Enhancement of the antiangiogenic activity of interleukin-12 by peptide targeted delivery of the cytokine to alphavbeta3 integrin.

We engineered a fusion protein, mrIL-12vp [mouse recombinant interleukin (IL)-12 linked to vascular peptide], linking the vascular homing peptide CDCRGDCFC (RGD-4C), a ligand for α v β 3 integrin, to mrlL-12 to target IL-12 directly to tumor neovasculature. The fusion protein stimulated IFN-y production in vitro and in vivo, indicating its biological activity was consistent with mrlL-12. Immunofluorescence techniques showed mrlL-12vp specifically bound to α v β 3 integrin-positive cells but not to α v β 3 integrin-negative cells. In corneal angiogenesis assays using BALB/c mice treated with either 0.5 μg/mouse/d of mrlL-12vp or mrlL-12 delivered by subcutaneous continuous infusion, mrlL-12vp inhibited corneal neovascularization by 67% compared with only a slight reduction (13%) in angiogenesis in the mrlL-12-treated animals (P = 0.008). IL-12 receptor knockout mice given mrlL-12vp showed a marked decrease in the area of corneal neovascularization compared with mice treated with mrlL-12. These results Indicate that mrlL-12vp inhibits angiogenesis through IL-12-dependent and IL-12-independent mechanisms, and its augmented antiangiogenic activity may be due to suppression of endothelial cell signaling pathways by the RGD-4C portion of the fusion protein. Mice injected with NXS2 neuroblastoma cells and treated with mrlL-12vp showed significant suppression of tumor growth compared with mice treated with mrlL-12 (P = 0.03). Mice did not show signs of IL-12 toxicity when treated with mrlL-12vp, although hepatic necrosis was present in mrlL-12-treated mice. Localization of IL-12 to neovasculature significantly enhances the antiangiogenic effect, augments antitumor activity, and decreases toxicity of IL-12, offering a promising strategy for expanding development of IL-12 for treatment of cancer patients.