Abstract 3315: Synergistic direct and distant bystander effects contribute to the efficiency of a new suicide gene therapy against cancer.

Abstract

Abstract Gene-directed enzyme prodrug therapy (GDEPT) systems have been developed for killing cancer cells by introducing a gene coding for a protein able to convert a harmless prodrug into a cytotoxic one. We built a highly efficient therapeutic suicide gene which metabolizes the prodrug cyclophosphamide (CPA) into cytotoxic metabolites. This gene was formed by the fusion of two human genes: a triple mutant of CYP2B6 (CYP2B6TM) modified by site-directed mutagenesis and the soluble part of NADPH cytochrome P450 reductase (RED). Moreover the linker between both genes was optimized to express a high RED activity to improve electron transfer required for CYP2B6TM catalytic activity. Expression of this fusion gene (CYP2B6TM-RED), by a recombinant lentivirus (LV), allowed switching of a resistant human pulmonary cell line (A549) into a sensitive cell line to CPA. After 48 hours of CPA treatment, cytotoxic tests showed that A549 cells expressing CYP2B6TM-RED had an IC50 of about 0.5 mM approximately 13-fold lower than that of cells expressing CYP2B6 wild type-RED. Our strategy was tested in tumor models using immunocompetent C57Bl/6 mice and the mouse epithelial pulmonary cell line (TC1). In vitro, this CPA resistant cell line became sensitive to CPA after expression of CYP2B6TM-RED gene with an IC50 comparable to that observed in transduced A549 cells.TC1 cells previously infected or not by LV-CYP2B6TM-RED were injected sub-cutaneously into C57Bl/6 mice. CPA was intraperitonealy administered each week at a dose of 140 mg/Kg when the tumor volume reached about 500 mm3. Mice with TC1-CYP2B6TM-RED tumors presented an important decrease of the tumor volume immediately after the first injection of CPA and only four injections of CPA were necessary to observe a complete disappearance of the tumors. Similar results were obtained when the tumor cells were composed of 25% of TC1 expressing CYP2B6TM-RED and 75% of non-infected cells demonstrating in vivo the contribution of a direct bystander effect to the efficiency of our strategy. Moreover cured mice were rechallenged with uninfected TC1 cells. In some cases a complete eradication of the tumor that was related to a specific cellular immune response against tumor cells was observed 7 days after cell inoculation. This result demonstrated an efficient distant bystander effect that would lead to a long-term protection against potential metastases. We are now testing CYP2B6TM-RED fusion gene either by intratumoral lentiviral injection or by infection of mesenchymal stem cells used as delivery strategy, in view of clinical human trials. Citation Format: Walid Touati, Johanne Seguin, Thi Tran, Eric Tartour, Claude Baillou, François M. Lemoine, Philippe Beaune, Isabelle de Waziers. Synergistic direct and distant bystander effects contribute to the efficiency of a new suicide gene therapy against cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3315. doi:10.1158/1538-7445.AM2013-3315