Daniel Hogan

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

Toca 511 and Toca FC show promising results in treating recurrent high-grade glioma, and a specific molecular signature correlates with treatment-related survival. Tag-team attack on glioma Toca FC (extended-release 5-fluorocytosine) and Toca 511 (vocimagene amiretrorepvec) are an investigational therapeutic combination for glioma, consisting of two parts: a prodrug that is inactive on its own and a modified virus that infects the tumor and delivers an enzyme, which then activates the drug and allows it to kill the glioma cells. Cloughesy et al. tested this therapy in 45 human patients with recurrent or progressive high-grade glioma and discovered that the treatment was well tolerated and improved survival compared to an external control group. In addition, the authors identified a gene signature that correlated with response to the treatment, which may help identify the patients most likely to benefit from this approach. 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).

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

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

MethylMeter®: bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples

AIM Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. MATERIALS & METHODS Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. RESULTS MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. CONCLUSION MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

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

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

Abstract 5630: 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)

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 naive, 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 patient9s 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.

403. Toca Retroviral Replicating Vector in Tumor and Blood from Clinical Trial Subjects with Recurrent High Grade Glioma

Toca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector (RRV) based on an amphotropic murine gammaretrovirus, that encodes a cytosine deaminase transgene that allows infected cells to selectively convert the antifungal drug 5-fluorocytosine (5-FC) into the antineoplastic drug 5-fluorouracil (5-FU). Toca 511 can be delivered by multiple routes, selectively infects and spreads in tumor cells and through multiple mechanisms can elicit an anti-tumor immune response. Thus, clinically, treatment with Toca 511 and extended-release 5-FC (Toca FC) is expected to selectively destroy tumor cells within the body, while leaving healthy cells unharmed. Toca 511 and Toca FC have been administered to >120 high grade glioma subjects in three phase I studies (NCT01156584, NCT01470794, NCT01985256) and, based on results from these trials, a phase 2/3 trial (Toca 5) has recently started recruitment (NCT02414165). Although Toca 511 plus Toca FC has been well tolerated, we investigated potential off-target viral infection and integration, and whether such off-target events have potential toxicity; we also investigated the stability profile of the Toca 511 genome after infection of tumor targets and off-target tissue (blood). To date, few subjects (approximately 10-25%) show quantifiable viral signal (LOQ approximately 4000 copies/mL) in blood after initial clearance, following Toca 511 injection into the brain tumor or tumor bed. In all cases, quantifiable virus RNA and DNA signal was cleared from blood. In order to better understand interactions among Toca 511 and subjects’ tumor and blood, we systematically mapped Toca 511 integration sites from subject samples with detectable virus and also sequenced, at high depth, integrated Toca 511 genomes, and in some cases RNA genomes, from these samples. Toca 511 integration profiles display a preference for integration near active transcription start sites, as has been seen generally for gammaretroviruses. There was no evidence for clonal expansion of Toca 511 integrated sites/cells or preferential retrieval of sites nearby oncogenes. Toca 511 sequences display an array of mutations in the tumor and blood, including transitions consistent with cytosine deaminase activity. The data provide a molecular correlate to the clinical safety profile of Toca 511 and Toca FC treatment as well as molecular characterization of RRV after infection, replication and therapeutic use in humans.

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.