We have developed an in vivo screening method in which ligand motifs that home to specific vascular beds can be selected after intravenous administration of a phage display random peptide library. This work has uncovered a vascular address system that allows organ-specific targeting to normal blood vessels and angiogenesis-related targeting to blood vessels of tumors. Our data show that tissue-specific vascular addresses in organs and angiogenesis-related vascular addresses in tumors may be used for targeted diagnosis and therapy. We also proposed a set of ethics guidelines for disease-oriented end-of-life research. Here, we propose to isolate ligand-receptor pairs by in vivo phage display in cancer patients and test them for targeted delivery to blood vessels.
Our specific aims are (i) to select peptides for vascular targeting of primary and metastatic cancer in patients. We will screen phage display random peptide libraries in human subjects. Peptide motifs will be selected as circulating ligands in vivo. We will outline the cellular distribution of the selected peptides in human blood vessels; molecular diversity within specialized structures in each tissue will be probed by laser capture microdissection (LCM); (ii) we will identify the vascular receptors for peptide ligands targeting different human tissues by using biochemical (affinity chromatography and protein arrays) and genetic (expression cloning) methods; (iii) we will image the vascular morphology of normal tissues and tissue representing progressive stages of cancer. Finally, we will study the structural requirements for the interaction between specific ligands and receptors by NMR and use this information as the basis to develop peptidomimetics. We will accomplish that by using methods developed for characterizing the microcirculation and the architecture, cellular composition, permeability, and extracellular matrix (ECM) of microvessels. The identification of novel receptors targeted by circulating ligands will shed light on the complex cellular and molecular diversity of the human vasculature. A unique and highly attractive feature of these functional assays is that they detect the availability of targets based not only on tissue expression level but also on accessibility to a circulating probe. We will validate these probes as delivery vehicles for targeted therapies and imaging agents. We expect that the striking molecular diversity of blood vessels will be translated into clinical applications. If successful, our efforts will accelerate the development of targeted anticancer therapies. ? ?
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