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 alphavbeta3 integrin, to mrIL-12 to target IL-12 directly to tumor neovasculature. The fusion protein stimulated IFN-gamma production in vitro and in vivo, indicating its biological activity was consistent with mrIL-12. Immunofluorescence techniques showed mrIL-12vp specifically bound to alphavbeta3 integrin-positive cells but not to alphavbeta3 integrin-negative cells. In corneal angiogenesis assays using BALB/c mice treated with either 0.5 microg/mouse/d of mrIL-12vp or mrIL-12 delivered by subcutaneous continuous infusion, mrIL-12vp inhibited corneal neovascularization by 67% compared with only a slight reduction (13%) in angiogenesis in the mrIL-12-treated animals (P = 0.008). IL-12 receptor knockout mice given mrIL-12vp showed a marked decrease in the area of corneal neovascularization compared with mice treated with mrIL-12. These results indicate that mrIL-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 mrIL-12vp showed significant suppression of tumor growth compared with mice treated with mrIL-12 (P = 0.03). Mice did not show signs of IL-12 toxicity when treated with mrIL-12vp, although hepatic necrosis was present in mrIL-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.
Dickerson
Erin B
EB
Department of Medical Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin, USA.
Akhtar
Nasim
N
Steinberg
Howard
H
Wang
Zun-Yi
ZY
Lindstrom
Mary J
MJ
Padilla
Marcia L
ML
Auerbach
Robert
R
Helfand
Stuart C
SC
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D006224:Cricetinae; D005434:Flow Cytometry; D039302:Integrin alphaVbeta3 / Q000378:metabolism*; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D018664:Interleukin-12 / Q000008:administration & dosage / Q000494:pharmacology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008811:Mice, Inbred DBA; D018345:Mice, Knockout; D008822:Mice, Transgenic; D009336:Necrosis / Q000473:pathology; D009368:Neoplasm Transplantation; D009389:Neovascularization, Pathologic; D009842:Oligopeptides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010957:Plasmids / Q000378:metabolism; D015202:Protein Engineering; D020033:Protein Isoforms; D018123:Receptors, Interleukin / Q000235:genetics; D053707:Receptors, Interleukin-12; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection
D020533:Angiogenesis Inhibitors; D039302:Integrin alphaVbeta3; D009842:Oligopeptides; D010455:Peptides; D020033:Protein Isoforms; D018123:Receptors, Interleukin; D053707:Receptors, Interleukin-12; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D018664:Interleukin-12; C047981:arginyl-glycyl-aspartic acid; D007371:Interferon-gamma