A Suicide Gene Therapy Combining the Improvement of Cyclophosphamide Tumor Cytotoxicity and the Development of an Anti-Tumor Immune Response

Abstract

Gene-directed enzyme prodrug therapy (GDEPT) consists in targeted delivery to tumor cells of a suicide gene responsible for in situ conversion of a prodrug into cytotoxic metabolites. One of the major limitations of this strategy in clinical application was the poor prodrug activation capacity of suicide gene. We built a highly efficient suicide gene capable of bioactivating the prodrug cyclophosphamide (CPA) by fusing a CYP2B6 triple mutant with NADPH cytochrome P450 reductase (CYP2B6TM-RED). Expression of this fusion gene via a recombinant lentivirus (LV) vector converted resistant human (A549) and murine (TC1) pulmonary cell lines into CPA-susceptible cell lines. We tested the efficiency of our GDEPT strategy in C57Bl/6 immunocompetent mice, using TC1 cells expressing the HPV-16 E6/E7 oncoproteins. In mice bearing tumors composed only of TC1-CYP2B6TM-RED cells, four CPA injections (140 mg/Kg once a week) completely eradicated the tumors for more than two months. Tumors having only 25% of TC1-CYP2B6TM-RED cells were also completely eradicated by five CPA injections, demonstrating a major in vivo bystander effect. Moreover, surviving mice were rechallenged with parental TC1 cells. The tumors regressed spontaneously 7 days after cell inoculation or grew more slowly than in control naive mice due to a strong immune response mediated by anti-E7CD8(+)T cells. These data suggest that combining the CYPB6TM-RED gene with CPA may hold promise as a highly effective treatment for solid tumors in humans.

Immunogenic cell death in a combined synergic gene- and immune-therapy against cancer

“…9,10 In our mouse models, we observed a specific decrease of Tregs upon a weekly administration of CPA. 3 This Treg inhibition might explain, at least in part, the ability of our strategy to induce specific anti-tumor CD8 + T cells.…”

“…Our approach aims at introducing, specifically into tumors, a modified gene which has been constructed and patented in our laboratory (CYP2B6TM-RED = CYP2B6*). [1][2][3][4] This gene transforms very efficiently, inside the tumor, a prodrug (cyclophosphamide, CPA) into toxic metabolites.…”

“…In immuno-competent mouse models, this suicide-gene, expressed in a tumor, allows CPA to completely eradicate the tumor, without any recurrence more than 24 months after stopping the treatment. 3,5 In cured mice, re-challenge experiments with tumor cells, not expressing CYP2B6*, demonstrated a sustainable immune response against tumor cells mediated by cytotoxic CD8 + T lymphocytes. 3,5 This immune response contributes to the efficacy of this approach.…”

“…3,5 In cured mice, re-challenge experiments with tumor cells, not expressing CYP2B6*, demonstrated a sustainable immune response against tumor cells mediated by cytotoxic CD8 + T lymphocytes. 3,5 This immune response contributes to the efficacy of this approach. It protects against recurrence of the primary tumor as well as secondary metastases.…”

Progress and problems with the use of suicide genes for targeted cancer therapy

“…At present, several groups are working to engineer new mutants of CYP2B6 in order to improve the affinity towards CPA. For example, Touati et al (2014), reported the structure of a triple mutant CYP2B6 in fusion with NADPH cytochrome P450 reductase that showed superior ability in converting CPA into its cytotoxic form [130]. While this system has shown great promise at the preclinical level, its safety and efficacy in clinical trials remains to be determined.…”