Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma.

PURPOSE: A strain of Salmonella typhimurium (VNP20009), attenuated by chromosomal deletion of the purI and msbB genes, was found to target to tumor and inhibit tumor growth in mice. These findings led to the present phase I study of the intravenous infusion of VNP20009 to patients with metastatic cancer. PATIENTS AND METHODS: In cohorts consisting of three to six patients, 24 patients with metastatic melanoma and one patient with metastatic renal cell carcinoma received 30-minute intravenous bolus infusions containing 10(6) to 10(9) cfu/m(2) of VNP20009. Patients were evaluated for dose-related toxicities, selective replication within tumors, and antitumor effects. RESULTS: The maximum-tolerated dose was 3 x 10(8) cfu/m(2). Dose-limiting toxicity was observed in patients receiving 1 x 10(9) cfu/m(2), which included thrombocytopenia, anemia, persistent bacteremia, hyperbilirubinemia, diarrhea, vomiting, nausea, elevated alkaline phosphatase, and hypophosphatemia. VNP20009 induced a dose-related increase in the circulation of proinflammatory cytokines, such as interleukin (IL)-1beta, tumor necrosis factor alpha, IL-6, and IL-12. Focal tumor colonization was observed in two patients receiving 1 x 10(9) cfu/m(2) and in one patient receiving 3 x 10(8) cfu/m(2). None of the patients experienced objective tumor regression, including those patients with colonized tumors. CONCLUSION: The VNP20009 strain of Salmonella typhimurium can be safely administered to patients, and at the highest tolerated dose, some tumor colonization was observed. No antitumor effects were seen, and additional studies are required to reduce dose-related toxicity and improve tumor localization.     

Salmonella-mediated tumor-targeting TRAIL gene therapy significantly suppresses melanoma growth in mouse model

Attenuated Salmonella typhimurium (S. typhimurium) strains can selectively grow and express exogenous genes in tumors for targeted therapy. We engineered S. typhimurium strain VNP20009 to secrete tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) under the control of a hypoxia-induced nirB promoter and examined the efficacy of Salmonella-mediated targeted expression of TRAIL in mice bearing melanoma tumor and in TRAIL-resistant RM-1 tumor. We found that VNP preferentially accumulated in tumor tissues and the nirB promoter effectively drove targeted expression of TRAIL. Compared with recombinant TRAIL protein and VNP20009 combination therapy, VNP20009 expressing TRAIL significantly suppressed melanoma growth but failed to suppress RM-1 tumor growth. Furthermore, we confirmed that VNP20009 expressing TRAIL yielded its antitumor effect by inducing melanoma apoptosis. Our findings indicate that Salmonella-mediated tumor-targeted therapy with TRAIL could reduce tumor growth and extend host survival.     

Systemic administration of an attenuated, tumor-targeting Salmonella typhimurium to dogs with spontaneous neoplasia: phase I evaluation.

PURPOSE: Genetically modified bacteria are a potentially powerful anticancer therapy due to their tumor targeting capacity, inherent antitumor activity, and ability to serve as efficient vectors for gene delivery. This study sought to characterize the acute and short-term toxicities and tumor colonization rates of a genetically modified Salmonella typhimurium (VNP20009) in dogs with spontaneous tumors, in the context of a phase I dose escalation trial. EXPERIMENTAL DESIGN: Forty-one pet dogs with a variety of malignant tumors received weekly or biweekly i.v. infusions of VNP20009, at doses ranging from 1.5 x 10(5) to 1 x 10(8) cfu/kg. Vital signs and clinicopathologic variables were monitored regularly. Incisional biopsies were obtained before and 1 week following the first infusion for histopathology and bacterial culture. RESULTS: The nominal maximum tolerated dose was 3 x 10(7) cfu/kg, with refractory fever and vomiting being the dose-limiting toxicities. One treatment-related acute death occurred. Bacteria were cultured from tumor tissue in 42% of cases. Thirty-five patients were evaluable for antitumor response. Major antitumor responses were seen in 15% (4 complete response and 2 partial response), and disease stabilization for at least 6 weeks in 10%. CONCLUSIONS: Administration of VNP20009 at doses with acceptable toxicity results in detectable bacterial colonization of tumor tissue and significant antitumor activity in tumor-bearing dogs.     

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.     

Oral delivery of tumor-targeting Salmonella for cancer therapy in murine tumor models

Tumor-targeting bacteria have been investigated intensively in recent years as anticancer agents. To ensure the reliability of infection, bacteria have conventionally been injected intravenously or intraperitoneally into animals or humans. However, systemic infection of bacteria is rather inconvenient and carries the risk of obvious toxicity. Here we tested whether Salmonella typhimurium VNP20009, a tumor-targeting strain, could be administrated orally for tumor therapy. Tumor-targeting potential, antitumor effects, as well as toxicity of orally administrated VNP20009 were investigated in this study. Oral delivery of VNP20009 not only exhibited high tumor-targeting potential, but also led to a significant anticancer effect by delaying tumor growth and prolonging survival in murine tumor models. As part of combination therapy, orally administrated bacteria notably improved the antitumor effect of cyclophosphamide. In vitro and in vivo studies showed that VNP20009 significantly induced tumor cell apoptosis. No obvious toxicity was observed during the treatments with oral inoculation of VNP20009. Comparative analysis of toxicity in tumor-bearing and tumor-free mice further revealed that orally administrated Salmonella had high safety compared to conventional systemic infection of bacteria. The findings indicated that oral administration of tumor-targeting bacteria is effective and safe. This approach provides a novel avenue in the application of bacteria as a potential antitumor agent.     

Enhanced therapeutic effect by combination of tumor-targeting Salmonella and endostatin in murine melanoma model

The growth of tumor is angiogenesis-dependent and it often contains hypoxia and necrotic areas. Salmonella VNP20009 could target and replicate in hypoxia and necrotic areas within tumor and induce antitumor effect. Angiogenesis inhibitor endostatin could reduce tumor angiogenesis and inhibit its growth. However, in the phase I trials of VNP20009 and endostatin at the maximum-tolerated dose, no antitumor effects for bacteria therapy and minor therapeutic effects for endostatin treatment were seen. The ineffectiveness of these agents in clinical trials suggests that the combination of these agents with synergic modalities might be necessary. Here we described antitumor effects mediated by the combination of VNP20009 with recombinant human endostatin in B16F10 murine melanoma model with the aim to exploit tumor-targeting of bacteria and anti-angiogenesis strategy to enhance therapeutic efficacy. Combination therapy of these agents significantly enhanced antitumor effects by inducing greater tumor growth inhibition, more severe tumor tissue necrosis as well as less blood vessel density than those induced by either of treatments. The findings suggest that the combination of tumor-targeting bacteria with angiogenesis inhibitor might be of value for the treatment of solid tumors.     

Comparison of the selective targeting efficacy of Salmonella typhimurium A1-R and VNP20009 on the Lewis lung carcinoma in nude mice

Salmonella typhimurium A1-R is auxotrophic for arg and leu, which attenuates growth in normal tissue but allows high tumor targeting and virulence. A1-R is effective against metastatic human prostate, breast, and pancreatic cancer as well as osteosarcoma, fibrosarcoma, and glioma in clinically-relevant mouse models. VNP20009 is also a genetically-modified strain of Salmonella typhimurium that has been tested in Phase I clinical trials, but is more attenuated than S. typhimurium A1-R and in addition of multiple amino-acid auxotrophs, is purine auxotropic with the purI mutation. In the present study, mouse Lewis lung carcinoma-bearing nude mouse models were treated with S. typhimurium A1-R or VNP20009. S. typhimurium A1-R and VNP20009 were both eliminated from the liver and spleen approximately 3-5 days after administration via the tail vein. However, A1-R showed higher tumor targeting and inhibited the Lewis lung carcinoma to a greater extent than VNP20009, with less body weight loss. The mice tolerated S. typhimurium A1-R to at a least 2-fold higher dose than VNP20009 when the bacteria were administered iv. The results of the present study suggest that S. typhimurium A1-R has greater clinical potential than VNP20009.     

Oral delivery of tumor-targeting Salmonella exhibits promising therapeutic efficacy and low toxicity

Tumor‐targeting bacteria have been developed as powerful anticancer agents. Salmonella typhimurium VNP20009, a representative tumor‐targeting strain, has been systemically administered as a single‐agent therapy at doses of 1 × 10⁶ to 3 × 10⁶ colony‐forming unit (cfu)/mouse, or in combination with other antitumor agents at doses of 1 × 10⁴ to 2 × 10⁵ cfu/mouse. Recently, we reported that oral delivery of VNP20009 at the dose of 1 × 10⁹ cfu/mouse induced significant anticancer effects comparable to that induced by systemic administration of this strain at 1 × 10⁴ cfu/mouse. To further address the efficacy and safety of oral administration of bacteria, here we performed a systemically comparative analysis of anticancer efficacy and toxicity of VNP20009 administered: (i) orally at a dose of 1 × 10⁹ cfu/mouse (VNP9‐oral); (ii) intraperitoneally at a dose of 1 × 10⁴ cfu/mouse (VNP4‐i.p.); or (iii) intraperitoneally at a dose of 1 × 10⁶ cfu/mouse in tumor‐free and tumor‐bearing murine models. The results showed that VNP9‐oral, similar to VNP4‐i.p., induced significant tumor growth inhibition whereas VNP6‐i.p. induced better anticancer effect in the B16F10 melanoma model. Among three treatments, VNP9‐oral induced the mildest and reversible toxicity whereas VNP6‐i.p. resulted in the most serious and irreversible toxicities when compared to other two treatments. Moreover, the combination of VNP9‐oral with a low dose of chemotherapeutics produced comparable antitumor effects but displayed significantly reduced toxicity when compared to VNP6‐i.p. The findings demonstrated that oral administration, as a novel avenue in the application of bacteria, is highly safe and effective. Moreover, the present preclinical study should facilitate the optimization of bacterial therapies with improved anticancer efficacy and reduced adverse effects in future clinical trials. (Cancer Sci 2009; 100: 2437–2443)     

重组沙门氏菌VNP20009-M对骨肉瘤的治疗作用

目的 研究携带甲硫氨酸酶基因的减毒沙门氏菌VNP20009-M对骨肉瘤的治疗作用及其机制。方法 将重组沙门氏菌VNP20009-M与骨肉瘤细胞MNNG-HOS共培养;骨肉瘤细胞MNNGHOS、U2OS及SaoS-2均高表达甲硫氨酸酶基因后探索细胞增殖、迁移及凋亡能力的变化;构建MNNG-HOS裸鼠皮下荷瘤模型,评价不同剂量的VNP20009-M在动物模型中的治疗效果。结果 通过质粒PCR验证甲硫氨酸酶基因只存在于目的菌株VNP20009-M中,且具有较高的甲硫氨酸酶活性,重组沙门氏菌构建成功。VNP20009-M显著诱导骨肉瘤细胞MNNG-HOS的凋亡。相比对照组,甲硫氨酸酶基因过表达的骨肉瘤细胞增殖、迁移能力被显著抑制。VNP20009-M治疗后小鼠皮下肿瘤生长被显著抑制。结论 VNP20009-M可诱导骨肉瘤细胞凋亡并抑制癌细胞增殖和迁移,可以作为一种新型高效的药物为临床提供新的治疗方式。     

A Salmonella Typhimurium mutant strain capable of RNAi delivery: Higher tumor-targeting and lower toxicity

Bacteria are highly versatile and useful tools that could deliver short interfering RNA. In this study, a phoP/phoQ double-deleted Salmonella Typhimurium named VNP(PhoP/Q⁻) based on the genetic background of VNP20009. The biological safety and function of VNP(PhoP/Q⁻) were also analyzed. Our study revealed the following results: (1) VNP(PhoP/Q⁻) exhibited lower titers in tumor-free livers and spleens than VNP20009, (2) The survival of VNP(PhoP/Q⁻) in macrophages and 4T1 tumor cells was significantly reduced compared with that of VNP20009, (3) The tumor-targeting ability of VNP(PhoP/Q⁻) was significantly enhanced compared with that of VNP20009, and the anticancer effects of VNP(pPhoP/Q⁻) and VNP20009 on tumor-bearing mice were similar, (4) VNP(PhoP/Q⁻) could release an shRNA-expressing plasmid and express the EGFP reporter gene in tumor tissue. Therefore, VNP(PhoP/Q⁻) exhibited a better safety level in tumor-free mice and elicited an anti-tumor effect on tumor-bearing mice. Moreover, VNP(PhoP/Q⁻) could release an shRNA-expressing plasmid into the cytoplasm of host cells to silence targeted genes.     

Tumor-specifically hypoxia-induced therapy of SPRY1/2 displayed differential therapeutic efficacy for melanoma

Activation of receptor tyrosine kinase (RTK) signalling pathways is frequently correlated to cancer cell proliferation, angiogenesis and cell survival. Sprouty (SPRY) proteins function as a physiological endogenous inhibitor of RTK signalling pathways, have been shown to be deregulated in most cancer cells. Here, we demonstrated that over-expression of SPRY1 and SPRY2 inhibited B16F10 cell proliferation through G1 phase arrest in vitro, and SPRY2 showed more potent inhibitory effects than SPRY1. In order to tumor-specific delivery of SPRY1/2 in vivo, two strains of attenuated Salmonella typhimurium VNP20009 (VNP-PQE-SPRY1 and VNP-PQE-SPRY2) were constructed to specifically express SPRY1 or SPRY2 under the control of a hypoxia-induced nirB promoter. The efficiency and specificity of the recombinant strains were validated in both bacteria and animal tumor models. SPRY1 and SPRY2 gene could be specifically driven by the nirB promoter under hypoxia, but not normoxia conditions. In addition, the tumor-targeting ability of VNP-PQE-SPRY1 or VNP-PQE-SPRY2 was similar with VNP. VNP-PQE-SPRY2 significantly suppressed melanoma growth in vivo, suggesting that SPRY2 is a more efficient agent for melanoma therapy. Moreover, the antitumor effect of VNP-SPRY2 is mainly mediated through the inhibition of ERK1/2 phosphorylation, which leads to the inhibition of proliferation in melanoma. Taken together, our results indicated that SPRY2 displayed more potent melanoma suppression than SPRY1 both in vitro and in vivo, and the hypoxia-induced tumor-specific gene therapy of SPRY2 delivered by VNP20009 is a promising strategy for melanoma therapy.     

Suppression of pancreatic ductal adenocarcinoma growth by intratumoral delivery of attenuated Salmonella typhimurium using a dual fluorescent live tracking system

Pancreatic ductal adenocarcinoma (PDAC) has the poorest prognosis among all malignancies and is resistant to almost all current therapies. Attenuated Salmonella typhimurium strain VNP20009 has been deployed as powerful anticancer agent in a variety of animal cancer models, and previous phase 1 clinical trials have proven its safety profiles. However, thus far, little is known about its effect on PDAC. Here, we established CFPAC-1 cell lines expressing an mKate2 protein and thus emitting far-red fluorescence in the subsequent xenograft implant. VNP20009 strain was further engineered to carry a luciferase cDNA, which catalyzes the light-emitting reaction to allow the observation of salmonella distribution and accumulation within tumor with live imaging. Using such VNP20009 strain and intratumoral delivery, we could reduce the growth of pancreatic cancer by inducing apoptosis and severe necrosis in a dosage dependent manner. Consistent with this finding, intratumoral delivery of VNP20009 also increase caspase-3 activity and the expression of Bax protein. In summary, we revealed that VNP20009 is a promising bacterial agent for the treatment of PDAC, and that we have established a dual fluorescent imaging system as a valuable tool for noninvasive live imaging of solid tumor and engineered bacterial drug.     

Synergistic effects of arsenic trioxide combined with Salmonella typhimurium in treating the advanced hepatocellular carcinoma in rat models

BackgroundTo evaluate the safety and efficacy of arsenic trioxide (ATO) combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 in treating the advanced hepatocellular carcinoma (HCC).MethodsThe proliferation assay, migration assay and real-time PCR analyses were performed to assess the impact of ATO combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 on the McA-RH7777 cells. Forty Buffalo rats were orthotopically implanted with HCC in the livers and randomly divided into four groups: (A) ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009; (B) ATO; (C) {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009; and (D) control. ATO (2 mg/kg) was administered by peritoneal injection once a day and continued for five days. {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 (about 1×10⁷ CFU) was directly injected into the tail vein. MRI examinations were performed to access the tumor responses one and 2 weeks later, respectively. Micro CT scans of chest were performed to assess the lung metastases. Hematoxylin-eosin (HE) staining and immunohistochemical analyses were performed to analyze the tumor tissues.ResultsIn the in vitro experiments, {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 suppressed the proliferation of McA-RH7777 cells, attenuated their migration ability, and weakened the potential of metastases. MRI examinations showed that the mean residual tumor volumes of ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group on the 7th day and 14th day after the administration of ATO combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 were significantly smaller than those of other groups. Micro CT scans revealed that the lung metastases rates of ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group and {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group were significantly lower than those of other groups. Immunohistochemical analyses displayed that the levels of VEGF and Vimentin in the tumors of ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group were obviously lower than those of other groups. The median survival of rats in the ATO plus {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 group was longer than those of other groups.ConclusionsThe strategy of ATO combined with {"type":"entrez-protein","attrs":{"text":"VNP20009","term_id":"1666609276","term_text":"VNP20009"}}VNP20009 was safe and had a potential to inhibit tumor growth, decrease the lung metastases, and prolong the overall survival in treating the advanced HCC. The two complementary interventions may have synergistic effects.     

Tumor-targeting Salmonella typhimurium improves cyclophosphamide chemotherapy at maximum tolerated dose and low-dose metronomic regimens in a murine melanoma model

Chemotherapy for cancer is partly limited by the inability of drugs to act on poorly vascularized or avascularized areas of tumors. Tumor-targeting bacteria are capable of preferentially replicating in these poorly perfused regions. Some strains have been combined with chemotherapeutic agents and the results have been promising. However, no systematic work has been carried out to test the effect of bacteria on clinical modes of chemotherapy, such as standard maximum tolerated dose (MTD) and novel low-dose metronomic (LDM) chemotherapy. Here Salmonella typhimurium VNP20009 was combined with cyclophosphamide (CTX) at both MTD and LDM schedules in a murine melanoma model. The results showed that VNP20009 significantly improved the effects of all forms of CTX treatments. The combination of VNP20009 and CTX led to a more significant decrease in tumor microvessel density and serum vascular endothelial growth factor (VEGF) level, compared with either treatment alone. Furthermore, combination therapy remarkably increased the number of bacteria within tumors when compared with bacteria treatment alone. These findings suggest that tumor-targeting bacteria, in conjunction with CTX at standard MTD and LDM regimens, might be of clinical value for the treatment of melanoma.     

Antitumor effect of Cepharanthin in the double grafted tumor system

The antitumor effect of Cepharanthin (CR) in a new experimental mouse model was studied. Intratumoral administration of CR strongly inhibited the growth of Meth-A solid tumors in male BALB/c mice and led to a complete regression of tumors and resistance to reinoculated tumor. Subsequently, the antimetastatic effect of CR was examined in the double grafted tumor system, in which mice first received simultaneous intradermal inoculations of Meth-A in both right (10(6) cells) and left (2 x 10(5) cells) flanks and were then injected with 0.5 mg of CR in the right tumor on days 3, 4 and 5. CR inhibited the growth of not only the right but also the left, nontreated tumor. Immunized spleen cells were taken from mice which had been cured with the intratumoral administration of CR. Adoptive transfer of CR immunized spleen cells caused the complete regression of Meth-A tumors. The effector cell activity was lost only after treatment with anti-Lyt-1 antibody. These results suggest that intratumoral administration of CR might induce Lyt-1 positive cytotoxic cells in the spleen and the left, non-treated tumor. In BALB/c nude mice, CR inhibited the growth of the right tumor but did not the left tumor. Therefore, the antitumor activity of CR on the left tumor in the double grafted tumor system is associated with a sequential immune mechanism in which T cells may play an important role. The antitumor effect of CR on the right tumor is direct cytotoxic effect. TILs (tumor infiltrating lymphocytes) obtained from left and right sides tumors treated with CR were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice. TILs from both sides clearly inhibited the growth of admixed Meth-A cells and seems to play an important role on antitumor effect in both tumors.     

Strain differences in the antitumor activity of an immunopotentiator, Nocardia rubra cell-wall skeleton, in B10 congenic and recombinant mice

An immunogenetic evaluation of the antitumor activity of the immunopotentiator Nocardia rubra cell-wall skeleton (N. rubra-CWS) has been performed using non-virus-producing tumors induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV) in B10 congenic and recombinant mice. Live tumor cells mixed with either N. rubra-CWS or a placebo control were inoculated intradermally into the right flank of syngeneic mice. With N. rubra-CWS, the development and growth of B10 mouse tumors, S1018(B10) and B10SA2F, in B10(H-2b) mice were completely inhibited in all test mice. B10.A(5R) mouse tumor, S322(5R), was completely suppressed in 23 of 25 B10.A(5R)(H-2i5) mice, and B10.BR mouse tumor, S623(BR), was completely suppressed in 7 of 14 B10.BR(H-2k) mice. However, B10.D2 mouse tumor, S908(D2), in B10.D2(H-2d) mice and B10.A mouse tumors, S826(BA) and B10ABr1F, in B10.A(H-2a) mice were not suppressed at all by N. rubra-CWS. N. rubra-CWS showed a remarkable antitumor effect in B10 mice, not only in mixed inoculation with the tumor cells but also with intratumoral administration. These effects were not seen in B10.A mice. Peritoneal exudate cells (PEC) from B10 and B10.A strains previously treated intraperitoneally with N. rubra-CWS and/or MMC-treated tumor cells showed cytolytic and cytostatic activities on tumor cells derived from both strains. These results did not reflect the strain difference seen in the in vivo studies. Mitogenic activity of N. rubra-CWS on spleen cells, however, was significantly different among B10 congenic mice; N. rubra-CWS induced greater blastogenesis of spleen cells from B10 mice than of those from B10.A mice, corresponding to the in vivo results showing strain difference. These results suggest that the host's immunogenetic background may play an important role in cancer therapy with immunopotentiators.     

Attenuated Salmonella targets prodrug activating enzyme carboxypeptidase G2 to mouse melanoma and human breast and colon carcinomas for effective suicide gene therapy

PURPOSE: We engineered the oncolytic Salmonella typhimurium-derived bacterium VNP20009 as a vector to target delivery to tumors of the prodrug-activating enzyme carboxypeptidase G2 (CPG2) and to show enhanced antitumor efficacy on administration of different prodrugs. EXPERIMENTAL DESIGN: We characterized CPG2 expression in vectors by immunoblotting, immunofluorescence, and enzyme activity. We assessed prodrug activation by high-performance liquid chromatography. Target human tumor cell and bacterial vector cell cytotoxicity was measured by flow cytometry and colony-forming assays. Therapy was shown in two human tumor xenografts and one mouse allograft with postmortem analysis of bacterial and CPG2 concentration in the tumors. RESULTS: CPG2 is expressed within the bacterial periplasm. It activates prodrugs and induces cytotoxicity in human tumor cells but not in host bacteria. Following systemic administration, bacteria multiply within xenografts reaching 2 x 10(7)/g to 2 x 10(8)/g at 40 days postinoculation. The concentration of CPG2 in these tumors increases steadily to therapeutic levels of 1 to 6 units/g. The bacteria alone reduce the growth of the tumors. Subsequent administration of prodrugs further reduces significantly the growth of the xenografts. CONCLUSIONS: The bacteria multiply within tumors, resulting in a selective expression of CPG2. The CPG2-expressing bacteria alone reduce the growth of tumors. However, in the presence of prodrugs activated by CPG2, this oncolytic effect is greatly increased. We conclude that bacterial oncolytic therapy, combined with CPG2-mediated prodrug activation, has great potential in the treatment of a range of cancers.     

Antitumor effect of PSK at a distant site: tumor-specific immunity and combination with other chemotherapeutic agents.

The antitumor effect of PSK, a Coriolus preparation, was analyzed with the double grafted tumor system in which BALB/c mice received intradermal inoculations of syngeneic Meth-A fibrosarcoma in the right (primary tumor, 10(6) cells) and left (distant tumor, 2 x 10(5) cells) flanks. Intratumoral administration of PSK significantly inhibited the growth of not only the right but also the left tumor. PSK also inhibited the growth of a methylcholanthrene-induced fibrosarcoma BAMC-1, and a methylurethane-induced adenocarcinoma Colon 26 in the double grafted tumor system of syngeneic BALB/c mice. However, when the left distant tumor was different from the right Meth-A tumor, the intratumoral administration of PSK in the right tumor was unable to inhibit the growth of the left BAMC-1 or RL male-1 tumor. The PSK-induced immunity, therefore, is tumor-specific and T lymphocytes may play an important role in antitumor memory function. The enhancement of concomitant immunity by PSK treatment was completely impaired by previous intravenous administration of an alkylating agent, cyclophosphamide (CY). The enhancement of sinecomitant immunity by PSK treatment was also impaired by previous CY intravenous administration. The antitumor effect of PSK was suppressed by previous intravenous administration of another alkylating agent, ACNU. It is possible that alkylating agents suppress the function of effector T cells and granulocytes which are very important for the antitumor immune cascade reaction due to PSK treatment. On the other hand, the antitumor effect of PSK was enhanced by previous intravenous administration of an anti-metabolite, 5-fluorouracil.     

Antitumor Activity of Oenothein B, a Unique Macrocyclic Ellagitannin

The antitumor effect of oenothein B, a macrocyclic ellagitannin from Oenothera erythrosepala Bordas, on rodent tumors was studied, Oenothein B exhibited a strong antitumor activity against MM2 ascites tumors upon intraperitoneal administration to the mice before or after the tumor inoculation. The tannin also inhibited the growth of Meth-A solid type tumor in mice. This antitumor effect of the tannin could not be attributed to its direct cytotoxic action on tumor cells, because the cytotoxicity was very weak in the presence of serum protein. When oenothein B was injected into the peritoneal cavity of mice, peritoneal exudate cells, including cytostatic macrophages, were induced. Furthermore, in the in vitro treatment of macrophages from mice and humans, the tannin stimulated release of an interleukin 1 (IL-1)-like activity and IL-1β from the cells. These results suggest that oenothein B exerts its antitumor effect through potentiation of the host-immune defense via activation of macrophages.     

Anti-tumor efficacy of Cloretazine (VNP40101M) alone and in combination with fludarabine in murine tumor and human xenograft tumor models

Cloretazine (VNP40101M), a new sulfonylhydrazine alkylating agent, has demonstrated broad-spectrum anti-tumor activity in preclinical studies. In this study, Cloretazine was evaluated both as a monotherapy and in combination with fludarabine in murine tumor and human tumor xenograft models. Cloretazine significantly inhibited the growth of subcutaneously implanted tumors, including B16F10 murine melanoma in C57BL/6 mice, and H460 human lung carcinoma and WiDr human colon carcinoma in athymic nude CD1 mice. The inhibition of tumor growth by Cloretazine was dose dependent, increasing from 42.2 to 87% as the dose escalated from 100 to 150 mg/kg. Cloretazine showed equivalent efficacy but lower toxicity compared to cyclophosphamide in these models. The combination therapy, consisting of a single dose of 10 mg/kg Cloretazine plus five doses of 70 mg/kg fludarabine, given every other day intraperitoneally, significantly increased the long-term survival of BDF1 mice bearing the L1210 murine leukemia. On Day 65 post-tumor implantation, the combination therapy yielded a 90% survival rate compared to 40% for Cloretazine alone and 0% for fludarabine alone.