Carlos E. Ibanez

Blockade of Type I Interferon (IFN) Production by Retroviral Replicating Vectors and Reduced Tumor Cell Responses to IFN Likely Contribute To Tumor Selectivity

ABSTRACT 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.

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 (As) in the J-K bifurcation domain sometimes serially added As. Therefore, vectors with 4-12 As 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 As gave roughly comparable levels, 4 and 8 As were within about 4-5-fold of the 6 As, whereas 10 and 12 As were marked lower. In terms of stability, after 10 infection cycles, expansion of As appeared to be a more frequent event affecting transgene protein expression than viral genome deletions or rearrangement: 4 and 5 As appeared completely stable; 6, 7, and particularly 8 As showed some level of expansion in the A bulge; 10 and 12 As underwent both expansion and transgene deletion. The strong relative translational activity of the 5 As 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.

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).

205. Cytotoxic and Immunotherapeutic Effects of Toca 511 and 5-Fluorocytosine in an Intraperitoneal Model of Metastatic Colorectal Cancer

Toca 511 (vocimagene amiretrorepvec), a gamma retroviral replicating vector encoding an optimized yeast cytosine deaminase (CD) gene, selectively replicates and spreads in tumors. In infected cells, CD enzyme is expressed and converts 5-FC (5-fluorocytosine, an orally-available anti-fungal drug) to 5-FU (5-fluorouracil), leading to both direct tumor cytotoxicity and extended immunotherapeutic effects in preclinical models. Toca 511 in combination with Toca FC (extended-release 5-FC) recently entered a Phase 2/3 trial (NCT02414165) in patients with recurrent HGG (high grade glioma). Toca 511 is also under investigation delivered via intravenous (IV) infusion followed by injection into the wall of the resection cavity (NCT01985256), followed by Toca FC, in patients with recurrent HGG. IV administration of vectors is minimally invasive, can easily be repeated if desired, and may be applicable to other tumor types including metastatic colorectal cancer (mCRC). Previously we have shown that IV delivery of Toca 511 in a mouse syngeneic mCRC liver model resulted in expression of CD in tumor foci, but not in adjacent normal liver, and followed by courses of 5-FC resulted in direct tumor response, improved survival, and a systemic anti-tumor immune response. Elevated circulating myeloid-derived suppressor cells (MDSC) are associated with advanced disease stages in CRC patients and failure of immunotherapeutic strategies. We further investigated the effect of intralesional administration of Toca 511 with 5-FC treatment on tumor recurrence and immune infiltrates in a model of CRC brain metastases. A significant decrease in MDSC in spleens and tumors was observed with Toca 511 and 5-FC compared to controls, via in situ production of 5-FU (p=0.03, p<0.0001; respectively). Currently, we are investigating the efficacy of Toca 511 and 5-FC in a mouse syngeneic intraperitoneal (IP) model. We identified the optimal delivery method for treatment of IP metastases, and evaluated survival after IV and IP vector delivery followed by courses of 5-FC. Treatment with Toca 511 and 5-FC led to an improved median survival compared to control. Systemic 5-FU has hematological toxicity even at low doses (20 mg/kg) in both naive and CRC tumor-bearing mice which could have an adverse effect on anti-tumor immune responses. We further evaluated Toca 511 and 5-FC treatment compared to systemic 5-FU in terms of efficacy, hematologic toxicity, and induction of anti-tumor immune responses in the IP mCRC model. The effect of Toca 511 and 5-FC compared to systemic 5-FU on MDSC and other immune cell populations will be presented. Our data provides support for the development of Toca 511 and 5-FC as a unique approach targeting both the tumor and the immune system for the treatment of metastatic cancers such as mCRC. A phase 1 study of IV Toca 511 and Toca FC in solid tumors, including mCRC, is planned (NCT02576665).

531. Intravenous Delivery of Toca 511 Gene Therapy in Combination with 5-Fluorocytosine for Intratumoral Production of 5-Fluorouracil in a Colon Cancer Metastasis Model

Despite advances in screening, colorectal cancer (CRC) remains the fourth most commonly diagnosed cancer and the second leading cause of cancer death for both men and women in the US. Approximately one half of patients with CRC develop liver metastases (mCRC). The standard treatment for mCRC is 5-fluorouracil (5-FU) based combination chemotherapy. 5-FU combination chemotherapy has extended the median survival of these patients from 6 to >20 months.We are pursuing a unique investigational approach to treat cancer via in situ production of 5-FU. Toca 511 (vocimagene amiretrorepvec), a retroviral replicating vector (RRV), selectively replicates and spreads in malignant cells and encodes an optimized yeast cytosine deaminase (CD) gene. Within infected cells, the CD enzyme is expressed and converts 5-FC (flucytosine, an orally available anti-fungal drug) to the anti-cancer drug 5-FU. Both a direct cytotoxic effect and an extended immunotherapeutic effect have been reported using this approach.Toca 511, in conjunction with subsequent oral extended-release 5-fluorocytosine (Toca FC), is currently under investigation in patients with recurrent high grade glioma. In these studies, Toca 511 is delivered either intratumorally (NCT01156584), by injection into the surgical resection bed (NCT01470794), or intravenously (NCT01985256) in subjects scheduled for subsequent resection. We tested the suitability of the intravenous approach for the treatment of mCRC in a mouse syngeneic liver metastasis model. CT-26-luciferase colon carcinoma cells were delivered via intrasplenic injection producing multiple tumor foci within the liver. Intravenous delivery of RRV resulted in expression of the vector encoded transgene in tumor foci but not in adjacent normal liver tissue. Intravenous delivery of Toca 511 followed by courses of 5-FC resulted in shrinkage or elimination of tumor foci and improved survival in this model of mCRC. The data is supportive of future clinical trials of intravenous Toca 511 followed by cycles of Toca FC in metastatic CRC.

61. Ascending Dose Trials of a Retroviral Replicating Vector (Toca 511) in Patients with Recurrent High-Grade Glioma: Clinical Update, Molecular Analyses, and Proposed Mechanism of Action

We have conducted Phase 1 studies in patients with high grade glioma of a retroviral replicating vector (RRV), Toca 511 (vocimagene amiretrorepvec), based on an amphotropic murine gamma retrovirus encoding an optimized cytosine deaminase. The vector appears highly selective for tumor cells. Infected cells convert the antifungal drug 5-fluorocytosine (5-FC) delivered as an orally available extended-release formulation (Toca FC), into the antineoplastic drug 5-fluorouracil (5-FU). Toca 511 has been delivered by intratumoral injection (NCT01156584), injection into the tumor bed post-resection (NCT01470794), or by IV administration (NCT01985256). In all cases the treatment is well-tolerated. In animal models extensive infection of tumors (close to 100%) leads to control of tumor growth both in xenograft and syngeneic models. Limited access to tumor tissue post Toca 511 treatment in trial subjects, and lack of good markers of human glioblastoma cells in these heterogeneous primary tumors make it difficult to determine the extent of cancer cell infection but there is good evidence of selective tumor infection by PCR, RTPCR and IHC against the CD protein. Clinical data in both the first two trials (intratumoral and resection bed administration with 39 and 43 evaluable subjects respectively) show a favorable safety profile and extended overall survival (OS) compared to historical controls. In the resection study, for example, median OS was 13.6 months compared to 7-8 months in matched historical controls, and the OS at 24 months was 32%. In addition an RNA expression signature in untreated resected tumors that predicts long term survival in trial subjects has been identified from subjects that subsequently underwent tumor bed administration of Toca 511 and Toca FC. This signature does not normally correlate with survival in available public data sets. In immune competent orthotopic animal models, Toca 511 and 5-FC treatment leads to apparent tumor elimination and induction of strong antitumor immune responses by several mechanisms, including local elimination of myeloid derived suppressor cells. Subcutaneous re-implantation of the same tumors did not lead to tumor growth in animals treated up to a year before, whereas tumors did develop in control naive animals. Available data in the human trials are consistent with the immune response playing a significant role in the apparent clinical efficacy. Thus, clinically, treatment with Toca 511 and extended-release 5-FC (Toca FC) appears to selectively destroy tumor cells within the body, while leaving healthy cells unharmed. Toca 511 and Toca FC have been administered to more than 120 high grade glioma subjects in the three studies and, based on results from these trials, a phase 2/3 trial (Toca 5 has recently started recruitment (NCT02414165). The combination of clinical and preclinical data supporting this decision will be reviewed.

Abstract 1413: Treatment of mouse liver and brain colon cancer metastases with Toca 511 and 5-fluorocytosine for intratumoral production of 5-fluorouracil leads to increased survival, induction of antitumor immune responses, and reduction of MDSC

Approximately 50% of patients with colorectal cancer (CRC) develop metastases (mCRC) during the course of disease, with liver the most frequent site. Standard treatment for mCRC is 5-fluorouracil (5-FU) based combination therapy, with median survival now >20 months. As a result of prolonged survival with metastatic disease, incidence of brain metastases from colorectal cancer is increasing. Approved immunotherapeutic agents have had limited effect in mCRC except in the subset of subjects (3-6%) with deficient mismatch repair. Recent studies suggest myeloid-derived suppressor cells (MDSC) contribute to cancer immune evasion by suppressing T cell anti-tumor functions and modulating innate immune responses. We are pursuing a unique approach to treat cancer via in situ production of 5-FU. Toca 511 (vocimagene amiretrorepvec), a retroviral replicating vector, selectively replicates and spreads in malignant cells and encodes an optimized yeast cytosine deaminase (CD) protein. In infected cells, CD enzyme is expressed and converts 5-FC (5-fluorocytosine, an oral anti-fungal drug) to 5-FU. Direct tumor cytotoxicity and extended immunotherapeutic effects have been reported using this approach. Toca 511 and Toca FC (extended release 5-FC) is under investigation in subjects with primary brain tumors, delivered intratumorally (NCT01156584), by injection into the resection bed (NCT01470794, NCT02414165), or intravenously (NCT01985256). We tested the intravenous (IV) approach for the treatment of mCRC in a mouse syngeneic liver metastasis model. CT-26-luciferase colon carcinoma cells were delivered via intrasplenic injection producing multiple tumor foci in the liver. IV delivery of Toca 511 followed by courses of 5-FC resulted in tumor response, improved survival, and immune mediated inhibition of rechallenge with tumor. IV delivery resulted in expression of vector encoded transgene in tumor foci but not in adjacent normal liver. Similar published results have been obtained in a number of brain tumor models with Toca 511+5-FC including a CT26 colon carcinoma brain metastasis model. We further investigated the effect of Toca 511+5-FC treatment on CRC tumor-induced MDSC in the CT-26 brain metastasis model. Intracranial cell implantation and intratumoral Toca 511 vector injections were followed by one course of 5-FC. Expansion of MDSC occurred in brain tumors and spleens of tumor bearing animals. A significant decrease in MDSC in spleens and tumors was observed with Toca 511+5-FC treatment compared to control (p = 0.03, p The results reported here support the development of Toca 511 and Toca FC as a novel immunotherapeutic approach for patients with mCRC. A phase 1 study of IV Toca 511 and Toca FC in solid tumors, including mCRC, is planned (NCT02576665). Citation Format: Maria Rodriguez-Aguirre, Kader Yagiz, Fernando Lopez Espinoza, Daniel Mendoza, Tiffany T. Huang, Carlos Ibanez, Harry E. Gruber, Douglas J. Jolly, Joan Robbins. Treatment of mouse liver and brain colon cancer metastases with Toca 511 and 5-fluorocytosine for intratumoral production of 5-fluorouracil leads to increased survival, induction of antitumor immune responses, and reduction of MDSC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1413.

227. Cancer Cell Susceptibility To Treatment With a Retroviral Replicating Vector Encoding Cytosine Deaminase and 5-FC Is Likely To Be Common

We are currently investigating the clinical utility of a Retroviral Replicating Vector (RRV), Toca 511 (vocimagene amiretrorepvec) which encodes an optimized yeast cytosine deaminase (yCD2) transgene, in recurrent high grade glioma (NCT01470794, NCT01156584 & NCT01985256). Toca 511, which is based on amphotropic murine gamma retroviruses, selectively infects tumors without immediate cell killing. Expression of virally-delivered yCD2 converts orally administered 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) in infected tumor cells. There are several variables that could affect how well this combination therapy works in different tumors. By integrating a combination of RNA-seq, Liquid Chromatography/Mass Spectrophotometry (LC/MS), and PCR analysis with biochemical and virological assays, key criteria have been identified that help predict how Tocagens Toca 511 RRV gene therapy will perform in several different tumor types. Efficient production and secretion of 5-FU by Toca511 infected tumor cells are expected to kill other uninfected cells in the immediate tumor microenvironment (the so-called “bystander” effect) and may, thereby, also influence the extent of induction of anti-tumor immune responses in animal models and patients.A panel of 9 established human tumors derived from 3 different tissue types (brain, colon and breast), each with unique histology, growth characteristics and morphology, was used to evaluate parameters central to effective antitumor activity in different indications. Parameters measured included: the rate of cell division; the rate of infection; the levels of CD gene integration and expression; intracellular and extracellular concentrations of 5-FC, 5-FU and downstream metabolites; gene expression levels of viral susceptibility genes and genes for pyrimidine metabolism. The data showed that: 1) all the tumor lines tested infected quite readily; 2) the rate of infection (varied 7-fold) was not dependent on the rate of cell division; 3) the expression of CD mRNA and protein were highly correlative with conversion of 5-FC to 5-FU; 4) all tumors were readily able to import 5-FC; 5) while LC/MS analysis established all tumors were readily able to import 5-FC, RNA-seq analysis correlated intracellular 5-FC levels with expression of SLC29A1, which codes for the main 5-FC transporter. Further, in vitro data from infected cell extracts, showed that the production of 5-FU could be augmented by increasing the 5-FC concentrations up to 5mg/mL. This suggests that infected tumor cells in vivo can kill uninfected nearby tumor cells better with higher doses of 5-FC. In summary, these studies identified multiple factors that influence Toca 511 gene therapy in the 9 tumor cell lines in culture. Data from this initial integrative approach further supports the conclusion that Toca 511 therapy is likely to be effective in killing multipletumortypes.

Abstract 708: Toca 511 gene therapy in combination with 5-fluorocytosine for intratumoral production of 5-fluorouracil in a colon cancer metastasis model

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Despite advances in screening, colorectal cancer (CRC) remains the fourth most commonly diagnosed cancer and the second leading cause of cancer death for both men and women in the US. Approximately one half of patients with CRC develop liver metastases (mCRC). The standard treatment for mCRC is 5-fluorouracil (5-FU) based combination chemotherapy. 5-FU combination chemotherapy has extended the median survival of these patients from 6 to >20 months. We are pursuing a unique investigational approach to treat cancer via intratumoral production of 5-FU. Patients are first administered Toca 511 (vocimagene amiretrorepvec), a retroviral replicating vector (RRV) which selectively replicates and spreads in malignant cells and encodes an optimized yeast cytosine deaminase (CD) gene. In infected cells, the CD enzyme is expressed and converts 5-FC (flucytosine, an orally available anti-fungal drug) to the anti-cancer drug 5-FU in situ. The combination of Toca 511 with oral extended release 5-FC (Toca FC) is currently in clinical trials for recurrent High Grade Glioma ([NCT01156584][1], [NCT01470794][2], and [NCT01985256][3]). We tested the suitability of this approach for the treatment of mCRC in a mouse syngeneic liver metastasis model. CT-26-luciferase colon carcinoma cells were delivered via intrasplenic injection producing multiple tumor foci within the liver. Toca 511 was then delivered either by intrasplenic or intravenous injection followed by courses of 5-FC. Vector delivery to tumor was monitored by PCR. 5-FC and 5-FU were detected using LC/MS. The effect of treatment with Toca 511 and 5-FC in this mouse model of mCRC will be presented. Citation Format: Maria E. Rodriguez-Aguirre, Fernando Lopez Espinoza, Bryan Martin, Kader Yadiz, Tiffany T. Huang, Carlos E. Ibanez, Derek G. Ostertag, Noriyuki Kasahara, Harry E. Gruber, Douglas J. Jolly, Joan M. Robbins. Toca 511 gene therapy in combination with 5-fluorocytosine for intratumoral production of 5-fluorouracil in a colon cancer metastasis model. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 708. doi:10.1158/1538-7445.AM2014-708 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01156584&atom=%2Fcanres%2F74%2F19_Supplement%2F708.atom [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01470794&atom=%2Fcanres%2F74%2F19_Supplement%2F708.atom [3]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01985256&atom=%2Fcanres%2F74%2F19_Supplement%2F708.atom