Prodrug Activator Gene Research Articles

Replication-competent retrovirus vector-mediated prodrug activator gene therapy in experimental models of human malignant mesothelioma

Replication-competent retrovirus (RCR) vectors have been shown to achieve significantly enhanced tumor transduction efficiency and therapeutic efficacy in various cancer models. In the present study, we investigated RCR vector-mediated prodrug activator gene therapy for the treatment of malignant mesothelioma, a highly aggressive tumor with poor prognosis. RCR vector expressing the green fluorescent protein marker gene (RCR-GFP) successfully infected and efficiently replicated in human malignant mesothelioma cell lines, as compared to non-malignant mesothelial cells in vitro. In mice with pre-established subcutaneous tumor xenografts, RCR-GFP vector showed robust spread throughout entire tumor masses after intratumoral administration. Next, RCR-CD, expressing the yeast cytosine deaminase (CD) prodrug activator gene, showed efficient transmission of the prodrug activator gene associated with replicative spread of the virus, resulting in efficient killing of malignant mesothelioma cells in a prodrug 5-fluorocytosine (5FC)-dose dependent manner in vitro. After a single intratumoral injection of RCR-CD followed by intraperitoneal administration of 5FC, RCR vector-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous tumor growth, and significantly prolonged survival in the disseminated peritoneal model of malignant mesothelioma. These data indicate the potential utility of RCR vector-mediated prodrug activator gene therapy in the treatment of malignant mesothelioma.

Abstract 5393: Prodrug activator gene therapy mediated by prostate-specific replicating retrovirus vectors

Abstract Replicating murine leukemia virus (RMLV)-based vectors can propagate specifically within actively dividing cancer cells and achieve highly efficient and tumor-selective gene delivery. However, in view of the known potential for retroviral integration-mediated genotoxicity, additional mechanisms to target the virus specifically to cancer cells are desirable. To further improve their tumor specificity and safety profile, we have developed RMLV vectors regulated by two different tissue-specific promoters. We generated GFP-expressing RMLV vectors ACE-emd (wild type), ACE-At-emd (regulated by ARR2PB promoter, a synthetic variant of the probasin promoter which exhibits high specificity for androgen receptor (AR)-positive prostate cancer cells) and ACE-PSES-emd (regulated by PSES promoter, an androgen-independent promoter which is highly active in PSA/PSMA-positive prostate cancer cells, both in the presence and absence of androgen). We also developed ACE-yCD, ACE-At-yCD and ACE-PSES-yCD carrying the yeast cytosine deaminase (yCD) prodrug-activator gene, which converts the nontoxic prodrug 5-fluorocytosine (5FC) into the chemotoxin 5-fluorouracil. These vectors were first in vitro tested in both prostate (LNCaP, MDA PCa 2b and PC-3) and non-prostate (293T) cell lines in the presence and absence of androgen. RMLV vector ACE-At-emd exhibited high specificity and robust replication in AR-positive prostate cancer cell lines, but only in the presence of androgen. RMLV vector ACE-PSES-emd exhibited high specificity for PSA/PSMA-positive prostate cancer cell line both in the presence and absence of androgen. For in vivo studies, male nude mice were inoculated subcutaneously with LNCaP cells (AR-positive, PSA/PSMA-positive) in Matrigel. RMLV vectors expressing GFP were injected intratumorally, and tumors were analyzed by flow cytometry 2 and 4 weeks after vector injection. The results showed that both ARR2PB- and PSES-regulated RMLV vectors achieve efficient gene delivery to LNCaP s.c. tumors in vivo. Cytotoxic activity of RMLV-yCD vectors was examined in vitro in LNCaP cells in the presence and absence of 5FC after virus infection. Cell-specific expression of each vector correlated with reduced cell viability compared to uninfected cells after 5FC treatment. We are now conducting studies to evaluate therapeutic efficacy vs. potential genotoxicity of these vectors for prodrug-activator gene therapy in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5393. doi:10.1158/1538-7445.AM2011-5393

Abstract 5386: Therapeutic efficacy of RMLV vectors in human and murine glioma models

Abstract Unique among replicating viruses being developed as oncolytic agents, replicating murine leukemia virus (RMLV) retroviruses propagate without immediate lysis of host cells and maintain viral persistence through stable integration. Due to its intrinsic inability to infect quiescent post-mitotic cells and susceptibility to innate antiviral defenses that exist in normal cells but are inactivated in many cancers, RMLV vector replication has been shown to be highly tumor-selective. We have previously demonstrated that RMLV vectors are capable of highly efficient replication in tumor cells, and we have found significantly enhanced survival benefit in a variety of cancer models in vivo when RMLV vectors are employed for delivery of prodrug activator genes such as yeast cytosine deaminase (yCD), which mediates the conversion of inactive prodrug 5-fluorocytosine (5FC) to the anti-cancer drug, 5-fluorouracil (5FU). In the present study, we tested a newly developed RMLV vector with modified virus backbone sequences and a codon-optimized yCD gene (Toca511). First, we examined in vitro cytotoxicity using human (U87) and murine (TU-2449) glioma cell lines by MTS assay. The cell viability of Toca511-transduced cells was reduced by >80% after 5 or 8 days of exposure to the prodrug, respectively. Second, we examined the therapeutic efficiency of this newly developed vector in subcutaneous U87 human glioblastoma models and TU2449 syngeneic mouse models. Toca511-mediated prodrug activator therapy strongly inhibited tumor growth in both subcutaneous models. Third, therapeutic efficacy was evaluated, and the minimum effective doses of 5FC prodrug and Toca511 vector determined in an intracranial U87 xenograft model. Systemic administration of 5FC resulted in significant inhibition of bioluminescent signals in mice whose tumors had been infected with Toca511 but not in control mice, associated with significant survival benefit even after injecting only 1×1e3 total vector units (p=0.0015). These data suggest that we have been successful in developing an improved retrovirus vector that shows strong therapeutic efficacy when used for prodrug activator gene therapy in both subcutaneous and intracranial xenograft models of human and/or murine glioma. This vector has now entered Phase I/II clinical trials for glioblastoma. Now, with the use of RMLV vectors such as Toca511, the original promise of retrovirus-mediated gene therapy strategies for cancer may finally be fulfilled. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5386. doi:10.1158/1538-7445.AM2011-5386

Adenovirus–Retrovirus Hybrid Vectors Achieve Highly Enhanced Tumor Transduction and Antitumor Efficacy In Vivo

Murine leukemia virus (MLV)-based replication-competent retrovirus (RCR) vectors have been shown to mediate efficient, selective, and persistent tumor transduction, thereby achieving significant therapeutic benefit in a wide variety of cancer models. To further augment the efficiency of this strategy, we have developed a delivery method employing a gutted adenovirus encoding an RCR vector (AdRCR); thus, tumor cells transduced with the adenoviral vector transiently become RCR vector producer cells in situ. As expected, high-titer AdRCR achieved significantly higher initial transduction levels in human cancer cells both in vitro and in vivo, as compared to the original RCR vector itself. Notably, even at equivalent initial transduction levels, more secondary RCR progeny were produced from AdRCR-transduced cells as compared to RCR-transduced cells, resulting in further acceleration of subsequent RCR replication kinetics. In pre-established tumor models in vivo, prodrug activator gene therapy with high-titer AdRCR could achieve enhanced efficacy compared to RCR alone, in a dose-dependent manner. Thus, AdRCR hybrid vectors offer the advantages of high production titers characteristic of adenovirus and secondary production of RCR in situ, which not only accelerates subsequent vector spread and progressive tumor transduction, but can also significantly enhance the therapeutic efficacy of RCR-mediated prodrug activator gene therapy.

Prodrug-activating Gene Therapy with Rabbit Cytochrome P450 4B1/4-Ipomeanol or 2-Aminoanthracene System in Glioma Cells.

We determined the cytotoxic properties of cytochrome P450 4B1 (CYP4B1) activated 4-ipomeanol (4-ipo) and 2-aminoanthracene (2-AA) in rat glioma to verify the CYP4B1/4-ipo or 2-AA system for prodrug-activating gene therapy. The cyp4B1 cDNA was cloned into pcDNA3.1/Hygro from rabbit lung total RNA (pcDNA-cyp4B1). Lentiviral vector encoding firefly luciferase (fLuc) was infected into C6 (rat glioma), and the fLuc-expressing cell was selected (C6-L). After transfection with pcDNA-cyp4B1 vector into C6-L, the single clone expressing cyp4B1 gene was selected (C6-CL). Prodrug for various concentrations of 4-ipo or 2-AA was treated for 72h and 96h. The cell survival rate of C6-CL was determined using MTT assay and trypan-blue dye exclusion methods. By RT-PCR analysis, fLuc and CYP4B1 expression was detected in C6-CL, but not in C6. MTT assay and trypan-blue dye exclusion showed that IC50 of C6-CL was 0.3mM and <0.01mM after 4-ipo or 2-AA treatment at 96h or 72h exposure, respectively. Cell survivals of C6-CL were more rapidly reduced after treatment with 4-ipo or 2-AA than those of C6-L cells. The cell survival rate with MTT and trypan-blue dye exclusion assay was well correlated with fLuc activity in C6-CL cells. CYP4B1-based prodrug-activating gene therapy may have the potential to treat glioma and the cytotoxic effects of CYP4B1 enzyme activated 4-ipo or 2-AA in C6, and could be clearly determined by bioluminescent activity in C6-CL.

Abstract B33: Intratumoral conversion of 5-fluorocytosine to 5-fluorouracil using a novel prodrug activator gene transfer technology in an immune-competent model of glioblastoma

Abstract Background: Patients with the most common and aggressive form of brain cancer, glioblastoma multiforme (GBM), have poor prognosis and current treatments remain only palliative. Previous gene therapy approaches using replication-deficient viral vectors have demonstrated limited efficacy likely due to poor gene delivery to the tumor. Methods: We used a novel replication-competent retroviral vector (Toca 511) designed and engineered to efficiently deliver a modified cytosine deaminase (CD) prodrug activating gene to glioma cells. Previous studies showed that such a replicating vector can preferentially infect intracranial human xenografts in nude mice (Tai et al. Mol Ther. 2005;12:842) and lead to increased survival in treatment groups compared to controls. In preparation for clinical trials we have improved the CD gene activity and further stabilized the vector structure to create Toca 511. We investigated the functionality of Toca 511 using stereotactic, intratumoral delivery in a syngeneic, orthotopic glioma model in immune-competent B6C3F1 mice. Freshly excised tumors were analyzed by quantitative PCR, Western, and HPLC. Results: Brain tumors isolated from mice treated with Toca 511 and fluorocytosine (5FC) had observable expression of CD protein and vector copy numbers per genome that demonstrate efficient tumor infection. Extensive tumor specific viral spread with almost no spread of vector from the site of injection was observed in the first 24 days. Highly efficient intratumoral conversion of the 5FC prodrug into the anticancer drug 5-fluorouracil (5FU) was demonstrated after intraperitoneal or oral dosing with 5FC. Studies with Toca 511 at high and mid dose levels in combination with 5FC resulted in prolonged survival compared to controls (&amp;gt; 5 months, p&amp;lt;0.0036 and &amp;lt;0.0354 respectively) in this syngeneic glioma model. The highest Toca 511 dose level tested in combination with 5-FC yielded no safety related findings in the mice. Conclusions: Toca 511 efficiently delivers a functional CD gene for expression in glioma cells in a syngeneic, orthotopic model in immune-competent mice. Toca 511 increases survival through the delivery of CD gene and subsequent efficient conversion of 5FC into 5FU within the brain tumor with no safety related findings. A phase 1 ascending dose trial investigating the safety and tolerability of Toca 511 in combination with 5FC in patients with recurrent GBM is about to initiate. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B33.

831. Preclinical Studies Support the Efficacy of Prodrug Activator Gene Transfer in Experimental Glioma Models by Enhanced Replication-Competent Retrovirus Vector

831. Preclinical Studies Support the Efficacy of Prodrug Activator Gene Transfer in Experimental Glioma Models by Enhanced Replication-Competent Retrovirus Vector

492. Optimizing the Genomic Stability, Transgene Expression, and Therapeutic Efficacy of Replication-Competent Retrovirus Vectors for Prodrug Activator Gene Transfer

492. Optimizing the Genomic Stability, Transgene Expression, and Therapeutic Efficacy of Replication-Competent Retrovirus Vectors for Prodrug Activator Gene Transfer

Testing double mutants of the enzyme nitroreductase for enhanced cell sensitisation to prodrugs: Effects of combining beneficial single mutations

Prodrug activation gene therapy for cancer involves expressing prodrug-activating enzymes in tumour cells, so they can be selectively killed by systemically administered prodrug. For example, Escherichia colinfsB nitroreductase (E.C. 1.6.99.7)(NTR), sensitises cells to the prodrug CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide), which it converts to a potent DNA-crosslinking agent. However, low catalytic efficiency with this non-natural substrate appears to limit the efficacy of this enzyme prodrug combination for eliminating the target cancer cells. To improve this, we aim to engineer NTR for improved prodrug activation. Previously, a number of single amino acid substitutions at six positions around the active site of the enzyme were found to increase activity, resulting in up to ∼5-fold enhanced cell sensitisation to CB1954. In this study we have made pairwise combinations among some of the best mutants at each of these 6 sites. A total of 53 double mutants were initially screened in E. coli, then the 7 most promising were inserted into an adenovirus vector and compared in SKOV3 human ovarian carcinoma cells for sensitisation to CB1954 and two alternative prodrugs. The most effective mutants, T41L/N71S and T41L/F70A, were 14–17-fold more potent than WT NTR at sensitising the cancer cells to CB1954. The best mutant for activation of the dinitrobenzamide mustard prodrug SN23862 was T41L/F70A (4.8-fold improvement); and S40A/F124M showed 1.7-fold improvement over WT with the nitrobenzylphosphoramide mustard prodrug LH7. In two tumour xenograft models using SKOV3 or human prostate carcinoma PC3, T41L/N71S NTR demonstrated greater CB1954-dependent anti-tumour activity than WT NTR.

Steady-State and Stopped-Flow Kinetic Studies of Three Escherichia coli NfsB Mutants with Enhanced Activity for the Prodrug CB1954

The enzyme nitroreductase, NfsB, from Escherichia coli has entered clinical trials for cancer gene therapy with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, CB1954 is a poor substrate for the enzyme. Previously we made several NfsB mutants that show better activity with CB1954 in a cell-killing assay in E. coli. Here we compare the kinetic parameters of wild-type NfsB with CB1954 to those of the most active single, double, and triple mutants isolated to date. For wild-type NfsB the global kinetic parameters for both k(cat) and K(m) for CB1954 are about 20-fold higher than previously estimated; however, the measured specificity constant, k(cat)/K(m) is the same. All of the mutants are more active with CB1954 than the wild-type enzyme, the most active mutant showing about 100-fold improved specificity constant with CB1954 over the wild-type protein with little effect on k(cat). This enhancement in specificity constants for the mutants is not seen with the antibiotic nitrofurazone as substrate, leading to reversed nitroaromatic substrate selectivity for the double and triple mutants. However, similar enhancements in specificity constants are found with the quinone menadione. Stopped-flow kinetic studies suggest that the rate-determining step of the reaction is likely to be the release of products. The most active mutant is also selective for the 4-nitro group of CB1954, rather than the 2-nitro group, giving the more cytotoxic reduction product. The double and triple mutants should be much more effective enzymes for use with CB1954 in prodrug-activation gene therapy.

E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954

Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO2 or 4-NO2 positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO2 group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

Phase Ib Trial of Oncolytic Herpes Virus G207 Shows Safety of Multiple Injections and Documents Viral Replication

The prognosis for patients with glioblastoma remains poor despite numerous phase I–II clinical trials on systemic chemotherapies such as the vascular endothelial growth factor–neutralizing antibody bevacizumab.1 Indeed, only two chemotherapies—implantable carmustine-containing Gliadel wafers and temozolomide2—have been approved for glioblastoma by the Food and Drug Administration, because these two agents showed benefit in phase III clinical trials by prolonging median survival by approximately 8 weeks. Therefore, there is a continued need to explore new treatments or to further refine and optimize older treatments for this type of brain cancer.

Optimized clostridium-directed enzyme prodrug therapy improves the antitumor activity of the novel DNA cross-linking agent PR-104.

We have previously shown that spores of the nonpathogenic clostridial strain C. sporogenes genetically engineered to express the E. coli-derived cytosine deaminase gene are effective in converting systemically injected nontoxic 5-fluorocytosine into the toxic anticancer drug 5-fluorouracil, thereby producing tumor-specific antitumor activity. To improve the expression of E. coli-derived genes with this system, we first replaced the original fdP promoter in the vector with one of two powerful endogenous clostridial promoters: that of the thiolase gene (thlP) and that for the clostridial transcription factor abrB310 (abrBP). These substitutions improved protein expression levels of the prodrug-activating genes by 2- to 3-fold in comparison with fdP-driven expression. However, despite these strong promoters, we found much higher expression of the nitroreductase (NTR) protein in the E. coli host compared with the clostridial host, which we hypothesized could be the result of different codon use between the two organisms. To test this, we constructed new expression vectors with an artificially synthesized NTR gene using optimized clostridial codons (sNTR). Results from both enzymatic assays and Western blots of cell extracts from clostridial transformants harboring plasmid constructs of thlP-sNTR and abrBP-sNTR showed that the expression and activity of the NTR gene product was increased by approximately 20-fold compared with the original construct. In vivo studies with i.v. administered sNTR-expressing C. sporogenes spores in SiHa tumor-bearing mice showed significantly improved antitumor efficacy when combined with either 5-aziridinyl-2,4-dinitrobenzamide (CB1954) or the novel dinitrobenzamide mustard prodrug, PR-104.

The nitroreductase prodrug SN 28343 enhances the potency of systemically administered armed oncolytic adenovirus ONYX-411NTR

Conditionally replicating adenoviruses (CRAd) 'armed' with prodrug-activating genes have the potential to augment the efficacy of virotherapy. An Escherichia coli nitroreductase (NTR) gene (nfsB) was introduced into the E3B region of the systemically active CRAd ONYX-411, to produce ONYX-411(NTR), which had single agent oncolytic activity equivalent to unarmed virus in vitro and in vivo. A fluorogenic probe (SN 29884) developed to monitor NTR expression revealed robust, durable NTR expression in ONYX-411(NTR) infected neoplastic but not primary human cell lines. NTR expression occurred >24 h post-infection in parallel with fiber and was sensitive to ara-C indicating transcriptional linkage to viral replication. A novel NTR prodrug, the 3,5-dinitrobenzamide-2-bromomustard SN 27686, was shown to be more dose potent and selective than CB 1954 and provided a superior bystander effect in 3D multicellular layer cultures. Its water-soluble phosphate ester SN 28343 was substantially more active than CB 1954 against xenografts containing a minority of stable NTR-expressing cells. A single intravenous dose of ONYX-411(NTR) (10(8) PFU) to nude mice bearing large H1299 xenografts (>350 mm(3)) resulted in tumor-specific NTR expression which increased over time. Despite extensive viral spread by day 14, this conservative virus dose and schedule was unable to control such well-established tumors. However, subsequent administration of SN 28343 resulted in the majority of mice (62.5%) being tumor-free on day 120.

Collaboration between hepatic and intratumoral prodrug activation in a P450 prodrug-activation gene therapy model for cancer treatment

Presently, we investigate the mechanisms whereby intratumoral expression of a cyclophosphamide-activating hepatic cytochrome P450 gene enhances therapeutic activity when cyclophosphamide is given on an every 6-day (metronomic) schedule. In P450-deficient 9L gliosarcomas grown in severe combined immunodeficient mice, metronomic cyclophosphamide substantially decreased tumor microvessel density and induced a approximately 70% loss of endothelial cells that began after the second cyclophosphamide treatment. These responses were accompanied by increased expression of the endogenous angiogenesis inhibitor thrombospondin-1 in tumor-associated host cells but by decreased expression in 9L tumor cells. These antiangiogenic responses preceded tumor regression and are likely key to the therapeutic activity of metronomic cyclophosphamide. Unexpectedly, 9L/2B11 tumors, grown from 9L cells infected with retrovirus encoding the cyclophosphamide-activating P450 2B11, exhibited antiangiogenic responses very similar to 9L tumors. This indicates that the tumor endothelial cell population is well exposed to liver-activated cyclophosphamide metabolites and that intratumoral P450 confers limited additional anti-endothelial cell bystander activity. In contrast, an increase in apoptosis, which preceded the antiangiogenic response, was substantially enhanced by intratumoral P450 2B11 expression. 9L/2B11 tumor regression was accompanied by an overall loss of tumor cellularity and by substantial enlargement of remaining P450-immunoreactive tumor cells as the number of P450-positive tumor cell decreased and the P450 protein content declined with cyclophosphamide treatment. We conclude that metronomic cyclophosphamide regresses P450-expressing tumors by two independent but complementary mechanisms: increased tumor cell killing via intratumoral P450-catalyzed prodrug activation, coupled with strong antiangiogenic activity, which is primarily associated with hepatic prodrug activation.

OncoVEX: A family of oncolytic herpes simplex viruses optimised for therapeutic use

13139 HSV in which ICP34.5 is deleted directs tumour selective cell lysis and has proven safe in Phase I clinical trials. To produce oncolytic HSV with enhanced anti-tumour properties, we have deleted ICP34.5 from a clinical isolate of HSV-1, which enhances the tumour cell killing capabilities of the virus, deleted ICP47 (which blocks antigen presentation), and inserted the gene encoding GM-CSF. This aimed to maximize anti-tumour immune responses following intra-tumoural injection and provide an in situ, patient-specific, anti-tumour vaccine, combined with oncolysis. In vivo, both injected and non-injected tumours could be cured and animals were then protected against tumour cell challenge. A Phase I clinical trial with the virus (OncoVEXGM-CSF) has been conducted including patients with cutaneous or sub-cutaneous deposits of a number of tumour types (Lead Investigator: Professor Charles Coombes, Hammersmith Hospital, London). This demonstrated the virus to have a good safety profile, the main side effects being ‘flu-like symptoms, similar to those which have previously been observed with other oncolytic products. Virus replication and GM-CSF expression was demonstrated together with inflammation, flattening and necrosis of injected lesions which was in some cases considerable and which was also in some cases observed in lesions which had not themselves been injected. In all cases where necrosis was observed in biopsies, this correlated with areas of staining for HSV, suggesting the virus had caused the effect. Following this promising data, Phase II studies are underway in multiple tumour types. In addition to OncoVEXGM-CSF, further versions of OncoVEX expressing other active genes have been constructed and tested in pre-clinical models. These include a virus expressing TNF∝, intended to be synergistic with radiotherapy, and versions of the virus expressing a pro-drug activating gene combined with the delivery of a fusogenic glycoprotein designed to maximize the properties of the virus for local tumour control. Each of these have shown promising results in pre-clinical tumour models, including in combination with chemotherapy and radiotherapy where benefits which are at least additive have been demonstrated. [Table: see text]

166. OncoVEX: A Family of Oncolytic Herpes Simplex Viruses Optimised for Therapeutic Use

HSV in which the neurovirulence factor ICP34.5 is inactivated directs tumour selective cell lysis and has proven safe in the Phase I clinical studies conducted so far. To produce oncolytic HSV with enhanced anti-tumour properties, we have deleted ICP34.5 from a clinical isolate of HSV-1, which enhances the tumour cell killing capabilities of the virus, and also deleted ICP47 (which blocks antigen presentation in HSV infected cells) and inserted the gene encoding GM-CSF. This aimed to maximize anti-tumour immune responses following intra-tumoural injection and provide an in situ, patient- specific, anti-tumour vaccine, combined with oncolysis. In vivo, both injected and non-injected tumours could be cured with this virus and animals were then protected against further tumour cell challenge. Following this promising data a Phase I clinical trial with the virus (OncoVEXGM-CSF) was conducted including patients with cutaneous or sub-cutaneous deposits of a number of tumour types (Lead Investigator: Professor Charles Coombes, Hammersmith Hospital, London). This demonstrated the virus to have a good safety profile, the main side effects observed being ‘flu-like symptoms, similar to those which have previously been observed with other oncolytic products and cancer vaccines, combined with evidence of biological activity including virus replication and GM- CSF expression. In addition inflammation, tumour flattening and necrosis was observed in injected lesions which was in some cases considerable and which was also in some cases observed in lesions which had not themselves been injected. In all cases where necrosis was observed in biopsy material, this correlated with areas of staining for HSV, suggesting the virus had caused the effect. Following this promising data, preparation for Phase II studies is underway. In addition to OncoVEXGM-CSF, further versions of the OncoVEX virus expressing other active genes have been constructed and tested in pre-clinical models. These include a virus expressing TNF|[vprop]|, intended to be synergistic with radiotherapy, and versions of the virus expressing a pro-drug activating gene combined with the delivery of a fusogenic glycoprotein designed to maximize the properties of the virus for local tumour control. Each of these have shown promising results in pre-clinical tumour models, including in combination with chemotherapy and radiotherapy where benefits which are at least additive have been demonstrated.

Engineering conditionally replication-competent adenoviral vectors carrying the cytosine deaminase gene increases the infectivity and therapeutic effect for breast cancer gene therapy

We constructed a conditionally replication-competent adenoviral vector Ad.Lp-CD-IRES-E1A(control) in which the expression of both the prodrug-activating cytosine deaminase gene and the viral replication E1A gene were driven by the L-plastin tumor-specific promoter. In order to overcome the low infectivity of the adenoviral vectors for breast cancer cells, and to increase the safety and efficacy for cancer gene therapy, this vector was further modified on a transductional level by simultaneously ablating the native tropism of the vector to the primary CAR receptor and inserting a RGD-4C peptide into the HI loop of the fiber, which allows the vector to use the alphavbeta3 and alphavbeta5 receptors as alternative receptors. The resulting vector was named Ad.Lp-CD-IRES-E1A(MRGD). The transduction efficiency of the vector for breast cancer cell lines which have low expression level of CAR was increased both in vitro and in vivo. The Ad.Lp-CD-IRES-E1A(MRGD) vector produces a higher vector particle yield and a greater cytotoxic effect in tumor cells which have a low expression level of CAR, than did the Ad.Lp-CD-IRES-E1A(control) vector. Intratumoral injection of the Ad.Lp-CD-IRES-E1A(MRGD) vector following the intraperitoneal injection of 5FC into xenotransplanted human breast cancer cell lines which have low expression level of CAR led to greater degree of tumor regression in vivo than did the intratumoral injection of control adenoviral vectors not so modified.

Cancer Scene Investigation: How a Cold Virus Became a Tumor Killer

Oncolytic therapy is a novel anticancer treatment with attenuated lytic viruses such as adenovirus (Ad). These viruses kill the host cells through their lytic replication cycle and are thus distinct from classical gene therapy viruses, which serve as gene delivery agents and do not replicate. Oncolytic Ads are genetically engineered so as to replicate only in cancer cells. Their replication cycle leads to viral multiplication, the killing of the host cells and spreading of the infection throughout the tumor. Following success in preclinical studies, their anti-tumor potential is now being evaluated in the clinic. Three oncolytic Ads (dl1520, Ad5-CD/TKrep, and CV706) have completed Phase I and II clinical trials in cancer patients where their administration via multiple routes and in combination with chemo- or radiotherapies, has demonstrated overall safety. These viruses are being re-engineered to arm them with additional therapeutic genes, bolstering their oncolytic activity with a bystander effect. For example, Ad5-CD/TKrep delivers a therapeutic prodrug-activating (suicide) gene. These data indicate that oncolytic Ads are a promising novel cancer treatment approach that can be combined with other modalities, such as gene therapy and classical chemo- and radiotherapies. Further improvements to enhance their specificity, targeting and oncolytic activity are needed however, as these first-generation viruses showed modest anti-tumor activity. To improve their efficacy in the clinic, it will be important to devise and incorporate novel monitoring techniques in the clinical trials, such as analysis of viral replication in biopsies and through the use of creative noninvasive imaging technologies.

Avian adenovirus vector CELO-TK displays anticancer activity in human cancer cells and suppresses established murine melanoma tumors

Avian adenovirus CELO is a novel adenovirus vector system with the advantages of efficient production, high virion stability, and the absence of crossreactivity with Ad5-neutralizing antibodies. In this study, we evaluated the anticancer efficacy of a CELO vector encoding the herpes simplex virus type 1 thymidine kinase, a prodrug-activating therapeutic gene. Vectors carrying the gene for HSV-tk or EGFP under the control of the HCMV promoter in place of the "nonessential" region of the CELO genome were constructed. Anticancer activity of the CELO-TK vector was studied in vitro, in human and murine tumor cells in cell culture, and in vivo, in established subcutaneous murine B16 melanoma tumors in C57BL/6 mice. The CELO-TK vector mediated delivery of functional HSV-tk to tumor cell lines in cell culture. Comparison of the CELO-TK vector to a first-generation human adenovirus type 5 vector Ad5-TK in cultured H1299 cells showed equal levels of functional activity at increasing multiplicities of infection with CELO-based vector. CELO vectors allowed for transduction and expression of EGFP and HSV-tk genes in subcutaneous melanoma tumors in C57BL/6 mice. Intratumoral injections of CELO-TK followed by ganciclovir administration resulted in suppression of tumor growth and significantly increased the median of survival. The results of the study demonstrated the efficacy of CELO vector as a vehicle for the delivery of prodrug-activating genes such as HSV-tk to tumor cells in vitro and in vivo.