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.

Clinical development of retroviral replicating vector Toca 511 for gene therapy of cancer.

INTRODUCTION: The use of tumor-selectively replicating viruses is a rapidly expanding field that is showing considerable promise for cancer treatment. Retroviral replicating vectors (RRV) are unique among the various replication-competent viruses currently being investigated for potential clinical utility, because they permanently integrate into the cancer cell genome and are capable of long-term persistence within tumors. RRV can mediate efficient tumor-specific delivery of prodrug activator genes, and subsequent prodrug treatment leads to synchronized cell killing of infected cancer cells, as well as activation of antitumor immune responses. AREAS COVERED: Here we review preclinical studies supporting bench-to-bedside translation of Toca 511, an optimized RRV for prodrug activator gene therapy, the results from Phase I through III clinical trials to date, and potential future directions for this therapy as well as other clinical candidate RRV. EXPERT OPINION: Toca 511 has shown highly promising results in early-stage clinical trials. This vector progressed to a registrational Phase III trial, but the results announced in late 2019 appeared negative overall. However, the median prodrug dosing schedule was not optimal, and promising possible efficacy was observed in some prespecified subgroups. Further clinical investigation, as well as development of RRV with other transgene payloads, is merited.

Molecular and Immunologic Signatures are Related to Clinical Benefit from Treatment with Vocimagene Amiretrorepvec (Toca 511) and 5-Fluorocytosine (Toca FC) in Patients with Glioma.

PURPOSE: High-grade gliomas (HGGs) are central nervous system tumors with poor prognoses and limited treatment options. Vocimagene amiretrorepvec (Toca 511) is a retroviral replicating vector encoding cytosine deaminase, which converts extended release 5-fluorocytosine (Toca FC) into the anticancer agent, 5-fluorouracil. According to preclinical studies, this therapy kills cancer cells and immunosuppressive myeloid cells in the tumor microenvironment, leading to T-cell-mediated antitumor immune activity. Therefore, we sought to elucidate this immune-related mechanism of action in humans, and to investigate potential molecular and immunologic indicators of clinical benefit from therapy. PATIENTS AND METHODS: In a phase I clinical trial (NCT01470794), patients with recurrent HGG treated with Toca 511 and Toca FC showed improved survival relative to historical controls, and some had durable complete responses to therapy. As a part of this trial, we performed whole-exome DNA sequencing, RNA-sequencing, and multiplex digital ELISA measurements on tumor and blood samples. RESULTS: Genetic analyses suggest mutations, copy-number variations, and neoantigens are linked to survival. Quantities of tumor immune infiltrates estimated by transcript abundance may potentially predict clinical outcomes. Peak values of cytokines in peripheral blood samples collected during and after therapy could indicate response. CONCLUSIONS: These results support an immune-related mechanism of action for Toca 511 and Toca FC, and suggest that molecular and immunologic signatures are related to clinical benefit from treatment.

PD-L1 checkpoint blockade delivered by retroviral replicating vector confers anti-tumor efficacy in murine tumor models.

Immune checkpoint inhibitors (CPIs) are associated with a number of immune-related adverse events and low response rates. We provide preclinical evidence for use of a retroviral replicating vector (RRV) selective to cancer cells, to deliver CPI agents that may circumvent such issues and increase efficacy. An RRV, RRV-scFv-PDL1, encoding a secreted single chain variable fragment targeting PD-L1 can effectively compete with PD-1 for PD-L1 occupancy. Cell binding assays showed trans-binding activity on 100% of cells in culture when infection was limited to 5% RRV-scFv-PDL1 infected tumor cells. Further, the ability of scFv PD-L1 to rescue PD-1/PD-L1 mediated immune suppression was demonstrated in a co-culture system consisting of human-derived immune cells and further demonstrated in several syngeneic mouse models including an intracranial tumor model. These tumor models showed that tumors infected with RRV-scFv-PD-L1 conferred robust and durable immune-mediated anti-tumor activity comparable or superior to systemically administered anti-PD-1 or anti PD-L1 monoclonal antibodies. Importantly, the nominal level of scFv-PD-L1 detected in serum is ∼50-150 fold less than reported for systemically administered therapeutic antibodies targeting immune checkpoints. These results support the concept that RRV-scFv-PDL1 CPI strategy may provide an improved safety and efficacy profile compared to systemic monoclonal antibodies of currently approved therapies.

Molecular Analyses Support the Safety and Activity of Retroviral Replicating Vector Toca 511 in Patients.

Purpose: Toca 511 is a gammaretroviral replicating vector encoding cytosine deaminase that selectively infects tumor cells and converts the antifungal drug 5-fluorocytosine into the antineoplastic drug 5-fluorouracil, which directly kills tumor cells and stimulates antitumor immune responses. As part of clinical monitoring of phase I clinical trials in recurrent high-grade glioma, we have performed extensive molecular analyses of patient specimens to track vector fate.Patients and Methods: Toca 511 and Toca FC (extended-release 5-fluorocytosine) have been administered to 127 high-grade glioma patients across three phase I studies. We measured Toca 511 RNA and DNA levels in available body fluids and tumor samples from patients to assess tumor specificity. We mapped Toca 511 integration sites and sequenced integrated Toca 511 genomes from patient samples with detectable virus. We measured Toca 511 levels in a diverse set of tissue samples from one patient.Results: Integrated Toca 511 is commonly detected in tumor samples and is only transiently detected in blood in a small fraction of patients. There was no believable evidence for clonal expansion of cells with integrated Toca 511 DNA, or preferential retrieval of integration sites near oncogenes. Toca 511 sequence profiles suggest most mutations are caused by APOBEC cytidine deaminases acting during reverse transcription. Tissue samples from a single whole-body autopsy affirm Toca 511 tumor selectivity.Conclusions: Toca 511 and Toca FC treatment was not associated with inappropriate integration sites and clonal expansion. The vector is tumor-selective and persistent in patients who received Toca 511 injections. Clin Cancer Res; 24(19); 4680-93. ©2018 AACR.

Durable complete responses in some recurrent high-grade glioma patients treated with Toca 511 + Toca FC.

Background: Vocimagene amiretrorepvec (Toca 511) is an investigational gamma-retroviral replicating vector encoding cytosine deaminase that, when used in combination with extended-release 5-fluorocytosine (Toca FC), results preclinically in local production of 5-fluorouracil, depletion of immune-suppressive myeloid cells, and subsequent induction of antitumor immunity. Recurrent high-grade glioma (rHGG) patients have a high unmet need for effective therapies that produce durable responses lasting more than 6 months. In this setting, relapse is nearly universal and most responses are transient. Methods: In this Toca 511 ascending-dose phase I trial (NCT01470794), HGG patients who recurred after standard of care underwent surgical resection and received Toca 511 injected into the resection cavity wall, followed by orally administered cycles of Toca FC. Results: Among 56 patients, durable complete responses were observed. A subgroup was identified based on Toca 511 dose and entry requirements for the follow-up phase III study. In this subgroup, which included both isocitrate dehydrogenase 1 (IDH1) mutant and wild-type tumors, the durable response rate is 21.7%. Median duration of follow-up for responders is 35.7+ months. As of August 25, 2017, all responders remain in response and are alive 33.9+ to 52.2+ months after Toca 511 administration, suggesting a positive association of durable response with overall survival. Conclusions: Multiyear durable responses have been observed in rHGG patients treated with Toca 511 + Toca FC in a phase I trial, and the treatment will be further evaluated in a randomized phase III trial. Among IDH1 mutant patients treated at first recurrence, there may be an enrichment of complete responders.

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.

A Retroviral Replicating Vector Encoding Cytosine Deaminase and 5-FC Induces Immune Memory in Metastatic Colorectal Cancer Models.

Treatment of tumors with Toca 511, a gamma retroviral replicating vector encoding cytosine deaminase, followed by 5-fluorocytosine (5-FC) kills tumors by local production of 5-fluorouracil (5-FU). In brain tumor models, this treatment induces systemic anti-tumor immune responses and long-term immune-mediated survival. Phase 1 Toca 511 and Toca FC (extended-release 5-FC) clinical trials in patients with recurrent high-grade glioma show durable complete responses and promising survival data compared to historic controls. The work described herein served to expand on our earlier findings in two models of metastatic colorectal carcinoma (mCRC). Intravenous (i.v.) delivery of Toca 511 resulted in substantial tumor-selective uptake of vector into metastatic lesions. Subsequent treatment with 5-FC resulted in tumor shrinkage, improved survival, and immune memory against future rechallenge with the same CT26 CRC cell line. Similar results were seen in a brain metastasis model of mCRC. Of note, 5-FC treatment resulted in a significant decrease in myeloid-derived suppressor cells (MDSCs) in mCRC tumors in both the liver and brain. These results support the development of Toca 511 and Toca FC as a novel immunotherapeutic approach for patients with mCRC. A phase 1 study of i.v. Toca 511 and Toca FC in solid tumors, including mCRC, is currently underway (NCT02576665).

Efficient Therapeutic Protein Expression Using Retroviral Replicating Vector with 2A Peptide in Cancer Models.

Toca 511, a retroviral replicating vector (RRV), uses an internal ribosomal entry site (IRES) to express an optimized yeast cytosine deaminase (yCD2), which converts 5-fluorocytosine to 5-fluorouracil. This configuration is genetically stable in both preclinical mouse models and human clinical trials. However, the use of IRES (∼600 bp) restricts choices of therapeutic transgenes due to limits in RRV genome size. This study replaced IRES with 2A peptides derived from picornaviruses with or without a GSG linker. The data show that GSG-linked 2A (g2A) peptide resulted in higher polyprotein separation efficiency than non-GSG linked 2A peptide. The study also shows that RRV can tolerate insertion of two separate 2A peptides to allow expression of two transgenes without compromising the assembly and function of the virus in addition to insertion of a single 2A peptide to confirm genetic stability with yCD2, green fluorescent protein, and HSV-1 thymidine kinase. In a parallel comparison of the RRV-IRES-yCD2 and RRV-g2A-yCD2 configurations, the study shows the yCD2 protein expressed from RRV-g2A-yCD2 has higher activity, resulting in a higher survival benefit in an intracranial tumor mouse model. These data enable a wider range of potential product candidates that could be developed using the RRV platform.

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.

Toca 511 gene transfer and treatment with the prodrug, 5-fluorocytosine, promotes durable antitumor immunity in a mouse glioma model.

BACKGROUND: Toca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD). Tumor-selective expression of CD converts the prodrug, 5-fluorocytosine (5-FC), into the active chemotherapeutic, 5-fluorouracil (5-FU). This therapeutic approach is being tested in a randomized phase II/III trial in recurrent glioblastoma and anaplastic astrocytoma (NCT0241416). The aim of this study was to identify the immune cell subsets contributing to antitumor immune responses following treatment with 5-FC in Toca 511-expressing gliomas in a syngeneic mouse model. METHODS: Flow cytometry was utilized to monitor and characterize the immune cell infiltrate in subcutaneous Tu-2449 gliomas in B6C3F1 mice treated with Toca 511 and 5-FC. RESULTS: Tumor-bearing animals treated with Toca 511 and 5-FC display alterations in immune cell populations within the tumor that result in antitumor immune protection. Attenuated immune subsets were exclusive to immunosuppressive cells of myeloid origin. Depletion of immunosuppressive cells temporally preceded a second event which included expansion of T cells which were polarized away from Th2 and Th17 in the CD4+ T cell compartment with concomitant expansion of interferon gamma-expressing CD8+ T cells. Immune alterations correlated with clearance of Tu-2449 subcutaneous tumors and T cell-dependent protection from future tumor challenge. CONCLUSIONS: Treatment with Toca 511 and 5-FC has a concentrated effect at the site of the tumor which causes direct tumor cell death and alterations in immune cell infiltrate, resulting in a tumor microenvironment that is more permissive to establishment of a T cell mediated antitumor immune response.

Retroviral Replicating Vector Delivery of miR-PDL1 Inhibits Immune Checkpoint PDL1 and Enhances Immune Responses In Vitro.

Tumor cells express a number of immunosuppressive molecules that can suppress anti-tumor immune responses. Efficient delivery of small interfering RNAs to treat a wide range of diseases including cancers remains a challenge. Retroviral replicating vectors (RRV) can be used to stably and selectively introduce genetic material into cancer cells. Here, we designed RRV to express shRNA (RRV-shPDL1) or microRNA30-derived shRNA (RRV-miRPDL1) using Pol II or Pol III promoters to downregulate PDL1 in human cancer cells. We also designed RRV expressing cytosine deaminase (yCD2) and miRPDL1 for potential combinatorial therapy. Among various configurations tested, we showed that RRV-miRPDL1 vectors with Pol II or Pol III promoter replicated efficiently and exhibited sustained downregulation of PDL1 protein expression by more than 75% in human cancer cell lines with high expression of PDL1. Immunologic effects of RRV-miRPDL1 were assessed by a trans-suppression lymphocyte assay. In vitro data showed downregulation of PDL1+ tumor cells restored activation of CD8+ T cells and bio-equivalency compared to anti-PDL1 antibody treatment. These results suggest RRV-miRPDL1 may be an alternative therapeutic approach to enhance anti-tumor immunity by overcoming PDL1-induced immune suppression from within cancer cells and this approach may also be applicable to other cancer targets.

Phase 1 trial of vocimagene amiretrorepvec and 5-fluorocytosine for recurrent high-grade glioma.

Toca 511 (vocimagene amiretrorepvec) is an investigational nonlytic, retroviral replicating vector (RRV) that delivers a yeast cytosine deaminase, which converts subsequently administered courses of the investigational prodrug Toca FC (extended-release 5-fluorocytosine) into the antimetabolite 5-fluorouracil. Forty-five subjects with recurrent or progressive high-grade glioma were treated. The end points of this phase 1, open-label, ascending dose, multicenter trial included safety, efficacy, and molecular profiling; survival was compared to a matching subgroup from an external control. Overall survival for recurrent high-grade glioma was 13.6 months (95% confidence interval, 10.8 to 20.0) and was statistically improved relative to an external control (hazard ratio, 0.45; P = 0.003). Tumor samples from subjects surviving more than 52 weeks after Toca 511 delivery disproportionately displayed a survival-related mRNA expression signature, identifying a potential molecular signature that may correlate with treatment-related survival rather than being prognostic. Toca 511 and Toca FC show excellent tolerability, with RRV persisting in the tumor and RRV control systemically. The favorable assessment of Toca 511 and Toca FC supports confirmation in a randomized phase 2/3 trial (NCT02414165).

Toca 511 plus 5-fluorocytosine in combination with lomustine shows chemotoxic and immunotherapeutic activity with no additive toxicity in rodent glioblastoma models.

BACKGROUND: Toca 511, a gamma retroviral replicating vector encoding cytosine deaminase, used in combination with 5-fluorocytosine (5-FC) kills tumor by local production of 5-fluorouracil (5-FU), inducing local and systemic immunotherapeutic response resulting in long-term survival after cessation of 5-FC. Toca 511 and Toca FC (oral extended-release 5-FC) are under investigation in patients with recurrent high-grade glioma. Lomustine is a treatment option for patients with high-grade glioma. METHODS: We investigated the effects of lomustine combined with Toca 511 + 5-FC in syngeneic orthotopic glioma models. Safety and survival were evaluated in immune-competent rat F98 and mouse Tu-2449 models comparing Toca 511 + 5-FC to lomustine + 5-FC or the combination of Toca 511 + 5-FC + lomustine. After intracranial implantation of tumor, Toca 511 was delivered transcranially followed by cycles of intraperitoneal 5-FC with or without lomustine at the first or fourth cycle. RESULTS: Coadministration of 5-FC with lomustine was well tolerated. In F98, combination Toca 511 + 5-FC and lomustine increased median survival, but "cures" were not achieved. In Tu-2449, combination Toca 511 + 5-FC and lomustine increased median survival and resulted in high numbers of cure. Rejection of tumor rechallenge occurred after treatment with Toca 511 + 5-FC or combined with lomustine, but not with lomustine + 5-FC. Mixed lymphocyte-tumor cell reactions using splenocytes from cured animals showed robust killing of target cells in an effector:target ratio-dependent manner with Toca 511 + 5-FC and Toca 511 + 5-FC + lomustine day 10. CONCLUSION: The combination of Toca 511 + 5-FC and lomustine shows promising efficacy with no additive toxicity in murine glioma models. Immunotherapeutic responses resulting in long-term survival were preserved despite lomustine-related myelosuppression.

Extensive Replication of a Retroviral Replicating Vector Can Expand the A Bulge in the Encephalomyocarditis Virus Internal Ribosome Entry Site and Change Translation Efficiency of the Downstream Transgene.

We have developed retroviral replicating vectors (RRV) derived from Moloney murine gammaretrovirus with an amphotropic envelope and an encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES)-transgene cassette downstream of the env gene. During long-term (180 days) replication of the vector in animals, a bulge of 7 adenosine residues (A's) in the J-K bifurcation domain sometimes serially added A's. Therefore, vectors with 4-12 A's in the A bulge in the J-K bifurcation domain were generated, and the impact of the variants on transgene protein expression, vector stability, and IRES sequence upon multiple infection cycles was assessed in RRV encoding yeast-derived cytosine deaminase and green fluorescent protein in vitro. For transgene protein expression, after multiple infection cycles, RRV-IRES with 5-7 A's gave roughly comparable levels, 4 and 8 A's were within about 4-5-fold of the 6 A's, whereas 10 and 12 A's were marked lower. In terms of stability, after 10 infection cycles, expansion of A's appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 A's appeared completely stable; 6, 7, and particularly 8 A's showed some level of expansion in the A bulge; 10 and 12 A's underwent both expansion and transgene deletion. The strong relative translational activity of the 5 A's in the EMCV IRES has not been reported previously. The 5A RRV-IRES may have utility for preclinical and clinical applications where extended replication is required.

Retroviral Replicating Vectors Deliver Cytosine Deaminase Leading to Targeted 5-Fluorouracil-Mediated Cytotoxicity in Multiple Human Cancer Types.

Toca 511 is a modified retroviral replicating vector based on Moloney γ-retrovirus with an amphotropic envelope. As an investigational cancer treatment, Toca 511 preferentially infects cancer cells without direct cell lysis and encodes an enhanced yeast cytosine deaminase that converts the antifungal drug 5-fluorocytosine to the anticancer drug, 5-fluorouracil. A panel of established human cancer cell lines, derived from glioblastoma, colon, and breast cancer tissue, was used to evaluate parameters critical for effective anticancer activity. Gene transfer, cytosine deaminase production, conversion of 5-fluorocytosine to 5-fluorouracil, and subsequent cell killing occurred in all lines tested. We observed >50% infection within 25 days in all lines and 5-fluorocytosine LD50 values between 0.02 and 6 µg/ml. Although we did not identify a small number of key criteria, these studies do provide a straightforward approach to rapidly gauge the probability of a Toca 511 and 5-fluorocytosine treatment effect in various cancer indications: a single MTS assay of maximally infected cancer cell lines to determine 5-fluorocytosine LD50. The data suggest that, although there can be variation in susceptibility to Toca 511 and 5-fluorocytosine because of multiple mechanistic factors, this therapy may be applicable to a broad range of cancer types and individuals.

Intravenous administration of retroviral replicating vector, Toca 511, demonstrates therapeutic efficacy in orthotopic immune-competent mouse glioma model.

Toca 511 (vocimagene amiretrorepvec), a nonlytic, amphotropic retroviral replicating vector (RRV), encodes and delivers a functionally optimized yeast cytosine deaminase (CD) gene to tumors. In orthotopic glioma models treated with Toca 511 and 5-fluorocytosine (5-FC) the CD enzyme within infected cells converts 5-FC to 5-fluorouracil (5-FU), resulting in tumor killing. Toca 511, delivered locally either by intratumoral injection or by injection into the resection bed, in combination with subsequent oral extended-release 5-FC (Toca FC), is under clinical investigation in patients with recurrent high-grade glioma (HGG). If feasible, intravenous administration of vectors is less invasive, can easily be repeated if desired, and may be applicable to other tumor types. Here, we present preclinical data that support the development of an intravenous administration protocol. First we show that intravenous administration of Toca 511 in a preclinical model did not lead to widespread or uncontrolled replication of the RVV. No, or low, viral DNA was found in the blood and most of the tissues examined 180 days after Toca 511 administration. We also show that RRV administered intravenously leads to efficient infection and spread of the vector carrying the green fluorescent protein (GFP)-encoding gene (Toca GFP) through tumors in both immune-competent and immune-compromised animal models. However, initial vector localization within the tumor appeared to depend on the mode of administration. Long-term survival was observed in immune-competent mice when Toca 511 was administered intravenously or intracranially in combination with 5-FC treatment, and this combination was well tolerated in the preclinical models. Enhanced survival could also be achieved in animals with preexisting immune response to vector, supporting the potential for repeated administration. On the basis of these and other supporting data, a clinical trial investigating intravenous administration of Toca 511 in patients with recurrent HGG is currently open and enrolling.

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 and 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 were confirmed in the highly aggressive, radioresistant human glioblastoma cell line U87EGFRvIII 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.

Blockade of type I interferon (IFN) production by retroviral replicating vectors and reduced tumor cell responses to IFN likely contribute to tumor selectivity.

UNLABELLED: We developed a Moloney mouse leukemia virus (MLV)-based retroviral replicating vector (RRV), Toca 511, which has displayed tumor specificity in resected brain tumor material and blood in clinical trials. Here, we investigated the interaction between Toca 511 and human host cells, and we show that RRVs do not induce type I interferon (IFN) responses in cultured human tumor cells or cultured human primary cells. However, exogenous type I IFN inhibited RRV replication in tumor cells and induced IFN-regulated genes, albeit at a lower level than in primary cells. Unexpectedly, RRVs did not induce IFN-α production upon incubation in vitro with human plasmacytoid dendritic cells (pDCs), whereas lentivirus vector and heat-treated RRVs did. Coincubation of RRVs with heat-treated RRVs or with lentivirus vector suppressed IFN-α production in pDCs, suggesting that native RRV has a dominant inhibitory effect on type I IFN induction. This effect is sensitive to trypsin treatment. In addition, heat treatment inactivated that activity but exposed an immune-stimulatory activity. The immune-stimulating component is sensitive to deglycosidases, trypsin, and phospholipase C treatment. Experiments with retroviral nonreplicating vectors and virus-like particles demonstrated that the immunosuppressive activity is not associated with the amphotropic envelope or the glyco-Gag protein. In summary, our data provide evidence that RRVs do not directly trigger type I IFN responses in IFN-responsive tumor cells. Moreover, RRVs appear to carry a heat-labile component that actively suppresses activation of cellular innate immune responses in pDCs. Inhibition of IFN induction by RRVs and the reduced response to IFN should facilitate tumor-specific infection in vivo. IMPORTANCE: RRVs have a convincing preference for replicating in tumor cells in animal models, and we observed similar preferences in the initial treatment of human glioblastoma patients. This study investigates the basis for the interaction between RRV and human host cells (tumor versus nontumor) in vitro. We found that RRVs do not trigger an IFN-α/ß response in tumor cells, but the cells are capable of responding to type I IFNs and of producing them when stimulated with known agonists. Surprisingly, the data show that RRVs can actively inhibit induction of cellular innate immunity and that this inhibitory activity is heat labile and trypsin sensitive and not attributable to the envelope protein. These data partially explain the observed in vivo tumor specificity.

MicroRNA 142-3p attenuates spread of replicating retroviral vector in hematopoietic lineage-derived cells while maintaining an antiviral immune response.

We are developing a retroviral replicating vector (RRV) encoding cytosine deaminase as an anticancer agent for gliomas. Despite its demonstrated natural selectivity for tumors, and other safety features, such a virus could potentially cause off-target effects by productively infecting healthy tissues. Here, we investigated whether incorporation of a hematopoietic lineage-specific microRNA target sequence in RRV further restricts replication in hematopoietic lineage-derived human cells in vitro and in murine lymphoid tissues in vivo. One or four copies of a sequence perfectly complementary to the guide strand of microRNA 142-3p were inserted into the 3' untranslated region of the RRV genome expressing the transgene encoding green fluorescent protein (GFP). Viral spread and GFP expression of these vectors in hematopoietic lineage cells in vitro and in vivo were measured by qPCR, qRT-PCR, and flow cytometry. In hematopoietic lineage-derived human cell lines and primary human stimulated peripheral blood mononuclear cells, vectors carrying the 142-3pT sequence showed a remarkable decrease in GFP expression relative to the parental vector, and viral spread was not observed over time. In a syngeneic subcutaneous mouse tumor model, RRVs with and without the 142-3pT sequences spread equally well in tumor cells; were strongly repressed in blood, bone marrow, and spleen; and generated antiviral immune responses. In an immune-deficient mouse model, RRVs with 142-3pT sequences were strongly repressed in blood, bone marrow, and spleen compared with unmodified RRV. Tissue-specific microRNA-based selective attenuation of RRV replication can maintain antiviral immunity, and if needed, provide an additional safeguard to this delivery platform for gene therapy applications.