Li-Mou Zheng

Use of preferentially replicating bacteria for the treatment of cancer

The past several years have seen renewed interest in the treatment of cancer with live microorganisms, based on the observation that some microorganisms display selective replication or preferential accumulation in the tumor microenvironment. Preferential replication offers great potential to amplify the therapeutic effect of the microorganism while sparing normal tissues from toxicity. Much of the current research intended to achieve selective replication within, and lysis of, tumor cells has focused on viruses, but recent observations in murine models with facultative anaerobic bacteria (1), as well as data generated more than 30 years ago with obligate anaerobic bacteria (2), indicate that some bacterial species can also preferentially replicate and accumulate within tumors. In contrast to viruses, the bacteria reside primarily in the extracellular tumor microenvironment (3) and possess certain features that may be advantageous in the treatment of cancer. Thus, bacteria are motile, which facilitates their spread throughout the tumor and can help target systemic disease. Because of their large genome size, bacteria can readily express multiple therapeutic transgenes, such as cytokines or pro–drug-converting enzymes, and their spread can be controlled with antibiotics if necessary.

Antitumor effect of VNP20009, an attenuated Salmonella, in murine tumor models.

VNP20009, a genetically modified strain of Salmonella typhimurium with deletions in the msbB and purI loci, exhibited antitumor activities when given systemically to tumor-bearing mice. VNP20009 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 10(4) to 3 x 10(6) cfu/mouse, produced tumor growth inhibitions of 57-95%. Tumor volume doubling time, another indicator for tumor growth inhibition, also significantly increased in mice treated with VNP20009. Using mice with immune system deficiencies, we also demonstrated that the antitumor effects of VNP20009 did not depend on the presence of T and B cells. In addition, VNP20009, given intravenously, inhibited the growth of lung metastases in mice. Only live bacteria showed the antitumor effect.

Tumor amplified protein expression therapy : Salmonella as a tumor-selective protein delivery vector

Attenuated strains of Salmonella typhimurium, VNP20009 and YS7212, when injected systemically to tumor-bearing mice, accumulated preferentially in tumors at levels at least 200-fold and, more commonly, 1000-fold greater than in other normal tissues. This selectivity occurred in subcutaneously implanted murine tumors, including B16F10 melanoma, M27 lung carcinoma, and colon 38 carcinoma. The preferential accumulation was also manifested in animals bearing human tumor xenografts, including Lox, C8186, DLD1, SW620, HCT116, HTB177, DU145, MDA-MB-231, and Caki. Four to five days after a single IV injection of 1 x 10(6) colony-forming unit (cfu)/mouse, we routinely detected VNP20009 proliferation and accumulation at levels ranging from 1 x 10(8) to 2 x 10(9) cfu/g tumor. The amount of VNP20009 accumulated in the liver ranged from 3 x 10(4) to 2 x 10(6) cfu/g. The distribution of Salmonella in tumors was homogenous; YS7212 could be detected from the periphery to the interior portion of the tumors. Using mice with various immunodeficiencies, we also discovered the same preferential accumulation of Salmonella in tumors implanted in these mice. The use of Salmonella as a protein delivery vector was shown by IV administration of the bacteria expressing either green fluorescent protein (GFP) or cytosine deaminase (CD) into tumor-bearing mice. GFP and CD were detected in tumors, but not in livers, taken from mice inoculated with Salmonella carrying these genes. Bacteria accumulation and CD expression persisted in the tumors for up to 14 days after a single bolus IV administration of bacteria to tumor-bearing mice.

Salmonella-based tumor-targeted cancer therapy: tumor amplified protein expression therapy (TAPET™) for diagnostic imaging

In preclinical studies, genetically engineered Salmonella have the ability to localize, selectively accumulate, and persist within transplantable murine tumors, spontaneous murine tumors and human tumor xenographs, and can express therapeutic proteins at high levels. These strains of engineered non-virulent Salmonella typhimurium display the capacity to accumulate and grow selectively in a variety of tumor types and to inhibit the growth of primary and metastatic tumors following intravenous injection into tumor-bearing mice. One strain of the bacteria (VNP20009) which has endogenous antitumor activity is currently in Phase I clinical trials. The bacteria are highly attenuated and genetically stable. The combination of the lipid mutation and the purine auxotrophy attenuate the virulence of the bacteria by greater than 10000-fold and enhance the specificity of the bacteria for tumor tissue. These bacteria have been found to be safe in mice, pigs and monkeys when administered intravenously. Second-generation Salmonella vectors will be developed to include transgenes that will express therapeutic agents and reporter transgenes for non-invasive imaging. We have performed a preliminary study to demonstrate localization of [(14)C]FIAU in tumored mice pretreated with Salmonella expressing HSV1-TK. The [(14)C]FIAU radioactivity and bacterial count data strongly support a Salmonella(TK)-dependent [(14)C]FIAU accumulation of at least 30-fold higher in tumor tissue compared to muscle tissue. These data warrant further investigation on the use of genetically engineered Salmonella as a systemically administered tumor-specific agents for tumor therapy and delivery of diagnostic imaging markers.

Comparative evaluation of the acute toxic effects in monkeys, pigs and mice of a genetically engineered Salmonella strain (VNP20009) being developed as an antitumor agent

The objective of these studies was to perform a comparative evaluation of the acute toxicity of VNP20009, a genetically engineered Salmonella strain, in monkeys, pigs, and mice. It is hypothesized that mice would be more susceptible than other animal species to the toxic effects of VNP20009, because mice are the most sensitive natural host for the parental wild-type Salmonella typhimurium strain. These studies also compared the virulence of VNP20009 and the parental Salmonella in mice. In Cynomolgus monkeys and Yorkshire pigs (n = 2/dose), various doses (expressed as colony forming units [cfu] per animal) of VNP20009, or vehicle, were administered as a single IV injection (∼ 1 ml/min). The body weight, body temperature, clinical signs, clinical pathology (serum chemistry and hematology), and ophthalmic examinations (only in monkeys) were evaluated at various times. Necropsy was performed on day 15 in the pigs, and necropsy and histopathology on days 8 or 15 in the monkeys. In C57BL/6 mice (n = 10/dose), various doses of VNP20009, or the parental Salmonella, were administered as a single IV bolus injection. The mice were observed daily over 3 weeks. The results from monkeys showed that VNP20009-related changes in clinical pathology were primarily confined to fiver enzymes and fiver function tests (i.e., cholesterol, triglyceride, alanine aminotransferase, and aspartate aminotransferase levels). Significant toxicological changes occurred only at the dose of 1 × 1010 cfu/monkey, but not at the doses of 1 × 108 or 3 × 109 cfu/monkey. Gross necropsy and histology findings were primarily confined to the spleen (enlargement, weight increase, and reticuloendothelial hyperplasia), thymus (size and weight reduction and lymphoid depletion), mesenteric lymph node (enlargement), and lung (weight increase). Most of these necropsy and microscopic findings, which occurred mostly in the high-dose group, may be related to the physiological responses to infection, rather than related to the intrinsic toxicity of VNP20009. The results from pigs showed that VNP20009 induced toxicological effects only at the dose of 3 × 109 cfu/pig, but not at the doses of 3 × 108 or 3 × 1010 cfu/pig. Both pigs treated with 3 × 1010 cfu/pig died within the first 2 days post-treatment. Necropsy showed the presence of abdominal transudate fluid, skin blotching, and pulmonary-and gall bladder-associated edema. Therefore, the pig mortality may have been related to the physical damage induced by the sudden systemic presence of large amounts of suspension. The results from mice showed that VNP20009, at doses as high as 1 × 106 cfu/mouse, did not induce any mortality. A 30% mortality rate was induced by 3 × 106 cfu/mouse, and 100% mortality by 1 × 107 cfu/mouse. The parental Salmonella, at a dose of 1 × 102 or 3 × 102 cfu/mouse, induced a 100% mortality. In conclusion, the doses of VNP20009 that induced acute toxicity are very high, suggesting that VNP20009 may be a safe agent. The virulence is 50,000 × less in VNP20009 than the parental Salmonella.

Evaluation of the acute and subchronic toxic effects in mice, rats, and monkeys of the genetically engineered and Escherichia coli cytosine deaminase gene-incorporated Salmonella strain, TAPET-CD, being developed as an antitumor agent.

TAPET-CD, a genetically engineered Salmonella strain with chromosomal-incorporated cytosine deaminase (CD) gene, has been shown to selectively accumulate tumors, suppress tumor growth, and convert 5-fluorocytosine (5-FC, an antifungal agent) to the antitumor agent 5-fluorouracil (5-FU) in animals. The current studies investigated the safety of TAPET-CD, and TAPET-CD/5-FC combination, in animals. In C57BL/6 mice (n = 10 females/dose), the maximum nonlethal dose of TAPET-CD (intravenous [IV] bolus) was 1 x 10(6) colony-forming units (cfu)/mouse, or > 10,000 x that of wild-type Salmonella. In Sprague-Dawley rats (n = 4/sex/group), after treatment with 4 weekly cycles of TAPET-CD (an IV injection/cycle at 1 x 10(5), 3 x 10(5), 1 x 10(6), 3 x 10(6), or 1 x 10(7) cfu/rat on day 1) and 5-FC (per os twice daily [PO b.i.d.], 250 mg/kg on days 2-7/cycle), clinical signs and mortality were evaluated daily, body weight and clinical pathology weekly, and gross necropsy on day 29. No treatment-related toxicity, although occasional and mild clinical signs (e.g., dehydration), increased hepatic enzyme/function values and white blood cells, splenic enlargement, and bilateral red discoloration of the kidneys, were observed. In cynolmogus monkeys, Experiment 1 involved treatment with TAPET-CD (IV injection at 1 x 10(9) cfu/monkey). Clinical signs and mortality were evaluated daily, body weight weekly, and gross necropsy on days 2, 7, and 31 (1/sex/time point). Experiment 2 involved treatment with TAPET-CD (IV injection at 1 x 10(9) and 1 x 10(10) cfu/monkey in Groups 1 to 3 and Groups 4 to 6, respectively) on day 1 and 5-FC (PO b.i.d. at 250, 500, and 1000 mg/kg in Groups 1 to 3, and 500, 1500, and 0 mg/kg in Groups 4 to 6, respectively) on days 4 to 17 (n = 1/sex/group). Clinical signs and mortality were evaluated daily; body weight and clinical pathology on days 1, 2, 4, 14, and 18; body temperature on days 1, 4, and 18; ophthalmic examinations on days 3 and 17; and gross necropsy and histopathology on day 18. Experiment 1 indicated that TAPET-CD at 1 x 10(9) or 1 x 10(10) cfu/monkey was well tolerated, with only occasional mild clinical signs (i.e., emesis, vomiting, inappetance, loose/infrequent/absence of stool), increases in hepatic enzyme/function values, and splenic enlargement. Experiment 2 indicated that TAPET-CD/5-FC combination had a maximum tolerated dose (MTD) of 1 x 10(10) cfu/monkey for TAPET-CD and 500 mg/kg for 5-FC in monkeys. Supra-MTDs induced renal toxicity. In conclusion, TAPET-CD had a good safety profile (reflected by the extremely large amount of TAPET-CD needed to induce mortality or toxicity) in mice, rats, and monkeys. More adverse events were observed with TAPET-CD/5-FC combination when compared to TAPET-CD and these events were similar to the reported effects of 5-FU, suggesting the involvement of 5-FU.

Tumour-Selective Salmonella-Based Cancer Therapy

Cancer therapies fail for several primary reasons; lack ofdrug effect on the cancerous tissue~ lack of selectivity for the cancerous tissue, andlor inadequate delivery to the target tissue. Drug effect and selectivity can be improved by increased understanding of molecular and cellular differences between cancer and normal tissues, thus enabling the design of drugs that potently affect cancer-specific molecular targets associated with malignant behaviour. Another approach is to improve the selective delivery ofanti-cancer agents to tumours. One approach is to use carriers that bind to cancer...specific targets, such as antibodies (Hall, 1995). However, most targeting approaches, even if selective, tend not to deliver sufficiently high concentrations of the agent to the tumour to induce significant therapeutic effects. Recent findings suggest that the pathogenic bacterium Sallnonella, when genetically modified, can be used to selectively deliver therapeutic agents to solid tumours at high concentrations (Pawelek et ai., 1997; Low et ai., 1999a). These attenuated bacteria are administered either systemically or locally, whereupon they typically replicate 1000 times greater in the tumour than in other tissue. The basis for preferential colonization and accumulation of Salmonella in tumours appears to include some of the same characteristics of tumours that provide resistance to drug and immune-based therapies (Bermudes et aI., 2000a,b). Why tumours are susceptible to Sabnonella is not well understood and probably includes a variety of factors. Poor penetration of components of the immune system, including antibodies, complement, CD8+ T-cells, granulocytes and macrophages