Prodrug Activator Gene Research Articles

673. Therapeutic Efficacy of Retroviral Replicating Vector (RRV) -Mediated Prodrug Activator Gene Therapy for Pancreatic Cancer

Pancreatic ductal adenocarcinoma is one of the most lethal cancers, thus new therapeutic strategies for this disease are urgently needed. Retroviral replicating vectors (RRV)-mediated prodrug activator gene therapy with Toca 511 (vocimagene amiretrorepvec), an optimized RRV encoding yeast cytosine deaminase (yCD) which converts the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU), is showing promising clinical activity in patients with recurrent high grade glioma, and is now being evaluated in a multi-center Phase II/III clinical trial. In the present study, we evaluated the therapeutic efficacy of RRV-mediated prodrug activator gene therapy in preclinical models of pancreatic cancer. We first examined the replication kinetics of RRV expressing the GFP reporter gene (RRV-GFP) in murine (Pan02) and human (MIAPaCa-2, BxPC-3, PANC-1 and SUIT-2) pancreatic cancer cell lines by flow-cytometric analysis and quantitative PCR. In all of these pancreatic cancer lines, RRV-GFP inoculated at MOI=0.05 (~5% initial transduction levels) showed rapid viral replication subsequently resulting in high levels of transduction, with the majority of pancreatic cancer lines reaching >90% GFP-positive cells over time. Next, we tested in vitro cytotoxicity by MTS assay after prodrug treatment of pancreatic cancer cells (Pan02 and MIAPaCa-2) transduced with Toca 511. In RRV-transduced pancreatic cancer cells, significant (~90%) cytotoxicity was induced by exposure to 0.1-1.0 mM 5-FC prodrug for 4 days, compared to untransduced and RRV-GFP transduced controls. We then evaluated in vivo therapeutic efficacy of Toca 511/5-FC prodrug activator gene therapy in Pan02 pancreatic tumor models established subcutaneously in immunocompetent syngeneic C57BL/6 mice. While 5-FC treatment alone showed no obvious inhibition of tumor growth as well as the nontreated control group, the majority (n=7/8) of Toca 511-transduced tumors showed complete regression after 5-FC treatment. Notably, systemic biodistribution studies showed no detectable RRV signals in genomic DNA from normal tissues of treated mice. In orthotopic models using luciferase-marked MIAPaCa-2 tumors established in the pancreas in nude mice, significant inhibition of bioluminescent signals was observed by optical imaging after 5-FC administration in mice with Toca 511-infected tumors, as compared to untreated control tumors. Thus, RRVs are highly efficient vehicles for delivering prodrug activator genes such as yCD to pancreatic cancer cells, thereby achieving significant cell killing upon pro-drug administration. Further preclinical studies are ongoing toward translating this novel therapeutic strategy into clinical trials for patients with pancreatic cancer.

111In-DOTA-Annexin V for imaging of apoptosis during HSV1-tk/GCV prodrug activation gene therapy in mice with NG4TL4 sarcoma

IntroductionApoptosis has been suggested as a cytocidal mechanism of the HSV1-tk-expressing cells when exposed to ganciclovir (GCV). This study evaluated the efficacy of 111In-labeled Annexin V for monitoring tumor responses during prodrug activation gene therapy with HSV1-tk and GCV. Materials and methodsAnnexin V was conjugated to DOTA using N-hydroxysulfosuccinimide (sulfo-NHS) and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC), labeled with 111In-InCl3 and purified using size exclusion chromatography to give 111In-DOTA-Annexin V conjugate. The radiochemical yield and the radiochemical purity of 111In-DOTA-Annexin V were 74±12% and 98±3%, respectively (n=10). 111In-DOTA-BSA was prepared similarly. An in vitro study to demonstrate the apoptosis of NG4TL4-STK cells after GCV treatment has been performed. Mice bearing NG4TL4-STK and NG4TL4-WT tumors were treated with GCV (10mg/kg daily) by i.p. injection for 7 consecutive days. Before and during the GCV treatment, biodistribution studies and scintigraphic imaging were performed at 2h post injection of the radiotracers. ResultsThe uptake of 111In-DOTA-Annexin V in treated cells (13.41±1.30%) was 4.1 times higher than that in untreated cells (3.21±0.37%). The GCV-induced cell apoptosis in NG4TL4-STK tumor resulted in a significantly increasing accumulation of 111In-DOTA-Annexin V (1.92±0.32%ID/g at day 0, 4.79±0.86%ID/g at day 2, 4.56±0.58%ID/g at day 4) was observed, but not for that of 111In-DOTA-BSA. During consecutive GCV treatment, scintigraphic imaging with 111In-DOTA-Annexin V revealed high uptake in NG4TL4-STK tumor compared with that in NG4TL4-WT tumor. However, no specific 111In-DOTA-BSA accumulation in NG4TL4-STK and NG4TL4-WT tumors was observed throughout the course of GCV treatment. ConclusionsThis study demonstrated that 111In-DOTA-Annexin V can be used for monitoring tumor cell apoptosis during prodrug activation gene therapy with HSV1-tk and GCV for cancer treatment.

IMPS-19RETROVIRAL REPLICATING VECTOR-MEDIATED DELIVERY OF AN IMMUNODOMINANT NEO-ANTIGEN EPITOPE TARGET FOR VIRO-IMMUNOTHERAPY IN EXPERIMENTAL GLIOMA

Retroviral replicating vectors (RRV) show intrinsic tropism for actively dividing cells within the immunosuppressed tumor microenvironment, and viral replication significantly enhances transduction efficiency and therapeutic efficacy for cancer gene therapy. RRV-mediated prodrug activator gene therapy is currently being evaluated in clinical trials at multiple centers (www.clinicaltrials.gov, NCT01156584, NCT01470794) with highly encouraging results after local virus injection in patients with recurrent high grade glioma. In murine syngeneic intracranial glioma models, we have shown that RRV-mediated prodrug activator gene therapy can further induce immune responses leading to tumor clearance. We are now investigating the use of RRV for efficient and stable transduction of tumors in situ with an exogenous dominant foreign epitope, thereby xenogenizing the tumor. Here we developed a novel RRV displaying the lymphocytic choriomeningitis virus glycoprotein gp33-41 (gp33) epitope fused to the N-terminus of the viral env gene (RRV-gp33). We confirmed that insertion of the gp33 epitope does not affect RRV replication kinetics, genomic stability over serial passage, or transgene expression. In vitro cell viability measured by xCELLigence electrical impedance assay showed target cell-specific cytotoxicity by transgenic lymphocytes engineered with a T cell receptor recognizing the gp33 epitope (P14 cells) when co-cultured with glioma cells transduced with RRV-gp33. In vivo, adoptively transferred P14 cells selectively homed to RRV-gp33-transduced intracranial syngeneic gliomas in immunocompetent hosts, and mediated increased survival. These results indicate that RRV-mediated gene transfer can be effectively used to confer a new antigen to gliomas as a target for subsequent immunotherapy, and further studies are underway to investigate the therapeutic effect of combining RRV-mediated prodrug activator gene therapy and neo-antigen target delivery for adoptive immunotherapy.

536. Dual-Vector Prodrug Activator Gene Therapy Using Retroviral Replicating Vectors Derived from Amphotropic Murine Leukemia Virus and Gibbon Ape Leukemia Virus

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. An amphotropic murine leukemia virus (AMLV)-based RRV encoding the yeast cytosine deaminase (CD) prodrug activator gene, designated Toca 511 (vocimagene amiretrorepvec), is currently being investigated in combination with Toca FC (extended-release 5-FC) in multi-center clinical trials in the United States for patients with recurrent high-grade glioma (http://www.clinicaltrials.gov NCT01156584, NCT01985256, NCT01470794). This treatment has been safe and well tolerated and median overall survival exceed historical benchmarks. RRV-mediated prodrug activator gene therapy represents the ultimate form of ‘intracellular’ chemotherapy, generated selectively and directly from within the infected cancer cells themselves, without incurring systemic toxicity. Moreover, preclinical data support subsequent activation of the immune system selectively against the cancer. However, some patients may not respond to this treatment, therefore, combination with additional therapeutic agents may be desirable to optimize treatment outcomes. Here we evaluated two different RRVs derived from AMLV and gibbon ape leukemia virus (GALV), encoding two different prodrug activator genes, CD and herpes simplex virus thymidine kinase (TK) in Hep3B human hepatocellular carcinoma cells.Both RRVs expressing the green fluorescent protein gene (AMLV-GFP and GALV-GFP) efficiently replicated in Hep3B cells and spread in culture. Additionally, AMLV-GFP can spread in GALV-mCherry pretransduced Hep3B cells but not in AMLV-mCherry pretransduced cells. Similarly, GALV-GFP can spread in AMLV-mCherry pretransduced cells but not in GALV-mCherry pretransduced cells. This mutually exclusive infection pattern is likely due to receptor interference resulting in superinfection resistance when the same viral strain is used. Notably, however, replication and spread of either RRV in culture was not affected by pretransduction with the counterpart RRV encoated with the other envelope.In order to investigate the effect of combined prodrug-dependent cell killing vs. multiple vector copy transduction in vitro, Hep3B cells were transduced with the AMLV-CD (Ca), GALV-CD (Cg), AMLV-TK (Ta), GALV-TK (Tg), respectively or in combination (Ca/Cg, Ta/Tg, Ca/Tg, Cg/Ta). The resultant cells were used to evaluate the cytotoxic effect of RRV-mediated prodrug activator gene therapy with CD and TK in the presence of their respective prodrugs, 5-fluorocytosine (5-FC) and Ganciclovir (GCV). In vitro cytocidal effects obtained by combining different prodrug activator genes (Ca/Tg and Cg/Ta) were significantly greater than when the same prodrug activator genes (Ca/Cg and Ta/Tg) were delivered with two different vectors. These data indicate the potential utility of dual-vector prodrug activator gene therapy using two different RRVs carrying different prodrug activator genes.

433. Sequence-Optimized Nitroreductase for Retroviral Replicating Vector (RRV) Mediated Prodrug Activator Gene Therapy in Human Glioma Models

Our studies to date have demonstrated dramatic survival benefit when tumor-selective retroviral replicating vectors (RRV) are employed for prodrug activator gene therapy in a variety of preclinical cancer models. RRV-mediated gene therapy using yeast cytosine deaminase (CD) is under investigation in multi-center ascending dose trials in patients with recurrent high grade glioma in the United States. We have further developed an RRV encoding E.coli nitroreductase (NTR), a prodrug activator enzyme which converts CB1954 to a potent bifunctional alkylating agent. We constructed RRV encoding wild-type E.coli NTR genes (RRV-NfsA, RRV-NfsB) as well as NTR variants extensively modified to optimize human codon usage and vector stability (RRV-NAO, RRV-NBO). NTR transgene insertion did not affect vector replication, which resulted in increasing NTR expression over time in U87 human glioma cultures for all vectors, but sequence optimisation significantly increased genomic stability of the RRV-NAO and RRV-NBO vectors over serial passage. U87 cells fully transduced with the optimized vectors showed higher levels of NTR protein and increased levels of enzymatic activity compared with cells transduced with wild-type vectors. In vitro cytotoxicity was examined by MTS assay after CB1954 treatment of fully transduced U87 cells. Viability was reduced by >80% within 48 hrs in cells transduced with RRV-NAO, which showed the most potent cell killing efficiency and bystander effect among all vectors tested. Significant reduction in luminescence and inhibition of tumor growth was observed in subcutaneous U87-FLuc2 tumors initiated with 2% RRV-NAO transduction followed by intraperitoneal administration of CB1954. In intracerebral U87-FLuc2 orthotopic tumor models, stereotactic intratumoral injection of RRV-NAO and repeated cycles of prodrug treatment also resulted in significant luminescence reduction, and achieved prolonged survival benefit. These data indicate that we have been successful in developing an improved prodrug activator gene with therapeutic efficacy when delivered by RRV in experimental models of human glioma.

629. Preclinical Evaluation of Prodrug Activator Gene Therapy by Retroviral Replicating Vectors (RRV) in Experimental Models of Breast Cancer CNS Metastasis

Retroviral replicating vectors (RRV) are capable of highly efficient replication in cancer cells in vitro and in vivo. In a variety of cancer models this is associated with high levels of tumor-selective gene transfer and significantly enhanced survival benefit when employed for prodrug activator gene therapy. An RRV encoding a modified yeast cytosine deaminase (CD) prodrug activator gene (Toca 511) in combination with extended-release flucytosine (Toca FC) is under investigation in multi-center ascending-dose trials in patients with recurrent high grade glioma. In the present study, our goal is to establish the feasibility of also applying this novel gene therapy strategy to secondary brain tumors arising from metastasis of systemic cancers. In particular, brain metastases of breast cancer frequently arise from highly aggressive, treatment-refractory, “triple-negative” (ER(-), PR(-), HER2(-)) cells, and are associated with a dismal prognosis of 4-6 months survival. First, RRV encoding GFP (AC3-emd, RRV-GFP) showed robust replication activity in the human breast cancer tumor cell lines MDA-MB-231-BR and JC over time at both MOI = 0.01 and 0.1, resulting in high levels of transduction within 1-2 weeks. Next, we tested in vitro cytotoxicity after 5-FC treatment of MDA-MB-231BR and JC cells infected with an RRV expressing CD (AC3-yCD2, Toca 511). In both of the AC3-yCD2-transduced cell lines, cell viability was reduced approximately 70-85% after exposure to 12.9 mg/mL 5-FC and complete cell killing was observed with 129 mg/mL 5-FC (6 days 5-FC treatment for 231-BR; 4 days for JC). In a 231-BR intracranial xenograft model, intratumoral injection of 10^6 Transducing Units of RRV-GFP resulted in >80% transduction efficiency on Day 10 post-virus injection. In survival studies, animals treated with Toca 511 and 5-FC showed statistically significant (p<0.0001) survival benefit. These preclinical data support the advancement of RRV gene therapy towards clinical investigation in patients with CNS-metastatic breast cancer.

RRV-mediated prodrug activator gene therapy combined with alloreactive T cell immunotherapy for malignancies in the CNS

RRV-mediated prodrug activator gene therapy combined with alloreactive T cell immunotherapy for malignancies in the CNS

A novel in vivo chemoselection strategy for genetically modified hematopoietic stem cells

leukocyte antigens (HLA) displayed on cancer cells, but absent on normal neuroglia in the CNS. AlloCTL migrate through tumors, and at high effectorto-target (E:T) cell ratios achieve selective alloreactive killing, mediating a localized immunorejection effect. Adoptive alloCTL immunotherapy for glioma is also currently being evaluated in a Phase I study (www.clinicaltrials.gov, NCT01144247). Mixed lymphocyte-target cell assays demonstrated that alloCTL-based RRV producer cells (alloCTL/RRV) maintain migratory and effector function, and efficiently transmitted RRV to co-cultured cancer cells even at E:T ratios as low as 1:100. Combining alloCTL adoptive immunotherapy and RRV prodrug activator gene therapy in vivo showed both early tumor growth inhibition aswell as continued regression upon prodrug administration, achieving significantly improved therapeutic efficacy compared to either treatment alone in orthotopic brain tumor models. Thus, alloCTL/RRV can act both as immunotherapeutic effector cells that directly kill targets at high E:T ratios, and as motile cellular ‘carriers’ that release RRV and facilitate virus spread to unkilled targets at low E:T ratios.

ET-23 * THERAPEUTIC EFFICACY OF RETROVIRAL REPLICATING VECTORS IN HUMAN BREAST CANCER CNS METASTASIS MODEL

Retroviral replicating vectors (RRV) are capable of highly efficient replication in cancer cells in vitro and in vivo. In a variety of cancer models this is associated with high levels of tumor-selective gene transfer and significantly enhanced survival benefit when employed for prodrug activator gene therapy. An RRV encoding a modified yeast cytosine deaminase (CD) prodrug activator gene (Toca 511) in combination with extended-release flucytosine (Toca FC) is under investigation in multi-center ascending-dose trials in patients with recurrent high grade glioma. In the present study, our goal is to establish the feasibility of also applying this novel gene therapy strategy to secondary brain tumors arising from metastasis of systemic cancers. In particular, brain metastases of breast cancer frequently arise from highly aggressive, treatment-refractory, triple-negative (ER(-), PR(-), HER2(-)) cells, and are associated with a dismal prognosis of 4-6 months survival. First, RRV encoding GFP (AC3-emd, RRV-GFP) showed robust replication activity in the human breast cancer tumor cell lines MDA-MB-231-BR and JC over time at both MOI = 0.01 and 0.1, resulting in high levels of transduction within 1-2 weeks. Next, we tested in vitro cytotoxicity after 5-FC treatment of MDA-MB-231BR and JC cells infected with an RRV expressing CD (AC3-yCD2, Toca 511). In both of the AC3-yCD2-transduced cell lines, cell viability was reduced approximately 70-85% after exposure to 12.9 µg/mL 5-FC and complete cell killing was observed with 129 µg/mL 5-FC (6 days 5-FC treatment for 231-BR; 4 days for JC). In a 231-BR intracranial xenograft model, intratumoral injection of 10^6 Transducing Units of RRV-GFP resulted in >80% transduction efficiency on Day 10 post-virus injection. In survival studies, animals treated with Toca 511 and 5-FC showed statistically significant (p < 0.0001) survival benefit. These preclinical data support the advancement of RRV gene therapy towards clinical investigation in patients with CNS-metastatic breast cancer.

ET-13 * RETROVIRAL REPLICATING VECTOR (RRV)-MEDIATED PRODRUG ACTIVATOR GENE THERAPY WITH CODON-OPTIMIZED NITROREDUCTASE IN EXPERIMENTAL GLIOMA

Our studies to date have demonstrated dramatic survival benefit when tumor-selective retroviral replicating vectors (RRV) are employed for prodrug activator gene therapy in a variety of preclinical cancer models. RRV-mediated gene therapy using yeast cytosine deaminase (CD) is under investigation in multi-center ascending dose trials in patients with recurrent high grade glioma in the United States. We have further developed an RRV encoding E.coli nitroreductase (NTR), a prodrug activator enzyme which converts CB1954 to a potent bifunctional alkylating agent. We constructed RRV encoding wild-type E.coli NTR genes (RRV-NfsA, RRV-NfsB) as well as NTR variants extensively modified to optimize human codon usage and vector stability (RRV-NAO, RRV-NBO). NTR transgene insertion did not affect vector replication, which resulted in increasing NTR expression over time in U87 human glioma cultures for all vectors, but sequence optimisation significantly increased genomic stability of the RRV-NAO and RRV-NBO vectors over serial passage. U87 cells fully transduced with the optimized vectors showed higher levels of NTR protein and increased levels of enzymatic activity compared with cells transduced with wild-type vectors. In vitro cytotoxicity was examined by MTS assay after CB1954 treatment of fully transduced U87 cells. Viability was reduced by >80% within 48 hrs in cells transduced with RRV-NAO, which showed the most potent cell killing efficiency and bystander effect among all vectors tested. Significant reduction in luminescence and inhibition of tumor growth was observed in subcutaneous U87-FLuc2 tumors initiated with 2% RRV-NAO transduction followed by intraperitoneal administration of CB1954. In intracerebral U87-FLuc2 orthotopic tumor models, stereotactic intratumoral injection of RRV-NAO and repeated cycles of prodrug treatment also resulted in significant luminescence reduction, and achieved prolonged survival benefit. These data indicate that we have been successful in developing an improved prodrug activator gene with therapeutic efficacy when delivered by RRV in experimental models of human glioma.

Radiosensitization of gliomas by intracellular generation of 5-fluorouracil potentiates prodrug activator gene therapy with a retroviral replicating vector.

A tumor-selective non-lytic retroviral replicating vector (RRV), Toca 511, and an extended-release formulation of 5-fluorocytosine (5-FC), Toca FC, are currently being evaluated in clinical trials in patients with recurrent high-grade glioma (NCT01156584, NCT01470794, NCT01985256). Tumor-selective propagation of this RRV enables highly efficient transduction of glioma cells with cytosine deaminase (CD), which serves as a prodrug activator for conversion of the anti-fungal prodrug 5-FC to the anti-cancer drug 5-fluorouracil (5-FU) directly within the infected cells. We investigated whether, in addition to its direct cytotoxic effects, 5-FU generated intracellularly by RRV-mediated CD/5-FC prodrug activator gene therapy could also act as a radiosensitizing agent. Efficient transduction by RRV and expression of CD was confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87-EGFRvIII and its parental cell line U87MG (U87). RRV-transduced cells showed significant radiosensitization even after transient exposure to 5-FC. This was confirmed both in vitro by a clonogenic colony survival assay and in vivo by bioluminescence imaging analysis. These results provide a convincing rationale for development of tumor-targeted radiosensitization strategies utilizing the tumor-selective replicative capability of RRV, and incorporation of radiation therapy into future clinical trials evaluating Toca 511 and Toca FC in brain tumor patients.

Adenoviral Vectors for Prodrug Activation-based Gene Therapy for Cancer

Cancer cell heterogeneity is a common feature - both between patients diagnosed with the same cancer and within an individual patient's tumor - and leads to widely different response rates to cancer therapies and the potential for the emergence of drug resistance. Diverse therapeutic approaches have been developed to combat the complexity of cancer, including individual treatment modalities designed to target tumor heterogeneity. This review discusses adenoviral vectors and how they can be modified to replicate in a cancer-specific manner and deliver therapeutic genes under multi-tiered regulation to target tumor heterogeneity, including heterogeneity associated with cancer stem cell-like subpopulations. Strategies that allow for combination of prodrug-activation gene therapy with a novel replication-conditional, heterogeneous tumor-targeting adenovirus are discussed, as are the benefits of using adenoviral vectors as tumor-targeting oncolytic vectors. While the anticancer activity of many adenoviral vectors has been well established in preclinical studies, only limited successes have been achieved in the clinic, indicating a need for further improvements in activity, specificity, tumor cell delivery and avoidance of immunogenicity.

Highly efficient tumor transduction and antitumor efficacy in experimental human malignant mesothelioma using replicating gibbon ape leukemia virus

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefit in a wide variety of cancer models. Here we evaluated two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), in human malignant mesothelioma cells. In vitro, both RRVs expressing the green fluorescent protein gene efficiently replicated in most mesothelioma cell lines tested, but not in normal mesothelial cells. Notably, in ACC-MESO-1 mesothelioma cells that were not permissive for AMLV-RRV, the GALV-RRV could spread efficiently in culture and in mice with subcutaneous xenografts by in vivo fluorescence imaging. Next, GALV-RRV expressing the cytosine deaminase prodrug activator gene showed efficient killing of ACC-MESO-1 cells in a prodrug 5-fluorocytosine dose-dependent manner, compared with AMLV-RRV. GALV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous ACC-MESO-1 tumor growth in nude mice. Quantitative reverse transcription PCR demonstrated that ACC-MESO-1 cells express higher PiT-1 (GALV receptor) and lower PiT-2 (AMLV receptor) compared with normal mesothelial cells and other mesothelioma cells, presumably accounting for the distinctive finding that GALV-RRV replicates much more robustly than AMLV-RRV in these cells. These data indicate the potential utility of GALV-RRV-mediated prodrug activator gene therapy in the treatment of mesothelioma.

Combined Alloreactive CTL Cellular Therapy with Prodrug Activator Gene Therapy in a Model of Breast Cancer Metastatic to the Brain

Individual or combined strategies of cellular therapy with alloreactive CTLs (alloCTL) and gene therapy using retroviral replicating vectors (RRV) encoding a suicide prodrug activating gene were explored for the treatment of breast tumors metastatic to the brain. AlloCTL, sensitized to the HLA of MDA-MB-231 breast cancer cells, were examined in vitro for antitumor functionality toward breast cancer targets. RRV encoding the yeast cytosine deaminase (CD) gene was tested in vivo for virus spread, ability to infect, and kill breast cancer targets when exposed to 5-fluorocytosine (5-FC). Individual and combination treatments were tested in subcutaneous and intracranial xenograft models with 231BR, a brain tropic variant. AlloCTL preparations were cytotoxic, proliferated, and produced IFN-γ when coincubated with target cells displaying relevant HLA. In vivo, intratumorally placed alloCTL trafficked through one established intracranial 231BR focus to another in contralateral brain and induced tumor cell apoptosis. RRV-CD efficiently spread in vivo, infected 231BR and induced their apoptosis upon 5-FC exposure. Subcutaneous tumor volumes were significantly reduced in alloCTL and/or gene therapy-treated groups compared to control groups. Mice with established intracranial 231BR tumors treated with combined alloCTL and RRV-CD had a median survival of 97.5 days compared with single modalities (50-83 days); all experimental treatment groups survived significantly longer than sham-treated groups (median survivals 31.5 or 40 days) and exhibited good safety/toxicity profiles. The results indicate combining cellular and suicide gene therapies is a viable strategy for the treatment of established breast tumors in the brain.

70. Retroviral Replicating Vector (RRV)-Mediated Prodrug-Activator Gene Therapy Transcriptionally Targeted to Prostate Cancer

70. Retroviral Replicating Vector (RRV)-Mediated Prodrug-Activator Gene Therapy Transcriptionally Targeted to Prostate Cancer

Toxicology and Biodistribution Studies for MGH2.1, an Oncolytic Virus that Expresses Two Prodrug-activating Genes, in Combination with Prodrugs

MGH2.1 is a herpes simplex virus type 1 (HSV1) oncolytic virus that expresses two prodrug-activating transgenes: the cyclophosphamide (CPA)-activating cytochrome P4502B1 (CYP2B1) and the CPT11-activating secreted human intestinal carboxylesterase (shiCE). Toxicology and biodistribution of MGH2.1 in the presence/absence of prodrugs was evaluated in mice. MGH2.1 ± prodrugs was cytotoxic to human glioma cells, but not to normal cells. Pharmacokinetically, intracranial MGH2.1 did not significantly alter the metabolism of intraperitoneally (i.p.) administered prodrugs in mouse plasma, brain, or liver. MGH2.1 did not induce an acute inflammatory reaction. MGH2.1 DNA was detected in brains of mice inoculated with 108 pfus for up to 60 days. However, only one animal showed evidence of viral gene expression at this time. Expression of virally encoded genes was restricted to brain. Intracranial inoculation of MGH2.1 did not induce lethality at 108 pfus in the absence of prodrugs and at 106 pfus in the presence of prodrugs. This study provides safety and toxicology data justifying a possible clinical trial of intratumoral injection of MGH2.1 with peripheral administration of CPA and/or CPT11 prodrugs in humans with malignant gliomas.

Ascending dose trials of the safety and tolerability of Toca 511, a retroviral replicating vector encoding cytosine deaminase, in patients with recurrent high-grade glioma.

2101 Background: Patients with the most common and aggressive form of brain cancer, glioblastoma multiforme (GBM), have a poor prognosis and current treatments are only palliative. Gene therapy approaches using replication-deficient viral vectors have been investigated previously for GBM with limited efficacy, likely due to poor gene delivery throughout the tumor. Methods: Toca 511 is a novel retroviral replicating vector (RRV) that is engineered to deliver a modified cytosine deaminase prodrug activator gene (CD) selectively to cancer cells. In those cancer cells expressing the CD gene, the antifungal prodrug 5-FC is converted to 5-FU. We are currently evaluating the safety and tolerability of a single dose of Toca 511 administered by direct intratumoral injection to subjects with recurrent high grade glioma (rHGG). After a period of time during which the vector is allowed to spread through the tumor, the patient begins oral 5-FC which is repeated cyclically. Results: Three subjects have been enrolled in each of the first two dose groups. No dose limiting toxicities (DLTs) have been identified in these groups and the treatment was well tolerated. There have been no grade 3 or 4 AEs considered related to either Toca 511 or 5-FC. A further 9 subjects were then enrolled at the 3rd dose level and are being followed. A second study has been initiated in which Toca 511 is injected into the walls of the resection cavity at the time of surgery for rHGG and followed by cyclic treatment with oral 5-FC. Conclusions: These clinical studies are the first to investigate a RRV to deliver genes to human brain tumors and further dose escalation is anticipated.

Combination of a fusogenic glycoprotein, pro-drug activation and oncolytic HSV as an intravesical therapy for superficial bladder cancer

Background:There are still no effective treatments for superficial bladder cancer (SBC)/non-muscle invasive bladder cancer. Following treatment, 20% of patients still develop metastatic disease. Superficial bladder cancer is often multifocal, has high recurrences after surgical resection and recurs after intravesical live Bacillus Calmette–Guérin. OncovexGALV/CD, an oncolytic herpes simplex virus-1, has shown enhanced local tumour control by combining oncolysis with the expression of a highly potent pro-drug activating gene and the fusogenic glycoprotein.Methods:In vitro fusion/prodrug/apoptotic cell-based assays. In vivo orthotopic bladder tumour model, visualised by computed microtomography.Results:Treatment of seven human bladder carcinoma cell lines with the virus resulted in tumour cell killing through oncolysis, pro-drug activation and glycoprotein fusion. OncovexGALV/CD and mitomycin C showed a synergistic effect, whereas the co-administration with cisplatin or gemcitabine showed an antagonistic effect in vitro. Transitional cell cancer (TCC) cells follow an apoptotic cell death pathway after infection with OncovexGALV/CD with or without 5-FC. In vivo results showed that intravesical treatment with OncovexGALV/CD + prodrug (5-FC) reduced the average tumour volume by over 95% compared with controls.Discussion:Our in vitro and in vivo results indicate that OncovexGALV/CD can improve local tumour control within the bladder, and potentially alter its natural history.

Blocking effects of siRNA on VEGF expression in human triple negetive breast l cancer cell line

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 coli nfsB 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.

Adenovirus-mediated hypoxia-targeted gene therapy using HSV thymidine kinase and bacterial nitroreductase prodrug-activating genes in vitro and in vivo

Hypoxia is an important factor in tumor growth. It is associated with resistance to conventional anticancer treatments. Gene therapy targeting hypoxic tumor cells therefore has the potential to enhance the efficacy of treatment of solid tumors. Transfection of a panel of tumor cell lines with plasmid constructs containing hypoxia-responsive promoter elements from the genes, vascular endothelial growth factor (VEGF) and erythropoietin, linked to the minimal cytomegalovirus (mCMV) or minimal interleukin-2 (mIL-2) promoters showed optimum hypoxia-inducible luciferase reporter gene expression with five repeats of VEGF hypoxic-response element linked to the mCMV promoter. Adenoviral vectors using this hypoxia-inducible promoter to drive therapeutic transgenes produced hypoxia-specific cell kill of HT1080 and HCT116 cells in the presence of prodrug with both herpes simplex virus thymidine kinase/ganciclovir and nitroreductase (NTR)/CB1954 prodrug-activating systems. Significant cytotoxic effects were also observed in patient-derived human ovarian cancer cells. The NTR/CB1954 system provided more readily controllable transgene expression and so was used for in vivo experiments of human HCT116 xenografts in nude mice. Subjects treated intratumorally with Ad-VEGFmCMV-NTR and intraperitoneal injection of CB1954 demonstrated a statistically significant reduction in tumor growth. Immunohistochemistry of treated xenografts showed a good correlation between transgene expression and hypoxic areas. Further investigation of these hypoxia-inducible adenoviral vectors, alone or in combination with existing modalities of cancer therapy, may aid in the future development of successful Gene-Directed Enzyme Prodrug Therapy systems, which are much needed for targeting solid tumors.