Prodrug Activator Gene Research Articles

Therapeutic Efficacy of Prodrug Activator Gene Therapy Using Retroviral Replicating Vectors for Human Ovarian Cancer.

Retroviral replicating vectors (RRV) have exhibited efficient tumor transduction and improved therapeutic benefits in a variety of cancer models. In this study, we validated two RRV created from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which use different cell receptors for virus entry, in human ovarian cancer (OC) cells. Expression levels of the receptors for AMLV (PiT-2) and GALV (PiT-1) in human OC cell lines (A2780, Caov3, RMG-1, SKOV-3), fibroblasts and HEK293 cells were evaluated using quantitative RT-PCR. In vitro RRV-GFP replication was monitored using flow cytometry, and cytotoxicity quantitated using AlamarBlue assay after 5-fluorocytosine treatment of OC cells transduced with RRV expressing the yeast cytosine deaminase prodrug activator gene. In vivo antitumor effect of RRV-mediated prodrug activator gene therapy was investigated in a SKOV-3 subcutaneous tumor model. Quantitative RT-PCR analysis revealed high expression levels of PiT-2 (AMLV receptor) and PiT-1 (GALV receptor) in the RMG-1 and SKOV3 OC cell lines, compared with their levels in non-malignant cells. In RMG-1 and SKOV3 cells, both RRV showed highly efficient RRV replication and spread leading to over 90% transduction by Days 10-13. Additionally, both RRV that express the yeast cytosine deaminase gene demonstrated effective cell killing of RMG-1 and SKOV-3 cells upon treatment with the prodrug 5-fluorocytosine. Notably, RRV-mediated prodrug activator gene therapy showed significant inhibition of subcutaneous SKOV-3 tumor growth in nude mice. RRV-mediated prodrug activator gene therapy may be used for treating PiT-expressing human OC.

Retroviral Replicating Vectors Mediated Prodrug Activator Gene Therapy in a Gastric Cancer Model.

Retroviral replicating vectors (RRVs) selectively replicate and can specifically introduce prodrug-activating genes into tumor cells, whereby subsequent prodrug administration induces the death of the infected tumor cells. We assessed the ability of two distinct RRVs generated from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV), which infect cells via type-III sodium-dependent phosphate transporters, PiT-2 and PiT-1, respectively, to infect human gastric cancer (GC) cells. A quantitative RT-PCR showed that all tested GC cell lines had higher expression levels of PiT-2 than PiT-1. Accordingly, AMLV, encoding a green fluorescent protein gene, infected and replicated more efficiently than GALV in most GC cell lines, whereas both RRVs had a low infection rate in human fibroblasts. RRV encoding a cytosine deaminase prodrug activator gene, which converts the prodrug 5-flucytosine (5-FC) to the active drug 5-fluorouracil, showed that AMLV promoted superior 5-FC-induced cytotoxicity compared with GALV, which correlated with the viral receptor expression level and viral spread. In MKN-74 subcutaneous xenograft models, AMLV had significant antitumor effects compared with GALV. Furthermore, in the MKN-74 recurrent tumor model in which 5-FC was discontinued, the resumption of 5-FC administration reduced the tumor volume. Thus, RRV-mediated prodrug activator gene therapy might be beneficial for treating human GC.

Inhibition of human oral squamous cell carcinoma proliferation and migration by prodrug-activating suicide gene therapies.

Head and neck squamous cell carcinoma (HNSCC), which originates from mucosal epithelium in the oral cavity, pharynx and larynx, is the sixth most common malignancy in the world. The prognosis of HNSCC is not satisfactory due to metastasis, resulting in 5-year survival rates ranging from 65.9 to 67.2%. Previously, we developed a method to evaluate the effect prodrug-activating suicide gene (PA-SG) therapy on the proliferation of HNSCC. The present study investigated PA-SG therapy on metastatic HNSCC by wound-healing assay and our previously established method. HSC-3 cells with stable expression of suicide genes thymidine kinase (TK) or cytosine deaminase (CD) were treated with prodrugs ganciclovir (GCV) or 5-fluorocytosine (5-FC), respectively. Both GCV and 5-FC inhibited HSC-3 proliferation while the bystander effect of CD/5-FC was greater compared with that of TK/GCV. GCV showed a greater anti-migration effect compared with that of 5-FC. To the best of our knowledge, the present study is the first to evaluate the anti-migratory and anti-proliferative effects of PA-SG therapies on metastatic HNSCC. This may also serve as a general method to quantify other types of PA-SC therapy. The present results demonstrated that PA-SG therapy is a promising treatment for anti-metastatic HNSCC therapy development.

Retroviral Replicating Vector Toca 511 (Vocimagene Amiretrorepvec) for Prodrug Activator Gene Therapy of Lung Cancer.

Therapeutic efficacy of retroviral replicating vector (RRV)-mediated prodrug activator gene therapy has been demonstrated in a variety of tumor models, but clinical investigation of this approach has so far been restricted to glioma and gastrointestinal malignancies. In the present study, we evaluated replication kinetics, transduction efficiency, and therapeutic efficacy of RRV in experimental models of lung cancer. RRV delivering GFP as a reporter gene showed rapid viral replication in a panel of lung cancer cells in vitro, as well as robust intratumoral replication and high levels of tumor transduction in subcutaneous and orthotopic pleural dissemination models of lung cancer in vivo. Toca 511 (vocimagene amiretrorepvec), a clinical-stage RRV encoding optimized yeast cytosine deaminase (yCD) which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil (5-FU), showed potent cytotoxicity in lung cancer cells upon exposure to 5-FC prodrug. In vivo, Toca 511 achieved significant tumor growth inhibition following 5-FC treatment in subcutaneous and orthotopic pleural dissemination models of lung cancer in both immunodeficient and immunocompetent hosts, resulting in significantly increased overall survival. This study demonstrates that RRV can serve as highly efficient vehicles for gene delivery to lung cancer, and indicates the translational potential of RRV-mediated prodrug activator gene therapy with Toca 511/5-FC as a novel therapeutic strategy for pulmonary malignancies.

EXTH-33. RETROVIRAL REPLICATING VECTORS PSEUDOTYPED WITH GIBBON APE LEUKEMIA VIRUS ENVELOPE FOR PRODRUG ACTIVATOR GENE THERAPY IN PRECLINICAL GLIOMA MODELS

Abstract Amphotropic retroviral replicating vector (RRV) Toca 511, expressing the yeast cytosine deaminase (CD) prodrug activator gene, showed promising evidence of therapeutic benefit and increased survival in early-phase trials for recurrent high-grade glioma. While a multi-center Phase 3 trial did not meet its overall endpoints, highly statistically significant survival was observed within predetermined patient subgroups compared to matched randomized control patients, and clinical investigation is on-going. Hence it is worthwhile to consider strategies aimed at enhancing therapeutic efficacy, such as delivering combinations of multiple transgenes. However, RRVs encoated with the same envelope compete for the same cancer cell surface receptors. We have now developed novel RRV encoated (‘pseudotyped’) with a heterologous envelope derived from Gibbon ape leukemia virus (GALV), which utilizes a different cell surface receptor from the native amphotropic retrovirus envelope for cellular entry. RRV(GALV) vectors expressing either GFP or HSV thymidine kinase (TK) were constructed, and efficient replication and transgene expression was observed in > 90% of both established and primary human glioblastoma cells within 14 days after initial infection at 0.01 (1%) multiplicity of infection (MOI). Genomic stability of RRV(GALV) vectors was also confirmed over prolonged propagation. Established and primary human glioblastoma cells infected with RRV(GALV)-TK vector showed ≥ 50%-90% reduction in cell viability after exposure to Ganciclovir prodrug in the range of 1µM-100µM for 5 days, as compared to uninfected control cells or cells infected with RRV(GALV)-GFP control vector. Furthermore, dual infection with RRV(GALV)-TK and amphotropic RRV-CD (Toca 511) resulted in synergistic cytotoxicity upon simultaneous exposure to their respective prodrugs. Further data will be presented from on-going studies evaluating these vectors in intracerebral glioblastoma models. These results indicate that GALV envelope-pseudotyped RRV can efficiently deliver prodrug activator gene therapy in experimental glioma models, and open the door to combinatorial gene therapy regimens with this vector platform.

THER-06. THERAPEUTIC EFFICACY OF RRV-MEDIATED PRODRUG ACTIVATOR GENE THERAPY IN CLINICAL TRIALS OF RECURRENT HIGH-GRADE GLIOMA AND IN MURINE ORTHOTOPIC MODELS OF INTRACEREBRAL GLIOMA AND INTRACEREBELLAR MEDULLOBLASTOMA

Toca 511, a clinical-stage tumor-selective retroviral replicating vector (RRV), encodes optimized yeast cytosine deaminase (CD), which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil (5-FU) within infected cancer cells. In preclinical models of intracerebral glioblastoma, 5-FU generated locally by Toca 511 (RRV-CD) prodrug activator gene therapy has also been shown to kill immunosuppressive myeloid cells in the tumor microenvironment, leading to anti-cancer immune activation and long-term survival. Early-phase clinical trials of Toca 511 in recurrent high-grade glioma showed highly promising evidence of therapeutic benefit, leading to a Phase III trial completed in late 2019 (n=400 patients, randomized 1:1 vs. standard chemotherapy), which appeared to show negative results overall. However, additional analysis showed possible efficacy in prespecified subgroups, and further clinical investigation is being pursued. In preclinical studies, we have also evaluated RRV for use in medulloblastoma, the most common malignant tumor of the pediatric nervous system. Both established and primary human medulloblastoma cell lines supported efficient RRV replication in vitro, with spread to >90% of cells by day 10 post-inoculation, and RRV-CD-transduced medulloblastoma cells showed significant dose-dependent reduction of viability upon exposure to 5-FC, compared to controls. In an intracerebellar HDMB03 medulloblastoma model, RRV-CD-treated mice exhibited long-term survival while on sequential cycles of 5-FC prodrug, until prodrug treatment was stopped, after which 25% long-term survival was observed (median survival 110 days) as compared to controls (median survival 28 days, 100% lethality) (p=0.00007). These results support further evaluation of RRV-mediated prodrug activator gene therapy for pediatric brain tumors.

EXTH-44. DEVELOPMENT OF RETROVIRAL REPLICATING VECTORS ENCODING SEQUENCE-OPTIMIZED NITROREDUCTASE FOR PRODRUG ACTIVATOR GENE THERAPY IN HUMAN GLIOMA MODELS

Abstract Early-phase clinical trials of Toca 511, a retroviral replicating vector (RRV) expressing an optimized form of yeast cytosine deaminase for prodrug activator gene therapy, showed highly promising evidence of therapeutic benefit and increased survival in recurrent high-grade glioma, but an international Phase 3 trial failed to meet primary and secondary endpoints. Nonetheless, promising results observed within predetermined subgroups, combined with the highly favorable safety profile of this approach, make it worthwhile to consider alternative prodrug-activating enzymes in order to increase therapeutic efficacy. In this study, we employed RRV to deliver modified versions of another prodrug activator gene, E.coli nitroreductase (NTR), which activates the prodrug CB1954 to generate a potent bifunctional alkylating agent. We constructed RRV encoding two different isoforms of wild-type E.coli NTR genes (NfsA, NfsB) as well as variants of both isoforms extensively modified to optimize human codon usage and vector stability. Further efforts to improve the potency of these prodrug activator enzymes included the construction of additional new vectors incorporating point mutations at the active site which have previously been demonstrated to improve prodrug conversion efficiency. NTR transgene insertion did not affect RRV replication, which resulted in increasing NTR expression over time in human glioma cells in culture. Sequence optimization resulted in enhanced genomic stability of the vectors upon serial passage and also in significantly reduced cell viability in vitro on treatment of fully transduced U87 and U87-EGFRviii glioma cells with CB1954 prodrug. In orthotopic U87-EGFRviii intracerebral tumor models, stereotactic intratumoral injection of sequence-optimized RRV-NTR and repeated cycles of prodrug treatment resulted in rapid and significant reduction of tumor burden as monitored by bioluminescence imaging, leading to prolonged survival benefit. These data indicate that improved therapeutic efficacy can be achieved with optimized NTR prodrug activator gene therapy delivered by RRV in experimental glioma models.

Efficient Prodrug Activator Gene Therapy by Retroviral Replicating Vectors Prolongs Survival in an Immune-Competent Intracerebral Glioma Model.

Prodrug activator gene therapy mediated by murine leukemia virus (MLV)-based retroviral replicating vectors (RRV) was previously shown to be highly effective in killing glioma cells both in culture and in vivo. To avoid receptor interference and enable dual vector co-infection with MLV-RRV, we have developed another RRV based on gibbon ape leukemia virus (GALV) that also shows robust replicative spread in a wide variety of tumor cells. We evaluated the potential of GALV-based RRV as a cancer therapeutic agent by incorporating yeast cytosine deaminase (CD) and E. coli nitroreductase (NTR) prodrug activator genes into the vector. The expression of CD and NTR genes from GALV-RRV achieved highly efficient delivery of these prodrug activator genes to RG-2 glioma cells, resulting in enhanced cytotoxicity after administering their respective prodrugs 5-fluorocytosine and CB1954 in vitro. In an immune-competent intracerebral RG-2 glioma model, GALV-mediated CD and NTR gene therapy both significantly suppressed tumor growth with CB1954 administration after a single injection of vector supernatant. However, NTR showed greater potency than CD, with control animals receiving GALV-NTR vector alone (i.e., without CB1954 prodrug) showing extensive tumor growth with a median survival time of 17.5 days, while animals receiving GALV-NTR and CB1954 showed significantly prolonged survival with a median survival time of 30 days. In conclusion, GALV-RRV enabled high-efficiency gene transfer and persistent expression of NTR, resulting in efficient cell killing, suppression of tumor growth, and prolonged survival upon CB1954 administration. This validates the use of therapeutic strategies employing this prodrug activator gene to arm GALV-RRV, and opens the door to the possibility of future combination gene therapy with CD-armed MLV-RRV, as the latter vector is currently being evaluated in clinical trials.

Targeted cell ablation in zebrafish using optogenetic transcriptional control.

Cell ablation is a powerful method for elucidating the contributions of individual cell populations to embryonic development and tissue regeneration. Targeted cell loss in whole organisms has been typically achieved through expression of a cytotoxic or prodrug-activating gene product in the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s). To address this limitation, we have used the light-inducible GAVPO transactivator in combination with two genetically encoded cell-ablation technologies: the nitroreductase/nitrofuran system and a cytotoxic variant of the M2 ion channel. Our studies establish ablative methods that provide the tissue specificity afforded by cis-regulatory elements and the conditionality of optogenetics. Our studies also demonstrate differences between the nitroreductase and M2 systems that influence their efficacies for specific applications. Using this integrative approach, we have ablated cells in zebrafish embryos with both spatial and temporal control.

Dual-vector prodrug activator gene therapy using retroviral replicating vectors

Retroviral replicating vectors (RRVs) have been shown to achieve efficient tumor transduction and enhanced therapeutic benefits in a variety of cancer models. In the present study, we evaluated a possible combinatorial effect of prodrug activator genes delivered by two different RRVs derived from amphotropic murine leukemia virus (AMLV) and gibbon ape leukemia virus (GALV) on human hepatocellular carcinoma Hep3B cells. Both RRVs showed efficient replicative spread in culture and can overcame superinfection resistance each other. Notably, the replication and spread of each RRV in culture remained unaffected by pretransduction with the counterpart RRV. We further transduced cells with RRVs which individually possessed the prodrug activator genes yeast cytosine deaminase (CD) and herpes simplex virus thymidine kinase (TK) alone or in combination, and evaluated the cytotoxic effects of RRV-mediated gene therapy with CD and TK in the presence of the respective prodrugs, 5-fluorocytosine and ganciclovir. All combinations of the two prodrug activator genes produced synergistic cytocidal effects, but the combined effects of the different genes were significantly greater than those of the same genes when delivered by two different vectors. The present findings indicate the potential utility of dual-vector gene therapy using two different RRVs carrying different prodrug activator genes.

Efficient tumor transduction and antitumor efficacy in experimental human osteosarcoma using retroviral replicating vectors

Retroviral replicating vectors (RRVs) have achieved 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), which utilize different cellular receptors (PiT-2 and PiT-1, respectively) for viral entry, in human osteosarcoma cells. Quantitative RT-PCR showed that low levels of expression of both receptors were observed in normal and non-malignant cells. However, high PiT-2 (for AMLV) and low PiT-1 (for GALV) expression was observed in most osteosarcoma cell lines. Accordingly, AMLV expressing the green fluorescent protein gene infected and replicated more efficiently than GALV in most osteosarcoma cell lines. Furthermore, RRVs expressing the cytosine deaminase prodrug activator gene showed differential cytotoxicity that correlated with the results of viral spread. AMLV-RRV-mediated prodrug activator gene therapy achieved significant inhibition of subcutaneous MG-63 tumor growth over GALV in nude mice. These data indicate that AMLV vectors predominate over GALV in human osteosarcoma cells. Moreover, our findings support the potential utility of the two RRVs in personalized cancer virotherapy on the basis of receptor expression.

Abstract 5927: Assessing the efficacy of a novel gene therapy approach for treating ovarian cancer: Combination RRV-mediated prodrug activated suicide gene therapy

Abstract Introduction/Aim: Retroviral replicating vectors (RRVs) afford a unique strategy for gene therapy targeting cancer cells. By “activating” RRVs with prodrug activator suicide genes, these RRVs become strong gene delivery vehicles that replicate selectively in cancer cells, and stably persist through proviral genomic integration, leading to inducible cell death upon prodrug administration. This strategy has shown highly promising clinical results in early phase trials (published), and is currently being evaluated in an international Phase III clinical trial for patients with recurrent high-grade glioma. Recently, we have also applied RRV-mediated gene therapy to preclinical models of ovarian cancer. The aim of this current study was to evaluate feasibility and efficacy of combination suicide gene therapy using two RRVs encoated (‘pseudotyped') with different envelope proteins and delivering different prodrug activators for the treatment of ovarian cancer. Methods: Viral spread was monitored in established and primary patient-derived ovarian cancer cell lines (SKOV3, OCI-P5X, OCI-C5X) over the course of ≥27 days using amphotropic murine leukemia virus (AMLV) envelope- and gibbon ape leukemia virus (GALV) envelope-pseudotyped RRVs delivering either fluorescent protein reporter genes (GFP, mStrawberry) or prodrug activator genes (yeast cytosine deaminase, E. coli nitroreductase), either individually or in combination, at various multiplicities of infection (MOI). Flow cytometry for GFP, mStrawberry, and Gag viral protein was used to determine transduction levels and transgene expression levels of RRVs at serial time points. Each therapeutic RRV was tested by MTS assay for its ability to induce cytotoxicity upon exposure to different concentrations prodrug. To determine viral integration and vector stability, PCR of genomic DNA extracted from infected cells was performed using RRV specific primers, and qRT-PCR was performed to determine vector copy number per cell. Results: All ovarian cancer cell lines tested could be >80% transduced by AMLV- and GALV-pseudotyped RRVs, both individually and in combination, within 6-24 days even after initial inoculation at MOI 0.001, with replication kinetics varying between different cell lines. Both RRVs could stably integrate into the target cell genome without evidence of transgene deletion. MTS assays demonstrated that prodrug activated cytotoxicity led to efficient killing of RRV-transduced ovarian cancer cells in vitro, with synergistic cytotoxicity observed at lower prodrug concentrations after combined treatment. Conclusion: RRV-mediated prodrug activator gene therapy may be an effective approach for efficiently and selectively inducing cytotoxicity in ovarian cancer cells. These data support further evaluation of this strategy through translational studies in vivo. Citation Format: James Grosso, Sara Collins, Aki Inagaki, Brian Slomovitz, Noriyuki Kasahara. Assessing the efficacy of a novel gene therapy approach for treating ovarian cancer: Combination RRV-mediated prodrug activated suicide gene therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5927.

Therapeutic activity of retroviral replicating vector-mediated prodrug activator gene therapy for pancreatic cancer.

Toca 511, a retroviral replicating vector (RRV) encoding the yeast cytosine deaminase (yCD) prodrug activator gene, which mediates conversion of the prodrug 5-fluorocytosine (5-FC) to the anticancer drug 5-fluorouracil (5-FU), is currently being evaluated in Phase II/III clinical trials for glioma, and showing highly promising evidence of therapeutic activity. Here we evaluated RRV-mediated prodrug activator gene therapy as a new therapeutic approach for pancreatic ductal adenocarcinoma (PDAC). RRV spread rapidly and conferred significant cytotoxicity with prodrug in a panel of PDAC cells. Efficient intratumoral replication and complete inhibition of tumor growth upon 5-FC administration were observed in both immunodeficient and immunocompetent subcutaneous PDAC models. Biodistribution of RRV was highly restricted in normal tissues, especially in immunocompetent hosts. Tumor growth inhibition by Toca 511 followed by 5-FC was also confirmed in the orthotopic PDAC model. This study provides the first proof-of-concept for application of Toca 511 and Toca FC (extended release 5-FC) to the treatment of human PDAC, and provided support for inclusion of PDAC in a Phase I study evaluating Toca 511 in various systemic malignancies, (NCT02576665), which has recently been initiated.

Pseudotyped retroviral replicating vectors for combination prodrug activator gene therapy of ovarian cancer

Pseudotyped retroviral replicating vectors for combination prodrug activator gene therapy of ovarian cancer

Vocimagene amiretrorepvec (Toca 511) for prodrug activator gene therapy in ovarian cancer models

Objective: Ovarian cancer is the leading cause of death in women with gynecologic malignancies in the United Staes, with <50% overall 5-year survival. Our studies to date have demonstrated significant survival benefit when tumor-selective retroviral replicating vectors (RRV) are employed for gene therapy in a variety of preclinical cancer models. RRV-mediated prodrug activator gene therapy using yeast cytosine deaminase (RRV-CD, ‘Toca 511’) is now being evaluated in a registered phase IIB/III trial at multiple sites throughout the United States and Canada (NCT02414165) for recurrent glioma. As FDA approved initiation of clinical trials for intravenous delivery of RRV, it is timely to consider applying this approach also to systemic malignancies such as ovarian cancer. Accordingly, we have conducted the first preclinical studies to evaluate the feasibility, safety, and efficacy of RRV-mediated prodrug activator gene therapy for ovarian cancer.

Retroviral replicating vector-mediated gene therapy achieves long-term control of tumor recurrence and leads to durable anticancer immunity.

Background.Prodrug-activator gene therapy with Toca 511, a tumor-selective retroviral replicating vector (RRV) encoding yeast cytosine deaminase, is being evaluated in recurrent high-grade glioma patients. Nonlytic retroviral infection leads to permanent integration of RRV into the cancer cell genome, converting infected cancer cell and progeny into stable vector producer cells, enabling ongoing transduction and viral persistence within tumors. Cytosine deaminase in infected tumor cells converts the antifungal prodrug 5-fluorocytosine into the anticancer drug 5-fluorouracil, mediating local tumor destruction without significant systemic adverse effects.Methods.Here we investigated mechanisms underlying the therapeutic efficacy of this approach in orthotopic brain tumor models, employing both human glioma xenografts in immunodeficient hosts and syngeneic murine gliomas in immunocompetent hosts.Results.In both models, a single injection of replicating vector followed by prodrug administration achieved long-term survival benefit. In the immunodeficient model, tumors recurred repeatedly, but bioluminescence imaging of tumors enabled tailored scheduling of multicycle prodrug administration, continued control of disease burden, and long-term survival. In the immunocompetent model, complete loss of tumor signal was observed after only 1–2 cycles of prodrug, followed by long-term survival without recurrence for >300 days despite discontinuation of prodrug. Long-term survivors rejected challenge with uninfected glioma cells, indicating immunological responses against native tumor antigens, and immune cell depletion showed a critical role for CD4+ T cells.Conclusion.These results support dual mechanisms of action contributing to the efficacy of RRV-mediated prodrug-activator gene therapy: long-term tumor control by prodrug conversion-mediated cytoreduction, and induction of antitumor immunity.

Insights into molecular therapy of glioma: current challenges and next generation blueprint.

Glioma accounts for the majority of human brain tumors. With prevailing treatment regimens, the patients have poor survival rates. In spite of current development in mainstream glioma therapy, a cure for glioma appears to be out of reach. The infiltrative nature of glioma and acquired resistance substancially restrict the therapeutic options. Better elucidation of the complicated pathobiology of glioma and proteogenomic characterization might eventually open novel avenues for the design of more sophisticated and effective combination regimens. This could be accomplished by individually tailoring progressive neuroimaging techniques, terminating DNA synthesis with prodrug-activating genes, silencing gliomagenesis genes (gene therapy), targeting miRNA oncogenic activity (miRNA-mRNA interaction), combining Hedgehog-Gli/Akt inhibitors with stem cell therapy, employing tumor lysates as antigen sources for efficient depletion of tumor-specific cancer stem cells by cytotoxic T lymphocytes (dendritic cell vaccination), adoptive transfer of chimeric antigen receptor-modified T cells, and combining immune checkpoint inhibitors with conventional therapeutic modalities. Thus, the present review captures the latest trends associated with the molecular mechanisms involved in glial tumorigenesis as well as the limitations of surgery, radiation and chemotherapy. In this article we also critically discuss the next generation molecular therapeutic strategies and their mechanisms for the successful treatment of glioma.

Abstract A43: Therapeutic efficacy of retroviral replicating vectors in orthotopic models of breast cancer metastasis to the brain

Abstract Our studies to date have demonstrated that retroviral replicating vectors (RRV) are capable of highly efficient replication in cancer cells in vitro and in vivo, associated with high levels of tumor-selective gene transfer and significantly enhanced survival benefit when employed for prodrug activator (“suicide”) gene therapy in a variety of cancer models. Clinical testing of RRV-mediated gene therapy using the yeast cytosine deaminase (CD) prodrug activator gene, which mediates conversion of the prodrug 5-fluorocytosine (5-FC) to the anti-cancer drug, 5-fluorouracil (5-FU), is currently underway in multi-center clinical trials being conducted in the United States for patients with recurrent high-grade glioma. In the present study, our goal has been to establish the feasibility of also applying this novel gene therapy strategy to secondary brain tumors arising from metastasis of systemic cancers to the CNS, which occur 5-10 times more frequently than primary brain tumors. Moreover, brain metastases are often accompanied by neurological deficits, which deprive patients of their quality of life. In particular, brain-metastatic breast cancer frequently arises from highly aggressive, treatment-refractory, ‘triple-negative' (ER(-), PR(-), HER2(-)) cells, is associated with a dismal prognosis of 4-6 months, and as treatment options are largely palliative, is the main cause of death in half of these patients. Hence this condition represents an unmet medical need. We first examined the replication kinetics of RRV expressing the GFP reporter gene (AC3-emd) in human and murine breast cancer cell lines by flow cytometry. The replication kinetics of AC3-emd in the triple-negative human breast cancer cell line MDA-MB-231-BR, and in the murine breast cancer cell line JC, showed robust replication activity 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 by MTS assay after 5-FC treatment of 231BR and JC cells that had been fully transduced with RRV expressing the CD prodrug activator gene (clinical-grade vector AC3-yCD2, also designated “Toca 511”). In both of the AC3-yCD2-transduced cell lines, cell viability was reduced approximately 70-85% even after exposure to 0.1 mM 5-FC treatment, and complete cell killing was observed after 6 days (231-BR) or 4 days (JC) exposure to 1 mM 5-FC treatment. We then examined in vivo replication kinetics of AC3-emd in intracranial 231-BR xenograft models. Intratumoral injection of 10e6 AC3-emd on Day 7 after tumor implantation was confirmed to achieve &amp;gt;80% transduction efficiency by flow cytometry on Day 10 post-injection. In survival studies, animals treated with AC3-yCD2+5-FC prodrug activator gene therapy showed statistically significant (p&amp;lt;0.0001) survival benefit compared to the AC3-yCD2+PBS control group. These data provide preclinical validation for a new Phase I dose escalation trial to evaluate RRV-mediated prodrug activator gene therapy in CNS-metastatic breast cancer, as well as other types of metastatic cancers (TOCA6 trial: clinicaltrials.gov NCT02576665), planned for initiation at the University of Miami, Cleveland Clinic, and UCLA, in mid-2016. Citation Format: Akihito Inagaki, Sara Collins, Suzanne Matsuura, Noriyuki Kasahara. Therapeutic efficacy of retroviral replicating vectors in orthotopic models of breast cancer metastasis to the brain. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A43.

Abstract A42: Alternate pseudotypes overcome receptor interference and enable combination suicide gene therapy with retroviral replicating vectors

Abstract 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 (MLV)-based RRV encoding the yeast cytosine deaminase (CD) prodrug activator gene, designated Toca 511 (vocimagene amiretrorepvec), is now being investigated in combination with Toca FC (extended-release 5-FC) in multi-center Phase IIB / III clinical trials for patients with recurrent high-grade glioma (clinicaltrials.gov: NCT02414165). 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. We further propose that combination with additional therapies may be desirable to optimize treatment outcomes. Here we first evaluated two different RRVs derived from MLV and gibbon ape leukemia virus (GALV), and expressing either the same fluorescent reporter gene (MLV-GFP and GALV-GFP, respectively), or different reporter genes (MLV-GFP vs. GALV-RFP; MLV-RFP vs. GALV-GFP), in different types of human cancer cells. Individually, RRVs expressing the same reporter gene efficiently infected human glioma, prostate cancer, and ovarian cancer cells, and showed efficient replication and spread in culture. When marked with different fluorescent reporter genes, it was found that MLV-GFP can spread in cells that had been pre-transduced with GALV-RFP, but not in MLV-RFP pre-transduced cells. Similarly, GALV-GFP can spread in MLV-RFP pre-transduced cells, but not in GALV-RFP pre-transduced cells. This mutually exclusive infection pattern is likely due to the phenomenon of receptor interference, which occurs when vectors derived from the same viral strain compete for binding to cell surface receptors, resulting in superinfection resistance. Notably, however, replication and spread of either RRV in culture was not affected by pre-transduction with RRV derived from a different strain, indicating that this approach could overcome receptor interference. In order to investigate the effect of combined prodrug-dependent cell killing in vitro, cells were then transduced with MLV- or GALV-based RRV expressing the yeast cytosine deaminase suicide gene (MLV-CD, GALV-CD), which converts the prodrug 5-fluorocytosine (5-FC) into the active drug 5-fluorouracil (5-FU), or with RRV expressing the Herpes thymidine kinase gene (MLV-TK, GALV-TK) which converts Ganciclovir (GCV) to GCV-monophosphate, either individually or in combination. In vitro cytocidal effects obtained by combining different prodrug activator genes were significantly greater when these genes were delivered with RRV derived from two different strains. These data indicate the potential utility of using RRV pseudotyped with envelopes derived from different viral strains to overcome receptor interference leading to superinfection resistance, and achieve effective combined prodrug activator gene therapy. Citation Format: James Grosso, Sara Collins, Akihito Inagaki, Suzanne Matsuura, Brian Slomovitz, Tan Ince, Noriyuki Kasahara. Alternate pseudotypes overcome receptor interference and enable combination suicide gene therapy with retroviral replicating vectors. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A42.

Abstract B03: Development of retroviral replicating vectors expressing codon-optimized nitroreductase for prodrug activator gene therapy in human glioma models

Abstract Our studies to date have demonstrated dramatic survival benefit when tumor-selective retroviral replicating vectors (RRV) are employed for gene therapy in a variety of preclinical cancer models. RRV-mediated prodrug activator gene therapy using yeast cytosine deaminase (RRV-CD; “Toca 511”) has been evaluated in multi-center Phase I ascending dose trials in patients with recurrent high grade glioma (http://www. clinicaltrials.gov: NCT01156584, NCT01985256, NCT01470794), and based on highly promising evidence of therapeutic benefit, a registrational Phase IIB/III trial has recently been initiated at multiple sites in the United States and Canada (NCT02414165). Translational development of further RRV-based therapeutic agents is also on-going, and we have now 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 optimization 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 &amp;gt;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 results 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. Further studies are aimed at translational development of improved RRV-NTR vectors for future clinical use both as a stand-alone therapy and in combination with RRV-CD (Toca 511). Citation Format: Sara Collins, Akihito Inagaki, Mark Tangney, Noriyuki Kasahara. Development of retroviral replicating vectors expressing codon-optimized nitroreductase for prodrug activator gene therapy in human glioma models. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr B03.