Abstract 5081: Development and characterization of a new antiangiogenic peptide targeting tumor vessels.
Authors: Di Matteo Paola , Asperti Claudia , Porcellini Simona , Spitaleri Andrea , Bordignon Claudio , Musco Giovanna , Rizzardi Gian-Paolo , Traversari Catia 
Published: August 2014

Abstract
Vascular targeting is a promising approach for the treatment of angiogenesis-dependent diseases particularly for tumor treatment. Targeted drug-delivery to tumor vasculature preferentially localizes the drug at the tumor site, thus increasing efficacy and decreasing systemic side effects. In tumor neovasculature activated endothelial cells have been shown to express molecules characteristic of angiogenic vessels and virtually not expressed in normal vessels. Notably, tumor-associated endothelial cells express an isoform of aminopeptidase N (CD13) that binds to the cyclic peptide Cys-Asn-Gly-Arg-Cys (named NGR). Such “vascular addresses” allow the selective targeting of systemically administrated therapies to tumors, including chemotherapeutic agents and cytokines. In particular, the NGR-TNF conjugate has been shown to induce potent antitumor effects and is currently tested in phase II-III clinical trials. In this context, we developed and characterized a new tumor targeting peptide with a pathway of specificity comparable to that of NGR, but with a higher stability and affinity for the tumor vessels. In particular, we synthesized peptides containing the Cys-Arg-Gly-Asn-Cys cyclic motif made of D-aminoacids (i.e. crGnc, named rGn). The rGn peptide can be considered a partial retro-inverso of the NGR peptide, since a real retro-inverso petpide requires cyclization through the backbone, whereas in this case cyclization was obtained via the formation of the disulphide bond between the N and the C- terminal cysteins. The main advantage of retro-inverso peptides is their high metabolic stability, because the peptide bonds generated by D-aminoacids are stable to enzymatic cleavage. To characterize the biological activity and functions of the new peptide, we studied the rGn/CD13 interactions in vitro and in vivo on both tumour blood vessels and normal tissues, using quantum dots (Qd), whole-mount histology, and fluorescence/confocal microscopy. rGn-Qd showed the same recognition pattern of NGR-Qd on primary cell cultures of endothelial origin. Consistently, in vivo administered rGn-Qd and NGR-Qd bind to tumor blood vessels of several murine tumours. Of note, the binding of either rGn-Qd or NGR-Qd to CD13 is efficiently competed by an excess of either NGR or rGn peptide, respectively, both in in vitro and in vivo models. In particular, the rGn peptide showed a significantly higher affinity than NGR for its target cells in vitro. Moreover, silencing CD13 with specific shRNA prevented not only anti-CD13 MoAb binding, but also rGn-Qd binding. Finally, stressed experimental conditions that allowed deamidation of NGR to isoDGR, do not affect rGn structural integrity. In conclusion, the rGn peptide binds to CD13 expressed by angiogenic vessels with higher affinity and stability than NGR. Coupling rGn to cytotoxic/cytostatic molecules could lead to the generation of new potent antitumor compounds.
Citation Format: Paola Di Matteo, Claudia Asperti, Simona Porcellini, Andrea Spitaleri, Claudio Bordignon, Giovanna Musco, Gian-Paolo Rizzardi, Catia Traversari. Development and characterization of a new antiangiogenic peptide targeting tumor vessels. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5081. doi:10.1158/1538-7445.AM2013-5081