The EBV/lipoplex is a nonviral gene delivery system composed of a cationic
lipid and Epstein-Barr virus (EBV)-based plasmid vector that carries the EBV oriP and
EBV nuclear antigen 1 (EBNA1) gene. Because the EBNA1 supports retention, nuclear localization, and transcriptional upregulation of the oriP-bearing plasmid, cells transfected with the EBV/lipoplex express the transgene at a very high level. We hypothesized that
tumor cells genetically manipulated with the EBV/lipoplex may be used as a
tumor vaccine without
drug selection, strongly contributing to
immunotherapy of patients with
malignancies. The
cytokines interleukin (IL)-12 and
IL-18 exert a variety of immune-regulatory functions including
interferon (IFN)-gamma production and cytotoxic T lymphocyte (CTL) and natural killer (NK) activation. Here, we investigated the possible
therapeutic effects of an autologous
tumor cell
vaccine in the
B16 melanoma model. The
vaccine was engineered to secrete
IL-12 and
IL-18 by means of the EBV/lipoplex. B16 cells were subcutaneously implanted into syngenic mice followed by repetitive immunization with irradiated B16 cells that had been transfected 3 days earlier by TFL2-3, a novel cationic
lipid, with EBV-plasmid vectors encoding
IL-12 and/or
IL-18 genes (B16/
mIL-12, B16/
mIL-18, and B16/
mIL-12+mIL-18). The mice vaccinated with B16/
mIL-12 underwent strong
tumor suppression accompanied by a high IFN-gamma production. Both CTL and NK activities were significantly elevated in these mice. When the
tumor cell
vaccine was prepared by means of conventional (non-EBV) plasmid vectors combined with the same cationic
lipid, the therapeutic outcome was not as good, suggesting the superiority of the EBV-based plasmid in engineering these types of
tumor vaccines. Vaccination with B16/
mIL-18 was not effective in suppressing
tumors, whereas B16/
mIL-12+mIL-18 showed comparable antitumor therapeutic validity as B16/
mIL-12 did. When IFN-gamma mutant (IFN-gamma(-/-) mice were treated, B16/
mIL-12
vaccine did not show any therapeutic activity, suggesting the necessity of IFN-gamma in the anti-
melanoma immune responses. In contrast, the antitumor effect was not affected by NK depletion in mice that received repetitive
injections with anti-
asialo GM1 antibody. Furthermore, vaccination with B16/
mIL-12 significantly suppressed pulmonary
metastases in mice that had been intravenously injected with parental B16. Our results suggest that the EBV/lipoplex is quite useful in generating an autologous
tumor cell
vaccine and that
IL-12 is an important component of the
vaccine.