37Salmonella strains have recently been developed as antitumor agents capable of both preferentially amplifying within tumors and expressing prodrug-converting enzymes such as the herpes simplex thymidine kinase 1 . These bacteria were attenuated by auxotrophic mutations that limited their pathogenesis in normal tissues but retained high-level replication within the tumors following systemic administration. The auxotrophic requirements of these Salmonella are apparently met within the tumor environment where they then replicate, reaching up to more than 1000 times the concentration found in normal tissues.A significant limitation for safe use of systemically administered bacteria in humans is the ability of the bacteria to induce tumor necrosis factor α (TNFα)-mediated septic shock 2,3 . However, modifications in bacterial components responsible for eliciting host immune responses such as TNFα induction could interfere with tumor targeting or antitumor activity.Several mutations in lipid biosynthesis are known in Escherichia coli and Salmonella sp. that lower TNFα induction and render the bacteria nontoxic. Some mutations, such as kdo -result in the production of lipid IV A , which substantially lowers TNFα induction and acts as an antagonist to the TNFα response from wild-type lipid A 4,5 . However, these and most other lipid mutations are temperature-sensitive and conditionally lethal to the bacteria 6 , limiting the potential for tumor-based amplification seen in auxotrophic Salmonella 1 .In E. coli, the msbB (mlt) gene 7,8 is involved in the terminal myristoylation of lipid A 9,10 . Genetic disruption of this gene in E. coli results in a stable nonconditional mutation that lowers TNFα induction up to 10-fold by whole bacteria or up to 10,000-fold by purified lipopolysaccharide (LPS) 9 . A similar toxicity profile is observed when the msbB gene is disrupted in Salmonella 11 . We generated a deletion in the coding sequence of msbB within a hyperinvasive strain of Salmonella we previously used for tumor-targeting as well as the parental wild type, and examined the effect on virulence and TNFα production both in vitro and in vivo. Results indicate that msbB -mutant Salmonella retain the properties of tumor accumulation and tumor suppression in the absence of eliciting high levels of TNFα. Results Isolation and genetic disruption of the Salmonella msbB gene.DNA sequence analysis of Salmonella msbB clones obtained by DNA/DNA hybridization indicated the presence of an msbB homolog with flanking gene organization (orfU, msbB, pykA, and zwf) identical to E. coli 8 . The DNA homology of the Salmonella msbB and the E. coli msbB was determined to be 75%, and the amino acid homology 98%, confirming that the cloned Salmonella gene is an msbB homolog.Putative knockouts obtained by transformation of the linearized deletion construct were confirmed by several criteria using Southern blot analysis (Fig. 1): Two bands corresponding to the tetracycline gene were observed in the knockout construct and in the knockout clones and w...

VNP20009 is a genetically modified strain of Salmonella typhimurium possessing an excellent safety profile, including genetically stable attenuated virulence (a deletion in the purI gene), reduction of septic shock potential (a deletion in the msbB gene), and antibiotic susceptibility. VNP20009 is genetically stable after multiple generations in vitro and in vivo. In mice, VNP20009 is rapidly cleared from the blood from a peak level of 1x104 cfu/mL to undetectable levels in 24 h. In tumor-bearing mice, VNP20009 accumulates preferentially in tumors over livers at a ratio of 1000&rcolon;1. In nonhuman primates, VNP20009 was also rapidly cleared from the blood, from a peak level of 1.0x106 cfu/mL to undetectable levels in 24 h. VNP20009 was detected in the liver, spleen, and bone marrow of monkeys; the amount decreased over time, and VNP20009 was cleared from all organs by day 41; no VNP20009 could be detected in the urine or feces of the monkeys. VNP20009 is genetically stable after many generations of growth (>140) both in vitro and in vivo.

VNP2000 9, a genetically modified strain of Salmonella typhii?Wrium with deletions in the msbB and purl loci, exhibited antitumor activities when given systemically to tumor-bearing mice. VNP2000 9 inhibited the growth of subcutaneously implanted B16F10 murine melanoma, and the human tumor xenografts Lox, DLD-1, A549, WiDr, HTB177, and MDA MB-231. A single intravenous injection of VNP20009 , at doses ranging from 1 x Hf to 3 x 106 cfulrnouse, produced tumor growth inhibitions of 57-95%. Tumor volume doubling time, another indicator for tumor growth inhibition, also significantly increased in mice treated with VNP2000 9. Using mice with immune system deficiencies, we also demon• strated that the antitumor effects of VNP20009 did not depend on the presence ofT and B cells. In addition, VNP20009 , given intravenously, inhibited the growth of lung metastases in mice. Only live bacteria showed the antitumor effect.

The study was designed to evaluate whether TAPET-CD, an attenuated strain of Salmonella typhimurium ex pressing Escherichia coli cytosine deaminase (CD), was capable of converting nontoxic 5-fluorocytosine (5-FC) to the active antitumor agent 5-fluorouracil (5-FU). The antitumor effect of TAPET-CD plus 5-FC against subcutaneously implanted colon tumors was also evaluated. TAPET-CD was given to tumor-bearing mice by a single bolus intravenous administration followed with 5-FC by intraperitoneal administration. TAPET-CD accumulated in tumors at levels 1000-fold higher than that in normal tissues and high levels of 5-FU were de growth by 88%-96%, compared to TAPET-CD alone, which inhibited tumor growth by 38%-79%. These data suggest that tumor-targeting Salmonella could be used to deliver prodrug-converting enzyme selectively to tumors and produced anti-tumor effects when the corresponding prodrug was also given. These studies demonstrate the potential use of attenuated Salmonella as a tumor-selective protein delivery vector.