Isoaspartate-Glycine-Arginine: A New Tumor Vasculature-Targeting Motif

Department of Biological and Technological Research, CIGT Program and IIT Network Research Unit of Molecular Neuroscience, San Raffaele Scientific Institute, Milan, Italy.
Cancer Research (Impact Factor: 9.33). 10/2008; 68(17):7073-82. DOI: 10.1158/0008-5472.CAN-08-1272
Source: PubMed


Asparagine deamidation in peptides or in fibronectin fragments containing the asparagine-glycine-arginine sequence generates isoaspartate-glycine-arginine (isoDGR), a new alphavbeta3 integrin-binding motif. Because alphavbeta3 is expressed in angiogenic vessels, we hypothesized that isoDGR-containing peptides could be exploited as ligands for targeted delivery of drugs to tumor neovasculature. We found that a cyclic CisoDGRC peptide coupled to fluorescent nanoparticles (quantum dots) could bind alphavbeta3 integrin and colocalize with anti-CD31, anti-alphavbeta3, and anti-alpha5beta1 antibodies in human renal cell carcinoma tissue sections, indicating that this peptide could efficiently recognize endothelial cells of angiogenic vessels. Using CisoDGRC fused to tumor necrosis factor alpha (TNF) we observed that ultralow doses (1-10 pg) of this product (called isoDGR-TNF), but not of TNF or CDGRC-TNF fusion protein, were sufficient to induce antitumor effects when administered alone or in combination with chemotherapy to tumor-bearing mice. The antitumor activity of isoDGR-TNF was efficiently inhibited by coadministration with an excess of free CisoDGRC, as expected for ligand-directed targeting mechanisms. These results suggest that isoDGR is a novel tumor vasculature-targeting motif. Peptides containing isoDGR could be exploited as ligands for targeted delivery of drugs, imaging agents, or other compounds to tumor vasculature.

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Available from: Angelo Corti, Jan 20, 2014
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    • "As it has been shown that the cyclic form of NGR has approximately ten-fold greater targeting efficacy than linear configurations of the same peptide sequence for targeting different tumors (Colombo et al., 2002), a cyclic NGR motif containing radiotracer might lead to high diagnostic efficiency. On the other hand, in order to create a 68 Ga-labeled c(NGR) conjugate a chelator other than DOTA with the ability to efficiently chelate trivalent gallium-ion at room temperature must be chosen, as thermal stability of a DOTA-c(NGR) conjugate might be insufficient due to decomposition of the cycle or the elevated temperature can enhance the conversion of asparagine residue into isoaspartate and aspartate resulting in isoDGR and DGR sequence (Curnis et al., 2008). "
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    ABSTRACT: Aminopeptidase N (APN/CD13) plays an important role in tumor neoangiogenic process and the development of metastases. Furthermore, it may serve as a potential target for cancer diagnosis and therapy. Previous studies have already shown that asparagine-glycine-arginine (NGR) peptides specifically bind to APN/CD13. The aim of the study was to synthesize and investigate the APN/CD13 specificity of a novel (68)Ga-labeled NOTA-c(NGR) molecule in vivo using miniPET. c[KNGRE]-NH2 peptide was conjugated with p-SCN-Bn-NOTA and was labeled with Ga-68 ((68)Ga-NOTA-c(NGR)). Orthotopic and heterotopic transplanted mesoblastic nephroma (NeDe) bearing Fischer-344 rats were prepared, on which biodistribution studies and miniPET scans were performed for both (68)Ga-NOTA-c(NGR) and ανβ3 integrin selective (68)Ga-NODAGA-[c(RGD)]2 tracers. APN/CD13 receptor expression of NeDe tumors and metastases was analyzed by western blot. (68)Ga-NOTA-c(NGR) was produced with high specific activity (5.13-5.92GBq/μmol) and with excellent radiochemical purity (95%<), at all cases. Biodistribution studies in normal rats showed that uptake of the (68)Ga-NOTA-c(NGR) was significantly (p⩽0.05) lower in abdominal organs in comparison with (68)Ga-NODAGA-[c(RGD)]2. Both radiotracers were mainly excreted from the kidney. In NeDe tumor bearing rats higher (68)Ga-NOTA-c(NGR) accumulation was found in the tumors than that of the (68)Ga-NODAGA-[c(RGD)]2. Using orthotopic transplantation, metastases were developed which showed specific (68)Ga-NOTA-c(NGR) uptake. Western blot analysis confirmed the presence of APN/CD13 expression in NeDe tumors and metastases. Our novel radiotracer (68)Ga-NOTA-c(NGR) showed specific binding to the APN/CD13 expressed ortho- and heterotopic transplanted NeDe tumors. Therefore, (68)Ga-NOTA-c(NGR) is a suitable tracer for the detection of APN/CD13 positive tumors and metastases in vivo. Copyright © 2015. Published by Elsevier B.V.
    Full-text · Article · Jan 2015 · European journal of pharmaceutical sciences: official journal of the European Federation for Pharmaceutical Sciences
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    • "The structural and functional similarity with RGD suggests that isoDGR can also be exploited, like RGD, as a ligand for targeted delivery of drugs, imaging agents, or other compounds to tumors. Consistent with this, fluorescent nanoparticles coupled to a cyclic CisoDGRC peptide could bind αvβ3-integrin and colocalize with antibodies against this integrin in vessels of human renal cell carcinoma [110]. Furthermore, extremely low doses (1–10 pg) of a recombinant protein made up of CisoDGRC fused to TNF induced anti-tumor effects in tumor-bearing mice through specifically targeting TNF to tumor sites (Curnis et al. [110]). "
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    ABSTRACT: A growing body of evidence suggests that the efficacy of cytokines in cancer therapy can be increased by targeting strategies based on conjugation with ligands that recognize receptors expressed by tumor cells or elements of the tumor microenvironment, including the tumor vasculature. The targeting approach is generally conceived to permit administration of low, yet pharmacologically active, doses of drugs, thereby avoiding toxic reactions. However, it is becoming clear that, in the case of cytokines, this strategy has another inherent advantage, i.e. the possibility of administering extremely low doses that do not activate systemic counter-regulatory mechanisms, which may limit their potential therapeutic effects. This review is focused on the use of tumor vasculature-homing peptides as vehicles for targeted delivery of cytokines to tumor blood vessel. In particular, we provide an overview of peptide-cytokine conjugates made with peptides containing the NGR, RGD, isoDGR or RGR sequences and describe, in more details, the biological and pharmacological properties of NGR-hTNF, a peptide-tumor necrosis factor-α conjugate that is currently being tested in phase II and III clinical studies. The results of preclinical and clinical studies performed with these products suggest that peptide-mediated vascular-targeting is indeed a viable strategy for delivering bioactive amounts of cytokines to tumor endothelial cells without causing the activation of counter-regulatory mechanisms and toxic reactions.
    Full-text · Article · Jun 2013 · BioDrugs
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    • "αvβ3 is a type of integrin that overexpresses on endothelial cells and tumor cells and is responsible for adhering to and migrating endothelial cells [4], [5]. Studies have shown that isoDGR has a more efficient affinity with αvβ3 than Arg-Gly-Asp (RGD) as a well-known αvβ3-binding motif [6], [7], [8], [9], [10], [11]. isoDGR not only has the canonical RGD/αvβ3 contacts, but also establishes additional polar interactions [12]. "
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    ABSTRACT: BACKGROUD: isoAsp-Gly-Arg (isoDGR) is a derivative of the Asn-Gly-Arg (NGR) motif, which is used as a targeted delivery tool to aminopeptidase N (CD13) positive cells. Recent studies have shown that cyclic isoDGR (CisoDGRC) has a more efficient affinity with α(v)β(3), a type of integrin that overexpresses in tumor cells. Antimicrobial peptides (AMPs) are an efficient antitumor peptide that specifically kills tumor cells. In the present study, we designed antimicrobial peptides containing the CisoDGRC motif (CDAK) and assessed its antitumor activity for CD13(-)/α(v)β(3) (+) breast cancer cells (MCF-7 and MDA-MB-231) in vitro and in vivo. IN VITRO: We assessed the cytotoxicity of CDAK for MCF-7 and MDA-MB-231 breast cancer cells, the human umbilical vein endothelial cell (HUVEC), and human foreskin fibroblasts (HFF). We performed an apoptosis assay using Annexin-V/PI, DNA ladder, mitochondrial membrane potential, and Caspase-3 and Bcl-2. The effect on cell cycles and affinity with cell were tested using flow cytometry and fluorescent microscopy and the effect on invasion was analyzed using an invasion assay. CDAK was injected intravenously into tumor-bearing athymic nude mice in vivo experiment. CDAK showed cytotoxic activity in MCF-7 and MDA-MB-231 cells, whereas HUVEC and HFF were less sensitive to the peptides. CDAK induced apoptosis, reduced mitochondrial membrane potential, promoted Caspase-3, and inhibited Bcl-2 expression in the two breast cancer cell lines. In addition, CDAK inhibited proliferation of cancer cell through S phase arrest, and own selective affinity with MCF-7 and MDA-MB-231cells, inhibited the invasion of MDA-MB-231 cells. In vivo, CDAK significant inhibited the progression of the tumor and the generation of neovascularization. Antimicrobial peptides containing the CisoDGRC (CDAK) motif could efficiently exhibit the antitumor activity for CD13(-)/α(v)β(3) (+) breast cancer cells.
    Preview · Article · Jan 2013 · PLoS ONE
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