Respiratory epithelial cells direct the initial inflammatory response in the cystic fibrosis (CF) lung, which is characterized by a massive influx of neutrophils into the airways. Once there, the neutrophils release proteases, like neutrophil elastase, into the airway lumen. The fact that elastase is a cause of lung damage in patients with CF has led to proposals for treatment with anti-proteases, yet the rests of clinical studies examining the use of the anti-protease have been disappointing. The systemic administration of anti-proteases is inefficient, and the concentrations delivered to the airway by the intravenous administration of the anti-protease are insufficient to inhibit the overwhelming amount of neutrophil elastase in the lung of patients with CF. Alternatively, inhaled anti-proteases are in homogeneously distributed in the CF lung, and these agents are trapped in the mucus layer which blocks the delivery to the epithelial surface. The ideal delivery system would permit the intravenous administration of an anti-protease, thus avoiding the barriers to aerosol delivery in the diseased lung, that specifically targets the lungs. One such approach would be to modify anti-protease by introducing a ligand that would target the airways via the polymeric immunoglobulin receptor (pIgR), which is designed for the efficient, non-degradative transfer of macromolecules across epithelial immunoglobulin receptor (pIgR), which is designed for the efficient, non-degradative transfer of macromolecules across epithelial surfaces. We have constructed recombinant fusion proteins consisting of an Rv fragment of anti-human secretory component linked to human alpha/1-antitrypsin (A/1AT), and have shown that protein conjugates consisting of antibody directed at the polymeric immunoglobulin receptor (pIgR) and A/1AT can be transported across epithelial cells in a basolateral-to-apical direction. This approach provides us with the ability to deliver therapeutic proteins, like A/1AT or secretory leukoprotease inhibitor, directly to the apical surface of the epithelium by targeting the pIgR with an appropriate ligand. This proposal is directed at determining the feasibility of transporting fusion proteins to the apical surface. We plan to refine this strategy by determining the optimal sequence of fusion proteins designed to accomplish this purpose, demonstrating the versatility of the approach by coupling other therapeutic proteins to the targeting ligand. We will also examine the transport of such fusions across cells that express the pIgR, and analyze the trafficking of the conjugates to determine whether the delivery of proteins to the luminal surface could be affected by co- treatment with the natural ligand or drugs. Finally, we will then use these reagents to examine the delivery of fusions and if bifunctitonal proteins can prevent the inflammatory response in rodent models. This proposal should allow us to better understand this method of protein delivery to the lung, and deserve further consideration as an alternative approach to therapy.
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