Targeted drug delivery offers an opportunity for the development of safer and more effective therapies for the treatment of cancer. In this study, we sought to identify short, cell-internalizing peptide ligands that could serve as directive agents for specific drug delivery in hematologic malignancies. By screening of human leukemia cells with a combinatorial phage display peptide library, we isolated a peptide motif, sequence Phe-Phe/TyrAny-Leu-Arg-Ser (F F / Y XLRS), which bound to different leukemia cell lines and to patient-derived bone marrow samples. The motif was internalized through a receptor-mediated pathway, and we next identified the corresponding receptor as the transmembrane glycoprotein neuropilin-1 (NRP-1). Moreover, we observed a potent anti-leukemia cell effect when the targeting motif was synthesized in tandem to the pro-apoptotic sequence D (KLAKLAK) 2 . Finally, our results confirmed increased expression of NRP-1 in representative human leukemia and lymphoma cell lines and in a panel of bone marrow specimens obtained from patients with acute lymphoblastic leukemia or acute myelogenous leukemia compared with normal bone marrow. These results indicate that NRP-1 could potentially be used as a target for liganddirected therapy in human leukemias and lymphomas and that the prototype CGFYWLRSC-GG-D (KLAKLAK) 2 is a promising drug candidate in this setting. (Blood. 2011;117(3):920-927) IntroductionThe development of targeted drug-delivery strategies for safer and more effective therapy in human hematologic malignancies has been a long-standing goal for clinical and basic investigators. We reasoned that profiling of human leukemia-and lymphoma-derived cell lines with combinatorial libraries might yield ligand peptide sequences that bind to specific internalizing receptors on cell surfaces and may potentially lead to the discovery of new or unrecognized therapeutic targets. Such targeting motifs could also serve as vehicles for the preferential delivery of cytotoxic agents to leukemia and lymphoma cells.Several cell surface-binding peptides recognizing receptors in the membranes of lymphoma and leukemia cell lines have been reported. [1][2][3][4][5] The selected peptide ligands are readily internalized by cells and may therefore be potentially useful in ligand-directed drug delivery. Recently, we described an arginine-rich motif that is internalized into leukemia and lymphoma cells through the macropinocytotic pathway; however, the precise cell surface receptor has yet to be identified. 6 In effect, there is currently a relative lack of well-defined ligand-receptor systems for targeting human leukemia or lymphoma cells. The identification and validation of ligandreceptor pairs for these hematologic cancer cells relative to normal leukocytes would potentially represent a differential strategy and perhaps even improve disease outcomes.In this study, we used a combinatorial phage display-based subtractive selection 7-9 to identify ligand motifs that bind to specific cell surface receptors on human leuk...

Acute myelogenous leukemias (AMLs) are characterized by medullary and extramedullary invasion. We hypothesized that a supramolecular complex, the leukemiacell invadosome, which contains certain integrins, matrix metalloproteinases (MMPs), and other as-yet unidentified proteins, is essential for tissue invasion and may be central to the phenotypic diversity observed in the clinic. Here we show that the specific binding of MMP-9 to leukocyte surface ␤ 2 integrin is required for pericellular proteolysis and migration of AMLderived cells. An efficient antileukemia effect was obtained by the hexapeptide HFDDDE, a motif of the MMP-9 catalytic domain that mediates integrin binding: HFDDDE prevented proMMP-9 binding, transmigration through a human endothelial cell layer, and extracellular matrix degradation. Notably, the functional protein anchorage between ␤ 2 integrin and proMMP-9 described in this study does not involve the enzymatic active sites targeted by known MMP inhibitors. Taken

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