus Panton- Valentine Leukocidin (PVL) gene and the Yersinia enlerocohtica virulence factor (LcrV) gene

Within still further embodiments, genetically enhanced anaerobic bacteria may contain further mutations in one or more toxin gene(s), including one or more phosphohpase toxin gene(s), to improve safety An exemplary toxin gene mutated in the genetically enhanced anaerobic bacteria presented herein is the phosphohpase c (pic) toxin gene

Genetically enhanced anaerobic bacteria according to any of these embodiments may be employed for the delivery of one or more genes or gene products For example, within certain aspects, genetically enhanced anaerobic bacteria may further comprise one or more genes encoding a prodrug-activating enzyme selected from the group consisting of a cytosine deaminase (CD) that converts 5-fluorocytosine to 5-fluorouracil and a nitroreductase (NTR) that activates CB1954 to 5-Aziridinyl-4-hydroxylammo-2-nitrobenzamide Within other aspects, genetically enhanced anaerobic bactena may further comprise one or more genes encoding a therapeutic protein selected from the group consisting of TNF-α, IL-I, IL-6, IL-IO, IL-12, IL-15, IFN-o, IFN-γ, TRAIL, GM-CSF, FLT3-hgand, and E coll colicin E3 Within yet other aspects, genetically enhanced anaerobic bacteria may further compπse one or more exotoxin genes from heterologous bacterial strains wherein the exotoxin genes are selected from the group consisting of a Clostridia Beta-toxin (CPB), a Streptococcal Streptolysin O (SLO), and a Staphylococcal Staphylolysin A (STA)

Anaerobic bactena that may be employed to generate the genetically enhanced anaerobic bactena of the present disclosure include the obligate anaerobic Clostridium and Bifidobacterium species and the facultative anaerobic Salmonella species Suitable Clostridium species may be selected from the group consisting of Clostridium perfringens, Clostridium histolyticum, Clostridium novyi, Clostridium tetam, Clostridium acetobutylicum, and Clostridium butyricum Suitable Bifidobacterium species may be selected from the group consisting of Bifidobacterium bifidum, Bifidobacterium infantis, and Bifidobacterium longum Suitable Salmonella species may be selected from the group consisting of Salmonella typhimurtum and Salmonella choleraesuis Genetically enhanced oncopathic anaerobic bacteria presented herein may be suitably employed for the treatment of a wide range of cancers associated with solid tumors having avascular, hypoxic regions Exemplary such tumors include, but are not limited to, pancreatic tumors, lung tumors, colorectal tumors, prostate tumors, breast tumors, liver tumors, bladder tumors, melanoms, sarcomas, fibrosarcomas, and glioblastomas Additional tumors that may be advantageously treated with the genetically enhanced oncopathic anaerobic bacteria dislcosed herein include those solid tumors having avascular, hypoxic regions following treatment with one or more antiangiogenic agent as described below Compositions of the present disclosure, and methods based on those compositions, may be advantageously employed in combination with one or more chemotherapeutic and/or radiation treatment regimen selected from the group consisting of gemcitabine (GEMZAR®, Eh Lilly and Co , Indianapolis, IN), docetaxel (TAXOTERE®, Sanofϊ-Avenns, Bπdgewater, NJ), erlotinib (TARCEV A®, Hoffmann-LaRoche, Nutley, NJ), fluorouracil, external beam radiation, and a combination thereof

Alternatively or additionally, compositions disclosed herein may be used in combination with one or more antiangiogenic agent such as, for example, sorafemb (NEXA V AR®, Bayer Pharmaceuticals Corporation, West Haven, CT), bevacizumab (AVASTIN®, Genentech, San Francisco, CA), and/or sunitinib (SUTENT®, Pfizer Inc , New York, NY), ANGIOCEPT™ (Adnexus, Bristol-Myers Squibb R&D Company, New York, NY), AMG-386 (Amgen, Inc , Thousand Oaks, CA), cediranib (RECENTIN™, AstraZeneca, Wilmington, DE), vandetimib (ZACTIMA®, AstraZeneca), thalidomide derivatives (Celgene Corp , Summit, NJ), pazopanib (GlaxoSmithKhne, Brentford, Middlesex, UK), abegπn (Medlmmune, Inc , Gaithersburg, MD), cilengitide (Merck KGaA, Darmstadt, Germany), vatalanib (Novartis, Basel, Switzerland), volociximab (PDL BioPharma/Biogen Idee, Pπnceton, NJ, Cambridge, MA, & Redwood City, Calif), axitinib (Pfizer, Inc , New York, NY), afhbercept (Regeneron/Sanofi -Aventis, Tarrytown, NY & Pans, France), and/or CDP-791 (UCB SA, Brussels, Belgium)

These and other embodiments, features, and advantages of the disclosure will become apparent from the detailed descπption and the appended claims set forth herein below AU literature and patent references cited throughout the application are hereby incorporated by reference in their entireties

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 discloses a schematic representation of the process for constructing a homologous recombinant strain (Figure IA), Diagram of construction of a homologous recombination fragment (an exogenous DNA fragment that is flanked by regions of DNA that are identical to the sequences flanking the gene to be replaced) The upstream and downstream regions flanking the gene targeted for deletion are amplified by PCR with primers PA1/PB1 and PA2/PB2, respectively PA3 and PB3 contain sequences matching the end of the desired gene to be inserted into the chromosome in place of the targeted deletion gene P A3 and PB3 also contain sequences matching the ends of the upstream and downstream flanking regions, respectively The three fragments are assembled by overlapping PCR to generate one homologous recombination fragment For detection of homologous recombination frequency in each construct, the ampR (ampicillin resistance) gene is the desired insertion gene (Figure IB), Diagram of replacement of a targeted deletion gene through homologous recombination The homologous recombination fragment containing the desired insertion gene flanked by the arms with sequence identity is introduced into a Cp strain by optimized electroporation In recombinant cells, the targeted gene in the original bactenal genome DNA is replaced by the desired insertion gene Any side products are lost quickly in dividing cells Screening may use either phenotype based on the inserted gene or bacterial colony in situ hybridization to identify the recombinant colonies The specificity of the recombination is verified by sequencing Figure 2 A is a bar graph depicting LD50 of Cp and Cp/sod under aerobic stress Cp

(open bars) and Cp/sod (patterned bars) were exposed to various conditions of oxidative stress and the LD 50 values were analyzed statistically by the unpaired t-test normal air, plumbagin, H₂O₂ and t-butylhydroperoxide The LD50 values were determined and analyzed by the unpaired t-test to determine statistical significance Comparing with Cp, a significant reduction in LD 50 was found for Cp/sod- under all oxidative stress conditions p<0 05, p<0 01 , p<0 04, and p<0 02, respectively

Figure 2B discloses Kaplan-Meier survival curves of tumor-bearing animals treated with PBS, Cp, and Cp/sod Mice beanng orthotopic pancreatic cancer were injected intravenously with IxIO⁷ and IxIO⁶ spores of Cp/sod (squares and triangles, respectively, n=15), IxIO⁶ spores of Cp (circles, n=15), and PBS (line without symbol, n=10) Statistical differences between groups were analyzed using the log-rank test There was no statistically significant between the PBS group versus those treated with 1x10⁶ spores of either Cp (p>04) or Cp/sod- (p>0 3) Statistically significant survival advantage was observed in tumor- beaπng mice after treatment with IxIO⁷ Cp/sod spores vs PBS (p<002) and vs IxIO⁶ Cp or Cp/sod- spores (p<0 03 and p<0 03, respectively)

Figure 3A discloses intratumoral accumulation of inflammatory cells after Cp/sod spore treatment Tumor sections in mice treated with PBS (open bars) and IxIO⁷ Cp/sod spores (patterned bars) were collected at day 2 post spore injection and analyzed by immunohistochemical staining for neutrophils, macrophages/monocytes and NK cells The results were quantified by morphometry and analyzed statistically by the unpaired t-test Intratumoral accumulation induced by bacteπal spore injection were observed for neutrophils and macrophages/monocytes (p<0 006 and p<0 01, respectively), but not NK cells (p>0 6) Figure 3B discloses neutrophil, macrophage/monocyte, and NK cell counts (upper panel), bacterial titers (middle panel), and percent necrosis (lower panel) in tumors at day 2 post spore injection in mice that received inflammatory cell depletion treatments Tumor sections in mice treated with control liposomes or rat IgG, as well as clodronate-contaimng liposomes, neutrophil and NK cell depletion antibodies, were analyzed by immunohistochemical and H&E staining Statistical significance in morphometnc analysis of the stained sections and intratumoral bacteπa titers was determined by the unpaired t-test In neutrophil and macrophage/monocyte depletion groups, reduction in intratumoral proinflamatory cell content, elevation in intratumoral bactenal titers, and enhancement of tumor necrosis were statistically significant (p<0 001, <0 03, and <002 in neutrophil depletion, p<0 001, <0 05, and <0 04 in macrophage/monocyte depletion, respectively) Although the reduction in intratumoral NK cell content was statistically significant (p<003), there were no changes in intratumoral bacteπal titers and tumor necrosis (p>02 and 04, respectively)

Figure 4 discloses the construction of a recombination fragment in which a lacZ, PVL or LcrV gene insertion 3' to the pfoR or adhE gene that results in a polycistronic structure A cDNA fragment containing pfoR or adhE gene leader (promoter and SD nbosome binding sequence) and coding region was cloned, as were its down-stream sequence and the respective transgene coding regions The three fragments were linked by overlapping PCR The transgenes are driven by the native promoters of pfoR or adhE and expressed as a polycistronic mRNAs

Figure 5A is a schematic representation of the homologous recombination DNA fragment in which the PVL gene insertion follows the pfoR gene in a polycistronic structure A cDNA fragment of pfoR gene coding region was cloned, as well as its down-stream sequence and the two subunits of PVL gene, LukS and LukF coding region The four fragments were linked by overlapping PCR An additional stop codon TAA was designed in the overlapping PCR primers and inserted between each fragment The exogenous PVL gene is driven by the native promoters of pfoR gene and expressed as a polycistronic messenger Figure 5B discloses bacteπal proliferation, sporulation and germination efficiencies of Cp (solid triangles), Cp/sod (solid squares), and Cp/sod/PVL (solid circles) The results were analyzed statistically by the unpaired t-test between Cp, Cp/sod- and Cp/sod7PVL p>0 4 in bacterial growth, p>03 in sporulation efficiencies, and p>04 in spore germination efficiencies at all time points

Figure 5C discloses survival percentages of mouse peripheral neutrophils and monocytes after exposure to serially diluted supernatants of various bacterial cultures The results were analyzed statistically by the unpaired t-test and significant reductions in cell survival were seen in non-diluted (1 1) and 10-times diluted (1 10) culture supernatants from Cp/sod /PVL (solid circles) and parental S aureus (solid triangles) when compared with those from Cp/sod^" (solid squares) in monocytes (left panel, p<0 001 and <0 02) and neutrophils (right panel, ρ<001 and <0 01)

Figure 6 discloses intratumoral contents of inflammatory cells, bacteπal titers and necrotic areas at day 2 after intravenous injection of 1x10⁷ spores of Cp/sod or Cp/sod/PVL in tumor-bearing mice neutrophil (Figure 6A), macrophages/monocytes (Figure 6B), bacteπal titers (Figure 6C), and necrosis (Figure 6D) Tumor sections in mice treated with 1x10⁷ spores of Cp/sod (left-hand pictures) or Cp/sod/PVL (πght-hand pictures) were analyzed by immunohistochemical staining for neutrophils (Figure 6A), macrophages/monocytes (Figure 6B), Gram staining for bacteria (Figure 6C) and H&E staining for tumor necrosis (Figure 6D) Statistical significance in morphometric analyses of the stained sections and intratumoral bacteπal titers were determined by the unpaired t-test There were statistically significant differences in the reduction of intratumoral neutrophil contents (p<002) and macrophage/monocyte contents (p<0 04), which were correlated with statistically significant elevations in intratumoral bacteπal titers (p<0 03), and extents of tumor necrosis (p<0 04)

Figure 7 discloses the kinetics of inflammatory cell accumulation, bacterial replication and tumor response after intravenous injection of IxIO⁷ spores of Cp/sod /PVL (solid circles) versus Cp/sod (solid squares) in tumor-bearing mice intratumoral neutrophil contents (Figure 7A), intratumoral macrophage/monocyte contents (Figure 7B), intratumoral bacteπal titers (Figure 7C), and tumor necrosis (Figure 7D) at days 0, 1, 3, 7, and 14 post spore administration Tumor sections in treated mice were analyzed by immunohistochemical staining for intratumoral neutrophil and macrophage/monocyte contents, and by H&E staining for tumor response measurements Tumor extracts were used for assaying of bacteπal titers by cultunng in vitro Statistical significance in morphometnc analyses of the stained sections and intratumoral bacteπal titers was determined by the unpaired t-test Reduction of intratumoral neutrophil contents (Figure 7A) was found at days 1, 3 and 7 post spore injection (p<003, <0 02 and <004, respectively), and reduction in intratumoral macrophage/monocyte contents (Figure 7B) was found at days 1, 3, 7 and 14 (p<003, <004, <003 and <0 04, respectively) Elevation in intratumoral bacteπal titers (Figure 7C) was statistically significant at days 1, 3, 7 and 14 (p<0 04, <0 02, <0 01 and <0 02, respectively), as was enhancement of tumor necrosis (Figure 7D) at days 3, 7 and 14 (p<0 04, <0 02 and <004, respectively) Figure 8 discloses Kaplan-Meier survival curves of tumor-bearing animals treated with PBS (line without symbols), Cp/sod^' (solid squares), Cp/sod7PVL (solid circles) Mice beaπng orthotopic pancreatic tumors were injected intravenously with IxIO⁷ spores of Cp/sod (n=16) and Cp/sod /PVL (n=16) or PBS (n=10) Kaplan-Meier survival curves were established in these treatment groups and statistical differences between groups were compared using the log-rank test There were statistically significant differences between the PBS treated group versus those treated with Cp/sod spores (p<002) and Cp/sod /PVL spores (p<0 001) A statistically significant survival advantage was observed in tumor- beaπng mice after treatment with IxIO⁷ spores of Cp/sod7PVL vs Cp/sod^' (p<0 001)

Figure 9 discloses the evaluation of systemic toxicities (Figure 9A) and organ toxicities (Figure 9B) in tumor-beanng mice after Cp/sodVPVL spore injection (Figure 9A), according to the United States Medical Licensing Examination (USMLE) standard laboratory values (broken lines), ALT, AST, direct-bihrubin, lndirect-bilirubin, and BUN were not statistically significant and were all within their respective normal ranges, indicative of normal liver and kidney functions There were also no significant changes in WBC, RBC, hemoglobin and hematocrit, indicative of normal hematologic functions Transient elevation in serum IL- 12, IFN-gamma and TNF-alpha levels was observed in mice treated with bacteπal spores as expected, but did not reach their respective toxicity levels Lyke et al , Infect Immun 72 5630-7, 2004 and Wan et al , Int Immunopharmacol 6 750-8, 2006 Statistical significance was analyzed with Kruskal-Wallis one-way ANOVA by ranks The standard normal ranges in healthy hosts are shown as broken lines (Figure 9B), H&E stained sections of normal pancreas, liver and spleen at days 0, 1, 3 and 14 post spore injection showed no pathological changes Figure 10 discloses the in vitro characterization of a Cp/plc /sod /PVL strain The results of bacteπal proliferation (Figure 10A), sporulation (Figure 10B) and germination

(Figure 10C) efficiencies in Cp, Cp/sod7PVL, and Cp/plc /sodVPVL were analyzed statistically by the unpaired t-test among the three strains p>0 4 in bacterial growth curves at all time points, p>03 in sporulation efficiencies, p>06 in spore germination efficiencies

Figure 11 discloses the effects of Gemcitabine, Erlotimb, and Docetaxel on Cp/plc^" /sod /PVL proliferation, sporulation, and germination in vitro The drugs were added to bacteπal cultures at 1OX and IX of the respective peak plasma concentrations in pancreatic cancer patients undergoing chemotherapy The results were analyzed by unpaired t-test There were no statistically significant differences in bacterial growth (top panels, p>04, 03, and 04), sporulation efficiency (middle panels, p>0 6, 0 4, and 0 5) and germination efficiency (bottom panels, ρ>0 4, 06, and 04), between the chemotherapeutic drug treatment groups and the PBS control group

Figure 12 discloses the quantification of tumor necrosis after gemcitabine and Cp/plc /sod /PVL spore treatments Tumor samples in mice treated with PBS, gemcitabine and Cp/plc /sod /PVL spore (3 mice per group) were collected at 3 days post spore injection, and analyzed by H&E staining There was no statistically significant difference in tumor necrosis between the gemcitabine and PBS treatment groups (p>0 B) Tumor necrosis was significantly enhanced in the Cp/plc /sod /PVL spore treatment group vs both gemcitabine and PBS control groups (p<0 01 and 0 01 , respectively)

Figure 13 discloses the effect of gemcitabine in Cp/plc /sod /PVL proliferation (A), sporulation (B) and germination (C) in vitro The drugs were added to bacterial cultures at 10x and Ix of the respective peak plasma concentrations in pancreatic cancer patients undergoing chemotherapy The results were analyzed by unpaired t-test There were no statistically significant differences in Cp bacterial growth (panel A, p>0 5), sporulation efficiency (panel B, p>0 6) and germination efficiency (panel C, p>0 6), between the gemcitabine added groups and the PBS control group

Figure 14 discloses Kaplan-Meier survival curves in treatment with Cp/plc /sod /PVL spores, gemcitabine, and combination of both agents, in orthotopic PANC02 tumor-beaπng mice Mice bearing PANC02 pancreatic cancer were treated with IxIO⁷ spores of Cp/plc /sod/PVL followed by gemcitabine (30 mice), IxIO⁷ spores of Cp/sod /PVL alone (30 mice), gemcitabine alone (36 mice), or PBS control (36 mice) The chemotherapy treatment was initiated at 4 hours post bacterial spore administration, at 125 mg/Kg, i p , twice weekly The mice were followed for survival and statistical differences between groups were compared by log-rank test Both Cp/plc /sod/PVL spore and gemcitabine alone treatments showed significant survival prolongation vs PBS (/KO 001 and 004, respectively) Cp/plc /sod/PVL alone showed significant survival advantage vs gemcitabine alone (p<0 001) Also, the combination treatment exhibited significant survival prolongation versus Cp/plc /sod/PVL or Gemcitabine alone and PBS control (p<0 03, 0 001 and 0 001, respectively)

DETAILED DESCRIPTION

The present disclosure is based upon the observation that genetically enhanced variants of the anaerobic bacterium Clostridium perfringens (Cp), a spore-forming, Gram- positive, obligate anaerobic bacteπum, are effective at selectively colonizing and inducing necrosis in orthotopic pancreatic tumors and, consequently, find utility as oncopathic agents for the treatment of a wide range of cancers associated with solid tumors having avascular, hypoxic regions Exemplary such tumors include, but are not limited to, pancreatic tumors, lung tumors, colorectal tumors, prostate tumors, breast tumors, liver tumors, bladder tumors, melanomas, sarcomas, fibrosarcomas, and glioblastomas Additional tumors that may be advantageously treated with the genetically enhanced oncopathic anaerobic bacteria disclosed herein include those solid tumors having avascular, hypoxic regions following treatment with one or more antiangiogenic agent as descπbed below

Tumors having avascular hypoxic regions that may be suitably treated by the genetically enhanced variants of the anaerobic bacteria disclosed herein are descπbed in the following references each of which is incorporated by reference in its entirety Chavaudra et al , Radiat Res 88 56-68, 1981, Flaten et al , Europ J Cancer V∑ 527-32, 1981, Guichard et al , Radiat Res 95 602-9, 1983, Guichard et al , J Nat Cancer Inst 58 1665-9, 1977, Rofstad, Radiat Res 87 670-83, 1981, Courtenay et al , Nature 263 771-2, 1976, Li et al , Cancer Gene Ther K) 105-111, 2003, Zhao et al , Proc Natl Acad Sci USA 102 755-760, 2005, Jain and Forbes, Proc Natl Acad Sci USA 98 14748-50, 2001, Ryan et al , BwEssays 28 84-94, 2006

In accordance with the present disclosure there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art Such techniques are explained fully in the literature See, eg , Sambrook, Fritsch & Mamatis, Molecular Cloning A Laboratory Manual, Second Edition (1989) Cold Spnng Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook et al "), DNA

Cloning A Practical Approach, Volumes I and II (D N Glover ed 1985), Oligonucleotide Synthesis (M J Gait ed 1984), Nucleic Acid Hybridization (B D Hames & S J Higgins eds (1985)), Transcription And Translation (B D Hames & S J Higgins, eds (1984)), Animal Cell Culture (R I Freshney, ed (1986)), Immobilized Cells And Enzymes (IRL Press, (1986)), B Perbal, A Practical Guide To Molecular Cloning (1984), F M Ausubel et al (eds ), Current Protocols in Molecular Biology, John Wiley & Sons, Inc ( 1994)

Pπor to setting forth the disclosure in more detail, it may be helpful to an understanding thereof to set forth definitions of certain terms to be used hereinafter

Definitions

As descπbed herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropπate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated As used herein, "about" or "comprising essentially of mean ± 15% of the indicated value or range, unless otherwise indicated The use of the alternative (e g , "or") should be understood to mean either one, both, or any combination thereof of the alternatives As used herein, the indefinite articles "a" and "an" refer to one or to more than one (/ e , at least one) of the grammatical object of the article By way of example, "a component" means one component or a plurality of components

The term "oncopathic bacteria," as used herein, refers to anaerobic bacteπa that may be employed to generate the genetically enhanced anaerobic bacteria of the present disclosure include species of the obligate anaerobic Clostridium and Bifidobacterium bacteria and species of the facultative anaerobic Salmonella bacteπa Suitable Clostridium species may be selected from the group consisting of Clostridium perfringens, Clostridium histolyticum, Clostridium novyi, Clostridium tetani, Clostridium acetobutyhcum, and Clostridium butyricum Suitable Bifidobacterium species may be selected from the group consisting of Bifidobacterium bifidum, Bifidobacterium infantis, and Bifidobacterium longum Suitable Salmonella species may be selected from the group consisting of Salmonella typhimurium and Salmonella choleraesuis

The term "anaerobic bacteria," as used herein, refers collectively to both facultative anaerobic bacteπa {e g , Salmonella species) and to obligate anaerobic bacteπa {e g , Bifidobacteπum and Clostridium species)

The term "oxygen tolerance gene," as used herein, refers to one or more gene selected from the group consisting of a superoxide dismutase {sod) gene, a glutathione peroxidase (gpo) gene, a rubrerythnn (rbr) gene, and an alcohol dehydrogenase family member (ydaD) gene

The term "inflammation suppressive gene," as used herein, refers to one or more gene that expresses a protein product capable of suppressing an inflammatory cellular responses (Cote, Microb Pathog 37 169-75, 2004) Typically, inflammation suppressive genes are isolated from one or more heterologous microbes and are exemplified in the present disclosure by the Staphylococcus aureus Panton- Valentine Leukocidin (PVL) gene and the

Yersinia enlerocolitica virulence factor (LcrV) gene

The term "toxin gene," as used herein, refers to bacterial genes that enhance or cause m vivo cellular toxicity in a host organism An exemplary toxin gene employed in genetically enhanced anaerobic bacteπa presented herein is the phosphohpase c (pic) toxin gene

The terms "polypeptide" and "protein" may be used herein interchangeably to refer to the product (or corresponding synthetic product) encoded by a particular gene, such as a nucleocapsid protein or RNA-dependent RNA polymerase polypeptide The term "protein" may also refer specifically to the polypeptide as expressed in cells A "peptide" refers to a polypeptide often amino acids or less

The term "gene" is used herein to refer to a portion of an RNA or DNA molecule that includes a polypeptide coding sequence operatively associated with expression control sequences Thus, a gene includes both transcribed and untranscπbed regions The transcribed region may include introns, which are spliced out of the mRNA, and 5'- and 3'- untranslated (UTR) sequences along with protein coding sequences In one embodiment, the gene can be a genomic or partial genomic sequence, in that it contains one or more introns

In another embodiment, the term gene may refer to a complementary DNA (cDNA) molecule (i e , the coding sequence lacking introns) In yet another embodiment, the term gene may refer to expression control sequences, such as a promoter, an internal πbosome entry site

(IRES), or an enhancer sequence

A "promoter sequence" is an RNA or DNA regulatory region capable of binding

RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence For purposes of defining the present invention, the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background Within the promoter sequence will be found a transcription initiation site (conveniently defined for example, by mapping with nuclease Sl), as well as protein binding domains (consensus sequences) recognized and bound to by RNA polymerase

"Sequence-conservative variants" of a polynucleotide sequence are those in which a change of one or more nucleotides in a given codon position results in no alteration in the amino acid encoded at that position

"Function-conservative variants" are those in which a given amino acid residue in a protein or enzyme has been changed without altering the overall conformation and function of the polypeptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like) Amino acids with similar properties are well known in the art For example, arginine, histidine and lysine are hydrophihc-basic amino acids and may be interchangeable Similarly, isoleucine, a hydrophobic amino acid, may be replaced with leucine, methionine or valine Such changes are expected to have little or no effect on the apparent molecular weight or isoelectric point of the protein or polypeptide

Amino acids other than those indicated as conserved may differ in a protein or enzyme so that the percent protein or amino acid sequence similarity between any two proteins of similar function may vary and may be, for example, from 70% to 99% as determined according to an alignment scheme such as by the Cluster Method, wherein similarity is based on the MEGALIGN algorithm A "variant" also includes a polypeptide or enzyme which has at least 60 % amino acid identity as determined by BLAST or FASTA algorithms, preferably at least 75%, most preferably at least 85%, and even more preferably at least 90%, and still more preferably at least 95%, and which has the same or substantially similar properties or functions as the native or parent protein or enzyme to which it is compared The change in amino acid residue can be replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, hydrophobic, aromatic, and the like) or different properties

As used herein, the term "homologous" in all its grammatical forms and spelling variations refers to the relationship between proteins that possess a "common evolutionary origin," including proteins from superfamilies (eg , the immunoglobulin superfamily) and homologous proteins from different species (e g , myosin light chain, etc ) Reeck el al , Cell 50 667 (1987) Such proteins (and their encoding nucleic acid sequences) have sequence homology, as reflected by their sequence identity, whether in terms of percent identity or similarity, or the presence of specific residues or motifs at conserved positions.

Accordingly, the term "sequence similarity" in all its grammatical forms refers to the degree of identity or correspondence between nucleic acid or amino acid sequences of proteins that may or may not share a common evolutionary origin (see Reeck et al., supra). However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and may or may not relate to a common evolutionary origin.

In a specific embodiment, two nucleic acid sequences are "substantially homologous" or "substantially identical" when at least about 80%, and most preferably at least about 90 or at least 95%, of the nucleotides match over the defined length of the nucleic acid sequence, as determined by sequence comparison algorithms, such as BLAST, FASTA, DNA Strider, etc. Sequences that are substantially homologous can be identified by comparing the sequences using standard software available in sequence data banks, or in a Southern hybridization experiment under, for example, stringent conditions as defined for that particular system.

Similarly, in a particular embodiment, two amino acid sequences are "substantially homologous" or "substantially identical" when greater than 80% of the amino acids are identical, or greater than about 90% or 95% are similar (functionally identical). Preferably, the similar or homologous sequences are identified by alignment using, for example, the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of the programs described above (BLAST, FASTA, etc.).

A nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook et al.). The conditions of temperature and ionic strength determine the "stringency" of the hybridization. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a T_n, (melting temperature) of 55°C, can be used, e.g., SxSSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS). Moderate stringency hybridization conditions correspond to a higher T_n,, e.g., 40% formamide, with 5x or 6x SCC. High stringency hybridization conditions correspond to the highest T_1n, e.g., 50% formamide, 5x or 6x SCC. SCC is a 0.15M NaCl, 0.015M Na-citrate. Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, which are well known variables in the art The greater the degree of identity or homology between two nucleotide sequences, the greater the value of T_n, for hybrids of nucleic acids having those sequences The relative stability (corresponding to higher T_1n) of nucleic acid hybridizations decreases in the following order RNA RNA, DNA RNA, DNA DNA For hybπds of greater than 100 nucleotides in length, equations for calculating T_n, have been derived (see Sambrook el al , supra, 950-951) For hybridization with shorter nucleic acids, / e , oligonucleotides, the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al , supra, 11 7-11 8) In certain embodiments, a hybπdizable nucleic acid has a length of at least about 10 nucleotides, preferably at least about IS nucleotides, and more preferably at least about 20 nucleotides In a specific embodiment, the term "standard hybridization conditions" refers to a T_n, of 55°C, and utilizes conditions as set forth above In a preferred embodiment, the T_m is 6O⁰C, in a more preferred embodiment, the T_n, is 65⁰C In a specific embodiment, "high stringency" refers to hybridization and/or washing conditions at 68⁰C in 02x SSC, at 42°C in 50% formamide, 4x SSC, or under conditions that afford levels of hybridization equivalent to those observed under either of these two conditions

The terms "mutant" and "mutation" mean any detectable change in genetic material, e g , RNA, DNA, or any process, mechanism, or result of such a change When compared to a control material, such change may be referred to as an "abnormality" This includes gene mutations in which the structure (eg , RNA or DNA sequence) of a gene is altered, any gene or nucleic acid molecule aπsing from any mutation process, and any expression product (e g , protein or enzyme) expressed by a modified gene or nucleic acid sequence The term "variant" may also be used to indicate a modified or altered gene, RNA or DNA sequence, enzyme, cell, etc , ι e , any kind of mutant

"Amplification" of nucleic acid sequences, as used herein, encompasses the use of polymerase chain reaction (PCR) to increase the concentration of a specific nucleic acid sequence within a mixture of nucleic acid sequences For a descnption of PCR, see Saiki el al . Science 239 487 (1988) "Sequencing" of a nucleic acid includes chemical or enzymatic sequencing "Chemical sequencing" of DNA denotes methods such as that of Maxam and Gilbert (Maxam-Gilbert sequencing, Maxam and Gilbert, Proc Natl Acad Sa USA 21560 (1977)), in which DNA is randomly cleaved using individual base-specific reactions "Enzymatic sequencing" of DNA denotes methods such as that of Sanger (Sanger el al , Proc Nail Acad Sa US A 74 5463 (1977)), in which a single-stranded DNA is copied and randomly terminated using DNA polymerase, including variations thereof, which are well- known in the art Preferably, oligonucleotide sequencing is conducted using automatic, computeπzed equipment in a high-throughput setting, for example, microarray technology, as described herein Such high-throughput equipment are commercially available, and techniques well known in the art

A "probe" refers to a nucleic acid or oligonucleotide that forms a hybπd structure with a sequence in a target region due to complementarity of at least one sequence in the probe with a sequence in the target protein As used herein, the term "oligonucleotide" refers to a nucleic acid, generally of at least 10, preferably at least 15, and more preferably at least 20 nucleotides, preferably no more than 100 nucleotides, that is hybπdizable to a genomic DNA molecule, a cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or other nucleic acid of interest Oligonucleotides can be labeled, e g , with ³²P-nucleotides or nucleotides to which a label, such as biotin, has been covalently conjugated In one embodiment, a labeled oligonucleotide can be used as a probe to detect the presence of a nucleic acid In another embodiment, oligonucleotides (one or both of which may be labeled) can be used as PCR primers, either for cloning full length or a fragment of a nucleic acid sequence of interest, or to detect the presence of nucleic acids encoding a polypeptide of interest In a further embodiment, an oligonucleotide of the invention can form a triple helix with a nucleic acid molecule of interest In still another embodiment, a library of oligonucleotides arranged on a solid support, such as a silicon wafer or chip, can be used to detect vaπous mutations of interest Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer Accordingly, oligonucleotides can be prepared with non-naturally occurring phosphoester analog bonds, such as thioester bonds, etc

Specific non-limiting examples of synthetic oligonucleotides envisioned for this invention include oligonucleotides that contain phosphorothioates, phosphotπesters, methyl phosphonates, short chain alkyl, or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages Most preferred are those with CH₂-NH-O-CH₂, CH₂- N(CH)₃-O-CH₂, CH₂-O-N(CH)₃-CH₂, CH₂-N(CH)₃-N(CH)₃-CH₂ and 0-N(CH)₃-CH₂-CH₂ backbones (where the phosphodiester is 0-PO₂-O-CH₂) U S Patent No 5,677,437 describes heteroaromatic oligonucleoside linkages Nitrogen linkers or groups containing nitrogen can also be used to prepare oligonucleotide mimics (U S Patent Nos 5,792,844 and 5,783,682) U S Patent No 5,637,684 describes phosphoramidate and phosphorothioamidate oligomeπc compounds Also envisioned are oligonucleotides having morpholino backbone structures (U S Patent No 5,034,506) In other embodiments, such as the peptide-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone Nielsen et al , Science 254 1497 (1991) Other synthetic oligonucleotides may contain substituted sugar moieties comprising one of the following at the 2' position OH, SH, SCH₃, F, OCN, O(CH₂)_nNH₂ or O(CH₂)_nCH₃ where n is from 1 to about 10, Ci to Cio lower alkyl, substituted lower alkyl, alkaryl or aralkyl, Cl, Br, CN, CF₃, OCF₃, O-, S-, or N-alkyl, O-, S-, or N-alkenyl, SOCH₃ , SO₂CH₃, ONO₂,NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, armnoalkylamino, polyalkylamino, substituted silyl, a fluorescein moiety, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide, or a group for improving the pharmacodynamic properties of an oligonucleotide, and other substituents having similar properties Oligonucleotides may also have sugar mimetics such as cyclobutyls or other carbocyclics in place of the pentofuranosyl group Nucleotide units having nucleosides other than adenosine, cytidine, guanosine, thymidine and uridine, such as inosine, may be used in an oligonucleotide molecule

The term "therapeutically effective amount" or "effective amount" refers to an amount of a recombinant oncopathic bacteπa composition sufficient to reduce, inhibit, or abrogate tumor cell growth, either in vitro or in a subject (e g , a dog or a pig or a cow) As noted herein, the reduction, inhibition, or abrogation of tumor cell growth may be the result of necrosis, apoptosis, or an immune response The amount of a recombinant oncopathic bacteπa composition that is therapeutically effective may vary depending on the particular oncopathic bacteπa used in the composition, the age and condition of the subject being treated, or the extent of tumor formation, and the like Oncopathic Bacteria

The present disclosure provides genetically enhanced anaerobic bacteria for the oncopathic therapy of cancers, including pancreatic cancers Genetically enhanced anaerobic bacteria disclosed herein are capable of colonizing and destroying hypoxic tumors and are less toxic m vivo as compared to the corresponding anaerobic bacteπa from which it derives Exemplified herein are genetically enhanced Clostridium perfingens, an oncopathic bacteπum that is able to selectively target hypoxic tumors

Other anaerobic bacteria may also be advantageously modified by the genetic enhancements disclosed herein such as, for example, incorporating one or more inflammatory suppressive genes to enhance anti-tumor efficacy, and mutating or otherwise disrupting one or more oxygen tolerant genes (sod gene) to enhance tumor selectivity and/or the pic toxin gene to improve safety

Anaerobic bacteπa that may be employed to generate the genetically enhanced anaerobic bacteπa of the present disclosure include species of the obligate anaerobic Clostridium and Bifidobacterium bacteria and species of the facultative anaerobic Salmonella bacteπa Suitable Clostridium species may be selected from the group consisting of Clostridium perfingens, Clostridium histofyticum, Clostridium novyi, Clostridium tetam, Clostridium acetobutylicum, and Clostridium butyricum Suitable Bifidobacterium species may be selected from the group consisting of Bifidobacterium bifidum, Bifidobacterium infantis, and Bifidobacterium longum Suitable Salmonella species may be selected from the group consisting of Salmonella typhimunum and Salmonella choleraesuis These anaerobic oncopathic bacteria are disclosed in the following references each of which is incorporated herein by reference in its entirety Parker et al , Proc Soc Exp Biol Med 66 461-467. 1947, Malmgren and Flanigan, Cancer Res .15 473-478, 1955, Mose and Mose, Cancer Res 24 212-216, 1964, Geπcke and Engelbart, Cancer Res 24 217-221, 1964, Thiele et al , Cancer Res 24 222-233, 1964, Carey et al , Eur J Cancer 3 37-46, 1967, Lemmon et al , Gene Ther 4 791-796, 1997, Theys et al , Cancer Gene Ther 8 294-297, 2001, Dang et al , Proc Natl Acad Set USA 98 15155-15160, 2001, Kohwi et al , Gann 69 613-618, 1978, Kimura et al , Cancer Res 40 2061-2068, 1980, Lee et al , J Gene Med 6 1382-1393, 2004, Lee et al , Cancer Gene Ther U 175-184, 2005, Yazawa et al , Cancer Gene Ther 7 269- 274, 2000, Yazawa et al , Breast Cancer Res Treat 66 165-170, 2001, Low et al , Nat Biotechnol J7 37-41, 1999, and Clairmont er al , J Infect Dis .181 1996-2002, 2000 Recombinant Oncopathic Bacteria Bearins an Inactive Oxygen Tolerance Gene

As part of the present disclosure, it was found that a Cp knock-out strain (Cp/sod), generated by deletion of the gene encoding superoxide dismutase {sod), a major gene associated with oxygen tolerance in Cp (Bπolat and Reysset, J Bacteriol JJ4 2333-43, 2002, Geissmann el al , J Bacteriology 181 7136-9. 1999, and Lehmann et al , J Bacteriology

178 7152-8, 1996), exhibited reduced oxygen tolerance in vitro and limited growth in normal tissues in vivo Intravenous infusion of Cp/sod in tumor-bearing mice led to enhanced intratumoral bacteria replication, tumor regression, and survival prolongation The genetically modified Cp/sod mutant strain showed reduced toxicity in normal tissues, elevated maximum tolerated dose (MTD) and statistically significant survival prolongation in tumor-bearing mice, compared with the wild type Cp

Thus, within certain embodiments, genetically enhanced anaerobic bactena contain one or more mutation(s) in one or more gene(s) associated with oxygen tolerance Exemplified herein are genetically enhanced anaerobic bacteria containing one or more mutation(s) is an oxygen tolerance gene selected from the group consisting of a superoxide dismutase (sod) gene, a glutathione peroxidase (gpo) gene, a rubrerythnn (rbr) gene, and an alcohol dehydrogenase family member (ydaD) gene

Sod is an oxidoreductase that catalyzes the reaction between superoxide anions and hydrogen to yield molecular oxygen and hydrogen peroxide This enzyme plays a major role in anaerobic organisms for protection against oxidative stress Gpo is a major peroxide scavenging enzyme that catalyzes the oxidation of glutathione to yield oxidized glutathione and water, and has been demonstrated to provide tolerance to oxygen radicals transformed E coli strains expressing gpo were examined for their ability to tolerate free oxygen radicals and the survival rate of the transformed bacteria was much higher than that of the wild-type bacteria Gpo participates in the enzymatic system aimed at scavenging free oxygen radicals There is no catalases have been found in Cp

In contrast, an alternative oxidative stress protection system, Rbr, has recently been identified in Cp Rbr is the terminal component of NADH peroxidase that catalyzes the reduction of hydrogen peroxide to water, and is believed to be a part of the anaerobic defense mechanism against oxidative stress based on its NADH peroxidase activity The advantage of the Rbr system is that there is no intracellular production of molecular oxygen, which probably acts as a signal for growth arrest under anaerobic conditions The ydaD gene encodes a NADPH dehydrogenase, and has been identified to be involved in the oxidative stress response by insertional mutagenesis and screening for resistance or sensitivity under various oxidative stress conditions Complementation and knock-out expeπments demonstrated that the ydaD gene is required for efficient resistance to oxidative stresses and is responsible for the production of NADPH, which is required for maintenance of the intracellular redox balance in growth-arrested cells

Therefore, a multi-knocking-out of these major oxygen tolerance genes (sod, gpo, rbr and ydaD genes) would result a sequential enhancement in tumor selectivity and treatment safety in the Clostridium perfingens and other bacteπal species, although the oxygen tolerance genes may vary in them individually

Each of these major oxygen tolerance genes (sod, gpo, rbr and ydaD genes) are described in further detail in the following references each of which is incorporated by reference herein in its entirety Bπolat and Reysset, J Bacterial JJ4 2333-43, 2002, Geissmann et al , J Bacteriology .18J.7136-7139, 1999, Lehmann et al , J Bacteriology 178 7152-7158, 1996, Knorpp et al , J Neural Transm JJ3 1145-55, 2006, Sun et al , Biochim Biophys Acta VH 199-204, 2005, Holland et al , FEBS Letters 337 52-55, 1994, Coulter et al , Inorg Chim Acta 292231-241, 2000, Pan and Imlay, MoI Microbiol 39 1562-1571, 2001

Recombinant Oncopathic Bacteria Comprising an Inflammation Suppressive Gene The tumor response of the Cp/sod mutant strain was modest and most treated animals ultimately succumbed to relapse over time Since microbial replication in immune- competent hosts is inhibited by inflammatory cellular responses (Cote, Microb Pathog 37 169-75, 2004), it was hypothesized that the replication potency of Cp/sod in tumors and treatment efficacy could be substantially elevated by constructing recombinants that express inflammation suppressive genes from heterologous microbes

Macrophages and neutrophils have been shown to play key roles m early host defenses against infection by microbial pathogens in the host Cote et al , Infection and Immunity 74 469-80, 2006 Neutrophils are first to accumulate in the infection sites and initiate the cytolytic process of invading microorganisms The local and blood-borne macrophages also migrate to the infection sites and initiate phagocytosis Neutrophils and macrophages are spoπcidal in vitro and have a protective role in the host Cote et al , Microb Pathog 32 169-75, 2004 and Welkos et al , Microb Pathog _ 1 15-36, 1989 As described in further detail herein, infra, it was demonstrated that neutrophils and macrophages/monocytes, but not NK cells, accumulate in tumors after Cp/sod spore administration Additionally, the reductions in neutrophils and macrophages were correlated with elevated intratumoral bacteria titers and tumor necrosis, suggesting their suppression in animals could substantially enhance treatment efficacy On the other hand, many invading microbes have evolved strategies for evasion and/or suppression of the host inflammatory responses Pathogenic bacteria are able to avoid phagocytic engulfment and killing Galan, J Experimental Medicine 201321-323, 2005 Panton- Valentine Leukocidin (PVL), produced by S aureus, can directly damage membranes of phagocytes including monocytes, macrophages and neutrophils Kato, Nippon Saikingaku Zasshi 36 445-57, 1981, Okumoto, lnt Ophthalmol Clin 25 133-142, 1985, and Genestier er al , J Clin Invest U5 3117-3127, 2005

It should be noted that tumor-associated macrophages positively influence the growth, survival, invasion and metastasis of tumor cells Witz, Cancer Res 68 9-13, 2008 Thus, inflammation suppressive genes such as PVL may act synergistically in promoting therapeutic bacterial survival while diminishing tumor-associated macrophage function

According to the present disclosure, it was found that the potency and treatment efficacy of Cplsod could be substantially elevated by constructing recombinant variants of Cp/sod that expressed one or more inflammation suppressive genes from heterologous microbes Indeed, the oncopathic effects of Cp/sod were amplified by vector-mediated expression of a heterologous inflammation inhibitory gene, Panton- Valentine Leukocidin (PVL) Cote et al , Microb Pathol 3J 169-75, 2004, Kato, Nippon Saihngaky Zasshi, 36 445-57, 1981, Okumoto, lnt Ophthalmol Clin 25 133-42, 1985, and Genestier et al , J Clin Invest 115 3117-27.2005 Thus, within certain embodiments, genetically enhanced anaerobic bacteria may further express one or more inflammation suppressive gene to enhance anti-tumor efficacy An exemplary inflammation suppressive gene employed in genetically enhanced anaerobic bacteπa presented herein is the recombinant Panton- Valentine Leukocidin (PVL) gene

The recombinant strain Cp/sod/PVL exhibited substantially enhanced oncopathic potency and survival prolongation in tumor-bearing mice without systemic and organ toxicities and, consequently, serves as an exemplary prototype for therapeutic agents for the treatment of pancreatic cancer and/or other poorly vascularized tumors The Cp/sod/PVL strain led to a significant reduction in intratumoral content of inflammatory cells, logarithmically elevated intratumoral bacteria titers, enhanced tumor necrosis, and substantially prolonged animal survival over those treated with Cp/sod Importantly, a substantial fraction of the treated mice remained alive after 100 days and there were no apparent systemic and organ toxicities associated with the systemic administration of Cp/sod /PVL spores at its effective dose Thus, the recombinant Cp/sod/PVL strain, with substantially elevated tumor selectivity and oncopathic potency, exemplifies safe and effective oncopathic agents for the treatment of patients with pancreatic cancer and other poorly vascularized tumors The scientific principle of bacteria-mediated expression of inflammation suppressive genes is generally applicable for the enhancement of oncopathic potency and efficacy of other oncopathic bacteria in cancer treatment

Gene Delivery by Genetically Enhanced Anaerobic Bacteria The genetically enhanced anaerobic bacteria described herein may further compπse one or more additional genetic modification(s) to permit the delivery of one or more gene(s) encoding a prodrug-activating enzyme Such further modified genetically enhanced bacteria may, for example, be used in methods comprising the coadministration of a prodrug Suitable prodrug-activating enzymes include, for example, a cytosine deaminase (CD) that converts 5-fluorocytosine to 5-fluorouracil and nitroreductase (NTR) that activates CB1954 to 5-Azindinyl-4-hydroxylamino-2-mtrobenzarnide See, for example, Theys et al , Cancer Detect Prey 25 548-557 (2001), Minton et al , FEMS Microbiol Rev 17 357-364 (1995), Lm et al , Gene Ther 9 291-296 (2002), Theys et al , Br J Cancer 95 1212-1219 (2006), Metharom et al , Cell MoI Immunol 2£4) 281-287 (2005), Li et al , Cancer Gene Ther 10 105-111 (2003), and Yazawa et al , Cancer Gene Ther 7 269-274 (2000)

Alternatively or additionally, the tumoπcidal activity of genetically enhanced anaerobic bacteria of the present disclosure may be enhanced by expressing one or more gene(s) encoding a therapeutic protein such as, for example, TNF-α, IL-I, IL-6, IL-10, IL-12, IL- 15, IFN- α, IFN-γ, TRAIL, GM-CSF, FLT3-hgand, E coli colicin E3 These anti-cancer agents have been widely investigated and demonstrated for their anti-tumor effect, and some of them have been employed in clinical trails Schlechte et al , Zentralbl Bakteriol Mikrobwl Hyg 268 347-356 (1988), Barbe et al , FEMS Microbiol Lett 246 67-73 (2005), Li et al , Cancer Gene Ther K) 105-111 (2003), Yazawa et al , Cancer Gene Ther 7 269- 274 (2000), Bach et al , Annu Rev Immunol 15 563-591 (1997), Dranoff et al , Proc Natl Acad Sa USA 90 3539-3543 (1993), Nishimoto et al , Blood 95 56-61 (2000), Qin et al ,

J Immunol 159 770-776 (1997), Brunda et al J Exp Med 178 1223-1230 (1993), Hazama et al , Br J Cancer 80 1420-1426 (1999), Biron, Immunity 14 661-664 (2001), Tracey & Cerami, Λ»/rø Rev Med 45 491-503 (1994), Walczak e/ α/ , Nature Med 5 157-163 (1999), and Lynch et al , Nature Med 3 625-631 (1997)

The tumor killing activity of genetically enhanced anaerobic bacteria can also be enhanced by the introduction of one or more exotoxin gene(s) from a heterologous bacterial strain such as, for example, Clostridia Beta-toxin (CPB), Streptococcal Streptolysin O (SLO), and/or Staphylococcal Staphylolysin A (STA) These general bacterial pathogenic factors are absent in the Cp strain and do not cause gas gangrene The expression of these heterologous exotoxin genes in Cp/sod are genetically stable and capable of inducing a substantial elevation in tumor cell killing The enhanced tumor response can be obtained without increased toxicity, as germination and growth of the recombinant Cp strains is restricted to the hypoxic tumor core regions Steinthorsdottir et al , Microbial Pathogenesis 28 45-50 (2000), Nagahama et al , J Biologic Chemi 278 36934-^1 (2003), Portnoy et al , Infection and Immunity 60(7) 2710-2717 (1992), Kehoe et al , Infection and Immunity 55(12) 3228-3232 (1987), and Essmann et al , Cell Death Differ K) 1260-72 (2003)

Combinations of a Genetically Enhanced Anaerobic Bacteria and one or more Second Therapeutic

Compositions of the present disclosure, and methods based on those compositions, may be advantageously employed in combination with one or more chemotherapeutic and/or radiation treatment regimen selected from the group consisting of gemcitabine (GEMZAR®, Eh Lilly and Co , Indianapolis, IN), docetaxel (TAXOTERE®, Sanofi-Avenus, Bπdgewater, NJ), erlotimb (TARCEV A®, Hoffmann-LaRoche, Nutley, NJ), fluorouracil, external beam radiation, and a combination thereof

Alternatively or additionally, compositions disclosed herein may be used in combination with one or more antiangiogenic agent such as, for example, sorafenib (NEXA VAR®, Bayer Pharmaceuticals Corporation, West Haven, CT), bevacizumab (AVASTIN®, Genentech, San Francisco, CA), and/or sunitinib (SUTENT®, Pfizer Inc , New York, NY), ANGIOCEPT™ (Adnexus, Bristol-Myers Squibb R&D Company, New York, NY), AMG-386 (Amgen, Inc , Thousand Oaks, CA), cediranib (RECENTIN™, AstraZeneca, Wilmington, DE), vandetimib (ZACTIMA®, AstraZeneca), thalidomide derivatives (Celgene Corp , Summit, NJ), pazopamb (GlaxoSmithKline, Brentford, Middlesex, UK), abegnn (Medlmmune, Inc , Gaithersburg, MD), cilengitide (Merck KGaA,

Darmstadt, Germany), vatalanib (Novartis, Basel, Switzerland), volociximab (PDL BioPharma/Biogen Idee, Pπnceton, NJ, Cambridge, MA, & Redwood City, Calif), axitinib (Pfizer, Inc , New York, NY), aflibercept (Regeneron/Sanofi -Aventis, Tarrytown, NY & Pans, France), and/or CDP-791 (UCB SA, Brussels, Belgium)

Formulations and Uses The genetically enhanced oncopathic anaerobic bacteria of this disclosure may be administered in a convenient manner such as by the oral, intravenous, lntra-arteπal, mtra- tumoral, intramuscular, subcutaneous, intranasal, intradermal, or suppository routes or by implantation (e g , using slow release molecules) Typically, genetically enhanced oncopathic anaerobic spore-forming bacteria, such as Clostridium species, may be administered as spores

Depending on the route of administration of an adjunctive therapy, like an immunotherapeutic agent, the agents contained therein may be required to be coated in a material to protect them from the action of enzymes, acids and other natural conditions which otherwise might inactivate the agents In order to administer the composition by other than parenteral administration, the agents will be coated by, or administered with, a mateπal to prevent inactivation The genetically enhanced oncopathic anaerobic bacteria of the present invention may also be administered parenterally or intraperitoneal^

As used herein "pharmaceutically acceptable earner and/or diluent" includes any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents and the like The use of such media and agents for biologically active substances is well known in the art Supplementary active ingredients can also be incorporated into the compositions

The carrier can be a solvent or dispersion medium containing, for example, water, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloπde Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin Stenle injectable solutions are prepared by incorporating the genetically enhanced oncopathic anaerobic bacteria of the present disclosure in the required amount of the appropriate solvent with various other ingredients enumerated herein, as required Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle that contains the basic dispersion medium and the required other ingredients from those enumerated above.

It may be advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutically or veterinary acceptable carrier.

Pharmaceutical compositions comprising the genetically enhanced oncopathic anaerobic bacteria of this disclosure may be manufactured by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical anaerobic oncopathic bacteria compositions may be formulated in conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries that facilitate formulating active genetically enhanced oncopathic anaerobic bacteria into preparations that can be used biologically or pharmaceutically. The genetically enhanced oncopathic anaerobic bacteria compositions can be combined with one or more biologically active agents and may be formulated with a pharmaceutically acceptable carrier, diluent or excipient to generate pharmaceutical or veterinary compositions of the instant disclosure. Pharmaceutically acceptable carriers, diluents or excipients for therapeutic use are well known in the pharmaceutical art, and are described herein and, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro, ed., 18^th Edition (1990)) and in CRC Handbook of Food, Drug, and Cosmetic Excipients, CRC Press LLC (S.C. Smolinski, ed. (1992)). In certain embodiments, genetically enhanced oncopathic anaerobic bacteria compositions may be formulated with a pharmaceutically or veterinary- acceptable carrier, diluent or excipient is aqueous, such as water or a mannitol solution (e.g., about 1% to about 20%), hydrophobic solution (e.g., oil or lipid), or a combination thereof (e.g., oil and water emulsions). In certain embodiments, any of the biological or pharmaceutical compositions described herein have a preservative or stabilizer. The biologic or pharmaceutical compositions of the present disclosure can be formulated to allow the genetically enhanced oncopathic anaerobic bacteria contained therein to be bioavailable upon administration of the composition to a subject. The level of genetically enhanced oncopathic anaerobic bacteria in tumors and other tissues after administration can be monitored by various well-established techniques, such as antibody-based assays (e.g., ELISA). In certain embodiments, genetically enhanced oncopathic anaerobic bacteria compositions are formulated for parenteral administration to a subject in need thereof (e.g., a subject having a tumor), such as a non-human animal or a human. Preferred routes of administration include intravenous, intra-arterial, subcutaneous, intratumoral, or intramuscular.

Proper formulation is dependent upon the route of administration chosen, as is known in the art. For example, systemic formulations are an embodiment that includes those designed for administration by injection, e.g. subcutaneous, intra-arterial, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for intratumoral, transdermal, transmucosal, oral, intranasal, or pulmonary administration. In embodiments for injection, the genetically enhanced oncopathic anaerobic bacteria compositions of the instant disclosure may be formulated in aqueous solutions, or in physiologically compatible solutions or buffers such as Hanks's solution, Ringer's solution, mannitol solutions or physiological saline buffer. In certain embodiments, any of the genetically enhanced oncopathic anaerobic bacteria compositions described herein may contain formulator agents, such as suspending, stabilizing or dispersing agents.

Administration can be achieved using a combination of routes, e.g., first administration using an intra-arterial route and subsequent administration via an intravenous or intratumoral route, or any combination thereof.

Methods of Use

In another aspect, the present disclosure provides methods of inhibiting the growth or promoting the killing of a tumor cell or treating cancer, such as pancreatic cancer, by administering a genetically enhanced oncopathic anaerobic bacteria according to the instant disclosure at a concentration sufficient to inhibit the growth of a tumor cell or to kill a tumor cell. In certain embodiments, the genetically enhanced oncopathic anaerobic bacteria is administered more than once, preferably twice, three times, or up to 10 times.

Genetically enhanced oncopathic anaerobic bacteria presented herein may be suitably employed for the treatment of a wide range of cancers associated with solid tumors having avascular, hypoxic regions. Exemplary such tumors include, but are not limited to, pancreatic tumors, colorectal tumors, prostate tumors, breast tumors, liver tumors, bladder tumors, melanomas, sarcomas, fibrosarcomas, glioblastomas, and combinations of these cancers. Additional tumors that may be advantageously treated with the genetically enhanced oncopathic anaerobic bactena dislcosed herein include those solid tumors having avascular, hypoxic regions following treatment with one or more antiangiogenic agent as described below

The following non-limiting examples are provided to illustrate vaπous aspects of the present disclosure All references, patents, patent applications, published patent applications, and the like are incorporated by reference in their entireties herein

EXAMPLES

EXAMPLE 1

BACTERIAL GROWTH, SPORE PURIFICATION, AND TRANSFORMATION IN CLOSTRIDIUM PERFRiNGENS

Clostridium perfringens (Cp) strain ATCC 13124 was grown anaerobically in ATCC recommended Reinforced Clostridial Medium (RCM) (Difco, BD Biosciences, San Jose, CA) The maximum yield of Cp spores occurred at five days following the transfer of vegetative bacteria to the sporulation medium Gill el al , Appl Environ Microbiol 4_1 90- 92, 1981 In vivo studies demand the use of highly purified spores that are free from other contaminants

The procedure for purification of Cp spores is as follows Bactena collected from the sporulation medium were incubated at 9O⁰C for 10 minutes, suspended in 70% ethanol for 20 minutes, and bacterial spores were extracted with 58% Renografin solution (Renocal-76, Princeton, N J ) To further separate the inert Cp spores from residual bactena and debns, the spore suspension was incubated with sahne-EDTA containing lysozyme (100 μg/ml) at

37°C for 45 minutes to degrade the bacterial cell wall peptidoglycan and nnsed five times with PBS to remove degradation products The spores were stored at 20 degrees

Bactenal proliferation, sporulation and germination profiles were determined at 0, 6, 12, 24, and 48 hours IxIO⁷ vegetative bacterial cells of each strain were transferred into sporulation medium and incubated for 5 days to test the maximum sporulation efficiency 1x10⁴ purified spores were transferred to RCM medium to test their germination efficiency

Cp is a Gram-positive bacterium and difficult to transform by conventional chemical- based procedures Electroporation was the most efficient method for the delivery of DNA fragments into Cp The optimized conditions were as follows Bactenal cells at IxIO⁸AnI and transforming DNA at 1 μg/ml in Bio-Rad Gene Pulser system, at a pulse capacity of 25 μF, 600 Ω, 6 KV/cm The optimal washing and electroporation buffers were 027 M sucrose, 5 mM Na₂HPO₄, 10 mM MgCl₂ (2 times/kept on ice 30 minutes) and 027 M sucrose, 5 mM Na₂HPO₄, 1 mM MgCl₂, respectively

EXAMPLE 2 ORTHOTOPIC PANCREATIC CANCER MODEL IN IMMUNE-COMPETENT BACTERIAL SPORE INJECTION, AND INFLAMMATORY CELL DEPLETION

To generate a primary and orthotopic pancreatic tumor model, the murine pancreatic cancer cell line PANC02 was maintained in DMEM supplemented with 10% heat- inactivated FBS and 100 U/ml penicillin-streptomycin Cells were harvested by brief trypsinization and resuspended in Earle's balanced salt solution (EBSS) The tumor cells (IxIO⁵) were directly implanted into the pancreas of 6-8 week old immune-competent and syngeneic female C57BL/6 mice with PBS as a negative control using a 100 μl Hamilton syringe and a 30- guage needle Laparotomy was performed to measure tumor sizes at vaπous times points post tumor cell implantation At 18 days post tumor implantation, tumor nodules larger than 5 mm x 5 mm were observed in the pancreata of greater than 90% of the animals Various doses of the purified bactenal spores were diluted in 02 ml PBS and systemically administered to mice bearing pancreatic tumors via the tail vein To determine the effect of neutrophils, macrophages, and NK cell depletion on bacterial replication and tumor response, monoclonal rat antibody RB6-8C5, (BD Biosciences Pharmingen, San Diego, CA, Rousseau et al , BMC Microbiology 1 17, 2001) was utilized against mouse polymorphonuclear neutrophils as well as a chemical depletion method for macrophages by hposome-mediated intracellular delivery of dichloromethylene-bisphosphonate (Clodronate) and a monoclonal rat anti-mouse NK cell antibody PK-136 (eBioscience), with control of rat IgG or liposome containing PBS Cote et al , Microb Palhog 37 169-75, 2004 and Rooyen et al , J lmmuno Meth 193 93-99, 1996

EXAMPLE 3

CONSTRUCTION AND CHARACTERIZATION OF CP STRAINS OF CLOSTRIDIUM PERFRINGENS HAVING DELETIONS IN OXYGEN TOLERANCE GENES

Recombinant Cp strains with reduced oxygen tolerance and enhanced oncopathic potency were constructed by homologous recombination Superoxide dismutase, a major oxygen tolerance gene in Cp, was knocked out to accentuate its oxygen sensitivity and tumor selectivity An inflammation suppressive gene from S aureus, Panton-Valentine Leukocidin, was knocked into Cp/sod to enhance its oncopathic potency Anti-tumor efficacy was determined by survival studies in an orthotopic model of pancreatic cancer in mice Systemic and organ toxicities were assessed by momtoπng serum chemistries and by histopathological examination The sod knock-out Cp strain was constructed by replacing sod with firefly luciferase

(LUC) by homologous recombination The 1 8 kb LUC gene was amplified from a commercially available plasmid by PCR with primers 5'-ATGGAAGACG CCAAAAAC-3' and 5'-CGCCGTGTAA TTCTAGAGTC-3' (Gene Link, Inc , Hawthorne, NY) The two flanking fragments of the sod gene of Cp were amplified by conventional PCR using the appropriate pπmers, 5'-ATTAAAAATT CATCCCTTG-3' and 5'-ACAGCTTTTC TCCCTAAG-3' for up-stream, 5'-CCTCCTCATA TTTGAAATA-3' and 5'- ATACCTTAAG CATATTAAG-3' for down-stream fragments (Gene Link, Inc ), respectively The recombinant DNA fragment was produced by an overlapping PCR based strategy and cloned into E coli The sequence of the recombination fragment was verified (Roche) Pate et at , Nucl Acid Res 21 1325-6, 1993 and Lm et al , Genome Res 7 389-98, 1997 The plasmid containing the correct product was amplified, purified, digested to yield the recombinant fragment, and verified by sequencing The gel-purified recombination fragment was transformed into Cp by electroporation (25 μF, 600 Ω, 6 KV/cm) The sod knock-out Cp strain (Cp/sod) was identified by colony hybridization using a LUC specific probe (5'-GGTGTTGGGC GCGTTATTTA TCGGAGTTGC AGTTGCG-3', Gene Link, Inc ), purified, and confirmed by DNA sequencing Ezaki et al , "Molecular Ecological Detection and Identification of Intestinal Microflora" (Japan Scientific Societies Press, ISBN 4-7622-296 IX, 2000)

There were no significant differences in bacterial growth rates, maximum yields of bacteπal spores, or spore germination efficiencies between the knock-out and wild type Cp strains in vitro Knocking out the sod gene does not affect anaerobic bacteπal growth characteristics To compare the oxygen tolerance of the mutant strain relative to wild type strain, sensitivities to various oxidative stresses were evaluated in vitro The results showed that the sod knock-out strain exhibited a significant reduction in bacterial survival under each of the oxidative stress conditions Fig 2A This indicated that sod is indeed a major oxygen tolerance gene in Cp Escalating doses of spores from the Cp/sod and Cp strains were then injected into the tail veins of mice bearing orthotopic pancreatic tumors There were 10 animals per group and observations were continued until day 14 after bacteπal spore inoculation The maximum tolerated doses (MTD) of Cp/sod and Cp spores without apparent toxic events were found to be IxIO⁷ and 1x10⁶, respectively These results indicated that there were reduced toxicities associated with the less oxygen-tolerant Cp/sod- strain in vivo A long-term survival study was performed to assess treatment efficacy using 1x10⁷ and IxIO⁶ Cp/sod- and 1x10⁶ Cp spores, with a PBS group as the negative control The survival curves were plotted according to the Kaplan-Meier method (Fig 2B)

The statistically significant survival prolongation over the PBS control group was observed in animals treated with Cp/sod at 1x10⁷ spores (p<002), but not in those treated with IxIO⁶ spores of Cp and Cp/sod (p>04 and p>0 3, respectively) Animals treated with 1x10⁷ spores of Cp/sod- also exhibited a statistically significant survival advantage over those treated with IxIO⁶ spores of Cp and Cp/sod (p<0 03 and 0<0 03, respectively) These results provided the proof of principle that mutant Cp strains with reduced oxygen tolerance could be administered intravenously at higher doses m vivo, leading to a prolongation of survival in mice beaπng orthotopic pancreatic tumors The extent of survival prolongation was modest, however, and most treated animals succumbed to relapse

Glutathione peroxidase (gpo), rubrerythπn (rbr) and an alcohol dehydrogenase family member, ydaD are other known major oxygen tolerance genes in Cp Bπolat and Reysset, 2002, Geissmann et al , 1999, and Lehmann et al , 1996 The respective genetic knock-out strains can be constructed through homologous recombination, as shown in Fig 1 To determine the recombination frequencies at each of these loci, DNA fragments were constructed carrying a promoterless ampR gene that replaced the coding regions of the target knock-out genes between their respective flanking sequences in the Cp genome, such that the recombinants can be selected by ampicilhn resistance The procedures depicted in Fig IA were as follows Using PCR, DNA sequences flanking each side of the target genes plus the coding sequence for the ampR gene were amplified such that they contained overlapping regions Overlapping PCR was used to link the fragments together and cloned the entire fragment (ampR surrounded on each side by Cp flanking sequences) into a plasmid for transformation into E coll The appropπate E coli transformants were selected by ampicilhn resistance and confirmed by sequence analysis The plasmids were digested with the appropriate restriction enzymes to release the Cp-ampR-Cp DNA fragments The recombinant fragment was transformed into wtCp by electroporation, and the recombinant bacterial strains were selected on agar plates containing 5 μg/ml Ampicilhn (Fig IB) The numbers of sod, gpo, rbr and ydaD knock-out mutants were 8, 6, 5 and 8 from 2 OxIO⁵ transformed cells, respectively

Bacteπal proliferation, sporulation and germination profiles were determined for the four mutant strains in which the major oxygen tolerance genes, sod, gpo, rbr ox ydaD, were individually replaced by ampR 3x10³ bacterial cells of the mutant strains and wild type strain were transferred to fresh RCM medium and incubated under anaerobic conditions Bacteπal cell numbers for each strain were determined at 0, 6, 12, 24 and 48 hours The maximum growth of vegetative bacteπa was found to be at day 1 in all strains There was no significant difference in bactenal growth between the knock-out and wtCp strains 1 xl O⁷ vegetative bacterial cells of each strain were transferred into sporulation medium and incubated for 5 days to test the maximum sporulation efficiency 1 x 10⁴ purified spores were transferred to RCM medium to test their germination efficiency There were no significant differences in the maximum yields of bacterial spores and spore germination efficiencies among all the strains tested The results indicate that knocking out the sod, gpo, rhr and ydaD gene does not affect anaerobic bacterial growth characteristics To compare the oxygen tolerance of these mutant strains relative to the wild type strain, their sensitivity to various oxidative stresses in vitro was evaluated using methods previously described by Bπolat and Reysset (2002) Bacteπal cells, Cp/sod, Cp/gpo , Cp/rbr, Cp/ydaD and wlCp, were diluted in PBS and cultured under various conditions of oxidative stress, including exposure to normal air and the addition of H₂O₂, t- butylhydroperoxide or plumbagin to the culture medium Bacterial cell survival was measured in normal air at 0, 1, 2, 3, 4, 5 and 6 hours and as a function of concentration (0, 1, 3 3, 10, 33, 100, 333 and 1000 μM) of plumbagin, H₂O₂ and t-butylhydroperoxide Percentages of bacteπal survival under these conditions were determined and compared with those obtained under standard anaerobic culture conditions These in vitro measurements were used to determine the reduction in oxygen tolerance after knocking out all four oxygen tolerant genes individually in Cp The results suggested that sod is a major oxygen tolerance gene in Cp because the sod knock-out strain exhibited a significant reduction in bacteπal survival under each oxidative stress condition It also suggested that the other three genes, gpo, rbr and ydaD, are contributing factors to oxygen tolerance because bacteπal survival in each knock-out strain was significantly reduced in normal air and reduced to varying degrees in response to the other oxidative stress conditions Thus, sequentially knocking out these genes in the Cp mutant should lead to recombinant strains with higher sensitivity to oxidative stress, which may result in elevated tumor selectivity and reduced toxicities

Most bacterial genes are expressed as polycistronic mRNAs A typical mRNA can have several coding regions with intervening non-translated intercistronic regions Lewin, "Genes V" (ISBN A- 19-269041-8, 1994) Protein synthesis in bacteria is very efficient and the polycistronic mRNAs are translated continuously by tightly packed πbosomes Unlike mammalian cells, there are no requirements for internal πbosomal entry sites in bacteπal polycistronic mRNAs for translation of the downstream coding sequences Lewin, "Genes V (ISBN A-19-269041-8, 1994) and Gold, Annu Rev Biochem 57 199-233, 1988

Recently, two genes in the anaerobic respiration and fermentation pathway in bacteπal energy metabolism have been shown to belong to the top 10 Predicted Highly Expressed (PHX) genes in Cp Karhn et al , Proc Nail Acad Sa USA 101 6182-7, 2004 As part of the present disclosure, it was found that the pyruvate ferredoxin oxidoreductase (pfoR) gene, one of the PHX genes, is highly expressed in Cp Accordingly, the pfoR was successfully utilized for the production of lacZ knock-in strains of Cp that have been screened for blue color on XOgal and molecularly characterized by sequencing The integration frequencies of the lacZ gene at the pfoR genetic loci was determined experimentally to be 5 OxIO^'4, and the inserted lacZ gene was driven by the endogenous promoters and successfully expressed from a polycistronic mRNA structure

The recombination DNA fragment was designed to insert the promoterless PVL coding sequences of S aureus following the translation termination codon of pfoR mRNA in order to generate a polycistronic structure, as shown in Fig 5A The PVL gene (from S aureus strain ATCC 49775) was inserted into the pfoR locus of Cp/sod to construct Cp/socT /PVL The pfoR coding and downstream sequences of Cp were PCR-amplifϊed using primers 5'-CCTTTAGCGTTTAAGTAGTC-3' and 5'-TCAATGGCAA TGAGAAAAAT G- 3', as well as its downstream sequences with primers 5'-GATGACCTCT CTTTCATTAA-3' and 5'-TTATGAGTAA AGTATCAGTA-3' (Gene Link, Inc ) The sequence of the two subunits, LukS and LukF in PVL gene was amplified from an S aureus strain ATCC 49775 with primers 5'-ATGTTTAAGA AACAAATG-3' and 5'-TTAATTATGT CCTTTC AC-3' for LukS, 5'-ATGAAAATGA AAAAATTAG-3' and 5'-TTAGTTGATT TAAACAA-3' for LukF, respectively

These coding sequences were linked to down-stream pfoR gene fragments by overlapping PCR to create the corresponding recombination fragment The overlapping PCR product was cloned into E cob and verified by sequencing (Roche) The recombination fragment was released from the plasmids, purified, and transformed into Cp/sod by electroporation The recombinant bactenal strains were by in situ colony hybridization using PVL specific probes (5'-CTCAAAATGT CCAATTCGAC TTTGTAAGAT A-3', Gene Link, Inc ) The recombinant strain, Cp/sod /PVL, was molecularly charactenzed by PCR sequencing The functional activity of PVL was assayed 2xlO⁷ bacteria/ml were serially diluted with RPMA-1640 medium (1 1, 1 10, 1 100, and 1 1000) and used in a cell killing assay using RPMI- 1640 as a negative control Mouse peripheral monocytes and neutrophils were isolated from whole blood using the immunomagnetic separation method (Cotter el al , American J of Pathol 154473-81, 2001), and seeded onto 96 well plates (IxIO⁶ cells/ml, 100 μl/well) The plates were incubated for 60 minutes at 37⁰C and leukocyte cytotoxicity was determined by MTT assays Dutta et al , Parasitol Internal 54 119-22, 2005

Cp/sod/PVL, a recombinant Cp/sod strain expressing the Panton-Valentine Leukocidin (PVL) gene from S aureus that is known to cause direct damage to phagocytic cell membranes, was constructed by homologous recombination Fig 3 A The proliferation, sporulation, and germination profiles of Cp/sod VPVL were determined in vitro, and shown to be equivalent to those of Cp and Cp/sod Fig 5B The results indicated that the insertion of the PVL gene into Cp/sod does not affect its anaerobic growth characteristics in vitro The functional activity of PVL was assayed by incubation of peπtoneal macrophage and polymorphonuclear cells with the bacterial culture supernatants in vitro, followed by an inflammatory cell viability assay For both macrophages/monocytes and neutrophils, statistically significant reductions in cell survival were observed after incubation with un-diluted (1 1) and 10-times diluted (1 10) supernatants from Cp/sod/PVL and parental S aureus, as compared to equivalently diluted culture supernatants from Cp/sod Fig 5C The results suggest that the PVL gene expressed from Cp/sod/PVL is functional

EXAMPLE 4

SENSITIVITY OF CLOSTRIDIUM PERFRINGENS TO OXIDATIVE STRESSES

To determine the reduction of oxygen tolerance in the mutant strain relative to the wild type strain, sensitivity to various oxidative stresses in vitro was evaluated using methods previously described Bπolat and Reysset, J Bacteriol JJ4 2333-43, 2002 Bactenal cells were diluted in PBS and cultured under various conditions of oxidative stress, including exposure to normal air and the addition of H₂O₂, t-butylhydroperoxide, or plumbagin to the culture medium Bacterial cell survival was measured in normal air at 0, 1, 2, 3, 4, 5 and 6 hours and as a function of concentration (0, 1, 3 3, 10, 33, 100, 333, and 1000 μM) of plumbagin, H2O2, and t-butylhydroperoxide Percentages of bacterial survival under these conditions were determined and compared with those obtained under standard anaerobic culture conditions

EXAMPLE 5

ENHANCED SAFETY AND EFFICACY OF A RECOMBINANT CP STRAIN WITH REDUCED OXYGEN TOLERANCE

The maximum tolerated dose (MTD) for each bacterial strain in tumor-bearing mice was determined by intravenous injection of buffer or escalating doses of spores that ranged from 10⁴ to 10^s at half-log increments (n=10/group) The endpoint was survival as defined by the time of death or by sacrifice when the animals appeared distressed as defined by significant weight loss, lethargy, or ruffled fur Statistical significance was analyzed using the One-tailed Fisher Exact Probability Test Anti-tumor efficacy was determined in a dose- controlled fashion after intravenous administration at their respective MTDs in mice beaπng orthotopic pancreatic cancer and the endpoint was survival The results were analyzed by the Kaplan-Meier method and compansons of survival curves between different groups were made by the log-rank test

Cp/sod showed reduced toxicities when spores were administered intravenously into tumor-bearing mice Animals treated with Cp/sod/PVL spores demonstrated a significant intralesional reduction in neutrophil and macrophage contents, which led to logarithmic elevation of intratumoral bacteπa titers, tumor necrosis and substantive survival prolongation, with a cure rate of 47% and without apparent systemic and organ toxicities

To determine the extent and kinetics of bacterial proliferation in tumors versus normal organs, tumor-bearing mice were treated with Cp/sod at its MTD (IxIO⁷) Animals were sacrificed at day 0 and day 3 post bacterial spore injection (4 mice per group) The tumors and major organs (pancreas, heart, lung, liver, kidney, spleen and bom marrow) were collected from the sacrificed animals Bacterial proliferation profiles in tumors versus major organs were determined by quantitative bacteπal culture of tissue extracts in vitro Bacterial titers in the tumors were 3- to 4-logs higher than those in the pancreas and blood samples, respectively (p<0 001), and were below detection in the other major organs Although there were recoverable bacteπa in the pancreas extracts, the titers were 3-logs below those of tumor extracts and it is not clear if these were derived from the pancreas or residual tumor tissues within the pancreas samples To determine the intratumoral distribution of the replicating bactena, Gram staining was performed on paraffin-embedded tumor sections collected at day 3, which was previously determined as the peak of bacterial replication in the lesions Vegetative bacteπal cells were found mainly in the necrotic tumor regions that bordered the viable tumors in the Cp/sod treated group and not the PBS group, and the results are suggestive of a correlative relationship between bacterial growth and tumor necrosis

To evaluate tumor response, H&E staining was performed on paraffin-embedded sections of tumors and major organs collected at day 3 post spore injection Background necrosis in tumors of mice treated with PBS was less than 10% However, about 55% tumor necrosis was observed in mice treated with Cp/sod, which was statistically significant as compared with the PBS control (p<001) These results demonstrated that the extent of tumor necrosis induced by bacterial spore treatment was dose-dependant and in agreement with survival analysis Importantly, there were no pathological changes found in any of the major organs including the pancreas, where a relatively low number of bacteπal spores was found

A long-term survival study was performed to assess treatment efficacy using doses of IxIO⁷ and IxIO⁶ spores for Cp/sod and IXlO⁶ for wtCp, with a PBS group as the negative control The survival curves were plotted according to the Kaplan-Meier method and statistical significance between the different treatment groups was determined by log-rank test Statistically significant survival prolongation over the PBS control group was observed in animals treated with Cp/sod at IxIO⁷ spores (p<0 02), but not in those treated with IxIO⁶ spores of either Cp/sod (p>02) or wtCp (p>03) Animals treated with IxIO⁷ spores of Cp/sod also exhibited a statistically significant survival advantage over those treated with 1x10⁶ spores of Cp/sod (p<0 04) or wlCp (p<0 04) These results demonstrated that recombinant Cp strains with reduced oxygen tolerance could be administered intravenously at higher doses in vivo, leading to a prolongation of survival in mice bearing orthotopic pancreatic tumors However the extent of survival prolongation was modest and most treated animals succumbed to relapse

Cp/sod/PVL provides a prototype for a novel class of oncopathic microbes for the effective and safe treatment of pancreatic cancer and other poorly vasculaπzed tumors in the future

The sod knock-out Cp strain, Cp/sod, was constructed by homologous recombination and confirmed by DNA sequencing There was no significant difference in bacteπal growth rates, spore yields and germination efficiencies between Cp/sod and Cp in vitro (data not shown). To compare the oxygen tolerance of the two strains, sensitivities to various conditions of oxidative stress were evaluated in vitro. The results showed that Cp/sod exhibited a significant reduction in bacterial survival under each of the oxidative stress conditions, indicating that it is indeed less oxygen tolerant. Escalating doses of spores from the Cp/sod and Cp were then injected into the tail veins of immune-competent and syngeneic mice bearing orthotopic pancreatic tumors. Their respective maximum tolerated doses (MTD) were determined to be IxIO⁷ and IxIO⁶ spores, respectively, indicating a significant improvement in safety of Cp/sod in vivo. A long-term survival study was performed to assess treatment efficacy using IxIO⁷ and IxIO⁶ Cp/sod, and IxIO⁶ Cp spores. A statistically significant survival prolongation over the PBS control group was observed only in animals treated with Cp/sod at IxIO⁷ spores (p<0.02), but not in those treated with IxIO⁶ spores of Cp and Cp/sod (p>0.4 and p>0.3, respectively). Animals treated with IxIO⁷ Cp/sod spores also exhibited a statistically significant survival advantage over those treated with IxIO⁶ spores of Cp and Cp/sod (p<0.03 and p<0.03, respectively). These results provided the proof of principle that recombinant Cp strains with reduced oxygen tolerance could be administered intravenously at higher doses without apparent toxicities in vivo, which could lead to a survival prolongation in mice bearing orthotopic pancreatic tumors. The extent of survival prolongation, however, was modest and most treated animals succumbed to relapse over time.

IxIO⁷ spores of ^' Cp/sod and Cp/sod/PVL were administered by tail vein injection into tumor-bearing mice, which were sacrificed on day 2 post spore administration. Tumors were collected and sections were analyzed by histology, Gram staining, and immunohistochemical staining for macrophages/monocytes and neutrophils, followed by morphometric and statistical analyses. Bacterial titers in tumors were also determined by quantitative bacterial culture from tumor extracts. Statistically significant reductions in intratumoral neutrophil (Fig. 6A) and macrophage/monocyte (Fig. 6B) contents were evident in Cp/sod/PVL treated mice. These results were also correlated with the significantly elevated intratumoral bacteria titers (Fig. 6C) and extents of tumor necrosis (Fig. 6D). Intratumoral proinflammatory cell contents, bacterial proliferation, and tumor response were determined in a kinetic study in tumor-bearing mice treated with 1x10⁷ spores of Cp/sod /PVL and Cp/sod. Animals were sacrificed at days 0, 1, 3, 7, and 14. Tumors, blood, and major organ samples were collected. Intratumoral contents of neutrophils in Cp/sod-/PVL treated mice showed significant reductions at days 1 3, and 7 post spore injection (Fig 7A), as well as intratumoral macrophage contents at days 1, 3, 7, and 14 (Fig 7B), vs Cp/sod- treated mice Corresponding to the reduction of intratumoral neutrophil and macrophage contents in Cp/sod/PVL treated mice, significant enhancement in intratumoral bacterial titers were found at days 1, 3, 7, and 14 (Fig 7C), as well as the extents of tumor necrosis at days 3, 7, and 14 (Fig 7D)

The effectiveness of Cp/sod/PVL in tumor treatment was evaluated by survival studies Fig 8 There was a statistically significance difference between the PBS group versus those treated with IxIO⁷ Cp/sod spores (p<0 02) Although the median survival was extended to 28 days from 21 days, all Cp/sod treated mice succumbed to tumor relapse within 50 days A substantial and statistically significant survival prolongation was observed in tumor-bearing mice after treatment with 1x10⁷ spores of Cp/sod/PVL vs Cp/sod (p<0 001) The median survival time was further extended to 61 days and 7 out of IS (47%) of the treated animals remained alive at 100 days Taken together, the results suggest that the PVL gene product was effective in suppressing the accumulation of neutrophils and macrophages/monocytes in the lesions, which permitted the bacteria to replicate to higher titers that led to enhanced tumor necrosis and substantially prolonged animal survival

EXAMPLE 6

ELEVATION OF THE ONCOPATHIC POTENCY OF CP/SOD- BY DEPLETION OF INFLAMMATORY CELLS IN THE HOST

Tumor-bearing mice were treated with bacteπal spores at their MTD Animals were sacrificed at various time points post spore injection (n=4 mice/group) The tumors and major organs (pancreas, heart, lung, liver, kidney, spleen, and bone marrow) were collected from the sacrificed animals Bacteπal proliferation profiles in tumors, major organs, and blood samples were determined with quantitative bacteπal culture and agar plating To determine the intratumoral distribution of the replicating bacteπa, Gram staining was performed on paraffin-embedded tumor sections To evaluate tumor response, H&E staining was performed on paraffin-embedded sections of tumors Additionally, tumor sections were used for immunohistochemical staining for neutrophils using rat anti-mouse Gr-I monoclonal antibodies (BD Pharmingen) and for macrophages/monocytes using rat anti-mouse F4/80 monoclonal antibodies (AbD Serotec) as well as H&E and Gram staining followed by morphometπc analyses The results were analyzed by the unpaired t-test To evaluate systemic toxicity, blood samples were collected from animals sacrificed at various time points post spore injection for determination of CBC, serum chemistries, and proinflammatory cytokine levels The major organs including heart, lung, spleen, liver, kidney, pancreas, and bone marrow were harvested for the preparation of paraffin-embedded sections H&E staining was performed on the tissue sections to assess histopathological changes

Since bacterial infection is known to induce a robust inflammatory response in an immune-competent host, the accumulation of inflammatory cells in the lesions of the treated mice was evaluated Tumor-bearing mice were injected with PBS or Cp/sod at its MTD (IxIO⁷ spores), and sacrificed on day 2 after treatment Tumor sections were analyzed by immunohistochemical staining for neutrophils, macrophages/monocytes and NK cells using rat anti-mouse monoclonal antibodies The stained inflammatory cells were quantified by morphometry and analyzed statistically by the unpaired t-test Fig 3A The accumulation of neutrophils and macrophages/monocytes in tumors after Cp/sod spore administration was statistically significant (p<0 006 and p<0 01, respectively), but not that of NK cells (p>06) These results indicated that neutrophils and macrophages/monocytes, but not NK cells, were rapidly recruited to the tumor sites in response to intravenous Cp/sod spore administration To determine the effect of macrophage/monocyte, neutrophil, and NK cell depletion on bacteπal replication and tumor response, tumor-bearing mice were injected with 1x10⁷ Cp/sod spores, together with clodronate-containing liposomes, which is a well- established depletion method for macrophages (Cote et al , Microb Pathog 37 169-75, 2004 and Rooijen et al , J Immuno Meth 193 93-99. 1996) and monoclonal rat anti-mouse neutrophil and NK antibodies On day 2 post treatment, tumor tissues were obtained from the treated mice by dissection and bacteπal titers in tumor extracts were determined by agar plating Additionally, tumor sections were used for immunohistochemical staining for neutrophils, macrophage/monocytes and NK cells, as well as Gram staining for bacteπa and H&E for tumor necrosis measurements The results were quantified by morphometry and analyzed statistical by the unpaired t-test

The respective depletion treatments effectively reduced intratumoral neutrophil, macrophage/monocyte and NK cell counts in tumors (Fig 3B, top panel) The reductions in neutrophils and macrophages were correlated with an elevation of intratumoral bacteria titers by approximately one log (Fig 3B, middle panel) and a statistically significant enhancement in tumor necrosis (Fig 3B, bottom panel) The successful depletion of NK cell was not, however, correlated with any enhancement of intratumoral bacteπal titers or tumor necrosis Taken collectively, the results suggest that neutrophils and macrophages/monocytes, but not NK cells, were rapidly recruited to the tumor sites in response to intravenous Cp/sod spore administration, and were the effector inflammatory cells in attenuating intratumoral bacteπal replication EXAMPLE 7

LACK OF SYSTEMIC AND ORGAN TOXICITIES IN TUMOR-BEARING MICE TREATED WITH CP/SOD-/PVL

To evaluate systemic toxicities of the bactenal spore treatment, blood samples collected from treated mice in the kinetic study above were analyzed for CBC, blood chemistries and serum pro-inflammatory cytokine levels Fig 9A According to the United States Medical Licensing Examination (USMLE) standard laboratory values, ALT, AST, BUN, direct-bilirubin and, indirect-bilirubin values all remained within their respective normal ranges at all time points, indicating that there were no apparent toxicities to the liver and kidneys RBC, WBC, hemoglobin and hematocπt values also remained within their normal ranges at all time points, indicating that there was no lymphopenia, hemolysis, or anemia Induction of IL- 12, IFN-gamma, and TNF-alpha, which are anticipated as initial inflammatory cytokines in response to bacterial infection, were found in animals treated with Cp/sod and Cp/sod /PVL The maximal serum levels of IL- 12, IFN-gamma, and TNF-alpha were all below their respective toxic levels (Lyke et al , Infect Immun 72 5630-7, 2004 and Wan et al , Int Immunopharmacol 6 750-8, 2006), and returned to the respective pre- treatment levels within 3 days All major organs including heart, lung, spleen, liver, kidney, pancreas and bone marrow were evaluated by H&E staining to determine their histopathology There were no apparent acute or long-term pathologies in the non-tumor bearing regions of the pancreas collected at days 0, 1, 3, and 14 as well as the liver and spleen, organs that are close to the pancreas Fig 9B There were also no clinically significant pathologic changes found in any organ at all of these time points These results indicated that there were no apparent systemic or organ toxicities in tumor-bearing mice after bacterial spore treatment

Hypoxic cores in poorly vascularized tumors are a major hindrance in cancer therapy, as they inhibit the effective delivery of therapeutic medications The presence of hypoxia in solid tumors however, also offers the potential for anaerobic bacterial colonization and tumor destruction Indeed beneficial effects of bacterial infection on tumors have been observed since the 18th century, and hundreds of cases of spontaneous regression of many types of malignancies following bacteπal infections had been recorded Critchley et al , Gene Therapy U. 1224-33, 2004 In recent years, a number of novel approaches to developing oncopathic bacteπa have been described, including Gram-negative Salmonella In contrast to obligate anaerobic bacteria such as Clostridia and Bifidobacteria, Salmonella are facultative anaerobic bacteria and have the potential to colonize oxygenated small metastatic lesions as well as large tumors with a hypoxic centre A major problem for the full exploitation of Salmonella in cancer treatment, however, is that it can induce TNF-alpha mediated septic shock Recently, attenuated Salmonella typhimurium strains, which are facultative anaerobes, have been described as anticancer agents Pawelek el al , Lancet Oncol 4 548-56, 2003 Bifidobacterium longum, a nonpathogenic Gram-positive anaerobic bacteπum, was shown to selectively germinate and grow in the hypoxic regions of solid tumors after intravenous injection Yazawa et al , Breast Cancer Res and Treatment 66 165-70, 2001 Vogelstem and coworkers investigated Clostridium novyi, an obligate anaerobic, for its capacity to grow within transplanted tumors Dang et al , Proc Natl Acad Sa USA 98 15155-60, 2001 A recombinant strain of C novyi devoid of its lethal toxin gene was engineered (C novyi-NT) and was shown to germinate within the avascular regions of tumors and destroy surrounding tumor cells in mice This treatment also caused acute lethality, however, in a significant fraction of mice Very recently, the same group showed that treatment of mice bearing large tumors with C nøvyz-NT plus a single dose of liposomal doxorubicin led to eradication of the tumors Cheong et al , Science 3J4 1308-11, 2006 The bacteπal factor responsible for the enhanced drug release was identified as a previously unrecognized protein termed "hposomase " Clostridia have also been genetically engineered to selectively deliver pro-drug-activating enzymes such as E coli cytosine deaminase (Theys et al , Cancer Gene Ther § 294-7, 2001) and nitroreductase (Lemmon et al , Gene Ther 4 791-6, 1997) that enhance treatment efficacy via a bystander effect Clostridium perfrmgens, an anaerobic, spore-forming, Gram-positive bacteπum, was evaluated as an oncopathic agent for cancer treatment After intravenous administration in immune-competent mice bearing orthotopic and syngeneic pancreatic cancer, the Cp spores were capable of preferential germination and proliferation within the hypoxic cores of tumors with oncopathic effects However, the bactena's residual tolerance to oxygen is such that they are still able to germinate and grow in normal tissues at reduced rates, which led to significant toxicities when administered intravenously at high doses in tumor-bearing mice The major gene associated with oxygen tolerance in Cp is the one encoding superoxide dismutase (sod) (Bπolat and Reysset, J Bacteriol 184 2333-43, 2002, Geissmann et al , J Bacteriology \%\_ 7136-9, 1999, and Lehmann et al , J Bacteriology 178 7152-8, 1996), and its knock-out strain (Cp/sod) exhibited reduced oxygen tolerance in vitro and limited growth in normal tissues in vivo, leading to a one-log elevation in its MTD in animals Intravenous infusion of Cp/sod in tumor-bearing mice led to enhanced intratumoral bacteria replication, tumor regression and survival prolongation However, the tumor response was modest and most treated animals succumbed to relapse over time, and more effective Cp strains will need to be developed

Macrophages and neutrophils have been shown to play key roles in early host defenses against infection by microbial pathogens in the host Cote et al , Infection and Immunity 74 469-80, 2006 Neutrophils are first to accumulate in the infection sites and initiate the cytolytic process of invading microorganisms The local and blood-borne macrophages also migrate to the infection sites and initiate phagocytosis Neutrophils and macrophages have been shown to be spoπcidal in vitro and have a protective role in the host Cote et al , Microb Pathog 37 169-75, 2004 and Welkos et al , Microb Pathog 7 15-36, 1989 In our studies, we demonstrated significant accumulation of neutrophils and macrophages/monocytes, but not NK cells, in tumors after Cp/sod spore administration Additionally, the reductions in neutrophils and macrophages were correlated with elevated intratumoral bacteria titers and tumor necrosis, suggesting their suppression in animals could substantially enhance treatment efficacy On the other hand, many invading microbes have evolved clever strategies for evasion and/or suppression of the host inflammatory responses Pathogenic bacteria are able to avoid phagocytic engulfment and killing Galan, J Exp Med 201 321-3, 2005 Panton- Valentine Leukocidin (PVL), produced by S aureus, can directly damage membranes of phagocytes including monocytes, macrophages and neutrophils Kato, Nippon Saikingaku Zasshi 36 445-57, 1981, Okumoto, Int Ophthalmol Clin 25 133- 42, 1985, and Genestier et al , J Clin Invest U5 3117-27, 2005

To enhance oncopathic potency and treatment efficacy, Cp/sod/PVL, a recombinant Cp/sod strain expressing PVL, was constructed The Cp/sod/PVL strain led to a significant reduction in intratumoral content of inflammatory cells, logarithmically elevated intratumoral bacteπa titers, enhanced tumor necrosis, and substantially prolonged animal survival over those treated with Cp/sod Importantly, a substantial fraction of the treated mice remained alive after 100 days and there were no apparent systemic and organ toxicities associated with the systemic administration of Cp/sod/PVL spores at its effective dose Thus this novel recombinant Cp/sod/PVL strain, with substantially elevated tumor selectivity and oncopathic potency, can be developed into a safe and effective oncopathic agent for the treatment of patients with pancreatic cancer and other poorly vascularized tumors in the future, and the scientific principle of bacteria-mediated expression of inflammation suppressive genes is generally applicable for the enhancement of oncopathic potency and efficacy of other oncopathic bacteria in cancer treatment

The MTD dose of Cp/sod/PVL in tumor-bearing mice was found to be IxIO⁷, which is the same as that of Cp/sod, suggesting that there were no extra toxicities associated with the PVL knocking-in strain in vivo 1x10⁷ spores of Cp/sod and Cp/sod/PVL were introduced by tail vein injection into tumor-bearing animals, which were sacrificed at day 2 after spore administration Tumors were collected and sections were analyzed by histology, Gram staining and rmmunohistochemical staining for macrophages and neutrophils, followed by morphometric and statistical analyses Bacterial titers in tumors also were determined by quantitative bacteπal culture from tumor extracts Statistically significant reductions in intratumoral neutrophil and macrophage contents were evident in Cp/sod/PVL treated mice These results were also correlated with significantly elevated intratumoral bacteπal titers and the extents of tumor necrosis The results suggested that the new strain could be a more effective agent for pancreatic cancer treatment, and are supportive of our hypothesis that the oncopathic potency of bacteria can be enhanced by vector-mediated expression of inflammation suppressive genes

To evaluate systemic toxicities of the bacterial spore treatment, blood samples collected from treated mice at various time points with 1x10⁷ spores of Cp/sod and Cp/sod /PVL were analyzed for CBC, blood chemistries and serum pro-inflammatory cytokine levels According to the United States Medical Licensing Examination (USMLE) standard laboratory values, ALT, AST, BUN, direct-bihrubin and lndirect-bilirubin values all remained within their respective normal ranges at all time points, indicating that there were no apparent toxicities to the liver and kidney RBC, WBC, hemoglobin and hematocrit values also remained within their normal ranges at all time points, indicating that there was no lymphopenia, hemolysis and anemia Induction of IL- 12, IFN -gamma and TNF-alpha, which are anticipated as initial inflammatory cytokine products in response to bacteπal infection, were found in animals treated with Cp/sod and Cp/sod/PVL However, the maximal serum levels of IL- 12, IFN-gamma and TNF-alpha were all below their respective toxic levels, and returned to the respective pre-treatment levels within 3 days All major organs including heart, lung, spleen, liver, kidney, pancreas and bone marrow were evaluated by H&E staining to determine their histopathology. There were no apparent acute and long term toxicities in the non-tumor bearing regions of the pancreas collected at days 0, 1, 3 and 14 as well as the liver and spleen, organs that are closest to the pancreas. There were also no significant pathologic changes found in any organ at all these time points. These results indicated that there were no apparent systemic or organ toxicities in tumor-bearing mice after the bacterial spore treatment.

EXAMPLE 8 Two HIGHLY EXPRESSED GENES IN CP FOR TRANSGENE INSERTION AND EXPRESSION The correlative relationship revealed by the results in the studies above suggest that suppression of neutrophils and macrophages in tumor-bearing animals can substantially enhance intratumoral bacteria replication, oncopathic potency and anti-tumor efficacy of the bacterial spore treatment. Recombinant Cp strains that express inflammation suppressive genes from heterologous bacteria are constructed for applications in tumor-bearing mice. Highly expressed genetic loci in Cp were identified for transgene insertion.

Recently, two genes in the anaerobic respiration and fermentation pathway in bacterial energy metabolism were shown to belong to the top 10 Predicted Highly Expressed (PHX) genes in Cp. Karlin et al., 2004. The pyruvate ferredoxin oxidoreductase (pfoR) gene catalyzes the final oxidative step in the fermentation of carbohydrates in anaerobic microorganisms that involves the oxidative decarboxylation of pyruvate to form acetyl-CoA + CO₂. The alcohol-acetaldehyde dehydrogenase (adfiE) gene converts acetyl coenzyme A (acetyl-CoA) to ethanol under anaerobic conditions. The pfoR and adhE genes exhibit stable gene expression at high levels in hypoxic solid tumors and are suitable candidate loci for the insertion of transgenes. The mRNA levels of the pfoR and adhE gene in Cp were analyzed by quantitative RT-PCR at the log phase of bacterial growth under anaerobic conditions, using a ribosomal protein (rpsD) gene as a reference. The expression of pfoR and adhE genes was about 1.4 and 1.2 times greater than that of the rpsD gene, respectively, and there was no statistically significant difference between the levels of pfoR and adhE gene expression. The knock-in frequencies at thepfoR and adhE loci was determined using lacZ as the reporter gene. The knock-in strains were constructed by homologous recombination, analogous to the construction of the knock-out mutant strains as described, supra. The DNA fragments were designed to insert the promoterless lacZ coding sequences following the translation termination codon of pfoR or adhE mRNA's in order to generate a polycistronic structure, which would not have any impact on the expression levels of the resident genes at the respective loci Fig 4 Bacteπal πbosomes are processive and will initiate translation at a downstream AUG codon on mRNA's without the need for an internal πbosome entry site The pfoR and adhE genes including their promoter and SD sequences were PCR-amplified with specific primers, respectively, as were their downstream sequences The lacZ fragment was linked to the pfoR and adhE gene fragments, and their downstream fragments were linked by overlapping PCR to create the corresponding recombination fragments as described in Fig 4 The overlapping PCR products were cloned in E colt and verified by sequencing The recombination fragments were released from the plasmids, purified and transformed into Cp by electroporation The lacZ knock-in Cp strains were screened for blue color on X-gal, and molecularly characterized by sequencing The knock-in Cp mutant numbers were found to be 4 recombinants m 2x10⁵ transformed cells at the pfoR locus and 4 recombinants in 3x10⁵ transformed cells at the adhE locus Thus, homologous recombination frequencies at the pfoR and adhE loci were 7 5XlO^"4 and 5 Ox 10⁴, respectively

Bacteπal proliferation, sporulation and germination profiles for the lacZ knock-in mutant strains were determined as descπbed above 3x10³ bacterial cells of the lacZ- expressing (Cp/pfoR-lacZ, Cp/adhE-lacZ) and wtQ? strains were transferred to fresh RCM medium and incubated under anaerobic conditions Bacteπal cell numbers in each strain at various time points were determined. The maximum growth of vegetative bactena was found to be at day 1 for all recombinant and wild-type Cp strains, and there was no significant difference between strains at all time points IxIO⁷ of vegetative bactenal cells of each strain were transferred into sporulation medium and incubated for 5 days to test the maximum sporulation efficiency, and 1x10⁴ of the purified spores were then transferred to RCM media to test their germination efficiency The maximum yields of bacteπal spores and spore germination efficiencies in the two lacZ-expressing knock-in and wild-type Cp strains showed no statistically significant differences in these parameters (not shown), and the results indicated that the knocking-in of exogenous genes into the pfoR and adhE loci as polycistronic mRNAs will not affect their anaerobic growth and germination characteristics

EXAMPLE 9

CONSTRUCTION AND CHARACTERIZATION OF PHOLPHOLIPASE C (PLC) DELETED STRAINS CP/PLC- AND CP/SOD-/PLC-

Since the 1950s it had been suspected that the alpha-toxin (phosphohpase C) was a major virulence determinant of C perfringens that can cause gas gangrene MacFarlane, 1955 It has since been demonstrated that the deletion of the ^JZC gene in Cp will eliminate its πsk in gas gangrene induction Titball, 2005 Each of the recombinant constructs disclosed, supra, contain a phosphohpase c (pic) gene, which is the alpha toxin gene of Cp known to be the causative agent of gas gangrene in animals and humans This major toxin gene was deleted from the Cp spores to decrease toxicity and, thus, enhance safety

To delete the pic alpha toxin gene from Cp, a recombinant DNA fragment containing Cp genomic DNA (Cp-GFP-Cp), where the pic gene was replaced by the GFP gene, was constructed and used to transform wt/Cp and Cp/sod using methods described herein, supra The transformed bacterial clones were screened by in situ colony hybridization with probes specific to GFP The recombinant strains, Cp/plc and Cp/sod VpIc, were molecularly characterized by PCR sequencing The proliferation, sporulation, and germination profiles of these recombinant strains were determined, and shown to be equivalent to those of Cp and Cp/sod The results indicated that the deletion of the pic gene in wtCp and Cp/sod did not affect bacterial growth characteristics in vitro To examine the gas gangrene causing capabilities of our recombinant strains, PBS and

1x10⁹ vegetative bacterial cells of Cp, Cp/sod, Cp/plc and Cp/sod IpIc in 0 1 ml were injected intramuscularly into the hind legs of normal mice (n=10) using 26-gauge needles as reported by Stevens et al, 1987 Gas gangrene in the injected mice developed swollen hemorrhagic thighs with crepitus, extensive tissue necrosis, and mortality within 24 h There were 7 and 2 cases of gas gangrene developed in wtCp and Cp/sod injected mice, respectively, suggesting that the less oxygen tolerant Cp/sod strain is safer than wtCp (p<003) More importantly, there were no gas gangrene development in all mice injected with Cp/plc and Cp/sod IpIc , confirming that pic is an essential gene in Cp that causes gas gangrene A primary and metastatic model of pancreatic cancer was established in mice by injection of PANC02 cells into the pancreas and the liver of the same animals IxIO⁵ and 3 3x10⁵ PANC02 cells were implanted respectively into the pancreas and the liver of 6-8 week old female C57BL/6 mice At 18 days post implantation, tumor nodules larger than 5 mm x 5 mm were observed in both the pancreas and the liver of most animals. Additionally, a firefly luciferase expressing PANC02 cell line was generated by transduction of the cells with a recombinant lentivrus vector expressing luciferase, and a high producer clone was identified by a bioluminescence assay. Using these cells for implantation in the pancreas and liver of mice, the primary and metastatic lesions can be monitored by non-invasive bioluminescence imaging in vivo.

The MTD dose of Cp/sod/plc^' in tumor-bearing mice was determined by a dose controlled study in tumor-bearing mice to be 1x10⁷ spores, which was the same as that of Cp/socT. To evaluate the effectiveness of the new Cp strain in cancer treatment, 1x10⁷ spores of Cp/sod and Cp/sod/plc were injected into the tail vein of mice bearing tumors in the pancreas and the liver, which were sacrificed at day 3 after spore administration. Bioluminescence imaging was performed at day -1 and day 3 before sacrifice. A statistically significant reduction in bioluminescence at day 3 was found in Cp/sod and Cp/sod/plc treated mice (p<0.03). Sections of pancreatic and hepatic lesions in mice at day 3 after spore administration were analyzed by Gram staining and there were ample bacteria in both, suggesting that the bacteria were capable of accessing both the primary and metastatic lesions for germination and growth. Bacterial titers in tumor extracts from the sacrificed animals were determined by quantitative bacterial culture, and there was no statistically significant difference between Cp/sod and Cp/sod/plc in the pancreatic and hepatic lesions, suggesting that the pic strain is equally efficient in intratumoral replication as the pic* strain. There were also no statistical differences in the bacterial titers of either strain between the pancreatic and the hepatic lesions, suggesting that the bacterial spores can germinate and grow in the primary and metastatic lesions with equal efficiency. Tumor sections were then analyzed by histological staining followed by morphometric and statistical analyses. While there were significant increases in the extent of tumor necrosis in the lesions of the spore treated mice versus PBS control, there was no significant difference between the pancreatic and hepatic lesions of the Cp/sod and Cp/sod /pic treated mice, suggesting that there was not a negative impact in anti-tumor treatment efficacy by knocking out the pic gene in Cp/sod.

Blood samples collected from treated mice were analyzed for CBC, blood chemistries and serum pro-inflammatory cytokine levels, and major organs were collected for histopathological examination. The results were all normal, indicating that there were no apparent systemic and organ toxicities of the Cp/sod/plc strain in tumor-bearing mice when administered at the MTD

In a related study, the pic gene in Cp/sod/PVL was knocked-out to create Cp/plc/sod /PVL and was shown to be incapable of inducing gas gangrene in mice PIc knock-out 5 strains {Cp/plc , Cp/plc /sod and Cp/plc /sod-/PVL) were constructed by replacing it with a 46 bp random and non-coding sequence by homologous recombination in the parental Cp, Cp/sod and Cp/sod/VPL strains descπbed in Li et al , J Nat Cancer Inst 100 (2008) To construct a homologous recombination fragment, the random sequence and the flanking regions (0 7 kb each) of the pic gene were amplified by PCR The three DNA fragments

10 were used to generate one homologous recombination fragment by overlapping PCR, which was inserted into pUC19 and cloned in E coll The fragment was released from the plasmid and transfected into the parental Cp strains by electroporation The transformants were screened by colony hybridization, using the random sequence probe and confirmed by DNA sequencing analysis Ezaki et al , Japan Scientific Societies Press (ISBN 4-7622-2961X, pp

15 214-218 (2000))

As with the Cp/sod and Cp/sod/plc strains, the Cp/sod/PVL and Cp/plcJsod /PVL strains also failed to exhibit any negative impact in anaerobic bacteπal growth characteristics in vitro and oncopathic potency in vivo Intravenous injection of Cp/plc /sod /PVL spores led to significant survival advantage in tumor-bearing mice with the same efficacy as Cp/sod

20 /PVL, indicating that the oncopathic potency of Cp is independent of a functional pic gene The treatment also did not lead to an attenuated immune response to a subsequent pathogen challenge Consequently, Cp/plc-/sod /PVL is a novel oncopathic bacteπal agent for the effective treatment of pancreatic cancer and other poorly vascularized tumors with a substantially enhanced safety profile See, also, Li et al , Human Gene Therapy, submitted

25 EXAMPLE 10

ENHANCEMENT OF TUMOR SELECTIVITY AND SAFETY OF CP/SOD-/PLC- BY SEQUENTIAL KNOCK-OUT OF ITS MAJOR OXYGEN TOLERANCE GENES

Recombinant anaerobic bacteπal spores exhibiting improved safety profiles and enhanced efficacy can be developed as oncopathic agents to treat primary and metastatic

30 pancreatic cancer, in combination with chemotherapy Recombinant Cp/sod/plc strains with minimal toxicity were constructed by knocking-out additional oxygen tolerant genes

The intratumoral replication potential of Cp/sod/plc was enhanced by insertion of genes from heterologous bacteria that suppress host inflammatory responses, which in turn elevates their oncopathic potency and treatment efficacy.

Each of the recombinant Cp strains are tested for efficacy and safety in immune- competent and syngeneic mice bearing pancreatic tumors in the pancreas and the liver. Combination treatment with existing chemotherapeutic drugs is tested to explore the potential of synergism in tumor response and survival prolongation that will be superior to chemotherapy alone.

The three remaining oxygen tolerance genes in Cp/sod/plc were deleted to further reduce oxygen tolerance and minimize toxicities by restricting bacteria from germination and growth in normal tissues. The recombinant Cp strains with multiple oxygen tolerance genes knocked out are oxygen intolerant and selectively localize to, germinate and grow in the hypoxic regions of primary and metastatic lesions of pancreatic cancer with minimal toxicities to the host.

As discussed herein, supra, spores of Cp/plc colonize and induce necrosis in an orthotopic pancreatic tumor model in mice. Because Cp is an aerotolerant anaerobe that is capable of surviving in soil or arterial blood, there were substantial systemic toxicities associated with the super-MTD doses after intravenous administration, which retained some level of oxygen tolerance and the ability to grow, albeit with substantially reduced efficiency, in normal tissues. Vegetative and stationary cells can also survive in a growth-arrested stage in the presence of oxygen and/or low concentrations of superoxide and hydroxyl radicals. Indeed, Cp possesses a complex oxidative stress response system that provides protection against the adverse effects of the reactive oxygen species encountered both in vivo and in vitro. Briolat and Reysset, 2002 and O'Brien and Melville, 2000.

The ability of Cp to survive in a wide variety of natural and accidental oxidative stress conditions suggests that multiple genes are involved in this adaptive response, as is the case with other aerobic or facultative anaerobic bacteria. Gille and Sigler, 1995. The major genes associated with oxygen tolerance in Cp include superoxide dismutase (sod), glutathione peroxidase (gpo), rubrerythrin (rbr), and an alcohol dehydrogenase family member (ydaD). Sod is an oxidoreductase that catalyzes the reaction between superoxide anions and hydrogen to yield molecular oxygen and hydrogen peroxide. The enzyme plays a major role in anaerobic organisms for protection against oxidative stress. Archibald and Fridovich, 1981 and Gruber et al., 1990. Genes encoding sod are found in a variety of anaerobes including Cp. Geissmann et al. , 1999. Gpo is a major peroxide scavenging enzyme that catalyzes the oxidation of glutathione to yield oxidized glutathione and water It provides tolerance to oxygen radicals Kappus and Sies, 1981 , Knorpp et al , 2006, and Sun et al , 2005 It has been suggested that Gpo participates in scavenging free oxygen radicals Holland et al , 1994 Rbr, identified in Cp by Lehmann el al , 1996, is the terminal component of NADH peroxidase that catalyzes the reduction of hydrogen peroxide to water, and is believed to be a part of the anaerobic defense mechanism against oxidative stress based on its NADH peroxidase activity Coulter et al , 1999 and Coulter et al , 2000

The ydaD gene encodes a NADPH dehydrogenase, which is required for maintenance of the intracellular redox balance in growth-arrested cells (Xiong et al , 2000), and has been identified to be involved in the oxidative stress response in Cp by insertional mutagenesis Bπolat and Reysset, 2002

Mutant Cp strains with knock-outs in each of the four oxygen tolerance genes were constructed and tested under various conditions of oxidative stress The in vitro results demonstrated that all four genes, sod, gpo, rbr and ydaD, contributed to oxygen tolerance of Cp to various degrees These experiments also suggested that knocking out these oxygen tolerant genes in Cp could lead to heightened sensitivity to oxidative stress, which would result in reduced toxicities while maintaining their ability to colonize and confer oncopathic effects to tumors in an anaerobic environment In one such strain, the sod gene in wtCp was replaced by the firefly luciferase gene Spores of this Cp/sod strain were tested by intravenous injection in the orthotopic pancreatic cancer model in mice and were shown both to be safer in the host, induced partial tumor necrosis and conferred a limited survival advantage over wtCp To further improve tumor selectivity and reduce toxicity to normal tissues, a seπes of oxygen-intolerant Cp strains is constructed by sequentially knocking out the remaining oxygen tolerance genes, gpo, rbr and ydaD, in the Cp/sod /pic strain

Individual oxygen tolerance gene knock-out mutants of Cp were constructed through homologous recombination and determined the respective recombination frequencies at the target loci Using the same methods, the three target oxygen tolerance genes (gpo, rbr and ydaD) are deleted in the Cp/sod /pic strain to generate a seπes of oxygen-intolerant Cp/plc (piCp/plc ) strains, followed by characterization in vitro to determine their sensitivities to oxidative stress and efficiencies for growth, sporulation and germination Their anti-tumor efficacy, bio-distnbution, and systemic and organ toxicities after intravenous administration are evaluated in mice bearing pancreatic and hepatic lesions of pancreatic cancer A series of random sequences (RS) are selected for replacement of the gpo, rbr and ydaD genes, as well as the inserted luciferase gene, in Cp/sod/plc Four such sequences (40 bp) were generated using the RANDNA software, a random DNA sequence generator (Piva F and Pπncipato G, 2006) RS-I, 5'-TAAGTAAGTA AGTAACTAAG AATCTCCGTC TTATCCGTCC-3', RS-2, 5'-TAACGCATAA GGAATAACGC TCTACCGCAG AGAACGTAAG-3', RS-3, 5'-TAAGACTTAA ATTGTCTTAA CATAAGCATT AGCGCTTGGG-3', RS-4, 5'-TAAGACACGA TAAGCATAAT TAAATACACT TTAGCCGTCG-3' The RSs were designed to contain a TAA stop codon, but not an ATG, in all three reading frames All four RSs were analyzed by BLAST and showed no homology to the genomic sequence of C perfringens These designer DNA oligonucleotides are synthesized from Gene Link, Inc , and are cloned and confirmed by sequencing The steps for constructing recombinant DNA fragments carrying these non-coding sequences to replace the coding regions of the target knocking-out genes are as descπbed in Figure 8 The RS-I fragment is used to replace the LUC gene, which was used originally for exchange of the sod gene in Cp/sod/plc , while RS-2, RS-3 and RS-4 is used to replace the gpo, rbr and ydaD genes, respectively The recombinant DNA fragments containing these hybrid sequences are cloned in E coll, released from the plasmids and transfected into Cp/sod/plc by electroporation Recombinant Cp strains with additional knockout genes are identified by in situ colony hybridization using probes specific to the respective RSs, and their identities are confirmed by sequencing Proceeding stepwise, a total of four oxygen-intolerant Cp/plc (oiCp/plc ) strains Cp/sod/plc , Cp/sod/plc /gpo , Cp/sod/plc /gpo /rbr and Cp/sod/plc /gpo /ydaD are generated

Bacterial proliferation, sporulation and germination efficiencies, as well as the antibiotic susceptibility profiles, of individual oiCp/plc strains are determined For the determination of growth efficiencies, 3x10³ bacterial cells of the individual oiCp/plc and wtCp/plc strains are transferred to fresh RCM media and incubated under anaerobic conditions Bacterial cells in each of the mutant strains at various time points are determined and the growth curves are compared to that of wtCp/plc 1x10⁷ of vegetative bacterial cells of each strain will be transferred into sporulation medium and incubated for 5 days to test the maximum sporulation efficiency 1x10⁴ of the purified spores are transferred to RCM media to test their germination efficiency To investigate oxygen tolerance of these oiCp/plc strains, their sensitivities to various conditions of oxidative stress are determined in vitro Bactenal cells from each of the sequential knock-out mutant strains, Cp/sod/plc , Cp/sod/plc/gpo , Cp/sod/plc/gpo/rbr and Cp/sod/plc /gpo /ydaD , as well as wtCp/plc , are diluted in PBS and exposed under various conditions of oxidative stress, including normal air, H₂O₂, t-butylhydroperoxide and plumbagin Bactenal cell survival is measured in normal air at 0, 1, 2, 3, 4, 5 and 6 hours, and as a function of concentration (0, 1, 3 3, 10, 33, 100, 333 and 1000 μM) of plumbagin, H₂O₂ and t-butylhydroperoxide Percentages of bactenal survival under these conditions are determined and compared with those obtained under anaerobic conditions The respective LD50s provide an in vitro determinant of the level of reduction in oxygen tolerance for each of the oiCp strains, and the results are analyzed statistically by unpaired t-test

The MTD for each of the four oiCp/plc strains is determined in mice beanng pancreatic and hepatic lesions of pancreatic cancer, which receive buffer or escalating doses of spores that range from 10⁴ to 10^s at half-log increments (n=10/group) The first endpoint is survival as defined by the time of death or by sacrifice when the animals appear distressed as defined by significant weight loss, lethargy or ruffled fur Statistical significance is analyzed using One-tailed Fisher Exact Probability Test Systemic and organ toxicities are the other endpoints Blood samples from the test animals are collected at 0, 1, 2, 3, 5, 7, 10 and 14 days post spore injection for determination of CBC, serum chemistry, proinflammatory cytokine levels and neutralizing antibody titers to the bactena The results are analyzed by Kruskal-Walhs one-way ANOVA by ranks Finally, the animals are sacrificed at day 14 and the major organs (heart, lung, spleen, liver, kidney, pancreas, bone marrow) are harvested for the preparation of both frozen and paraffin-embedded sections H&E and Gram staining is performed on the tissue sections to determine organ toxicities, if any, as descnbed in the preliminary studies The MTD doses for the four oiCp/plc strains are defined as the respective maximum dose that does not lead to any of the toxicity endpoints

To confirm that the oncopathic potency of the oiCp/plc strains are not compromised, anti-tumor efficacy for each of the four oiCp/plc strains is determined in a dose-controlled fashion (up to their respective MTD doses) after intravenous administration in mice beanng pancreatic and hepatic lesions of pancreatic cancer (n=15/group) PBS and wtCp/plc treatment groups are included as controls, and the animals are subjected to bioluimnescence imaging analyses before and after spore treatment at the weekly intervals to monitor tumor response A primary endpoint is survival and the results are analyzed by the Kaplan-Meier method, and comparisons of survival curves between different groups are made by the log- rank test

To examine intratumoral bacterial germination and growth, as well as tumor response, another set of tumor-bearing mice are treated with one or more oiCp/plc strain at its MTD and serially sacrificed at 0, 1, 2, 3, 5, 7, 10 and 14 days (n=5/group) Using the pancreatic and hepatic lesions recovered at the time of sacrifice, intraturomal bacteπa replication is determined by cultuπng the tumor extracts and by Gram staining of the tumor sections

Tumor response is monitored by H&E staining, and the extent of tumor necrosis is evaluated by morphometπc and statistical analyses by unpaired t-test

EXAMPLE 11

ENHANCEMENT OF INTRATUMORAL REPLICATION POTENCY OF CP/SOD-/PLC- BY INSERTION OF INFLAMMATION SUPPRESSIVE GENES FROM HETEROLOGOUS BACTERIA

Intratumoral bacteπal replication, and hence the oncopathic potency and anti-tumor efficacy, of Cp/sod /pic in hypoxic tumor regions can be substantially enhanced by insertion of inflammation suppressive genes from heterologous bacteπa

As discussed above, macrophages and neutrophils play key roles in early host defenses against infection by microbial pathogens m the host Cote et al , 2006 and Mayer- Scholl et al , 2004 While natural killer cells are critical components in immune inflammatory responses to intracellular pathogens, they are not the primary targets here as Cp is an extra-cellular pathogen When invading microbes penetrate the extracellular space of tissues, the cellular immune inflammatory response is immediately brought into play First to accumulate in the infection sites and initiate the cytolytic process of invading microorganisms are neutrophils The local and blood-borne macrophages also migrate to the infection site and initiate phagocytosis Neutrophils and macrophages are referred to as the professional inflammatory cells for their roles in these processes They have been shown to be sporicidal in vitro (Bozue et al , 2006 and Welkos et al , 2002) and have a protective role for the host infected with bacteria and bactenal spores at vivo Welkos el al , 1989 and Cote et al , 2004 Indeed, there were substantial accumulation of neutrophils and macrophages/monocytes in the lesions after bacterial spore administration

Antibody-mediated depletion of neutrophils and clodronate-mediated depletion of macrophages were performed in tumor-bearing mice and showed significant reduction of the respective inflammatory cells in the lesions, elevated intratumoral bactena titers and substantially enhanced tumor response

To survive and thrive in the infected hosts, many invading microorganisms have evolved strategies for evasion and/or suppression of the inflammatory responses in the host Pathogenic bactena have been shown to be able to avoid phagocytic engulfinent and killing Hornef et al , 2002 and Galan, 2005 The microbial strategies for survival include functionally inhibiting phagocytosis, as well as directly damaging or killing the phagocytic cells Rosenberger and Finlay, 2003 LcrV, a bacteπal virulence factor produced by Yersinia enterocolitica, was shown to be able to induce immunosuppressive cytokine production

Toll Like receptors (TLRs) are pathogen recognition receptors (PRRs) that respond to pathogen associated molecular patterns (PAMPs) duπng microbial invasion TLR-2 is expressed on the macrophage surface and mediates the response to various surface molecules of Gram-positive bactena, and has been observed in trafficking to phagosomal membranes Underhill el al , 1999 and Alvarez, 2005 Lcr V interacts with TLR-2 to modify macrophage cytokine production by increasing the secretion of IL-10, attenuating the inflammatory response and increasing bactenal survival Sing el al , 2006 and Giacomini et al , 2001 Ironically, bacterial interference with TLR2 signaling by LcrV makes TLR-based recognition a detriment to the host response to these bactena, as mice expressing TLR2 are more susceptible to infection by these bactena Sing et al , 2006

IL-10 is immunosuppressive in several ways, including the inhibition of macrophage activation, inhibition of PMN-denved chemokine expression, reduction of the half-life time of CC and CXC chemokines, suppression of proinflammatory cytokine production as well as down-regulation of the expression of MHC II that is required for antigen presentation Reithmeier et al , 2005, Geijtenbeek et al , 2003, and Moore et al , 1993 Thus, localized bactenal exploitation of host cell capacity to produce immunosuppressive cytokines provides an effective means for the invading microbes to modulate host defense mechanisms and evade immune recognition Hornef et al , 2002

Panton-Valentine Leukocidin (PVL), produced by S aureus, directly damages phagocytic cell membranes Kato, 1981 PVL is a secreted bicomponent pore-forming exotoxin and consists of two subunits (protein monomers), LukE (S class 32 2 kDa) and LukD (F class 34 3 kDa), which are expressed individually and act together to damage the cell membrane of phagocytes, including monocytes, macrophage and neutrophils Okumoto, 1985, Genestier el al , 2005 Only 2% of S aureus isolates express leukocidin, but nearly 90% of the strains isolated from severe dermonecrotic lesions express this lmmuno-toxin, which suggests that it is an important factor in necrotizing skin infections Kato, 1981 The lmmuno-toxin subunits bind to the leukocyte cell membrane, form a hetero-oligomeπc transmembrane pore composed of four LukF and four LukS subunits, thereby forming an octameric pore in the affected membrane Genestier et al , 2005 PVL exhibits cell specificity towards leukocytes, although there are other susceptible cell types Kamio, 1997 and Tomita and Kamio, 1997

The oncopathic potency and antitumor efficacy of Cp/plc can be substantively enhanced by insertion of the PVL and/or LcrV genes into its genome PVL is secreted by recombinant Cp/plc as it is a secreted product from a Gram-positive organism with a similar secretory system The LcrV gene product is an intracellular protein It can be fused to a signal sequence from the eglA gene of Clostridium acetobutylicum, which leads to the secretion of exogenous genes in Clostridial bacteria Barbe et al , 2005 and Theys et al , 1999)

A recombinant Cp/sod/PVL strain was constructed, see supra, and a substantial reduction in inflammatory cell contents was demonstrated in the lesions in treated mice that correlated with superior oncopathic activities than its parental Cp/sod strain This inflammation suppressive strain also did not exhibit apparent systemic and organ toxicities in the treated mice

Most bacterial genes are expressed as polycistronic mRNAs A typical mRNA can have several coding regions with intervening non-translated intercistromc regions Lewin, 1994 Protein synthesis in bacteπa is very efficient and the polycistronic mRNAs are translated continuously by tightly packed πbosomes Unlike mammalian cells, there are no requirements for internal πbosomal entry sites in bacteπal polycistronic mRNAs for translation of the downstream coding sequences Gold, 1988 and Lewin, 1994

The pyruvate ferredoxin oxidoreductase (pfoR) gene and the alcohol-acetaldehyde dehydrogenase (adfiE) gene were utilized for production of lacZ knock-in strains of Cp The integration frequencies of the lacZ gene at both genetic loci were determined experimentally to be 5 0-7 5 x 10^"4, and the inserted lacZ gene was driven by the endogenous promoters and successfully expressed from a polycistronic mRNA structure

Two lmmune-suppressive genes were introduced into the pfoR gene locus of Cp/sod /pic Bacterial strains carrying the desired LcrV and PVL genes are available from ATCC The recombinant DNA fragments (pfoR-PVL, pfoR-LcrV and pfoR-PVL/LcrV) carrying the proposed knock-in genes at the pfoR locus are constructed as described, supra The knock- in strains are constructed by homologous recombination, analogous to the construction of the recombinant mutant strains as descπbed in Fig 1 The recombination DNA fragments are designed to insert the promoterless LcrV and PVL coding sequences 3' to the termination codon of pfoR mRNA in order to generate a polycistronic structure Insertion of lacZ into this position of the pfoR locus did not have any impact on the expression levels of the resident gene or the growth characteristics of the recombinant bacteria The pfoR coding sequence and its downstream sequences are PCR-amphfied using specific pπmers The LcrV and PVL genes are amplified from plasmids available at the ATCC, and their coding sequences are linked to the pfoR gene fragments by overlapping PCR to create the corresponding recombination fragments as descπbed in Fig 1 The overlapping PCR products are cloned in E coli and verified by sequencing The recombination fragments are released from the plasmids, purified and transformed into Cp/sod/plc by electroporation The recombinant bacterial strains are screened by in situ colony hybridization with probes specific to LcrV or PVL (Ezaki at al , 2000)

The recombinant strains, Cp/sod/plc /PVL, Cp/sod/plc /LcrV, and Cp/sod/plc /PVL/LcrV, are molecularly characterized by PCR sequencing LcrV and PVL expression from the recombinant strains is assayed by Western blotting using monoclonal antibodies generated against the respective bacterial proteins produced from transformed E coli clones and purified according to the methods of Genestier et al (2005) and Solecki et al (2005), respectively

The PVL gene from S aureus strain ATCC 49775 and the LcrV gene from Y enterocolitica strain NCTC 22703 are PCR amplified and cloned in E coli with His-tags The recombinant proteins are purified by affinity chromatography on mtπlotriacetic acid columns and used to immunize mice, and hybπdoma clones expressing monoclonal antibodies to the respective recombinant proteins will be generated by the Hybπdoma Core at Mount Sinai Finally, the recombinantly expressed proteins isolated from the bacterial culture media are assayed for functional activities by incubation with peritoneal macrophage and polymorphonuclear cells in vitro, followed by a cell viability assay according to the methods of Sing et al , 2006

Bacterial proliferation, sporulation and germination efficiencies, as well as the antibiotic susceptibility profiles, of three recombinant Cp strains (Cp/sod/plc /PVL, Cp/sod /plc/LcrV and Cp/sod/plc /PVL-LcrV), are determined For the determination of growth efficiencies, 3x10³ bacteπal cells of the individual recombinant Cp strains are transferred to fresh RCM media and incubated under anaerobic conditions Bacteπal cell numbers in each of the mutant strains at various time points are determined and the growth curves are compared to that of Cp/sod/plc For the determination of sporulation efficiencies, IxIO⁷ vegetative bacteπal cells of each strain are transferred into sporulation media and incubated for 5 days to determine their maximum sporulation efficiencies For the determination of germination efficiencies, IxIO⁴ of the punfied spores from each strain are transferred to RCM media To examine oxygen tolerance of these recombinant Cp strains, their sensitivities to various conditions of oxidative stress are determined in vitro Bacteπal cells from each of the knock-in strains, Cp/sod/plc /PVL, Cp/sod/plc /LcrV, and Cp/sod/plc /PVL-LcrV, as well as wtCp/plc , are diluted in PBS and exposed under vaπous conditions of oxidative stress, including normal air, H₂O₂, t-butylhydroperoxide, and plumbagin Bacterial cell survival is measured in normal air at 0, 1, 2, 3, 4, 5 and 6 hours, and as a function of concentration (0, 1, 3 3, 10, 33, 100, 333 and 1000 μM) of plumbagin, H₂O₂, and t-butylhydroperoxide Percentages of bacteπal survival under these conditions are determined and compared with those obtained under anaerobic conditions The conditions that cause 50% bacteπal cell death (LD50) provide an in vitro determinant of the level of reduction in oxygen tolerance for each of the recombinant Cp strains and the results are analyzed by unpaired t-test to determine statistical significance

Since exogenous bacterial genes encoding secreted inflammation suppressive proteins are inserted into the recombinant Cp strains (Cp/sod/plc /PVL, Cp/sod/plc /LcrV and Cp/sod /pic /PVL/LcrV), the MTDs for each of the three recombinant Cp strains in mice beaπng pancreatic and hepatic lesions is determined As these recombinant strains do not have less toxicity than Cp/sod/plc, the animals receive buffer or descending doses of spores from the MTD dose of Cp/sod/plc to 10⁴ at half-log decrements (n=10/group) Survival is defined by the time of death or by sacrifice when the animals appear distressed as defined by significant weight loss, lethargy or ruffled fur Statistical significance is analyzed using One-tailed Fisher Exact Probability Test Systemic and organ toxicities are determined in blood samples from the test animals collected at 0, 1, 2, 3, 5, 7, 10 and 14 days post spore injection for determination of CBC, serum chemistry, proinflammatory cytokine levels and neutralizing antibody titers to the bacteπa. The results are analyzed by Kruskal-Wallis oneway ANOVA by ranks Finally, the animals are sacrificed at day 14 and the major organs (heart, lung, spleen, liver, kidney, pancreas, bone marrow) are harvested for the preparation of both frozen and paraffin-embedded sections H&E and Gram staining are performed on the sections to determine organ toxicities, if any, as described in the preliminary studies The MTD dose for each of the inflammation suppressive strains is defined as the respective maximum dose that does not lead to any of the toxicity endpoints

Anti-tumor efficacy for each of the inflammation suppressive strains is determined in a dose-controlled fashion (up to its respective MTD doses) after intravenous administration in mice bearing pancreatic and hepatic lesions of pancreatic cancer (n=15/group) PBS and Cp/sod/plc treatment groups are included as controls, and the animals are subjected to bioluminescence imaging analyses before and after spore treatment at the weekly intervals to monitor tumor response

The primary endpoint is survival, the results are analyzed by the Kaplan-Meier method and comparisons of survival curves between different groups are made by the log- rank test To examine intratumoral bacteπa germination and growth, as well as intratumoral expression of the recombinant proteins and tumor response, another set of tumor-bearing mice are treated with the most effective inflammation suppressive strain at its MTD and serially sacrificed at 0, 1, 2, 3, 5, 7, 10 and 14 days (n=5/group) Using the pancreatic and hepatic lesions recovered at the time of sacrifice, intraturomal bacteria replication is determined by cultuπng of the tumor extracts and by Gram staining of the tumor sections Intratumoral expression of PVL and LcrV is monitored by western blotting using monoclonal antibodies against the respective bacterial proteins Intratumoral contents of inflammatory cells are determined by immunohistochemical staining of tumor sections Tumor response is monitored by H&E staining of tumor sections, and necrosis within the lesions is evaluated by morphometπc and statistical analyses by unpaired t-test A recombinant strain that combines the optimal safety features of the oxygen intolerant strains (piCp) and the most effective inflammation suppressive gene combination (PVL+/-LcrV), as described supra, is constructed and characterized in vitro This novel recombinant strain (oiCp/plc /PVL+/-LcrV) is used to determine its antitumor efficacy and safety, in a dose-controlled fashion, in mice bearing pancreatic and hepatic lesions of pancreatic cancer It is expected that this recombinant strain will exhibit the greatest tumor selectivity and oncopathic potency, such that substantial tumor response and survival advantages in tumor-bearing mice can be achieved without toxicity Two additional inflammation suppressive genes from heterologous bacteria may be tested in Cp/sod/plc/PVL/LcrV bactena (the Chemotaxis Inhibitory Protein of

Staphylococcus aureus (CHIPS) and Exotoxin A from Pseudomonas aeruginosa (PEA))

CHIPS is a protein secreted by Staphylococcus aureus, which specifically inhibits C5a and fMLP-induced responses of neutrophils and monocytes, therefore inhibit early leukocyte migration Haas et al , 2004 PEA is considered to be a major virulence factor that is able to damage and kill cells by blocking protein synthesis It has been shown to act on phagocytes before bacterial ingestion, thereby impairing host defense Schultz et al , 2001,

Stuart et al , 1982, and Obana et al , 1977 These additional inflammation suppressive genes are inserted into the adhE locus of Cp/sod/plc /PVULcrV as a polycistronic mRNA

EXAMPLE 12

ENHANCEMENT OF ANTI-TUMOR EFFICACY OF CHEMOTHERAPY BY COMBINATION TREATMENT WITH RECOMBINANT CP SPORES

There are regions in poorly-vascularized tumors that are relatively oxygen-πch Tumor cells located in such regions are refractory to anaerobic bacteπal spore treatment By virtue of their access to oxygen and blood supply however, tumor cells in these regions are accessible to chemotherapeutic drugs distributed via circulation The ability of chemotherapeutic drugs to destroy tumor cells in the well-vasculaπzed and oxygen-πch regions, therefore, complements the ability of anaerobic bactena to destroy the hypoxic tumor core, leading to substantially enhanced tumor response and survival prolongation The anaerobic bacteπal spore treatment is combined with one or more chemotherapeutic drug(s) to treat pancreatic cancer in the mouse model system, descπbed supra, and tested for improved efficacy as compared to chemotherapy alone

The presence of oxygen-nch regions in poorly-vasculaπzed tumors may explain the absence of sustained tumor regression in animal models following treatment with anaerobic bactena Thus, the use of other agents that can target these oxygen-πch regions complements and enhances the tumor cytotoxicity of the spore treatment

Two chemotherapeutic regimens are used for this purpose Gemcitabine was approved by the FDA in 1997 for the treatment of pancreatic cancer (Burns et al , 1997), which has remained the standard treatment today It has a good safety profile with a low incidence of grade 3 or 4 toxicities (Aapro et al , 1998), and is also effective in delaying disease relapse when given as adjuvant treatment following curative resection in patients with localized pancreatic cancer Oettle et al , 2007 Moreover, the anti-tumor activity of gemcitabine is enhanced with survival prolongation (5 91 to 624 months) when combined with erlotinib, and the combination has been approved by the FDA in pancreatic cancer treatment Because of the modest improvement in survival over gemcitabine alone, however, current clinical investigation in various Phase III trials is targeted towards identifying drags that can significantly enhance the antitumor activity of gemcitabine

The addition of bacterial spore treatment to gemcitabine or gemcitabine + erlotinib is tested for synergistic destruction of pancreatic cancer as compared to chemotherapy alone It is envisioned that these chemotherapeutic drugs and the oncopathic bacteπa treatment are complementary, and that the outcome will yield supeπor efficacy as compared to chemotherapy alone

Cp/sod/plc is used in the presence and absence of gemcitabine at its clinical dose and schedule (240 mg/kg weekly for 3 weeks, i v ) or gemcitabine + erlotinib (15 mg/kg daily, p o ) Bornmann et al , 2007 PBS, gemcitabine, and gemcitabine + erlotinib treatment alone are included as controls Since these combination regimens do not have less toxicity than the spore treatment alone, mice bearing pancreatic and hepatic lesions of pancreatic cancer receive buffer or descending doses of spores from the MTD dose of Cp/sod/plc to 10⁴ at half-log decrements (n=10/group) The first endpoint is survival as defined by the time of death or by sacrifice when the animals appear distressed as defined by significant weight loss, lethargy or ruffled fur Statistical significance is analyzed using One-tailed Fisher Exact Probability Test Systemic and organ toxicities is the other endpoint

Blood samples from the test animals are collected at 0, 1, 3, 7, 14, 21 and 28 days post spore injection for determination of CBC, serum chemistry, proinflammatory cytokine levels, and neutralizing antibody titers to the bacteria The results are analyzed by Kruskal-Walhs one-way ANOVA by ranks The animals are sacnficed at day 28 and the major organs (heart, lung, spleen, liver, kidney, pancreas, bone marrow) are harvested for the preparation of both frozen and paraffin- embedded sections H&E and Gram staining are performed on the tissue sections to determine organ toxicities, if any The MTD dose for Cp/sod/plc in combination with the chemotherapeutic drugs is defined as the maximum dose that does not lead to any additional toxicity to chemotherapy alone

Anti-tumor efficacy of Cp/sod/plc is determined m a dose-controlled fashion (up to its MTD dose, n=15/group) by intravenous administration in mice bearing pancreatic and hepatic lesions, alone and in combination with gemcitabine and gemcitabine + erlotinib at their respective clinical doses and schedules (weekly treatments for three weeks with one week rest, three repeat cycles for the 3+ month survival studies) PBS, gemcitabine and gemcitabine + erlotinib treatment alone is included as controls The animals are subjected to bioluminescence imaging analyses before and after spore treatment at the weekly intervals to monitor tumor response The results are analyzed by the Kaplan-Meier method and comparisons of survival curves between different groups are made by the log-rank test

To examine intratumoral bacteria germination and growth, as well as tumor response, another set of tumor-bearing mice that have undergone combination chemotherapy and the most effective dose of Cp/sod/plc are serially sacrificed at 0, 1, 3, 7, 14, 21 and 28 days (n=5/group) Using the pancreatic and hepatic lesions recovered at the time of sacrifice, intraturomal bacteria replication will be determined by culturing of the tumor extracts and by Gram staining of the tumor sections Tumor response is monitored by H&E staining of tumor sections, and the extent of necrosis within the lesions will be evaluated by morphometry and statistical analyses by unpaired t-test Another chemotherapeutic drug that may be tested is docetaxel (10 mg/kg weekly for three weeks, i v ), which is a semisynthetic taxane acting as a microtubule de-stabilizer and is not expected to have an impact on bacterial growth Dang et al , 2004) Docetaxel is active as a single agent in phase II trials in pancreatic cancer, with a response rate of 15% (Dumontet et al , 1999) and median survival of 36 weeks (Androulakis et al , 1999) Microtubule destabilizing agents such as the vinca alkaloids, in contrast, disrupt blood flow to tumors but only at doses greater than the MTD in animal models Dang et al , 2004 The same expeπmental protocols described for gemcitabine, supra, are used in its combination treatment with the anaerobic bacteria spores

A recombinant strain that combines the optimal safety features of the oxygen intolerant strains and the most effective inflammation suppressive strains constructed (ι e oiCp/plc /PVL ^y/-LcrV) are used to determine in a dose-controlled fashion in mice bearing pancreatic and hepatic lesions of pancreatic cancer, together with an effective chemotherapy PBS and the chemotherapeutic drugs alone are included as controls The MTD dose of the optimal recombinant strain in combination with the most effective chemotherapy regimen is determined, and the anti-tumor efficacy of combination treatment with bacterial spores and chemotherapy is determined in mice bearing pancreatic and hepatic lesions of pancreatic cancer as descnbed herein, supra With the combination of gemcitabine, gemcitabine + erlotinib, or docetaxel, and the most effective recombinant Cp strain, the treatment regimens are expected to exhibit significant improvement on tumor response and survival prolongation in tumor-bearing mice over bactenal spore treatment and chemotherapy alone

Alternatively, the anti-tumor effect may be enhanced with genetic prodrug activation therapy by inserting one or more bacterial pro-drug-activating enzyme gene(s), including cytosine deaminase and nitroreductase, as has been developed in Clostridium strains Theys el al , 2001 and Lemmon el al , 1997 Additionally, administration of liposome mediated anti-tumor drug following Cp spore treatment may enhance treatment efficacy, which has been reported in a distinct Clostridium strain recently Cheong et al , 2006

Another alternative strategy is to inhibit neo-angiogenesis (Saif, 2006) in combination with oncopathic bacteria and chemotherapy The inhibition of angiogenesis in tumors reduces blood and oxygen supplies to the lesions, which escalates hypoxia within the tumors and enhances the proliferation of anaerobic bacteπa Pancreatic carcinoma shows over- expression of both vascular endothelial growth factor (VEGF) and its receptor (Seo et al , 2000) The recombinant humanized anti-VEGF monoclonal antibody suppresses the growth of pancreatic cancer Hurwitz et al , 2004, Willett et al , 2004 Additionally, over- expression of cyclooxygenase-2 (COX-2) is detected in 75% of resected pancreatic cancer that correlates with aggressive tumor biology, enhanced angiogenesis and invasiveness Wang et al , 2003 and Zhou et al , 2004 Celecoxib, a highly selective COX-2 specific inhibitor, has anti-tumor activity against a variety of cancers in animal models, including pancreatic cancer xenografts Raut et al , 2004 Thus, it is contemplated that the combination of bactenal spore treatment with chemotherapy and the monoclonal rat anti- mouse VEGFR2 antibody (Angio-Proteomie, Inc) and/or Celecoxib will further extend antitumor efficacy

EXAMPLE 13 CONSTRUCTION AND IN VITRO CHARACTERIZATION OF A CP/PLC /SOD /PVL STRAIN

A 40 bp random non-coding sequence (NCS) was generated with RANDNA software (Piva and Pπncipato, In Silico Biol 6£3} 253-8, 2006) and used for replacement of the pic gene in Cp/sod/PVL A recombinant DNA fragment containing Cp genomic DNA (Cp- NCS-Cp), where the pic gene was replaced by the NCS, was constructed and used to transform Cp/sod/PVL Bacterial clones were screened by in situ colony hybridization with probes specific to the NCS The pic knock-out strain was then molecularly characterized by PCR sequencing The proliferation, sporulation and germination profiles of Cp/plc /sod /PVL were determined, and shown to be equivalent to those of wild type Cp and Cp/sod/PVL (Fig 10) These data demonstrated that pic deletion in Cp/sod/PVLhad no negative impact on its anaerobic growth characteristics in vitro

EXAMPLE 14

PHARMACOLOGICAL CONCENTRATIONS OF CHEMOTHERAPEUTIC DRUGS DO NOT AFFECT BACTERIAL PROLIFERATION, SPORULATION, OR GERMINATION IN VITRO

This Example discloses the combination of a bacterial spore treatment with a chemotherapeutic drug in tumor-bearing mice

The effects of the following three chemotherapeutic drugs on Cp proliferation, spomlatioti, and germination were determined Gemcitabine (GEMZAR®, Eh Lilly and Co , Indianapolis, IN), Docetaxel (TAXOTERE®, Sanofi-Aventis, Bπdgewater, NJ), and Erlotinib (TARCEV A®, Hoffmann-LaRoche, Nutley, NJ) Each chemotherapeutic drug was added to the culture media of both Cp/sod/PVL and Cp/plc/sod/PVL, alone, with PBS as control The final drug concentrations in the bacteπal cultures were adjusted to 300 and 30 μg/ml for Gemcitabine, 40 and 4 μg/ml for Docetaxel, and 10 and 1 μg/ml for Erlotinib, which were 10- and 1-fold higher than their respective peak plasma concentrations determined in clinical pharmacokinetic studies of the drugs Czejka el al , Onkologie 28(6- TJ 318-22, 2005, Lunardi et al , Arm Oncol 13(21 280-5. 2002, and Masters et al , J Chromatogr B Analyt Technol Biomed Life Sa 848(2) 379-83, 2007 Incubation of bacteπal culture was performed at 37°C under anaerobic conditions As shown in Fig 11, there were no statistically significant differences in bacterial proliferation, sporulation, and germination efficiencies between the drug containing and the PBS groups These results demonstrate that the proposed chemotherapeutic agents did not negatively affect the proliferation of Cp/plc /sod/PVL in tumor-bearing mice at their respective clinical doses Administration of a chemotherapeutic agent may be initiated immediately following bacterial spore injection (on day 0) to maximize the combination treatment effects of these two therapeutic regimen

EXAMPLE 15

ENHANCED ANTI-TUMOR EFFICACY IN COMBINATION TREATMENT WITH GEMCITABINE AND CP/PLC-/SOD-/PVL BACTERIAL SPORES Gemcitabine is the first line chemotherapeutic drug in current clinical treatment for pancreatic cancer The treatment with bacteπal spores of the oncopathic anaerobic microbe, Cp/plc/sod/PVL, previously showed a substantially elevated tumor selectivity and safety, enhanced oncopathic potency and significantly prolonged animal survival, as an improvement which may lead to the development of safe and effective oncopathic treatment in pancreatic and other poorly vascularized tumors cancer However, the anaerobic bacteπa could not effectively replicate in the relatively oxygen-πch peritumoral regions, which resulted in tumor recurrence in half of the treated animals Whereas, tumor cells located in those well-vascularized regions are susceptible to chemotherapeutic drugs distributed via circulation Here we examined the anti tumor potency in treatment with Cp/plc /sod/PVL spores or genicitabrne alone, and a combination of both agents It was indicated that the anti cancer efficacy of Cp/plc /sod/PVL spores was significantly greater than gemcitabine, although gemcitabine treatment also provided a significantly survival advantage over PBS control Importantly, it was also demonstrated that the anti cancer efficacy in combination of Cp/plc /sod/PVL and gemcitabine showed substantial prolonged survival, comparing to each of them alone Therefore, the combinatorial use of the two complementary agents may be better able to target tumor cells in both the well- and poorly-oxygenated regions of the solid tumors and lead to substantially enhanced treatment efficacy EXAMPLE 16

EVALUATION OF TUMOR RESPONSE IN TREATMENTS WITH GEMCITABINE AND CP/PLC-/SOD-/PVL SPORES

The maximum tolerated dose (MTD) for the strain Cp/plc /sod/PVL in PANC02 tumor-bearing C57/BL6 mice was determined to be 1x10⁷ spores, which was the same as that of its parental strain Cp/sod/PVL Prior to evaluating the anti cancer effect of the chemotherapy, the toxicity profile of the chemotherapeutic drug, gemcitabine, was determined in normal mice at 125 mg/kg, i p , twice weekly for three weeks, with PBS treatment as control Animals were observed daily for their general appearance and blood was collected weekly for CBC and biochemistry analyses There were no statistically significant differences in RBC, hemoglobin, hematocrit, WBC, neutrophil, platelet, ALT, and AST between the chemotherapeutic drug treatment group and the PBS group (data not shown) To evaluate the oncopathic potency and effectiveness in cancer treatment, PBS control, 125 mg/kg of gemcitabine and IxIO⁷ spores of Cp/plc /sod/PVL were administrated into PANC02 tumor bearing C57BL/6 mice, which were sacrificed at day 3 after treatment Tumor sections were analyzed by histological staining followed by morphometnc and statistical analyses While there were significant increases in the extent of tumor necrosis in the lesions in Cp/plc /sod /PVL spore treated mice versus both gemcitabine and PBS control (p<001 and 001, respectively), there was no significant difference between the gemcitabine and PBS treated mice (p>0.S) (Fig. 12). These results suggested that the extent of tumor necrosis induced by Cp/plc/sod/PVL spores is much greater than that by gemcitabine, which is the current clinical treatment of pancreatic cancer.

EXAMPLE 17 THE EFFECTS OF GEMCITABINE ON CP BACTERIAL GROWTH CHARACTERISTICS

We expected that the anti-cancer efficacy might be substantially enhanced by a combination of Cp/plc/sod/PVL spore treatment with the chemotherapeutic drug, gemcitabine, to target the relatively well-oxygenized tumor cells in the peritumoral regions. To determine the effects of chemo-drug on Cp/plc/sod/PVL proliferation, sporulation and germination, gemcitabine (GEMZAR, Eli Lilly, Indianapolis, Indiana) was added to the culture media of Cp/plc/sod/PVL, with PBS as control. The final drug concentrations in the bacterial cultures were adjusted to 300 and 30 μg/ml, which were 10- and 1- fold higher than the peak plasma concentrations determined in clinical pharmacokinetic studies of the drugs. Incubation of bacterial culture was performed at 37^CC under anaerobic conditions. As shown in Fig. 13, there were no statistically significant differences in bacterial proliferation, sporulation and germination efficiencies between groups containing gemcitabine and PBS. This indicates that gemcitabine will not negatively affect bacterial germination and replication in tumors in vivo. Thus the chemotherapy could be initiated immediately after bacterial spore injection on day 0 to maximize their combination treatment effects. EXAMPLE 18

ANTI-CANCER EFFICACY IN TREATMENT WITH SINGLE AGENT OR COMBINATION OF GEMCITABINE AND CP/PLC-/SOD-PVL SPORES

To evaluate the anti tumor efficacy of Gemcitabine and combination with Cp/plc/sod /PVL spores in the orthotopic pancreatic cancer mouse model, a survival study was performed with gemcitabine (125 mg/kg, i.p., twice weekly, which are the safe and effective doses used in tumor-bearing mice (Solorzano et al., Clinical Cancer Research 9:1858-67, 2003; Nakazawa et al., Cancer Chemother Pharmacol 57:165-170, 2006), IxIO⁷ Cp/plc/sod/PVL spores, a combination of both, along with a PBS control group (Fig. 14). Both Cp/plc/sod /PVL spore and gemcitabine single treatments showed significant survival prolongation vs. PBS control (p<0.001 and 0.04, respectively). Importantly, Cp/plc/sod/PVL treatment also produced significantly greater survival advantage compared to that of gemcitabine treatment alone (/?<0.001). More importantly, the combination treatment exhibited significant survival prolongation over treatment with Cp/plc /sod/PVL spore or Gemcitabine alone and PBS control (p<0.03, 0.001 and 0.001, respectively). These results strongly suggest that the treatment outcome of bacterial spores is superior to that of the standard of care at present, and a combination of these treatment regimens could further elevate the anti-cancer efficacy.

Claims

CLAIMSWhat is claimed is:

1. A genetically enhanced anaerobic bacteria, said bacteria comprising a mutation in a gene associated with oxygen tolerance.

2. The genetically enhanced anaerobic bacteria of claim I wherein said anaerobic bacteria is an obligate anaerobic bacteria or a facultative anaerobic bacteria.

3. The genetically enhanced anaerobic bacteria of claim 2 wherein said obligate anaerobic bacteria is selected from the group consisting of a Clostridium species and a Bifidobacterium species.

4. The genetically enhanced anaerobic bacteria of claim 3 wherein said Clostridium species is selected from the group consisting of Clostridium perfrmgens, Clostridium histalyticum, Clostridium novyi, Clostridium telani, Clostridium acetobutylicum, and Clostridium butyricum.

5. The genetically enhanced anaerobic bacteria of claim 3 wherein said Bifidobacterium species is selected from the group consisting of Bifidobacterium bifidum.

Bifidobacterium infantis, and Bifidobacterium longum.

6. The genetically enhanced anaerobic bacteria of claim 2 wherein said facultative anaerobic bacteria is a Salmonella species.

7. The genetically enhanced anaerobic bacteria of claim 6 wherein said Salmonella species is selected from the group consisting of Salmonella typhimurium and Salmonella choleraesuis.

8. The genetically enhanced anaerobic bacteria of any one of claims 1-7 wherein said gene associated with oxygen tolerance is selected from the group consisting of a superoxide dismutase (sod) gene, a glutathione peroxidase igpo) gene, a rubrerytbrin (rbr) gene, and an alcohol dehydrogenase family member iydaD) gene.

9. The genetically enhanced anaerobic bacteria of any one of claims 1-7, further comprising an inflammation suppressive gene.

10. The genetically enhanced anaerobic bacteria of claim 9 wherein said inflammation suppressive gene is selected from the group consisting of the Staphylococcus aureus Panton- Valentine Leukoctdin (PVL) gene and the Yersinia enterocoUtica virulence factor (LcrV) gene.

11. The genetically enhanced anaerobic bacteria of any one of claims 1-7, further comprising a mutation in a toxin gene.

12. The genetically enhanced anaerobic bacteria of claim 11 wherein said toxin gene is a phospholipase toxin gene.

13. The genetically enhanced anaerobic bacteria of claim 12 wherein said pbospholipase toxin gene is the pbospholipase c {plc) toxin gene.

14. The genetically enhanced anaerobic bacteria of claim 9, further comprising a mutation in a toxin gene.

5 S. The genetically enhanced anaerobic bacteria of claim 14 wherein said toxin gene is a phospholipase toxin gene.

16. The genetically enhanced anaerobic bacteria of claim 15 wherein said phospholipase toxin gene is the phospholipase c (pic) toxin gene.

17. The genetically enhanced anaerobic bacteria of any one of claims 1-7, further comprising a gene encoding a prodrug-activating enzyme selected from the group consisting of a cytosine deaminase (CD) that converts 5-fluorocytosine to 5-fluorouracil and a nitroreductase (NTR) that activates CB 1954 to 5-Aziridinyl-4-hydroxylamino-2- nitrobenzamide.

18. The genetically enhanced anaerobic bacteria of claim 17, further comprising an inflammation suppressive gene.

19. The genetically enhanced anaerobic bacteria of claim 18, further comprising a mutation in a toxin gene.

20. The genetically enhanced anaerobic bacteria of any one oi claims ι-/, runner comprising a gene encoding a therapeutic protein selected from the group consisting of TNF- α, IL-I, IL-6, IL-IO, IL- 12, IL-15, IFN-α, IFN-γ, TRAIL, GM-CSF, FLT3-ϋgand, and E. colt colicin E3.

21. The genetically enhanced anaerobic bacteria of claim 20, further comprising an inflammation suppressive gene.

22. The genetically enhanced anaerobic bacteria of claim 21, further comprising a mutation in a toxin gene.

23. The genetically enhanced anaerobic bacteria of any one of claims 1-7, further comprising an exotoxin gene from a heterologous bacterial strain said exotoxin gene selected from the group consisting of a Clostridia Beta-toxin (CPB), a Streptococcal Streptolysin O (SLO), and a Staphylococcal Staphy lolysin A (STA).

24. The genetically enhanced anaerobic bacteria of claim 23, further comprising an inflammation suppressive gene.

25. The genetically enhanced anaerobic bacteria of claim 24, further comprising a mutation in a toxin gene.

26. A genetically enhanced anaerobic bacteria, said bacteria comprising (a) a mutation in a gene associated with oxygen tolerance, (b) an inflammation suppressive gene, and (c) a mutation in a toxin gene.

27. The genetically enhanced anaerobic bacteria of claim 26 wherein said anaerobic bacteria is Clostridium perfingens.

28. The genetically enhanced anaerobic bacteria of claim 27 wherein said oxygen tolerance gene is selected from the group consisting of a superoxide dismutase (sod) gene, a glutathione peroxidase (gpo) gene, a rubrerythrin (rbr) gene, and an alcohol dehydrogenase family member (ydaD) gene.

29. The genetically enhanced anaerobic bacteria of claim 27 wherein said inflammation suppressive gene is selected from die group consistin^g <*f tt"> Sinnhvlnmrcus aureus Panton- Valentine Leukocidin (PVL) gene and the Yersinia nee factor (LcrV) gene.

30. The genetically enhanced anaerobic bacteria of claim 27 wherein said toxin gene is a phospholipase toxin gene.

31. The genetically enhanced anaerobic bacteria of claim 27 wherein said oxygen tolerance gene is a superoxide dismutase (sod) gene, wherein said inflammation suppressive gene is the Staphylococcus aureus Panton- Valentine Leukocidin (PVL) gene, and wherein said toxin gene is the phospholipase c (pic) toxin gene.

32. The genetically enhanced anaerobic bacteria of claim 27, further comprising a gene encoding a prodrug-βctivating enzyme selected from the group consisting of a cytosine deaminase (CD) that converts 5-fluorocytosine to 5-fluorouracil and a nitroreductase (NTR) that activates CB 1954 to S-AaridinyM-hydroxylamino-2-nitrobenzamide.

33. The genetically enhanced anaerobic bacteria of claim 27, former comprising a gene encoding a therapeutic protein selected from the group consisting of TNF-α, IL-I, IL-6, IL-IO, IL- 12, IL-IS, IFN-α, IFN-γ, TRAIL, GM-CSF, FLT3-ligand, and E. cW/colicin E3.

34. The genetically enhanced anaerobic bacteria of claim 27, further comprising an exotoxin gene from a heterologous bacterial strain said exotoxin gene selected from the group consisting of a Clostridia Beta-toxin (CPB), a Streptococcal Streptolysin O (SLO), and a Staphylococcal Staphylolysin A (STA).

35. A genetically enhanced anaerobic bacteria, said bacteria comprising an inflammation suppressive gene.

36. The genetically enhanced anaerobic bacteria of claim 35 wherein said anaerobic bacteria is an obligate anaerobic bacteria or a facultative anaerobic bacteria.

37. The genetically enhanced anaerobic bacteria of claim 36 wherein said obligate anaerobic bacteria is selected from the group consisting of a Clostridium species and a Bifidobacterium species.

38. The genetically enhanced anaerobic bacteria of claim 37 wherein said

Clostridium species is selected from the group consisting of Clostridium perfrmgetis, Clostridium histofyticum, Clostridium novyt, Clostridium tetatil, Clostridium acetobutylicum, and Clostridium butyricum.

39. The genetically enhanced anaerobic bacteria of claim 37 wherein said Bifidobacterium species is selected from the group consisting of Bifidobacterium bifidum, Bifidobacterium infantis, and Bifidobacterium longμm.

40. The genetically enhanced anaerobic bacteria of claim 36 wherein said facultative anaerobic bacteria is a Salmonella species.

41. The genetically enhanced anaerobic bacteria of claim 40 wherein said Salmonella species is selected from the group consisting of Salmonella typhimurn^'an and Salmonella cholentesuis.

42. The genetically enhanced anaerobic bacteria of any one of claims 35-41 wherein said inflammation suppressive gene is selected from the group consisting of the

Staphylococcus aureus Panton-Valentine Leukocidin (PVL) gene and the Yersinia enterocolitis virulence factor (ixrV) gene.

43. The genetically enhanced anaerobic bacteria of claim 35, said bacteria further comprising a mutation is a gene associated with oxygen tolerance.

44. The genetically enhanced anaerobic bacteria of claim 43 wherein said gene associated with oxygen tolerance is selected from the group consisting of a superoxide dismutase (sod) gene, a glutathione peroxidase (gpo) gene, a rubrerymrin (rbr) gene, and an alcohol dehydrogenase family member (ydaD) gene.

45. The genetically enhanced anaerobic bacteria of claim 35 or claim 43, said bacteria further comprising a mutation in a toxin gene.

46. The genetically enhanced anaerobic bacteria of claim 45 wherein said toxin gene is a phospbolipase toxin gene.

47. The genetically enhanced anaerobic bacteria of claim 46 wherein said phospholipase toxin gene is the phospholipase c (pic) toxin gene.

48. The genetically enhanced anaerobic bacteria of claim 35, further comprising a gene encoding a prodrug-activating enzyme selected from the group consisting of a cytosme deaminase (CD) that converts 5-fluorocytosine to 5-fluoroυracil and a nitroreductase (NTR) that activates CB 1954 to 5-Aziridinyl-4^iydroxylamύio-2-nitrobenzamide.

49. The genetically enhanced anaerobic bacteria of claim 35, further comprising a gene encoding a therapeutic protein selected from the group consisting of TNF-α, IL-I , IL-6, IL-IO, IL- 12, DL-IS, IFN-α, IFN-γ, TRAIL, GM-CSF, FLT3-ligan<L and E. coll colicin £3.

50. The genetically enhanced anaerobic bacteria of claim 3S, further comprising an exotoxin gene from a heterologous bacteria) strain said exotoxin gene selected from Ae group consistg of a Clostridia Beta-toxin (CPB), a Streptococcal Streptolysin O (SLO), and a Staphylococcal Staphylolysin A (STA).

51. A method for the treatment of a solid tumor having an avascular hypoxic region in a patient, said method comprising the step of administering to said patient a genetically enhanced anaerobic bacteria comprising a mutation in a gene associated with oxygen tolerance.

52. The method of claim 51 wherein said anaerobic bacteria is an obligate anaerobic bacteria or a facultative anaerobic bacteria.

53. The method of claim 52 wherein said obligate anaerobic bacteria is selected from the group consisting of a Clostridium species and a Bifidobacterium species.

54. The method of claim S3 wherein said Clostridium species is selected from the group consisting of Clostridium perfrmgens, Clostridium histolyticum, Clostridium novyi, Clostridium tetanl, Clostridium acetobutylicum, and Clostridium buiyricum.

55. The method of claim 54 wherein said Clostridium species is administered as a spore.

56. The method of claim 53 wherein said Bifidobacterium species is selected from the group consisting of Bifidobacterium bifidum. Bifidobacterium infantis, and Bifidobacterium longum.

57. The method of claim 52 wherein said facultative anaerobic bacteria is a Salmonella species selected from the group consisting of Salmonella typhimurhtm and

Salmonella cholemesuis.

58. The method of any one of claims 51-57 wherein said gene associated with oxygen tolerance is selected from the group consisting of a superoxide dismutase (sod) gene, a glutathione peroxidase (gpo) gene, a rubrerythπn (rhr) gene, and an alcohol dehydrogenase family member (ydaD) gene.

59. The method of any one of claims 51-57 wherein said genetically enhanced anaerobic bacteria further comprises an inflammation suppressive gene.

60. The method of claim 59 wherein said inflammation suppressive gene is selected from the group consisting of the Staphylococcus aureus Panton- Valentine Leukocidin (PVlS) gene and the Yersinia enterocolitica virulence factor (IxrV) gene.

61. The method of any one of claims 51-57 wherein said genetically enhanced anaerobic bacteria further comprises a mutation in a toxin gene.

62. The method of claim 61 wherein said toxin gene is a phospholipase toxin gene.

63. The method of claim 62 wherein said phospholipase toxin gene is the phospholipase c {pic) toxin gene.

64. The method of claim 51 wherein said genetically enhanced anaerobic bacteria further comprises a gene encoding a prodrug-activating enzyme selected from the group consisting of a cytosine deaminase (CD) that converts 5-fluorocytosine to 5-fluorouracil and a nitroreductase (NTR) that activates CBl 954 to 5-Aziridinyl-44rydroxylamino-2- nitrobenzamide.

65. The method of claim 51 wherein said genetically enhanced anaerobic bacteria further comprises a gene encoding a therapeutic protein selected from the group consisting of TNF-α, IL-1, 1L-6, IL-IO, IL-12, 1L-15, IFN-α, IFN-γ, TRAIL, GM-CSF, FLT3-liganα\ and E. coli colicin E3.

66. The method of claim 51 wherein said genetically enhanced anaerobic bacteria further comprises an exotoxin gene from a heterologous bacterial strain said exotoxin gene selected from the group consisting of a Clostridia Beta-toxin (CPB), a Streptococcal Streptolysin O (SLO), and a Staphylococcal Staphy lolysin A ( STA).

67. A method for the treatment of a solid tumor having an avascular hypoxic region in a patient, said method comprising the step of administering to said patient a genetically enhanced anaerobic bacteria, said bacteria comprising (a) a mutation in a gene associated with oxygen tolerance, (b) an inflammation suppressive gene, and (c) a mutation in a toxin gene.

68. The method of claim 67 wherein said anaerobic bacteria is Clostridium perfingens.

69. The method of claim 68 wherein said oxygen tolerance gene is selected from the group consisting of a superoxide dismutase {sod) gene, a glutathione peroxidase (gpo) gene, a rubrerythrin (rbr) gene, and an alcohol dehydrogenase family member (ydaD) gene.

70. The method of claim 68 wherein said inflammation suppressive gene is selected from the group consisting of the Staphylococcus aureus Panton-Valenttne Leukocidm (PVL) gene and the Yersinia enterocolitica virulence factor (LcrV) gene.

71. The method of claim 68 wherein said toxin gene is a phospholipase toxin gene.

72. The method of claim 68 wherein said oxygen tolerance gene is a superoxide dismutase (sod) gene, wherein said inflammation suppressive gene is the Staphylococcus aureus Panton-Valentine Leukocidin (PVL) gene, and wherein said toxin gene is the phospbolipase c (pic) toxin gene.

73. The method of any one of claim 51 or claims 67-72, further comprising the step of administering to said patient a chemotberapeutic and/or radiation treatment regimen,

74. The method of claim 73 wherein the chemotherapeutic drug is selected from the group consisting of gemcitabine, fhiorouracil, docetaxol, and erlotinib.

75. The method of any one of claim Sl or claims 67-72, further comprising the step of administering to said patient an antiangiogenic agent selected from the group consisting of sorafenib, bevacizumab, sυnitinib, ANGIOCEPT™, AMG-386, cediranib, vandetimib, a thalidomide derivative, pazopanib, abegrin, cilengitide, vatalanib, volociximab, axitinib, aftibercept, and CDP-791.

76. The method of any one of claim 51 or claims 67-72 wherein said solid tumor having an avascular hypoxic region is selected from the group consisting of a pancreatic tumor, a colorectal tumor, a prostate tumor, a breast tumor, a liver tumor, a bladder tumor, a melanoma, a sarcoma, a fibrosarcoma, and a glioblastoma.

77. The method of claim 67 wherein said genetically enhanced anaerobic bacteria further comprises a gene encoding a prodrug-activating enzyme selected from the group consisting of a cytosine deaminase (CD) that converts 5-fluorocytosine to 5-fluorouracil and a nitroreductase (NTR) mat activates CB1954 to 5-Aziridinyl-4-hydroxylamino-2- nitrobenzamide.

78. The method of claim 67 wherein said genetically enhanced anaerobic bacteria further comprises a gene encoding a therapeutic protein selected from the group consisting of TNF-α, IL-1, 1L-6, IL-IO, IL-12, 1L-15, IFN-α, IFN-γ, TRAIL, GM-CSF, FLT3-ligand, and E. coli colicin E3.

79. The method of claim 67 wherein said genetically enhanced anaerobic bacteria further comprises an exotoxin gene from a heterologous bacterial strain said exotoxin gene selected from the group consisting of a Clostridia Beta-toxin (CPB), a Streptococcal Streptolysin O (SLO), and a Staphylococcal Staphylolysin A (STA).

80. A method for the treatment of a solid tumor having an avascular hypoxic region in a patient, said method comprising the step of administering to said patient a genetically enhanced anaerobic bacteria, said bacteria comprising an inflammation suppressive gene.

81. The method of chum 80 wherein said anaerobic bacteria is an obligate anaerobic bacteria or a facultative anaerobic bacteria.

82. The method of claim 81 wherein said obligate anaerobic bacteria is selected from the group consisting of a Clostridium species and a Bifidobacterium species.

83. The method of claim 82 wherein said Clostridium species is selected from the group consisting of Clostridium perftngens, Clostridium histolyilcum, Clostridium novyi, Clostridium telani, Clostridium acetobutylicum, and Clostridium butyricum.

84. The method of claim 82 wherein said Bifidobacterium species is selected from the group consisting of Bifidobacterium bifidum, Bifidobacterium infamis, and

Bifidobacterium longum.

85. The method of claim 81 wherein said facultative anaerobic bacteria is a Salmonella species.

86. The method of claim 85 wherein said Salmonella species is selected from the group consisting of Salmonella typhimurium and Salmonella choleraesuis.

87. The method of any one of claims 80-86 wherein said inflammation suppressive gene is selected from the group consisting of the Staphylococcus aureus Panton- Valentine Leukocidin (PVL) gene and the Yersinia enterocoliilca virulence factor (LcrV) gene.

88. The method of claim 80 wherein said genetically enhanced anaerobic bacteria further comprises a mutation is a gene associated with oxygen tolerance.

89. The method of claim 88 wherein said gene associated with oxygen tolerance is selected from the group consisting of a superoxide dismutase (sod) gene, a glutathione peroxidase (goo) gene, a rubrerythrin (rbr) gene, and an alcohol dehydrogenase family member (ydaD) gene.

90. The method of claim 80 or claim 88 wherein said genetically enhanced anaerobic bacteria further comprises a mutation in a toxin gene.

91. The method of claim 90 wherein said toxin gene is a phospholipase toxin gene.

92. The method of claim 91 wherein said pbospholipaβe toxin gene is the phospholipase c (pic) toxin gene.

93. The method of claim 80 wherein said genetically enhanced anaerobic bacteria further comprises a gene encoding a prodrug-activating enzyme selected from the group consisting of a cytosine deaminase (CD) that converts 5-f1uorocytosine to 5-fluorouracil and a nitroreductase (NTR) mat activates CB 1954 to 5-Aziri<ϋnyl-4-hydroxylamino-2- nhrobenzamide.

94. The method of claim 80 wherein said genetically enhanced anaerobic bacteria further comprises a gene encoding a therapeutic protein selected from die group consisting of TNF-α, IL-I, IL-6, IL-10, DL-12, IL-15, IFN-α, BFN-γ, TRAIL, GM-CSF, FLT3-ligand, and E. co// colicin E3.

95. The method of claim 80 wherein said genetically enhanced anaerobic bacteria further comprises an exotoxin gene from a heterologous bacterial strain said exotoxin gene selected from the group consisting of a Clostridia Beta-toxin (OPB), a Streptococcal Streptolysin O (SLO), and a Staphylococcal Staphylolysin A (STA).