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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.

Figure S9

Toca 511 qPCR detection results

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.

Tg 511-13-01 (Study 13) dosing table.

Supplementary Figure, Table & Dataset legends

Supplementary Figures S1 to S9 and Supplementary Table S1.

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 (

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.

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 anti-tumor 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 RRVs. 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.

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.

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.

Author(s): Cloughesy, Timothy F; Landolfi, Joseph; Vogelbaum, Michael A; Ostertag, Derek; Elder, James B; Bloomfield, Stephen; Carter, Bob; Chen, Clark C; Kalkanis, Steven N; Kesari, Santosh; Lai, Albert; Lee, Ian Y; Liau, Linda M; Mikkelsen, Tom; Nghiemphu, Phioanh; Piccioni, David; Accomando, William; Diago, Oscar R; Hogan, Daniel J; Gammon, Dawn; Kasahara, Noriyuki; Kheoh, Thian; Jolly, Douglas J; Gruber, Harry E; Das, Asha; Walbert, Tobias | Abstract: 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.

Author(s): Mitchell, Leah A; Lopez Espinoza, Fernando; Mendoza, Daniel; Kato, Yuki; Inagaki, Akihito; Hiraoka, Kei; Kasahara, Noriyuki; Gruber, Harry E; Jolly, Douglas J; Robbins, Joan M | Abstract: BackgroundToca 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.MethodsFlow 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.ResultsTumor-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.ConclusionsTreatment 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.

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. Keywords: gene therapy, RNA interference, cancer immunotherapy

Author(s): Inagaki, Akihito; Takahashi, Masamichi; Kamijima, Shuichi; Hiraoka, Kei; Tam, Quincy; Faure-Kumar, Emmanuelle; Treger, Janet; Bogan, Brooke; Hacke, Katrin; Collins, Sara; Jolly, Douglas; Kasahara, Noriyuki | Abstract: Abstract Toca 511, an improved retroviral replicating vectors (RRVs) expressing a codon-optimized cytosine deaminase, has shown highly promising evidence of therapeutic benefit in preclinical and clinical studies for gene therapy of glioma. In the present study, we engineered human mesenchymal stem cells (MSC) as tumor-homing cellular carriers that produce and release RRV, and evaluated the effect of this mode of virus delivery on the time course of intratumoral RRV dissemination. Human MSC isolates were engineered to produce RRV (MSC-RRV). Cytotoxicity assays confirmed efficient prodrug activator function in U-87 glioma cells transduced with vectors produced from MSC-RRV. To evaluate intratumoral migration activity and tumor-homing migration activity in vivo, individual MSC isolates were injected either directly into human glioma xenografts, or into the contralateral brain hemisphere. MSC-RRV isolates showing the highest levels of intratumoral migration activity were selected, and the efficiency of intratumoral dissemination and tumoricidal activity achieved by these isolates was compared against injection of RRV virus preparations in subcutaneous glioma models. Compared to virus injection, MSC-RRV achieved 1.6x-higher levels of tumor transduction (pl 0.05), and 2x-reduced tumor growth (p=0.018) at earlier time points. In short-term survival studies using lower doses of MSC-RRV cells vs. RRV virus injected 14 days post-establishment of intracranial U-87 gliomas, a small but statistically significant prolongation of median survival was seen with intracranial tumors treated with MSC-RRV as compared to RRV (26 vs. 20 days, pl 0.05) after only a single cycle of 5-FC prodrug. Thus, MSC can be employed as mobile tumor-homing RRV-producer cells, which release RRV as they migrate to tumor foci in vivo and actively penetrate into individual tumor masses, resulting in more rapid tumor transduction and earlier therapeutic efficacy, which may be advantageous particularly for multi-focal and metastatic disease. This study was funded by the California Institute for Regenerative Medicine (TR2-01791).

Toca 511, a clinical-stage tumor-selective retroviral replicating vector (RRV) encoding an optimized yeast cytosine deaminase (CD) prodrug activator gene, in combination with Toca FC (extended-release 5-fluorocytosine (5-FC)), produces 5-fluorouracil (5-FU) within infected cancer cells, which in preclinical models has also been shown to kill immunosuppressive myeloid cells in the tumor microenvironment, leading to anti-cancer immune activation and long-term survival. This combination treatment is under evaluation in a registrational Phase 3 trial in patients with recurrent high-grade glioma. Here, we investigated the feasibility of applying Toca 511 & Toca FC to medulloblastoma, the most common malignant tumor of the pediatric nervous system. Established (HDMB03, DAOY) and primary (PDX10) human medulloblastoma cells were transduced with RRVs encoding either Emerald green fluorescent protein reporter (RRV-EMD) or the CD enzyme (RRV-CD, i.e. Toca 511). RRV replication in vitro, examined by flow cytometry for EMD fluorescence, showed spread to >90% of cells in culture by day 10 post-inoculation. In contrast to non-transduced or RRV-EMD-transduced controls, RRV-CD-transduced medulloblastoma cells exhibited significant dose-dependent reduction of viability upon exposure to 5-FC, as measured by MTS assay. In vivo, stereotactic intracerebellar implantation of HDMB03 cells into nude mice without treatment resulted in cell dose-dependent median survival time and eventual 100% lethality at all cell doses tested, with median survival of 28 days after implantation of 1x10^5 tumor cells. In this orthotopic medulloblastoma model, mice with RRV-CD-transduced intracerebellar HDMB03 tumors exhibited long-term survival upon 5-FC treatment, as compared to mice treated with PBS or implanted with non-transduced cells (p=0.00007). No deaths occurred in these mice with RRV-CD-transduced HDMB03 tumors 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). The results of this proof-of-concept study support further preclinical investigation of RRV-CD-mediated prodrug activator gene therapy for this pediatric malignancy.

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.

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.

186 Background: Toca 511 (vocimagene amiretrorepvec) is a cancer-selective, retroviral replicating vector encoding yeast cytosine deaminase that converts 5-fluorocytosine (5-FC) into 5-fluorouracil in the tumor microenvironment (TME). In animal models, Toca 511 and 5-FC kill dividing cancer and nearby immunosuppressive cells, leading to antitumor immune activity. A Phase 1 study of Toca 511 & Toca FC (extended-release 5-FC) in patients with recurrent high grade glioma revealed results consistent with this proposed mechanism. A Phase 3 trial is ongoing. Methods: Toca 6 (NCT02576665) is a Phase 1b, single-arm, multicenter study designed to investigate immunological changes after Toca 511 & Toca FC treatment in patients with advanced solid tumors, including colorectal cancer (CRC). Patients received intravenous (IV) Toca 511 for 3 days, and underwent biopsy of metastatic tumor before and ~ 4 weeks after starting oral Toca FC. Toca FC was repeated every 4-6 weeks. Peripheral blood mononuclear cells and tumor biopsies were evaluated for treatment related immune responses. Results: 17 CRC patients with a median 5 lines of prior chemotherapy were enrolled. At last data cut-off, 9 patients were alive and the median overall survival was 9.4 months. A patient receiving concomitant panitumumab had a partial response. IV Toca 511 led to viral expression in tumor, which decreased post-Toca FC while maintaining a reservoir of virus in the remaining tumor. T cells shifted from naïve to effector phenotypes, CD4 memory T cells expanded, and/or B cells increased after Toca FC treatment in 36% of patients. Marked changes in tumor infiltrating cells (CD11b myeloid cells, Tregs, exhausted T cells and CD8 T cells) occurred after Toca FC treatment. Treatment has been generally well tolerated. We also plan to report insights gained from RNA analysis of TME and update on clinical finding. Conclusions: Clinical data suggest a signal of activity in these heavily pretreated CRC patients warranting further exploration. IV Toca 511 administration showed viral infection of CRC metastatic tumor. Toca 511 & Toca FC may be associated with T cell mediated immune activity in peripheral blood and metastatic tumor, consistent with pre-clinical data in multiple tumor types. Clinical trial information: NCT02576665.

Toca 511 (vocimagene amiretrorepvec) is a cancer selective, retroviral replicating vector encoding a codon optimized, heat stabilized cytosine deaminase that converts Toca FC (extended-release 5- fluorocytosine, 5-FC) into the anticancer agent 5-fluorouracil. Preclinical evidence demonstrates that the Toca 511 & Toca FC regimen kills cancer cells and immunosuppressive myeloid cells in the tumor microenvironment, leading to durable antitumor immune responses that can be adoptively transferred to untreated animals. In an ascending dose trial (NCT01470794) in patients with recurrent high grade glioma (rHGG), Toca 511 was injected into the resection cavity walls at the time of resection, and then multiple courses of oral Toca FC were administered. Multiyear durable and complete responses by independent radiology review have been reported. Human immune monitoring results support an immunologic mechanism of action and identify potential biomarkers related to patient outcomes. Measurements included the quantification of peripheral blood and tumor infiltrating leukocyte subsets by flow cytometry, immunohistochemistry, and deconvolution of DNA and RNA sequencing data. In addition, systemic cytokine levels were assessed in peripheral blood serum by multiplex digital ELISA. Univariate comparisons and multivariate models revealed immunologic trends associated with patient outcomes. Pre-treatment tumor infiltrating cell subsets, quantified via deconvolution of RNA sequencing data, were associated with both objective responses and survival. Subsequent exploratory models applied to selected patient data indicate that a combined biomarker using mRNA signatures from multiple leukocyte subsets may predict patient outcomes with high sensitivity and selectivity. In addition, post-treatment serum cytokine time-course results suggest that differences and temporal modulations are associated with both objective response and survival. These results support an immune-related mechanism of action for the Toca 511 & Toca FC regimen. Potentially predictive and/or prognostic biomarkers of patient outcomes will be evaluated in the ongoing randomized Phase 3 Toca 5 trial in patients with rHGG (NCT02414165).

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.

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.

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.

Toca 511 (vocimagene amiretroprepvec) is an investigational, conditionally lytic, retroviral replicating vector that encodes an optimized yeast cytosine deaminase (CD) gene. The CD gene converts the prodrug, Toca FC (investigational, extended-release 5-fluorocytosine), into the chemotherapeutic, 5-FU in infected tumors. In a Phase 1 study (NCT01985256), Toca 511 was injected intravenously for 1, 3, or 5days to patients with recurrent high grade glioma. Tumors were subsequently resected, and Toca 511 was injected into the resection cavity walls. At the time of resection, tissue from various regions of the tumor was collected and processed for quantitative PCR analysis of CD RNA and DNA. Tissue from corresponding locations was fixed for assessment of CD protein expression by immunohistochemistry (IHC). Expression of CD protein was quantified based on immunofluorescence signal and was shown to co-localize in cells with detectable levels of gag, a viral structural protein. CD protein expression by IHC was assessed in tissue from locations that corresponded with samples positive for CD by PCR and ranged from 1.16% to 10.4% area of the field. These data show that intravenous delivery of Toca 511 results in appreciable deposition of vector in the tumor. Further, we have observed an inverse correlation between T cell infiltrate in the tumor microenvironment and clinical benefit in a complementary Phase Istudy in which Toca 511 was solely delivered into the resection cavity. Therefore, the nature of the study design described herein, which uses IV delivery of Toca 511, provides a unique opportunity to assess the spatial relationship between CD protein expression and histological features of the tumor. Immunohistochemical assessment to determine spatial correlates between CD protein and T cells, T regulatory cells, and immunosuppressive myeloid cells will be presented. Updated clinical response data will also be presented.

A dose-escalation clinical study (NCT01470794) using a retroviral replicating vector (RRV), Toca 511 (vocimagene amiretrorepvec), in combination with oral Toca FC (extended-release 5-fluorocytosine, 5-FC) is ongoing to evaluate safety, mechanism of action, and preliminary efficacy of this investigational therapy in patients with recurrent HGG. Toca 511 encodes a yeast-derived, codon-optimized, heat-stabilized cytosine deaminase that converts 5-FC to the anti-cancer drug 5-FU in infected tumors. This virus replicates selectively in malignant tissue and may have broad application in cancer therapies. When Toca FC is administered, cytosine deaminase is designed to convert 5-FC to 5-FU resulting in cancer cell death. In preclinical tumor models, as infected cancer cells are killed, diffusible 5-FU also kills susceptible cells, including myeloid derived suppressor cells that contribute to immune-suppression in the tumor microenvironment. This action by Toca 511 and Toca FC in animal models generates durable anti-tumor immune responses that can be transferred to naïve, untreated animals. In the clinical study presented here, Toca 511 is injected into resection cavity walls at time of resection followed by multiple courses of oral Toca FC. Multi-year durable and ongoing objective responses are observed, including four complete responses and two partial responses. We plan to report temporal changes in peripheral immune cells between responding and non-responding patients, including baseline differences in peripheral blood cell populations as well as temporal shifts in leukocyte subsets, T-cell receptor clonality, and serum cytokine levels. Sustained markers of proliferation and immune activation are observed in patients with response or stable disease compared to those that progressed. Immunohistochemistry analysis of resident T-cells in patient tumors taken at time of resection show fewer T-cells in responding and stable disease patients before treatment compared to those that did not respond. Data are consistent with the induction of anti-tumor immune response after Toca 511 and Toca FC administration.

Toca 511, a clinical-stage retroviral replicating vector (RRV) encoding an optimized yeast cytosine deaminase (CD) prodrug activator gene, in combination with Toca FC (extended-release 5-fluorocytosine (5-FC)), is designed to produce 5-FU which kills cancer cells and immune-suppressive myeloid cells in the tumor microenvironment, leading to anti-cancer immune activation and long term survival. The combination treatment is currently under evaluation in an international Phase 2/3 trial in patients with recurrent high-grade glioma. In the present study, we investigated the feasibility of further applying Toca 511 to brain-metastatic breast cancer, which frequently arises from highly aggressive, treatment-refractory, “triple-negative” (ER(-) PR(-) HER2(-)) disease, and is associated with a dismal prognosis of 4-6 months survival. We first evaluated in vitro replication kinetics of RRV encoding the GFP reporter gene in MDA-MB-231-BR (human) and JC (murine) breast cancer cells. After virus inoculation at either M.O.I. of 0.01 or 0.1, high levels of transduction were achieved within 1-2 weeks as measured by flow cytometric quantitation of GFP fluorescence. Next, we tested in vitro cytotoxicity by MTS assay after 5-FC treatment of MDA-MB-231BR and JC cells transduced with Toca 511. In both Toca 511-transduced breast cancer lines, cell viability was reduced by approximately 70-85% after exposure to 0.1 mM 5-FC and complete cell killing was observed with 1 mM 5-FC within 4-6 days. In survival studies, animals treated with Toca 511 followed by 5-FC prodrug showed statistically significant (231-BR: p

Toca 511 (vocimagene amiretrorepvec) is an investigational retroviral replicating vector that selectively infects dividing cancer cells, integrates into the genome and replicates due to immune defects in tumors. Toca 511 spreads through tumors and stably delivers the gene encoding an optimized yeast cytosine deaminase that converts the prodrug Toca FC (an investigational, extended-release formulation of 5-fluorocytosine) into 5-fluorouracil. 5-fluorouracil kills infected cancer cells and surrounding cancer cells, myeloid derived suppressor cells and tumor associated macrophages, thus enabling immune activity against the tumor. In this phase 1 trial (NCT01470794), ascending doses of Toca 511 were injected into the resection bed of patients with rHGG, followed by multiple courses of oral Toca FC. Additional cohorts included combination of the investigational therapy with bevacizumab or lomustine. Objective responses (ORs) are observed in patients with IDH1 wildtype and mutant tumors, including 3 complete responses (CRs) and 2 partial responses with the investigational therapy, and 1 CR with the investigational therapy and bevacizumab. The IDH1-mutant patients treated at 1st recurrence all had CRs and the fact that a CR in rHGG is rare suggests that the investigational treatment may be playing a role. ORs are observed 6–19 months after Toca 511 injection, consistent with an immunologic mechanism. Median time to initial response is 9.2 months; median duration of response (mDoR) is 25.2 months. Excluding combination cohorts, mDoR is 26.7 months. All responders are alive 21.2+ to 42.6+ months, suggesting a correlation of durable responses (ORs lasting > 24 weeks) with overall survival. In a 24-patient subgroup who received the recommended Ph2 Toca 511 dose, a durable response rate of 20.8% was observed. Across the Phase 1 program, the safety profile remains favorable. Updated clinical benefit, safety, immune activity, and molecular profiles will be reported.

Toca 511 (vocimagene amiretrorepvec) is a gamma retroviral replicating vector that selectively infects cancer cells in vivo and encodes cytosine deaminase. In combination with the prodrug, 5-fluorocytosine (5-FC), Toca 511 produces 5-fluorouracil (5-FU) locally in the tumor microenvironment. This work aimed to determine if the addition of a checkpoint inhibitor, αCTLA-4 would provide therapeutic benefit to Toca 511 and 5-FC in a mouse model of glioma. Initially, we noted that Toca 511 and 5-FC was highly efficacious and that it provided little room for improvement and therefore combination with αCTLA-4 was not able to show additive benefit against the primary cancer. Further examination revealed that tumor associated Regulatory T cells were significantly reduced with αCTLA-4 treatment and long term memory was shown to be significantly improved with the combination. Adoptive transfer of immune cells from animals that cleared their primary tumor through Toca 511, 5-FC, and αCTLA-4 showed 100% survival benefit to animals bearing orthotopic gliomas; significantly greater than the ~50% survival seen with transfer from animals that cleared primary tumor through Toca 511 and 5-FC alone. Further, αCTLA-4 treatment during clearance of primary tumors resulted in a marked reduction of memory T regulatory cells in secondary tumors. Finally, we wanted to determine if αCTLA-4 in combination with Toca 511 and 5-FC significantly reduced tumor burden in a model using a submaximal infection level of Toca 511. Specifically, restricting Toca 511 infection to only 2% of tumor cells limited the activity of 5-FC in a subcutaneous setting and the loss of efficacy with 2% infection was rescued when 5-FC treatment was combined with αCTLA-4. These data suggest that αCTLA-4, and other compounds that target T regulatory cells, should be evaluated in patients receiving Toca 511 and Toca FC to determine if the combination confers additional clinical benefits.

Toca 511, a clinical-stage retroviral replicating vector (RRV) encoding an optimized yeast cytosine deaminase (CD) prodrug activator gene, in combination with Toca FC (extended-release 5-fluorocytosine (5-FC)), is designed to produce 5-FU which kills cancer cells and immune-suppressive myeloid cells in the tumor microenvironment, leading to anti-cancer immune activation and long-term survival. This combination treatment is currently under evaluation in an international Phase 2/3 trial in patients with recurrent high-grade glioma. In the present study, we investigated the feasibility of further applying Toca 511 & Toca FC in medulloblastoma, the most common malignant tumor of the pediatric nervous system. Aggressive chemotherapy regimens increase survival in children with this disease; however, systemic toxicity limits efficacy. Here we have initiated the first preclinical studies to evaluate RRV-mediated prodrug activator gene therapy in medulloblastoma. Human medulloblastoma cell lines (HDMB03, DAOY) or patient-derived primary medulloblastoma cells were transduced with RRVs encoding either Emerald green fluorescent reporter (RRV-EMD) or the CD enzyme (RRV-CD). RRV replication in vitro, examined by flow cytometry for EMD fluorescence, showed spread to >90% of cells in culture by day 10 post-inoculation. In contrast to non-transduced or RRV-EMD-transduced controls, RRV-CD-transduced medulloblastoma cells exhibited significant dose-dependent reduction of viability upon exposure to 5-FC, as measured by MTS assay. In vivo, stereotactic intracerebellar implantation of non-transduced HDMB03 cells into nude mice resulted in cell dose-dependent median survival time and eventual 100% lethality at all cell doses tested. In this orthotopic medulloblastoma model, mice with RRV-CD-transduced intracerebellar HDMB03 tumors demonstrated increased survival upon 5-FC treatment, as compared to mice treated with PBS or implanted with non-transduced cells. These results support further investigation of RRV-CD-mediated prodrug activator gene therapy for this pediatric malignancy.

Author(s): Hiraoka, Kei; Inagaki, Akihito; Kato, Yuki; Huang, Tiffany T; Mitchell, Leah A; Kamijima, Shuichi; Takahashi, Masamichi; Matsumoto, Hiroshi; Hacke, Katrin; Kruse, Carol A; Ostertag, Derek; Robbins, Joan M; Gruber, Harry E; Jolly, Douglas J; Kasahara, Noriyuki | Abstract: BackgroundProdrug-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.MethodsHere 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.ResultsIn 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 g300 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.ConclusionThese 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.

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/β 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.

Toca 511 (vocimagene amiretrorepvec) is an investigational, conditionally lytic, retroviral replicating vector that selectively infects cancer cells due to cell division requirements for virus integration into the genome, and defects in innate and adaptive immune responses found in malignant tissues that support virus replication. Toca 511 spreads through cancer cells and stably delivers optimized yeast cytosine deaminase (CD) that, upon administration of the prodrug, Toca FC (an investigational, extended-release version of 5-fluorocytosine [5-FC]), converts 5-FC into 5-fluorouracil (5-FU). 5-FU kills infected dividing cancer cells and diffuses to and kills surrounding cells in the tumor microenvironment, including immunosuppressive myeloid cells. In animal models, this depletion of immunosuppressive myeloid cells leads to therapeutically active immunity against tumors. A similarly derived antitumor response may occur in cancer patients, as local injection of Toca 511 into the tumor bed after resection of recurrent high-grade glioma followed by treatment with Toca FC has been associated with prolonged survival and durable complete responses (median duration of follow-up: 37.4+ months); responses were delayed in onset, consistent with time to response for immuno-oncology agents. The current phase 1b, multicenter, open-label study (Toca 6; NCT02576665) is designed to investigate changes in immune activity after treatment with Toca 511 and Toca FC in patients with advanced malignancies. Toca 511 is administered intravenously (IV) daily for 3 days and then as a single injection into metastatic or recurrent tumor. Oral Toca FC is started ~4 weeks later and repeated every 4-6 weeks. Biopsies are obtained prior to and following exposure to Toca 511 and Toca FC treatment to evaluate changes in immune activity, and peripheral blood is obtained contemporaneously for evaluation. The study has enrolled 19 patients to date (colorectal cancer: 15; sarcoma: 2; non-small cell lung and pancreas cancer: 1 each). Treatment has been well tolerated. Viral RNA, DNA, and CD protein expression are observed in tumor after IV delivery of Toca 511. We plan to report on tumor microenvironment remodeling that follows treatment with Toca 511 and Toca FC. Infiltrating T-cell subpopulations, B cells, and monocytes quantified by immunofluorescence from stained formalin-fixed, paraffin-embedded samples will be presented. Additionally, changes in peripheral blood, including T-cell effector, helper/memory, and regulatory populations, and myeloid lineage cells following exposure to Toca 511 alone and following subsequent exposure to Toca FC will be reported. Data from this study will inform future development of Toca 511 and Toca FC alone or in combination with other therapies in patients with solid tumors. Citation Format: Jaime Merchan, Jordi Rodon, Derek Ostertag, Shree Venkat, Arthur Donahue, Peder Horner, Dalissa Tijera, Thian Kheoh, Douglas J. Jolly, Harry E. Gruber, Jolene S. Shorr, Gerald S. Falchook. Effects of Toca 511 and Toca FC on tumor microenvironment and peripheral blood populations in patients with advanced malignancies [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A018.

In the present study, we compared the therapeutic effect of tumor-selective retroviral replicating vectors (RRV) expressing the yeast cytosine deaminase (CD) delivered by convection-enhanced delivery (CED) or simple injection, followed by systemic administration of the pro-drug, 5-fluorocytosine (5-FC). Treatment with RRV-CD and systemic 5-FC significantly increased survival in rodent U87MG glioma model in comparison with controls (P0.01). Interestingly, CED of RRV-CD followed by 5-FC further enhanced survival in this animal model in comparison with intra-tumoral injection of RRV-CD, followed by systemic 5-FC (P0.05). High expression levels of Ki-67 were found in untreated tumors compared with treated. Untreated tumors were also much larger than treated. CED resulted in excellent distribution of RRV while only partial distribution of RRV was obtained after injection. Furthermore, RRV-CD and CD were also found in tumors from treated rats at study end points. These results demonstrated that RRV vectors may efficiently transduce and stably propagate in malignant human glioma, thereby achieving a significant in situ amplification effect after initial administration. We conclude that delivery of RRV into the glioma by CED provides much wider vector distribution than simple injection, and this correlated with better therapeutic outcomes.

In pharmaceutical drug development, information on where a drug distributes within the body is helpful in understanding the overall chemical and/or biological characteristics of the compound. This is typically assessed preclinically in relevant animal species that are often used for the formal safety studies (toxicology, pharmacokinetics and drug metabolism. One accepted principle of animal studies is the application of the “3 R’s concept” (replacement, refinement and reduction http://www.nc3rs.org.uk/) to reduce animal use as far as makes sense. The focus of this article is to suggest that, for gene therapy biodistribution studies, reductions in animal use could be rationally achieved with no increase in clinical risk.

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).

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.

Retroviral replicating vectors (RRVs) are a nonlytic alternative to oncolytic replicating viruses as anticancer agents, being selective both for dividing cells and for cells that have defects in innate immunity and interferon responsiveness. Tumor cells fit both these descriptions. Previous publications have described a prototype based on an amphotropic murine leukemia virus (MLV), encoding yeast cytosine deaminase (CD) that converts the prodrug 5-fluorocytosine (5-FC) to the potent anticancer drug, 5-fluorouracil (5-FU) in an infected tumor. We report here the selection of one lead clinical candidate based on a general design goal to optimize the genetic stability of the virus and the CD activity produced by the delivered transgene. Vectors were tested for titer, genetic stability, CD protein and enzyme activity, ability to confer susceptibility to 5-FC, and preliminary in vivo antitumor activity and stability. One vector, Toca 511, (aka T5.0002) encoding an optimized CD, shows a threefold increased specific activity in infected cells over infection with the prototype RRV and shows markedly higher genetic stability. Animal testing demonstrated that Toca 511 replicates stably in human tumor xenografts and, after 5-FC administration, causes complete regression of such xenografts. Toca 511 (vocimagene amiretrorepvec) has been taken forward to preclinical and clinical trials.

Conventional interventions for treating glioblastoma (GBM) patients has had limited success, with a median overall survival of 15–17 months. Recently, immune checkpoint inhibitors (CPIs) showed long-term response rates from 20–30% in some tumors, but no consistent clinical benefit with these agents has been demonstrated so far in GBM patients. We propose and provide preliminary evidence for a strategy using retroviral replicating vectors (RRV) to deliver CPI agents selectively to cancer cells that may circumvent such issues. An RRV encoding a single-chain variable fragment targeting PD-L1 (RRV-scFv-PDL1, Toca 521) binds to both mouse and human PD-L1 by competitive ELISA and competes for target occupancy with a commercially available monoclonal antibody against cell surface PD-L1. A dose-dependent bystander effect is observed with scFv PD-L1 protein expressed from RRV-scFv-PDL1 infected tumor cells showing saturated receptor binding to the cell surface PD-L1 of bystander cells when co-cultured with as low as 10% scFv PD-L1 expressing cells. In addition, the immune functional activity of scFv PD-L1 to reverse PD-1/PD-L1 mediated immune suppression was observed in a co-culture system in vitro and further supported by in vivo mouse models. Such models included a syngeneic orthotopic glioma showing that tumors infected with RRV-scFv-PDL1 conferred robust and durable immune-mediated antitumor activity superior to systemically administered anti-PD-1/anti-PD-L1 monoclonal antibodies. These results support the concept that RRV-scFv-PDL1 CPI strategy may provide an improved safety and efficacy profile compared to systemic monoclonal antibodies. The superior anti-tumor activity of RRV-scFv-PDL1 may be due to consistent high levels of scFv PD-L1 within the tumor microenvironment. This localized delivery approach with less concern for autoimmune adverse events may be therapeutically beneficial as an immuno-oncology agent either alone or in combination.

Toca 511 (vocimagene amiretrorepvec) is an investigational, conditionally lytic retroviral replicating vector (RRV) that selectively infects cancer cells. Toca 511 spreads through tumors, stably delivering an optimized cytosine deaminase (CD) gene that converts the prodrug, Toca FC (investigational, extended-release 5-FC), into 5-FU. Based on preclinical studies, 5-FU kills infected dividing cancer cells and diffuses and kills surrounding cancer cells, myeloid derived suppressor cells, and tumor associated macrophages, thus reestablishing tumor immunity. In a previous Phase 1 study, intravenous (IV) injection of Toca 511 successfully delivered and expressed CD protein to high grade glioma tumors. In animal models of metastatic colorectal cancer, IV Toca 511 infected metastatic sites, and subsequent 5-FC treatment resulted in decreased tumor size and improved survival. Toca 6 is a Phase 1b, multicenter, open-label study in patients with advanced solid tumors or lymphoma (NCT02576665) designed to investigate changes in immune activity after treatment with Toca 511 & Toca FC. Vector deposition and transgene expression in tumor tissue following IV delivery of Toca 511 also is followed. Toca 511 is injected IV daily for 3 days, with subsequent injection either intratumorally after biopsy or, in the brain tumor setting, into resection cavity walls following resection. Oral Toca FC is started approximately 4 weeks later and repeated every 4-6 weeks. A total of 30 patients are planned to be enrolled. In patients treated to date, viral RNA, DNA, and CD protein expression after IV Toca 511 delivery have been detected in liver metastases from colorectal cancer and mesenteric lymph node metastases from pancreas cancer, demonstrating IV delivery of virus to and transgene expression in tumor. Preliminary data indicate treatment has been generally well tolerated to date, with few related adverse events. Data on changes from baseline in immune activity in tumor (infiltrating T-cell subpopulations, B cells, monocytes) and peripheral blood (effector, memory, Treg, and myeloid lineage cells), viral and CD protein expression in tumor, and safety will be presented. Citation Format: Jaime Merchan, Jordi Rodon, Gerald Falchook, Shree Venkat, Derek Ostertag, Dalissa Tejera, Thian Kheoh, Douglas J. Jolly, Harry E. Gruber, Jolene S. Shorr. A phase Ib study of Toca 511, a retroviral replicating vector, followed by Toca FC in patients with advanced cancer [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 CT067.

Toca 511 (vocimagene amiretrorepvec) is an investigational, conditionally lytic, retroviral replicating vector (RRV). RRVs selectively infect cancer cells due to defects in innate and adaptive immune responses found in cancers that support virus replication, and cell division requirements for virus integration into the genome. Toca 511 spreads through cancer cells, stably delivering an optimized cytosine deaminase (CD) gene that converts the prodrug Toca FC (investigational, extended-release 5-fluorocytosine) into 5-fluorouracil (5-FU), a canonical chemotherapeutic. In preclinical tumor models, as infected cancer cells are killed, diffusible 5-FU also kills nearby susceptible cells, including uninfected cancer cells, and myeloid derived suppressor cells (MDSC) that contribute to immune-suppression in the tumor microenvironment. This action by Toca 511 and Toca FC has been shown in animal models to generate a durable anti-tumor immune response that can be transferred to naïve, untreated animals. The Toca 511 and Toca FC immunotherapeutic are proposed to remodel the tumor microenvironment to break tumor tolerance resulting in induction of antitumor activity by the patient's immune system and durable complete responses. In a phase 1 clinical study for recurrent high grade glioma (NCT01470794), Toca 511 was injected into resection cavity walls at time of resection followed by multiple courses of oral Toca FC. We observed multi-year durable and objective responses; including 5 ongoing complete responses in a group of 23 patients in the higher dose single agent treatment cohorts given approximately the same Toca 511 doses and having the same entry criteria as an ongoing Phase 3 study in recurrent high grade glioma (NCT02414165). Patient tumors at time of resection were analyzed by exome sequencing, RNA sequencing, IHC, and TCR sequencing, before Toca 511 treatment. In this study, we report higher levels of tumor infiltrating T cells by TCR sequencing, before the start of treatment, were significantly associated with responding patients compared to patients whose disease progressed. The significance of this data is supported by the preclinical mechanism of action reported previously. Additionally, we plan to report on T cell, B cell, and myeloid populations in the tumor as measured by IHC and RNA sequencing and their relationship to clinical response. Data reported here will provide mechanistic context to the immunotherapeutic mode of action proposed to account for durable responses seen in treatment of brain tumors with Toca 511 and Toca FC. Citation Format: Derek Ostertag, William Accomando, Leah Mitchell, Maria Rodriguez-Aguirre, Daniel Hogan, Oscar Diago, Dawn Gammon, Ali Haghighi, Harry Gruber, Asha Das, Douglas Jolly. Immune profile of tumor microenvironment helps predict response in patients treated with an investigational immunotherapeutic consisting of a retroviral replicating vector (Toca 511) and an extended-release formulation of 5-fluorocytosine (Toca FC) [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 5630.

Toca 511 (vocimagene amiretrorepvec) is an investigational retroviral replicating vector that selectively infects dividing cancer cells, integrates into the genome and replicates due to immune defects in tumors. Toca 511 spreads through tumors and stably delivers the gene encoding an optimized yeast cytosine deaminase that converts the prodrug Toca FC (investigational, extended-release of 5-fluorocytosine) into 5-fluorouracil. In preclinical models, 5-fluorouracil kills infected dividing cancer cells, myeloid derived suppressor cells and tumor associated macrophages, enabling immune activation against the tumor. In this dose ascending Ph1 trial (NCT01470794), Toca 511 was injected into the resection cavity wall of patients with rHGG, followed by courses of oral Toca FC. Additional cohorts included combination with bevacizumab or lomustine. Across the Ph1 program, the safety profile remains favorable. Objective responses (ORs) were assessed by IRR using MRI scans prior to Toca FC treatment as baseline. ORs occurred 6-19 months after Toca 511 administration, suggesting an immunologic mechanism. The ORs were observed in 4 patients with IDH1-wildtype and 2 patients with IDH1-mutant tumors, including 5 complete responses (CRs) with the investigational therapy alone, and 1 CR in combination with bevacizumab. The median duration of response (mDoR) was 35.1+ months. As of AUG2017, all responders were CR and remain alive. In a 23-patient subgroup who received high doses of Toca 511 and met Ph3 trial criteria, mOS was 14.4 months, 3-year survival rate was 26.1%, and mDoR was 35.7+ months with a durable response rate of 21.7%. Data suggest a positive association of durable response with OS.

TPS2613Background: Toca 511 (vocimagene amiretrorepvec) is an investigational, conditionally lytic, retroviral replicating vector that selectively infects cancer cells. Toca 511 spreads through tum...

TPS880 Background: Toca 511 (vocimagene amiretrorepvec) is an investigational, conditionally lytic, retroviral replicating vector (RRV). RRVs selectively infect cancer cells due to defects in innate and adaptive immune responses found in cancers that support virus replication, and the requirement for cell division for virus integration into the genome. Toca 511 spreads through tumors, stably delivering an optimized cytosine deaminase (CD) gene that converts the prodrug, Toca FC (investigational, extended-release 5-FC) into 5-FU. 5-FU kills infected dividing cancer cells and diffuses and kills surrounding cancer cells, myeloid derived suppressor cells, and tumor associated macrophages, thus reestablishing tumor immunity (Cloughesy et al. Neuro Oncol 2016). In a Phase 1 study, resected high grade glioma tumors expressed CD protein following intravenous (IV) Toca 511.1 In animal models of metastatic colorectal cancer, IV Toca 511 infected metastatic sites, and subsequent 5-FC treatment resulted in decreased tumor size and improved survival (Yagiz et al. Mol Therapy 2015). Methods: Toca 6 is a Phase 1b, multicenter, open-label study (NCT02576665) that aims to investigate changes in immune activity after treatment with Toca 511 & Toca FC in patients with solid tumors, including gastrointestinal tumors. A total of 30 patients who have advanced malignancies, including colorectal and pancreatic cancer, with molecular characteristics that may increase sensitivity to 5-FU or viral infection, or IDH1 mutated solid tumors (e.g., intrahepatic cholangiocarcinoma) will be enrolled. In these patients, Toca 511 is injected IV daily for 3 days, then intratumorally after biopsy. Oral Toca FC is started ~4 weeks later and repeated every 4-6 weeks. Changes from baseline in intratumoral immune activity (infiltrating T-cell subpopulations, B cells, monocytes) at 4 weeks after start of Toca FC are assessed. Contemporaneous peripheral blood is analyzed for effector, memory, Treg, and myeloid lineage cells. Viral RNA, DNA, and CD protein expression in tumor after IV Toca 511 are measured. Safety and efficacy will be determined. Concomitant checkpoint inhibitor therapy may be given following immune activity assessments. The study has enrolled 3 patients. Clinical trial information: NCT02576665.

Toca 511 (vocimagene amiretrorepvec), in combination with Toca FC (5-FC; flucytosine extended-release tablets), is an investigational combination product currently under development for the treatment of high-grade glioma (HGG) and other solid tumors. Toca 511 is a gamma-retroviral replicating vector (RRV) delivering a cytosine deaminase transgene to infected cells to convert antifungal drug 5-fluorocytosine (5-FC) into antineoplastic drug 5-fluorouracil (5-FU). Toca 511 can be administered by multiple routes; selectively infects, spreads, and persists in cancer cells; and through multiple mechanisms of action can elicit an antitumor immune response. Treatment directly kills infected cancer cells. Diffusible 5-FU also kills neighboring susceptible cells that contribute to the immune-suppressed tumor microenvironment, especially myeloid immune suppressor cells. After multiple cycles of 5-FC, treated animals that clear tumor are resistant to tumor rechallenge. Adoptive transfer recipients of splenocytes from animals that previously cleared their tumor through treatment with Toca 511 and 5-FC experience a significant survival benefit compared to animals receiving spleen cells from naïve donors. Toca 511 and Toca FC have been administered to 126 recurrent high-grade glioma (rHGG) patients in three phase I studies (NCT01156584, NCT01470794, NCT01985256), and a phase 2/3 trial (Toca 5) is ongoing (NCT02414165). Toca 511 plus Toca FC has been well tolerated in these patients. In the clinical study presented, Toca 511 is injected into resection cavity walls at time of tumor resection, and followed with multiple courses of Toca FC. In a subset of phase I patients that mimic the phase 2/3 rHGG enrollment criteria, there were 3 complete responses and 2 partial responses out of 24 patients observed. The median duration of response is 26.7 months. These responders have an overall survival range from 24.0+ to 42.6+ months and ongoing. Data are consistent with induction of antitumor immune responses after Toca FC administration. Changes in peripheral immune populations as well as interrogation of immunogenic biomarkers in responding compared to nonresponding patients will be presented. Citation Format: Tiffany T. Montellano, Derek Ostertag, Daniel J. Hogan, Oscar R. Diago, Dawn Gammon, Ali Haghighi, William Accomando, Leah Mitchell, Asha Das, Harry Gruber, Douglas J. Jolly. Antitumor cellular immune response elicited by Toca 511 and Toca FC therapy in preclinical and clinical studies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B010.