Combinatorial ligand-directed lung targeting

Abstract

Phage display of random peptide libraries is a powerful, unbiased method frequently used to discover ligands for virtually any protein of interest and to reveal functional protein-protein interaction partners. Moreover, in vivo phage display permits selection of peptides that bind specifically to different vascular beds without any previous knowledge pertaining to the nature of their corresponding receptors. Vascular targeting exploits molecular differences inherent in blood vessels within given organs and tissues, as well as diversity between normal and angiogenic blood vessels. Over the years, our group has identified phage capable of homing to lung blood vessels based on screenings using immortalized lung endothelial cells combined with in vivo selections after intravenous administration of combinatorial libraries. Peptides targeting lung vasculature have been extensively characterized and a lead homing peptide has shown interesting biological properties, bringing novel insights as to the implications of lung endothelial cell apoptosis in the pathogenesis of emphysema. We have also designed and developed targeted nanoparticles with imaging capabilities and/or drug delivery functions by combining phage display technology and elemental gold (Au) nanoparticles, constituting a promising platform for the development of therapeutic agents for imaging and treatment of lung disorders. Given the important role of the endothelium in the pathogenesis and progression of several diseases associated with the airways, ligand-directed discovery of lung vascular markers is an important milestone toward the development of future targeted therapies.

Figure 1.

Framework for discovery and validation of organ-specific vascular markers. In the ex vivo steps, endothelial cells isolated from different organs of the H-2Kb-tsA58 mouse (termed ImmortoMouse; see Reference 51) are maintained in culture at permissive temperature. Using the in vitro phage display method BRASIL (biopanning and rapid analysis of selective interactive ligands; see Reference 40), peptides targeting these cell populations are selected, validated, and prioritized based on their binding profile. Peptide ligands are further validated by phage in vivo assays in which, after tail-vein injection, phage homing to the different vascular beds are quantified by colony count or immunostaining with anti-bacteriophage specific antibodies. The peptides targeting organ-specific vascular ligands then serve as a template for the design (in silico) and synthesis of organ targeting-agents. These designed agents can be delivered in vivo for targeted therapy applications, generation of animal models of degenerative diseases, or in vivo imaging of organ specific diseases.