Neuroblastoma, the most common solid tumor of infancy derived from the sympathetic nervous system, continues to present a formidable clinical challenge. Sterically stabilized immunoliposomes (SIL) have been shown to enhance the selective localization of entrapped drugs to solid tumors, with improvements in therapeutic indices. We showed that SIL loaded with doxorubicin (DXR) and targeted to the disialoganglioside receptor GD 2 [aGD 2 -SIL(DXR)] led to a selective inhibition of the metastatic growth of experimental models of human neuroblastoma. By coupling NGR peptides that target the angiogenic endothelial cell marker aminopeptidase N to the surface of DXR-loaded liposomes [NGR-SL(DXR)], we obtained tumor regression, pronounced destruction of the tumor vasculature, and prolonged survival of orthotopic neuroblastoma xenografts. Here, we showed good liposome stability, long circulation times, and enhanced timedependent tumor accumulation of both the carrier and the drug. Antivascular effects against animal models of lung and ovarian cancer were shown for formulations of NGR-SL(DXR). In the chick embryo chorioallantoic assay, NGR-SL(DXR) substantially reduced the angiogenic potential of various neuroblastoma xenografts, with synergistic inhibition observed for the combination of NGR-SL(DXR) with aGD 2 -SIL(DXR). A significant improvement in antitumor effects was seen in neuroblastoma-bearing animal models when treated with the combined formulations compared with control mice or mice treated with either tumor-or vascular-targeted liposomal formulations, administered separately. The combined treatment resulted in a dramatic inhibition of tumor endothelial cell density. Long-term survivors were obtained only in animals treated with the combined tumor-and vascular-targeted formulations, confirming the pivotal role of combination therapies in treating aggressive metastatic neuroblastoma.

Bortezomib is an effective inhibitor of neuroblastoma cell growth and angiogenesis. These findings provide the rationale for further clinical investigation of bortezomib in pediatric neuroblastoma.

Various NGR-containing peptides have been exploited for targeted delivery of drugs to CD13-positive tumor neovasculature. Recent studies have shown that compounds containing this motif can rapidly deamidate and generate isoaspartate-glycine-arginine (isoDGR), a ligand of ␣v␤3-integrin that can be also exploited for drug delivery to tumors. We have investigated the role of NGR and isoDGR peptide scaffolds on their biochemical and biological properties. Peptides containing the cyclic CNGRC sequence could bind CD13-positive endothelial cells more efficiently than those containing linear GNGRG. Peptide degradation studies showed that cyclic peptides mostly undergo NGR-to-isoDGR transition and CD13/integrin switching, whereas linear peptides mainly undergo degradation reactions involving the ␣-amino group, which generate non-functional six/seven-membered ring compounds, unable to bind ␣v␤3, and small amount of isoDGR. Structure-activity studies showed that cyclic isoDGR could bind ␣v␤3 with an affinity >100-fold higher than that of linear isoDGR and inhibited endothelial cell adhesion and tumor growth more efficiently. Cyclic isoDGR could also bind other integrins (␣v␤5, ␣v␤6, ␣v␤8, and ␣5␤1), although with 10 -100-fold lower affinity. Peptide linearization caused loss of affinity for all integrins and loss of specificity, whereas ␣-amino group acetylation increased the affinity for all tested integrins, but caused loss of specificity. These results highlight the critical role of molecular scaffold on the biological properties of NGR/isoDGR peptides. These findings may have important implications for the design and development of anticancer drugs or tumor neovasculature-imaging compounds, and for the potential function of different NGR/isoDGR sites in natural proteins.Various peptides containing the Asn-Gly-Arg (NGR) motif have been discovered by peptide-phage library panning in tumor-bearing mice (1). The tumor-homing properties of these peptides rely on the interaction with aminopeptidase N (CD13), a membrane protease expressed by the tumor neovasculature (2, 3). Because of this property, these peptides have been exploited for ligand-directed delivery of various drugs and particles to tumor vessels, in the attempt to increase their antitumor activity (4). For instance, we have shown that peptides containing cyclic CNGRC and linear GNGRG motives can be used for delivering tumor necrosis factor ␣ (TNF) 3 (5-7), interferon ␥ (8 -10), and liposomal doxorubicin (11, 12) to tumor neovasculature, improving their therapeutic properties. The CNGRC-TNF conjugate, called NGR-TNF, is currently tested in phase II clinical studies (13-15). Other investigators have used the NGR motif embedded in similar or different molecular scaffolds for delivering chemotherapeutic drugs, antiangiogenic drugs, tissue factor, viruses, and other compounds to tumor vessels (1, 16 -32). Recently, a CNGRC peptide with an acetylated N-terminal ␣-amino group has been successfully exploited also for quantitative molecular magnetic resonance imaging of ...