Use of transcriptional regulatory sequences of telomerase (hTER and hTERT) for selective killing of cancer cells.

Telomerase (hTER and hTERT) plays a crucial role in cellular immortalization and carcinogenesis. Telomerase activity can be detected in about 85% of different malignant tumors, but is absent in most normal cells. In situ hybridization analysis showed that high levels of hTER and hTERT expression are present in bladder cancer, while no signal was detected in normal tissue. Therefore, in this work we propose to use hTER and hTERT transcriptional regulatory sequences to control the expression of a cytotoxic gene in bladder tumor cells, resulting in the selective destruction of this cell population. Expression vectors containing the diphtheria toxin A-chain (DT-A) gene were linked to hTER and hTERT transcriptional regulatory sequences, respectively. Inhibition of protein synthesis occurred in bladder and hepatocellular carcinoma cells transfected with the plasmids containing the DT-A gene under the control of the hTER or hTERT promoters in correlation with their activity. These studies support the feasibility of using hTER and hTERT transcriptional regulatory sequences for targeted patient-oriented gene therapy of human cancer.

Combination therapy with lamivudine and adenovirus causes transient suppression of chronic woodchuck hepatitis virus infections.

Treatment of hepatitis B virus carriers with the nucleoside analog lamivudine suppresses virus replication. However, rather than completely eliminating the virus, long-term treatment often ends in the outgrowth of drug-resistant variants. Using woodchucks chronically infected with woodchuck hepatitis virus (WHV), we investigated the consequences of combining lamivudine treatment with immunotherapy mediated by an adenovirus superinfection. Eight infected woodchucks were treated with lamivudine and four were infected with approximately 10(13) particles of an adenovirus type 5 vector expressing beta-galactosidase. Serum samples and liver biopsies collected following the combination therapy revealed a 10- to 20-fold reduction in DNA replication intermediates in three of four woodchucks at 2 weeks after adenovirus infection. At the same time, covalently closed circular DNA (cccDNA) and viral mRNA levels both declined about two- to threefold in those woodchucks, while mRNA levels for gamma interferon and tumor necrosis factor alpha as well as for the T-cell markers CD4 and CD8 were elevated about twofold. Recovery from adenovirus infection was marked by elevation of sorbitol dehydrogenase, a marker for hepatocyte necrosis, as well as an 8- to 10-fold increase in expression of proliferating cell nuclear antigen, a marker for DNA synthesis, indicating significant hepatocyte turnover. The fact that replicative DNA levels declined more than cccDNA and mRNA levels following adenovirus infection suggests that the former decline either was cytokine induced or reflects instability of replicative DNA in regenerating hepatocytes. Virus titers in all four woodchucks were only transiently suppressed, suggesting that the effect of combination therapy is transient and, at least under the conditions used, does not cure chronic WHV infections.

Effect of preexisting anti-herpes immunity on the efficacy of herpes simplex viral therapy in a murine intraperitoneal tumor model.

HSV-1716, a replicating nonneurovirulent herpes simplex virus type 1, has shown efficacy in treating multiple types of human tumors in immunodeficient mice. Since the majority of the human population has been previously exposed to herpes simplex virus, the efficacy of HSV-based oncolytic therapy was investigated in an immunocompetent animal tumor model. EJ-6-2-Bam-6a, a tumor cell line derived from h-ras-transformed murine fibroblast, exhibit a diffuse growth pattern in the peritoneal cavity of BALB/c mice and replicate HSV-1716 to titers observed in human tumors. An established intraperitoneal (ip) tumor model of EJ-6-2-Bam-6a in naive and HSV-immunized mice was used to evaluate the efficacy of single or multiple ip administrations of HSV-1716 (4 x 10(6) pfu/treatment) or of carrier cells, which are irradiated, ex vivo virally infected EJ-6-2-Bam-6a cells that can amplify the viral load in situ. All treated groups significantly prolonged survival versus media control with an approximately 40% long-term survival rate (cure) in the multiply treated, HSV-naive animals. Prior immunization of the mice with HSV did not significantly decrease the median survival of the single or multiply treated HSV-1716 or the carrier cell-treated groups. These studies support the development of replication-selective herpes virus mutants for use in localized intraperitoneal malignancies.

Oncolytic herpes simplex virus-1 lacking ICP34.5 induces p53-independent death and is efficacious against chemotherapy-resistant ovarian cancer.

Replication-restricted herpes simplex virus-1 (HSV-1) strains lacking ICP34.5 are emerging as powerful anticancer agents against several solid tumors including epithelial ovarian cancer (EOC). Although chemotherapy-resistant tumors would be likely candidates for treatment with HSV-1 mutants lacking ICP34.5, the efficacy of these mutants on such tumors is unknown. In the present study, we investigated whether chemotherapy resistance affects the response of ovarian cancer cells to HSV-R3616, an ICP34.5-deficient, replication-restricted HSV-1. Primary EOC cultures obtained from patients who varied in their responses to platinum/paclitaxel induction chemotherapy displayed similar sensitivity to HSV-R3616. Similarly, chemotherapy-sensitive ovarian cancer cells A2780 and PA-1, possessing wild-type p53, and their respective chemotherapy-resistant clones A2780/200CP, lacking p53 function, and PA-1/E6, permanently expressing the HPV E6 gene, were equally sensitive to HSV oncolysis. Because wild-type HSV can kill cells by apoptosis and nonapoptotic mechanisms, we investigated the involvement of apoptosis and the role of the p53 tumor suppressor gene in oncolysis induced by HSV-R3616. Infection of ovarian cancer cell lines by HSV-R3616 was followed by cell death via apoptosis or nonapoptotic mechanisms as noted by morphology, cell cycle analysis, and in situ TUNEL assay. p53 protein levels remained unchanged, and Bax protein levels decreased in cells possessing intact p53 and that mainly underwent HSV-induced apoptosis. Loss of p53 function did not affect the frequency or rate of apoptosis or the sensitivity of EOC cells to the oncolytic effect of HSV-R3616. These results suggest that recombinant HSV-1 lacking ICP34.5 is capable of killing ovarian cancer cells that lack p53 function, resist apoptosis, and/or are chemotherapy resistant. These data support the hypothesis that HSV-based oncolytic therapy may be efficacious in chemotherapy-resistant tumors, including tumors that are deficient in p53.

Multi-attenuated herpes simplex virus-1 mutant G207 exerts cytotoxicity against epithelial ovarian cancer but not normal mesothelium and is suitable for intraperitoneal oncolytic therapy.

Recombinant strains of herpes simplex virus-1 (HSV-1) harboring mutations in the infected cell product (ICP)34.5 region lose their neurovirulence and replicate more efficiently in dividing tumor cells than stationary cells, becoming replication-selective oncolytic agents. Additional mutation of the ICP6 gene, which encodes ribonucleotide reductase, further impairs the ability of HSV-1 mutants to replicate in normal cells, enhancing tumor selectivity. The present study investigated the effect of HSV-G207, a recombinant HSV-1 lacking ICP34.5 and ICP6, against epithelial ovarian cancer (EOC) in vitro and in vivo in a mouse xenograft model. To assess the selectivity of multimutated HSV-G207 against malignant cells, HSV-G207 and wild-type HSV-F were comparatively tested against normal human peritoneal mesothelial cells and EOC cells in vitro. HSV-G207 infected both EOC cells and mesothelial cells; however, unlike EOC cells, mesothelial cells provided a poor substrate for replication of HSV-G207. In contrast to wild-type HSV-F, HSV-G207 exerted a potent oncolytic effect on EOC cells but spared normal mesothelial cells in vitro. Primary EOC cells were more sensitive to the virus than established EOC cell lines. A single intraperitoneal injection of HSV-G207 resulted in a significant reduction in tumor volume and tumor spread in vivo. HSV-G207 was shown to penetrate deeply within tumor nodules and caused no apparent intraperitoneal toxicity. Oncolytic therapy with multimutated replication-restricted HSV may offer a novel approach in the treatment of EOC.

A pilot study of systemic corticosteroid administration in conjunction with intrapleural adenoviral vector administration in patients with malignant pleural mesothelioma.

One of the primary limitations of adenoviral (Ad) -mediated gene therapy is the generation of anti-Ad inflammatory responses that can induce clinical toxicity and impair gene transfer efficacy. The effects of immunosuppression on these inflammatory responses, transgene expression, and toxicity have not yet been systematically examined in humans undergoing Ad-based gene therapy trials. We therefore conducted a pilot study investigating the use of systemic corticosteroids to mitigate antivector immune responses. In a previous phase I clinical trial, we demonstrated that Ad-mediated intrapleural delivery of the herpes simplex virus thymidine kinase gene (HSVtk) to patients with mesothelioma resulted in significant, but relatively superficial, HSVtk gene transfer and marked anti-Ad humoral and cellular immune responses. When a similar group of patients was treated with Ad.HSVtk and a brief course of corticosteroids, decreased clinical inflammatory responses were seen, but there was no demonstrable inhibition of anti -Ad antibody production or Ad-induced peripheral blood mononuclear cell activation. Corticosteroid administration also had no apparent effect on the presence of intratumoral gene transfer. Although limited by the small numbers of patients studied, our data suggest that systemic administration of steroids in the context of Ad-based gene delivery may limit acute clinical toxicity, but may not inhibit cellular and humoral responses to Ad vectors.

Combined therapy with chemotherapeutic agents and herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) in human non-small cell lung cancer.

A replication-selective herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) has shown efficacy both in vitro and in vivo against human non-small cell lung cancer (NSCLC) cell lines but complete eradication of tumor has not been accomplished with a single viral treatment in our murine xenograft models. Therefore, strategies to enhance the efficacy of this treatment were investigated. We determined the oncolytic activity of HSV-1716 in NCI-H460 cells in combination with each of four chemotherapeutic agents: mitomycin C (MMC), cis-platinum II (cis-DDP), methotrexate (MTX), or doxorubicin (ADR). Isobologram analysis was performed to evaluate the interaction between the viral and chemotherapeutic agents. The oncolytic effect of HSV-1716 in combination with MMC was synergistic in two of five NSCLC cell lines. In the other three cell lines, the combined effect appeared additive. No antagonism was observed. The in vivo effect of this combination was then examined in a murine xenograft model. NCI-H460 flank tumors were directly injected with HSV-1716 (4 x 106 PFU) followed by intravenous MMC administration (0.17 mg/kg) 24 hr later. After 3 weeks, the mean tumor weight in the combined treatment group was significantly less than either individual treatment in an additive manner. The synergistic dose of MMC neither augmented nor inhibited viral replication in vitro and HSV-1716 infection did not upregulate DT-diaphorase, which is the primary enzyme responsible for MMC activation. In summary, the combination of HSV-1716 with common chemotherapeutic agents may augment the effect of HSV-based therapy in the treatment of NSCLC.

Oncolytic therapy using a mutant type-1 herpes simplex virus and the role of the immune system.

BACKGROUND: Herpes simplex virus (HSV)-1716, a replication-restricted herpes simplex virus type 1, has shown efficacy as an oncolytic treatment for central nervous system tumors, breast cancer, ovarian cancer, and malignant mesothelioma. We evaluated the efficacy of HSV-1716 in a murine lung cancer model, Lewis lung carcinoma. METHODS: Lewis lung carcinoma cells were infected with HSV-1716 and implanted in the flanks of mice at varying ratios of infected to uninfected cells. Tumor burden was assessed by measurement of the weight of the tumor nodule. The role of the immune system was examined by performing experiments in both immunocompetent and SCID mice. Tumors were implanted in the opposite flank to evaluate the vaccine effect. RESULTS: In immunocompetent and SCID animals, ratio of 1:10 (infected-to-uninfected) cells completely prevented tumor formation and ratio of 1:100 suppressed tumor growth. Established tumors at a distant site in the groups receiving HSV-1716 infected cells showed no difference in size versus control, suggesting absence of a vaccine effect. CONCLUSIONS: We conclude that HSV-1716 may provide a oncolytic therapy for lung cancer even in the absence of immune system induction and a "carrier" cell could potentially deliver this vector.

Use of carrier cells to deliver a replication-selective herpes simplex virus-1 mutant for the intraperitoneal therapy of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) remains localized within the peritoneal cavity in a large number of patients, lending itself to i.p. approaches of therapy. In the present study, we investigated the effect of replication-selective herpes simplex virus-1 (HSV-1) used as an oncolytic agent against EOC and the use of human teratocarcinoma PA-1 as carrier cells for i.p. therapy. HSV-1716, a replication-competent attenuated strain lacking ICP34.5, caused a direct dose-dependent oncolytic effect on EOC cells in vitro. A single i.p. administration of 5 x 10(6) plaque-forming units resulted in a significant reduction of tumor volume and tumor spread and an increase in survival in a mouse xenograft model. PA-1 cells supported HSV replication in vitro and bound preferentially to human ovarian carcinoma surfaces compared with mesothelial surfaces in vitro and in vivo. In comparison with the administration of HSV-1716 alone, irradiated PA-1 cells, infected at two multiplicities of infection with HSV-1716 and injected i.p. at 5 x 10(6) cells/animal, led to a significant tumor reduction in the two models tested and the significant prolongation of mean survival in one model. Histological evaluation revealed extensive necrosis in tumor areas infected by HSV-1716. Immunohistochemistry against HSV-1 revealed areas of viral infection within tumor nodules, which persisted for several weeks after treatment. Administration of HSV-infected PA-1 carrier cells resulted in larger areas of tumor infected by the virus. Our results indicate that replication-competent attenuated HSV-1 exerts a potent oncolytic effect on EOC, which may be further enhanced by the utilization of a delivery system with carrier cells, based on amplification of the viral load and possibly on preferential binding of carrier cells to tumor surfaces.

Impact of preexisting and induced humoral and cellular immune responses in an adenovirus-based gene therapy phase I clinical trial for localized mesothelioma.

Little is known about the immune responses induced by recombinant adenoviral (Ad) vectors in humans. The humoral and cellular immune responses were therefore analyzed in 21 patients with localized malignancy (mesothelioma), who received intrapleurally high doses of a replication-defective Ad5 vector carrying a suicide gene. Eight of 21 patients had pretreatment titers of neutralizing antibodies (NAb) to Ad at > or =1:100. Peripheral blood mononuclear cells (PBMCs) proliferated in response to adenoviral 5 structural proteins before treatment in 17 of 21 patients. Preexisting humoral and cellular immunity did not preclude gene transfer. Vector instillation induced high titers of nonneutralizing and neutralizing anti-Ad antibody (4- to 341-fold increase in 18 of 20 patients) in a dose-dependent manner. Three patients generated antibodies to the transgene, herpes simplex virus thymidine kinase. Ad5-specific proliferation of PBMCs increased significantly (>3-fold) after vector administration in 12 of 21 patients in a dose-dependent manner. Thus, replication-defective Ad5 administered intrapleurally induced significant humoral and cellular immune responses that induced no obvious adverse clinical sequelae.

Adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir gene therapy in patients with localized malignancy: results of a phase I clinical trial in malignant mesothelioma.

Malignant pleural mesothelioma is a fatal neoplasm that is unresponsive to standard modalities of cancer therapy. We conducted a phase I dose-escalation clinical trial of adenoviral (Ad)-mediated intrapleural herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) gene therapy in patients with mesothelioma as a model for treatment of a localized malignancy. The goals of this phase I trial were to assess the safety, toxicity, and maximally tolerated dose of intrapleural Ad.HSVtk, to examine patient inflammatory response to the viral vector, and to evaluate the efficiency of intratumoral gene transfer. Twenty-one previously untreated patients were enrolled in this single-arm, dose-escalation study with viral doses ranging from 1 x 10(9) plaque-forming units (pfu) to 1 x 10(12) pfu. A replication-incompetent recombinant adenoviral vector containing the HSVtk gene under control of the Rous sarcoma virus (RSV) promoter-enhancer was introduced into the pleural cavity of patients with malignant mesothelioma followed by 2 weeks of systemic therapy with GCV at a dose of 5 mg/kg twice a day. The initial 15 patients underwent thoracoscopic pleural biopsy prior to, and 3 days after, vector delivery. The last six patients underwent only the post-vector instillation biopsy. Dose-limiting toxicity was not reached. Side effects were minimal and included fever, anemia, transient liver enzyme elevations, and bullous skin eruptions, as well as a temporary systemic inflammatory response in those receiving the highest dose. Strong intrapleural and intratumoral immune responses were generated. Using RNA PCR, in situ hybridization, immunohistochemistry, and immunoblotting, HSVtk gene transfer was documented in 11 of 20 evaluable patients in a dose-related fashion. This study demonstrates that intrapleural administration of an adenoviral vector containing the HSVtk gene is well tolerated and results in detectable gene transfer when delivered at high doses. Further development of therapeutic trials for treatment of localized malignancy using this vector is thus warranted.

The effect of promoter strength in adenoviral vectors containing herpes simplex virus thymidine kinase on cancer gene therapy in vitro and in vivo.

The use of adenoviral vectors to deliver the herpes simplex virus thymidine kinase (HSVtk) gene followed by treatment with the prodrug ganciclovir (GCV) has promise for a variety of applications where excess cell proliferation is detrimental such as treatment of tumors and vascular restenosis. Optimizing this system is thus an important goal. The purpose of this study was to determine if the induction of higher levels of HSVtk expression would augment the sensitivity to GCV. This was accomplished by generating adenoviral vectors that expressed HSVtk from promoters of different efficiencies (the CMV versus RSV promoters). Despite higher levels of HSVtk expression per cell with the CMV promoter, there was no significant enhancement of antitumor effects between RSV- and CMV-driven adenovirus vectors in in vitro and in vivo studies indicating that simply increasing HSVtk enzyme levels per cell above a minimal threshold level will not be effective in augmenting the HSVtk/GCV system. These results suggest that other strategies, e.g., the use of higher doses of GCV, augmentation of the "bystander effect," the generation of mutant HSVtk genes with higher substrate affinities, the discovery of improved vectors with increased transduction efficiencies, or the development of new prodrugs with higher affinities for HSVtk will therefore be needed to enhance therapeutic responses.

Use of a "replication-restricted" herpes virus to treat experimental human malignant mesothelioma.

Modified, nonneurovirulent herpes simplex viruses (HSVs) have shown promise in the treatment of brain tumors. However, HSV-1 can infect and lyse a wide range of cell types. In this report, we show that HSV-1716, a mutant lacking both copies of the gene coding ICP-34.5, can effectively treat a localized i.p. malignancy. Human malignant mesothelioma cells supported the growth of HSV-1716 and were efficiently lysed in vitro. i.p. injection of HSV-1716 into animals with established tumor nodules reduced tumor burden and significantly prolonged survival in an animal model of non-central nervous system-localized human malignancy without dissemination or persistence after i.p. injection into SCID mice bearing human tumors. These findings suggest that this virus may be efficacious and safe for use in localized human malignancies of nonneuronal origin such as malignant mesothelioma.

A putative sirolimus (rapamycin) effector protein.

Sirolimus (rapamycin), a new immunosuppressive drug, inhibits proliferation of a wide spectrum of T and B cells. The immunosuppressive mechanism of sirolimus is still unclear. We recently isolated a membrane associated protein with an apparent molecular weight of 210 kDa, p210, from cultured Molt 4 cells and BJAB cells and from normal human T cells using an affinity matrix method. The p210 binds to sirolimus:FKBP12 complex, but only at background levels to FKBP12 alone, to FK506:FKBP12 complex, or sirolimus-biotin alone. Among the sirolimus analogs tested, the binding ability of p210 to drug:FKBP12 complexes correlates with the immunosuppressive activity of the drugs, suggesting that p210 is the sirolimus effector protein.

Modulation of TNF alpha and IL-1 beta from endotoxin-stimulated monocytes by selective PDE isozyme inhibitors.

The effect of selective PDE isozyme inhibitors including vinpocetine (PDE-I), CI-930 and milrinone (PDE-III), rolipram and nitraquazone (PDE-IV) and zaprinast (PDE-V) on monocyte viability and production of tumor necrosis factor (TNF alpha) and interleukin-1 beta (IL-1 beta) elicited from endotoxin-stimulated human monocytes was investigated. None of the inhibitors affected monocyte viability at 10 microM or lower concentrations. PDE-IV inhibitors and to a lesser extent, PDE-III inhibitors suppressed TNF alpha production. Only high concentrations of PDE-IV inhibitors modestly suppressed IL-1 beta. Zaprinast stimulated IL-1 beta and to a lesser extent TNF alpha production. These data show that TNF alpha and IL-1 beta production are differentially regulated, and that PDE III, PDE-IV and PDE-V isozymes are functional in endotoxin-stimulated monocytes. Clinical trials will be needed to ascertain if PDE-IV inhibitors are able to suppress TNF alpha levels in man.

Strategies for the development of new antiarthritic agents.

Therapeutic advances in rheumatoid arthritis (RA) have largely focused on the development of non-steroidal antiinflammatory drugs (NSAIDs) with improved characteristics compared with aspirin [Brooks & Day, New Engl. J. Med., 324, 1716-1725 (1991)]. For example, greater potency, safety, improved tolerance in the elderly and reduced frequency of dosing have been achieved. However, these agents are generally considered to be palliative treating of the symptoms of the disease. The development of disease modifying drugs (DMD), also known as second line drugs, for RA has not been very successful. Most of the agents that are currently used in this category were originally used to treat other diseases such as malignancy (cyclophosphamide, methotrexate), Wilson's disease (d-penicillamine) and tuberculosis (gold salts) [Pullar, Br. J. clin. Pharmac., 30, 501-510 (1990)]. Unfortunately, none of the agents is ideal and each has potentially serious side-effects. There have been several attempts to develop agents with new mechanisms of action that hopefully will greatly improve these current therapies.

Differential regulation of TNF-alpha and IL-1beta production from endotoxin stimulated human monocytes by phosphodiesterase inhibitors.

The effect of selective PDE-I (vinpocetine), PDE-III (milrinone, CI-930), PDE-IV (rolipram, nitroquazone), and PDE-V (zaprinast) isozyme inhibitors on TNF-alpha and IL-1beta production from LPS stimulated human monocytes was investigated. The PDE-IV inhibitors caused a concentration dependent inhibition of TNF-alpha production, but only partially inhibited IL-1beta at high concentrations. High concentrations of the PDE-III inhibitors weakly inhibited TNF-alpha, but had no effect on IL-1beta production. PDE-V inhibition was associated with an augmentation of cytokine secretion. Studies with combinations of PDE isozyme inhibitors indicated that PDE-III and PDE-V inhibitors modulate rolipram's suppression of TNF production in an additive manner. These data confirm that TNF-alpha and IL-1beta production from LPS stimulated human monocytes are differentially regulated, and suggest that PDE-IV inhibitors have the potential to suppress TNF levels in man.