Over the past 15 years, the study of protein messengers within the immune and hematopoietic systems has lead to the identification of more than 25 distinct protein molecules possessing regulatory function. Interleukin -2 (IL-2) was one of the first immune system hormones to be discovered and has been one of the most intensely investigated. In addition, IL-2 is being used clinically for a variety of applications including anti - tumor and anti - HIV trials. Structure - activity relationships for the IL-2 - IL-2 receptor system have also been the focus of many studies. Nevertheless, no potent IL-2 antagonists or superagonists have been reported. This has been in part due to the complexity of the IL-2 receptor system, the third subunit of which was only recently identified, and the fact a long standing 3 Angstroms X-ray structure for IL-2 was also recently shown to be seriously in error. Despite these limitations, our laboratory has pursued structure function studies that have resulted in the preparation of IL-2 analogs with partial antagonist and superagonist properties. These effects are weak, however, when compared to the progress achieved in SAR studies of the related cytokine, Human Growth Hormone, in which more powerful methods such as phage display have been employed. Consequently, we have concentrated our recent efforts developing the required tools that will allow us to begin a new approach to the preparation of potent 1L-2 analogs using the same phage display methodology. We have successfully expressed all three receptor subunit ectodomains to serve as selection media. We have developed an entirely new approach to the design and preparation of soluble receptor complexes which bind IL-2 with cell surface-like affinity. These complexes may prove to be the key to the successful application of random library selection in the IL-2 receptor system. We have identified several templates from our previous studies that will be employed instead of wildtype IL-2, each template has a unique activity that should facilitate preparation of more potent analogs. Finally, we have developed a peptide template that will assume the same conformation in solution as on the surface of phage. Random libraries in the context of this template should lead to the selection of IL-2 antagonists in a smaller peptide format.
Our Specific Aims are: A. To exploit the versatility of """"""""phage display"""""""" methodology for the preparation of IL-2 analogs with greatly enhanced receptor binding properties as a route to both agonists and antagonists. B. To employ a stable helical hairpin peptide sequence as a template for phage display of random surface topology libraries capable of maintaining the displayed receptor binding conformation in solution.
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