Abstract A43: Therapeutic efficacy of retroviral replicating vectors in orthotopic models of breast cancer metastasis to the brain
Authors: Inagaki Akihito , Collins Sara , Matsuura Suzanne , Kasahara Noriyuki 
Published: January 2017

Abstract
Our studies to date have demonstrated that retroviral replicating vectors (RRV) are capable of highly efficient replication in cancer cells in vitro and in vivo, associated with high levels of tumor-selective gene transfer and significantly enhanced survival benefit when employed for prodrug activator (“suicide”) gene therapy in a variety of cancer models. Clinical testing of RRV-mediated gene therapy using the yeast cytosine deaminase (CD) prodrug activator gene, which mediates conversion of the prodrug 5-fluorocytosine (5-FC) to the anti-cancer drug, 5-fluorouracil (5-FU), is currently underway in multi-center clinical trials being conducted in the United States for patients with recurrent high-grade glioma. In the present study, our goal has been to establish the feasibility of also applying this novel gene therapy strategy to secondary brain tumors arising from metastasis of systemic cancers to the CNS, which occur 5-10 times more frequently than primary brain tumors. Moreover, brain metastases are often accompanied by neurological deficits, which deprive patients of their quality of life. In particular, brain-metastatic breast cancer frequently arises from highly aggressive, treatment-refractory, ‘triple-negative' (ER(-), PR(-), HER2(-)) cells, is associated with a dismal prognosis of 4-6 months, and as treatment options are largely palliative, is the main cause of death in half of these patients. Hence this condition represents an unmet medical need.
We first examined the replication kinetics of RRV expressing the GFP reporter gene (AC3-emd) in human and murine breast cancer cell lines by flow cytometry. The replication kinetics of AC3-emd in the triple-negative human breast cancer cell line MDA-MB-231-BR, and in the murine breast cancer cell line JC, showed robust replication activity over time at both MOI = 0.01 and 0.1, resulting in high levels of transduction within 1-2 weeks. Next, we tested in vitro cytotoxicity by MTS assay after 5-FC treatment of 231BR and JC cells that had been fully transduced with RRV expressing the CD prodrug activator gene (clinical-grade vector AC3-yCD2, also designated “Toca 511”). In both of the AC3-yCD2-transduced cell lines, cell viability was reduced approximately 70-85% even after exposure to 0.1 mM 5-FC treatment, and complete cell killing was observed after 6 days (231-BR) or 4 days (JC) exposure to 1 mM 5-FC treatment. We then examined in vivo replication kinetics of AC3-emd in intracranial 231-BR xenograft models. Intratumoral injection of 10e6 AC3-emd on Day 7 after tumor implantation was confirmed to achieve &gt;80% transduction efficiency by flow cytometry on Day 10 post-injection. In survival studies, animals treated with AC3-yCD2+5-FC prodrug activator gene therapy showed statistically significant (p&lt;0.0001) survival benefit compared to the AC3-yCD2+PBS control group. These data provide preclinical validation for a new Phase I dose escalation trial to evaluate RRV-mediated prodrug activator gene therapy in CNS-metastatic breast cancer, as well as other types of metastatic cancers (TOCA6 trial: clinicaltrials.gov NCT02576665), planned for initiation at the University of Miami, Cleveland Clinic, and UCLA, in mid-2016.
Citation Format: Akihito Inagaki, Sara Collins, Suzanne Matsuura, Noriyuki Kasahara. Therapeutic efficacy of retroviral replicating vectors in orthotopic models of breast cancer metastasis to the brain. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr A43.