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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.

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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.

Locations of Toca 511 Integration Sites

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

Tg 511-11-01 (Study 11) dosing table.

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Toca 511 signal in tumor and nonneoplastic brain samples taken at time of autopsy from a Study 8 patient.

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Supplementary Text

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Supplementary Figure, Table & Dataset legends

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Supplementary Figures S1 to S9 and Supplementary Table S1.

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 Tocagen's 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.