Abstract
Toca 511, a clinical-stage 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)), is designed to produce 5-FU which kills cancer cells and immune-suppressive myeloid cells in the tumor microenvironment, leading to anti-cancer immune activation and long term survival. The combination treatment is currently under evaluation in an international Phase 2/3 trial in patients with recurrent high-grade glioma. In the present study, we investigated the feasibility of further applying Toca 511 to brain-metastatic breast cancer, which frequently arises from highly aggressive, treatment-refractory, “triple-negative” (ER(-) PR(-) HER2(-)) disease, and is associated with a dismal prognosis of 4-6 months survival. We first evaluated
in vitro
replication kinetics of RRV encoding the GFP reporter gene in MDA-MB-231-BR (human) and JC (murine) breast cancer cells. After virus inoculation at either M.O.I. of 0.01 or 0.1, high levels of transduction were achieved within 1-2 weeks as measured by flow cytometric quantitation of GFP fluorescence. Next, we tested
in vitro
cytotoxicity by MTS assay after 5-FC treatment of MDA-MB-231BR and JC cells transduced with Toca 511. In both Toca 511-transduced breast cancer lines, cell viability was reduced by approximately 70-85% after exposure to 0.1 mM 5-FC and complete cell killing was observed with 1 mM 5-FC within 4-6 days. In survival studies, animals treated with Toca 511 followed by 5-FC prodrug showed statistically significant (231-BR: p<0.0001, JC: p=0.0003) survival benefit compared to the control group. These data provide preclinical validation for a new Phase Ib trial evaluating RRV-mediated immunotherapy in various types of metastatic malignancies, including CNS-metastatic breast cancer (TOCA 6 trial: clinicaltrials.gov NCT02576665), currently recruiting at the University of Miami.