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

Abstract

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.