Immuno-Angioprevention by IL-12 gene transfer Free

53

IL-12 is a Th1 cytokine that harbors anti-angiogenic effects thought to be mediated by an IFN-gamma and angiostatic CXCR3 chemokine ligand cytokine cascade. Naked DNA intra-muscular injection of an expression vector plasmid producing IL-12 resulted in low but significant, well-tolerated elevation of serum IL-12 levels. Injection of the IL-12 plasmid at least 2 days, and up to 20 days, before subcutaneous injection of matrigel with angiogenic factors resulted in strong prevention of angiogenesis in both C57/bl and nude mice. A single injection of the IL-12 plasmid contemporarily with the matrigel or 2 days after resulted in partial, statistically not significant inhibition. Control plasmid injection did not effect either angiogenesis or angiogenesis inhibition by IL-12 protein in vivo. Angiogenesis inhibition was observed in NK cell depleted C57/bl and nude mice as well as in IFN-gamma-/- and CXCR3-/- knock-out mice, indicating NK and/or T-cell initiated IFN-gamma-chemokine cascades were not involved in the angiogenesis inhibition observed in vivo. We then demonstrated that preventive IL-12 plasmid DNA gene transfer significantly reduced the growth and vascularization of highly angiogenic KS-Imm human Kaposi’s sarcoma and TS/A murine mammary carcinoma tumors in nude and/or syngeneic mice. To further study the effects of IL-12 angiogenesis inhibiton on tumor growth, KS-Imm or the murine uveal melanoma cell line 99E1 were transfected with a related pIRES IL-12 expression vector driven by the CMV promoter. G418 resistant clones were selected and the level of IL-12 mRNA expression and protein IL-12 protein release were analyzed, respectively, by RT-PCR and ELISA assays. IL-12 transfected KS-Imm cells were essentially unable to form tumors in nude mice; these animals showed high levels of serum IL-12 and toxicity. Mock-transduced 99E1 cells, which do not produce IL-12 on their own, formed tumors when injected subcutaneously in nude mice. In contrast, 99E1 cells transfected with the IL-12 vector formed small tumors, and showed well tolerated, intermediate levels of serum IL-12. Orthotopic intraoccular injection of the IL-12 transduced 99E1 cells rarely formed tumors as compared to the mock and wild-type controls. These data suggest that a preventive gene therapy approach using anti-angiogenic cytokines can effectively inhibit tumor angiogenesis and KS, representing an example of angio-immune-prevention.