The clinical use of tumor necrosis factor alpha (TNF) as an anticancer drug is limited to local treatments because of its dose-limiting systemic toxicity. We show here that murine TNF fused with CNGRC peptide (NGR-TNF), an aminopeptidase N (CD13) ligand that targets activated blood vessels in tumors, is 12-15 times more efficient than murine TNF in decreasing the tumor burden in lymphoma and melanoma animal models, whereas its toxicity is similar. Similarly, human NGR-TNF induced stronger antitumor effects than human TNF, even with 30 times lower doses. Coadministration of murine NGR-TNF with a CNGRC peptide or an anti-CD13 antibody markedly decreased its antitumor effects. Tumor regression, induced by doses of murine NGR-TNF lower than the LD50, was accompanied by protective immunity. In contrast, no cure was induced by TNF at any dose. These results suggest that targeted delivery of TNF to CD13 may enhance its immunotherapeutic properties. Moreover, these findings reveal the potential of tumor homing peptides to generate a new class of recombinant cytokines that compared to immunocytokines have a simpler structure, could be easier to produce and are potentially less immunogenic.

Isoaspartate formation in extracellular matrix proteins, by aspartate isomerization or asparagine deamidation, is generally viewed as a degradation reaction occurring in vivo during tissue aging. For instance, non-enzymatic isoaspartate formation at RGD-integrin binding sites causes loss of cell adhesion sites, which in turn can be enzymatically "repaired" to RGD by protein-L-isoAsp-O-methyltransferase. We show here that isoaspartate formation is also a mechanism for extracellular matrix activation. In particular, we show that deamidation of Asn 263 at the Asn-Gly-Arg (NGR) site in fibronectin N-terminal region generates an ␣ v ␤ 3 -integrin binding site containing the L-isoDGR sequence, which is enzymatically "deactivated" to DGR by protein-L-isoAsp-O-methyltransferase. Furthermore, rapid NGRto-isoDGR sequence transition in fibronectin fragments generates ␣ v ␤ 3 antagonists (named "isonectins") that competitively bind RGD binding sites and inhibit endothelial cell adhesion, proliferation, and tumor growth. Time-dependent generation of isoDGR may represent a sort of molecular clock for activating latent integrin binding sites in proteins.Fibronectins are adhesive proteins that mediate a variety of cellular interactions with extracellular matrix and play important roles in hemostasis, thrombosis, inflammation, wound repair, angiogenesis, and embryogenesis (1, 2). About 20 isoforms of human fibronectin can be generated as a result of alternative splicing of the primary transcript (1, 3). Fibronectins are large glycoproteins (ϳ450 kDa) composed of two nearly identical disulfide-bonded subunits present in most body fluids and extracellular matrix of many tissues. Each subunit consists of three types of repeating homologous modules termed FN-I, FN-II, and FN-III repeats. Alternatively spliced modules, called EDA, EDB, and IIICS, can also be present (1, 3). Single modules or groups of modules may contain binding sites for different molecules, including sulfated glycosaminoglycans, DNA, gelatin, heparin, and fibrin (1, 3, 4). Furthermore, fibronectins contain binding sites for about half of the known cell surface integrin receptors (5, 6). In particular, the FN-III 10 repeat contains an RGD site that can bind, and ␣II b ␤ 3 integrins, while the FN-III 9 repeat contains the so-called "synergy site" PHSRN that cooperates with RGD in the binding of ␣ 5 ␤ 1 and ␣II b ␤ 3 (1, 7).Primary and tertiary structure analysis of human fibronectin showed that this protein contains two GNGRG loops, located in FN-I 5 and FN-I 7 modules, that are conserved in bovine, murine, rat, amphibian, and fish (8). Two additional NGR sites, less conserved, are also present in human FN-II 1 and FN-III 9 (see Fig. 1). Recent experimental work showed that peptides containing the NGR motif can inhibit ␣ 5 ␤ 1 -and ␣ v ␤ 1 -mediated cell adhesion to fibronectin (9).These notions prompted us to investigate the functional role of NGR in fibronectin. We observed that the NGR sequence of FN-I 5 (residues 263-265) promotes endothelial cell adhesion via an...

Drug delivery and penetration into neoplastic cells distant from tumor vessels are critical for the effectiveness of solid-tumor chemotherapy. We have found that targeted delivery to tumor vessels of picogram doses of TNF-α (TNF), a cytokine able to alter endothelial barrier function and tumor interstitial pressure, enhances the penetration of doxorubicin in tumors in murine models. Vascular targeting was achieved by coupling TNF with CNGRC, a peptide that targets the tumor neovasculature. This treatment enhanced eight-to tenfold the therapeutic efficacy of doxorubicin, with no evidence of increased toxicity. Similarly, vascular targeting enhanced the efficacy of melphalan, a different chemotherapeutic drug. Synergy with chemotherapy was observed with 3-5 ng/kg of targeted TNF (intraperitoneally), about 10 6-fold lower than the LD 50 and 10 5-fold lower than the dose required for nontargeted TNF. In addition, we have also found that targeted delivery of low doses of TNF to tumor vessels does not induce the release of soluble TNF receptors into the circulation. The delivery of minute amounts of TNF to tumor vessels represents a new approach for avoiding negative feedback mechanisms and preserving its ability to alter drug-penetration barriers. Vascular targeting could be a novel strategy for increasing the therapeutic index of chemotherapeutic drugs.

IntroductionPhage display libraries are often used for discovering peptide sequences that interact with proteins differentially expressed in normal and pathologic tissues. 1,2 Indeed, in vivo panning of peptide-phage libraries in tumor-bearing animal models has proven useful for selecting peptides able to interact with proteins expressed within tumor-associated blood vessels and therefore to home to neoplastic tissues. 3 Among the ligands identified so far, cyclic and linear peptides containing the Asn-Gly-Arg (NGR) motif have been exploited for systemic, yet ligand-directed targeted delivery of therapeutic and imaging agents to angiogenic blood vessels, including cytotoxic drugs and cytokines, among other entities (such as viruses and nanoparticles). These findings highlight the value of NGR peptides in drug development. In this review we discuss the biochemical and biologic properties of NGR and NGR-derived compounds. Given that many native proteins contain the sequence NGR, we also address the emerging role of NGR as an unrecognized "molecular timer" due to the timedependent generation of isoAsp-Gly-Arg (isoDGR), a new integrinbinding motif that regulates a gain-of-function within the extracellular matrix protein fibronectin. 4 The discovery of the NGR motifIn vitro panning of several phage libraries against the ␣5␤1 integrin led to the selection of various RGD-containing peptides and also of the peptide NGRAHA. 5 These peptides (including NGRHA) inhibited cell attachment mediated by both ␣v␤3 and ␣v␤5 integrins. Moreover, 8 NGR-containing peptides were isolated upon screening of cyclic peptide libraries under similar experimental conditions. 6 Notably, one selected phage clone displayed the peptide CVLNGRMEC, which is similar to the sequence ALN-GREE found within the 9th type III repeat of human fibronectin. 7 Further studies based on in vitro selection of libraries on ␣v␤3, revealed several different peptides containing the NGR motif, such as NGRIPD, TNGRGP, NGRSFR, RSRNGR, NGRNTV. 8 In another line of investigation, in vivo phage-display screenings were performed to isolate tumor-homing peptides. Systemic administration of a phage library into nude mice bearing human breast carcinoma xenografts led to selection of a tumor vasculaturehoming phage carrying the sequence CNGRCVSGCAGRC. 3 Tumor homing was inhibited by co-injection with the CNGRC peptide (NGR-2C) indicating that this short cyclic loop is a functional tumor targeting peptide. Phage displaying the peptides NGRAHA or CVLNGRMEC, previously identified in vitro, also selectively localized to tumors. 3 The NGR receptor(s)In vivo phage display-based studies showed that also the peptide ACDCRGDCFC (RGD-4C), an ␣v␤3/␣v␤5 binding sequence, can bind to tumor neovasculature. 3,9 Cross-inhibition experiments of NGR-2C and RGD-4C-phage clones with synthetic RGD-4C and NGR-2C peptides showed that RGD-4C peptide does not compete the homing of NGR-2C-phage to tumors and vice versa. 3 This result suggests that NGR-2C and RGD-4C bind to distinct receptors in tumor ...

We demonstrate for the first time that CgA is produced by human myocardium and exerts negative inotropic and lusitropic effects on mammalian heart. CgA may represent a key player in neuroendocrine regulation of cardiac function and a potential therapeutic target in heart failure.

• Circulating chromogranin A and its fragments form a balance of anti-and proangiogenic factors regulated by thrombin-dependent cleavage.• The alteration of this balance could provide a new mechanism for triggering angiogenesis in cancer and other pathophysiologic conditions.Angiogenesis, the formation of blood vessels from pre-existing vasculature, is regulated by a complex interplay of anti and proangiogenic factors. We found that physiologic levels of circulating chromogranin A (CgA), a protein secreted by the neuroendocrine system, can inhibit angiogenesis in various in vitro and in vivo experimental models. Structure-activity studies showed that a functional antiangiogenic site is located in the C-terminal region, whereas a latent anti-angiogenic site, activated by cleavage of Q76-K77 bond, is present in the N-terminal domain. Cleavage of CgA by thrombin abrogated its anti-angiogenic activity and generated fragments (lacking the C-terminal region) endowed of potent proangiogenic activity. Hematologic studies showed that biologically relevant levels of forms of full-length CgA and CgA1-76 (anti-angiogenic) and lower levels of fragments lacking the C-terminal region (proangiogenic) are present in circulation in healthy subjects. Blood coagulation caused, in a thrombin-dependent manner, almost complete conversion of CgA into fragments lacking the C-terminal region. These results suggest that the CgA-related circulating polypeptides form a balance of anti and proangiogenic factors tightlyregulated byproteolysis. Thrombin-induced alteration of this balance could provide a novel mechanism for triggering angiogenesis in pathophysiologic conditions characterized by prothrombin activation. (Blood. 2013;121(2):392-402) IntroductionAngiogenesis, the process of formation of new blood vessels from pre-existing vessels, is tightly regulated by the coordinated action of anti and proangiogenic factors. [1][2][3] When this balance is disturbed, the result is either excessive or insufficient angiogenesis. Altered angiogenesis, causing excessive or insufficient blood vessel growth, is a common denominator underlying many pathologic conditions, including cardiovascular diseases, macular degeneration, skin diseases, diabetic ulcers, stroke, rheumatoid arthritis, cancer, and many others. [1][2][3] A growing body of evidence suggests that angiogenesis can be regulated by peptides derived from proteins released by neurons and neuroendocrine cells. For example, we have previously shown the recombinant fragment 1-78 of human chromogranin A (CgA), a 439-residue long protein stored in the secretory granules of many endocrine cells, neurons, and cardiomyocytes 4-6 can inhibit angiogenesis in experimental models. 7 Other investigators have shown that a synthetic fragment encompassing residues 352-372 (CgA352-372) can promote angiogenesis. 8 Studies on the mechanism of action have shown that CgA1-78 (also called vasostatin-1) can inhibit endothelial cell proliferation, migration, and invasion induced by vascular endothelial growth factor...

Cyclic and linear peptides containing the Asn-Gly-Arg (NGR) motif have proven useful for delivering various anti-tumor compounds and viral particles to tumor vessels. We have investigated the role of cyclic constraints on the structure and tumor-homing properties of NGR peptides using tumor necrosis factor-␣ (TNF) derivatives containing disulfide-bridged (CNGRC-TNF) and linear (GNGRG-TNF) NGR domains. Experiments carried out in animal models showed that both GNGRG and CNGRC can target TNF to tumors. However, the antitumor activity of CNGRC-TNF was >10-fold higher than that of GNGRG-TNF. Molecular dynamic simulation of cyclic CNGRC showed the presence of a bend geometry involving residues Gly 3 -Arg 4 . Molecular dynamic simulation of the same peptide without disulfide constraints showed that the most populated and thermodynamically favored configuration is characterized by the presence of a ␤-turn involving residues Gly 3 -Arg 4 and hydrogen bonding interactions between the backbone atoms of Asn 2 and Cys 5 . These results suggest that the NGR motif has a strong propensity to form ␤-turn in linear peptides and may explain the finding that GNGRG peptide can target TNF to tumors, albeit to a lower extent than CNGRC. The disulfide bridge constraint is critical for stabilizing the bent conformation and for increasing the tumor targeting efficiency.Phage display peptide libraries are commonly used to obtain peptide sequences interacting with proteins differentially expressed in normal and pathological tissues (1, 2). For instance, in vivo panning of phage libraries in tumor-bearing animals have proven useful for selecting peptides able to interact with proteins expressed within tumor-associated vessels and to home to neoplastic tissues (3). Among the various tumor targeting ligands identified so far, the CNGRC peptide have proven useful for delivering various anti-tumor compounds, like chemotherapeutic drugs, apoptotic peptides and cytokines, to tumor vessels (3-5). For example, we have recently shown that targeted delivery of tumor necrosis factor-␣ (TNF) 1 to tumor vasculature can be obtained by coupling its N terminus to the C terminus of the CNGRC peptide, by genetic engineering technology (5). This approach markedly improved the therapeutic index of TNF in animal models, either when used alone (5) or in combination with chemotherapeutic agents (6). Studies on the mechanism of action showed that the targeting domain of this TNF derivative (called NGR-TNF) binds an aminopeptidase (CD13) isoform expressed in tumor vessels, and not other isoforms expressed in normal epithelia or myeloid cells (7). Besides CNGRC, other tumor vasculature targeting peptides containing the NGR motif have been identified, such as linear NGRAHA and cyclic CVLNGRMEC (3). These and other linear and cyclic NGR peptides have been used for targeting viral particles to endothelial cells (8, 9). Although these findings may suggest that peptide cyclization is not necessary for the targeting properties of NGR peptides, the role of cysteines and th...

Abnormal tumor vasculature impairs T lymphocyte adhesion to endothelial cells and lymphocyte extravasation into neoplastic tissues, limiting the therapeutic potential of both active and adoptive immunotherapies. We have found that treatment of tumor-bearing mice with NGR-TNF, a Cys-Asn-Gly-Arg-Cys peptide-TNF fusion product capable of altering the endothelial barrier function and improving drug penetration in tumors, associated with the intratumor upregulation of leukocyte-endothelial cell adhesion molecules, the release of proinflammatory cytokines and chemokines, and the infiltration of tumor-specific effector CD8+ T cells. As a result, NGR-TNF enhanced the therapeutic activity of adoptive and active immunotherapy, delaying tumor growth and prolonging survival. Furthermore, we have found that therapeutic effects of these combinations can be further increased by the addition of chemotherapy. Thus, these findings might be relevant for the design of novel immunotherapeutic approaches for cancer patients.