Sungkwon Kang
Published: 2012
Total Pages: 462
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Inflammation is considered as a hallmark of host defense against infections and injuries. On the flipside, prolonged and non-resolving chronic inflammation is also associated with various pathological conditions. In the immune and inflammatory responses, it is the leukocyte integrins and their physiologic ligands that provide the essential molecular basis for cell adhesion and recognition. As important as these molecules are in maintaining healthy immune system, aberrant activities have been implicated in dysregulated inflammation, making integrins and their ligands a major therapeutic target. In this dissertation, I have developed and applied protein engineering techniques for modulating structure and function of integrins and their ligands, and thereby rendering opportunities for therapeutic development. Integrins have at least two distinct conformations, denoted as inactive or active. We have engineered the major ligand binding domains, or the inserted (I) domains, of leukocyte integrins into an activated state, competent for ligand binding. Possessing the ability to harness the I domains expressed in their inactive (wild-type) and active (high affinity mutants) states allowed us to discover neoepitope specific antibodies that preferentially bind to the active conformation of integrins. This was a streamlined process performed with the novel protein engineering platform, yeast surface two-hybrid, that we developed, which greatly facilitated the process of antigen engineering and novel antibody discovery. The discovered antibody potently inhibited leukocyte migration on ligand coated surfaces. Such antibodies specific against active conformation of integrins may be safer and administered at lower dosages, and result in better clinical outcomes. We also used the engineered I domains to create drug and gene delivery nanoparticles that mimic how leukocytes would bind and migrate selectively to inflammatory sites. More specifically, we used the I domain derived from the integrin lymphocyte function associated antigen-1 (LFA-1) for inflammation-specific accumulation of anti-inflammatory drugs, which otherwise would create systemic cytotoxicity. Delivery by the I domain was inflammation-specific because the physiological ligand of LFA-1, intercellular adhesion molecule-1 (ICAM-1) has a highly inducible expression on numerous cell types, including endothelial cells and immune cells. Specificity toward inflammation was dependent on the avidity of the I domain on delivery vehicles, and such optimally adjusted multimeric binding to ICAM-1 elicited rapid endocytosis. In this dissertation, I demonstrate that the use of the interaction between ICAM-1 and the engineered I domain provide a great opportunity to pierce through the barriers of gene delivery systems, with the addition of one more component for endosomal escape. Indeed, by using a cationic polymer previously known to elicit efficient endosomal escape, we were able to formulate nanoparticles that deliver genes like viruses, with improved gene transfer efficiency and for systemic applications. We anticipate that our virus-like particles may greatly contribute to a successful translation of such therapeutics into the clinics.