Anti-tumor effect induced by both DNA vaccine and oncolytic adenovirus expressing multi-target genes related to immunity in malignant melanoma

Abstract

Immunogene therapy is an immune system-mediated strategy for cancer treatment that involves the delivery of immune-modulating genes to the tumor site to induce an adaptive anti-tumor immune response in the host. This study was designed to develop a novel anti-cancer immunogene therapy effective against malignant melanoma in the C57BL/6 mouse model. A recombinant plasmid containing MART1, a human melanoma-specific tumor antigen, was used to induce an immune reaction against the mouse melan-A epitope in order to overcome the peripheral tolerance of the mouse to murine melan-A. In addition, mouse granulocyte macrophage-colony stimulating factor (mGM-CSF) and short hairpin RNA against mouse transforming growth factor-β2 (shmTGF-β2) genes were delivered together with MART1, because GM-CSF is known to be the most potent inducer of antitumor immunity, and TGF-β is known to be involved in tumor survival and host immune suppression. These genes were delivered to cancer cells by using an oncolytic adenovirus. A recombinant DNA expressing human MART1 (MART1 plasmid) and a recombinant adenovirus expressing human MART1 were investigated for their potential effects on priming and boosting immune responses.First, the expression of MART1 was increased in MART1 plasmid-transfected B16BL6 mouse melanoma cells in a dose-dependent manner in vitro. Notably, the cytotoxic activity of splenocytes isolated from MART1 plasmid-injected non-tumor-bearing mice was enhanced compared to those isolated from control plasmid-injected mice.Thus, recombinant oncolytic adenovirus expressing mGM-CSF and shmTGF-β2 were also investigated for their potential to stimulate the non-specific immune response and decrease the expression of signaling molecules involved in tumor cell survival and growth, respectively. To this end, the effect of recombinant oncolytic adenovirus expressing both mGM-CSF and

shmTGF-β2 (GT virus) was compared to that of the recombinant adenovirus expressing mGM-CSF only (G virus), following intratumoral injection of the virus into melanoma-bearing C57BL/6 mice. This investigation shows that administration of the G virus leads to delayed tumor growth compared to the empty viral control, while tumor growth in mice that received the GT virus was significantly decreased (P < 0.001) compared to both the control- and G virus-treated mice. Finally, an oncolytic adenovirus expressing MART1, mGM-CSF, and shmTGF-β2 (MGT virus) was constructed and administered to boost the immune response and cancer cell death. Administration of this virus induced a stronger and longer-lived immune response than that observed in the controls. Interestingly, none of the mice that received MART1 plasmid pre-treatment in addition to MGT viral injection showed any signs of tumor growth and 100% were viable 43 days after tumor cell injection. This study investigates the anti-tumor effects of repeated MART1 plasmid vaccination and immune stimulation/tumor cell lysis with an oncolytic adenovirus expressing MART1/mGM-CSF/shmTGF-β2. The results presented herein highlight the function of these genes during tumorigenesis as well as the possible therapeutic options of this treatment strategy. Additional work is necessary to further evaluate the clinical application of this combination therapy to treat malignant melanoma.