Abstract
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.