IL7 has been shown to stimulate the proliferation and induce signal transduction events in both pre-B and T cell progenitors, cytotoxic T cells, lymphokine activated killer cells, myeloid cells, and a variety of leukemia and lymphoma cells freshly isolated from patients. This application is being submitted as a continuation of the molecular genetic analysis of IL7. They propose to continue studies on the structure function relationships of IL7 with particular emphasis on receptor binding and signal transduction. Initial studies have led to the development of an atomic scale molecular model of IL7. This model employs constraints imposed by the introduction of disulfide bonds between cystein residues (Cys3-Cys142; Cys35-Cys90; Cys48-Cys93). Using this model, they introduced a series of site-directed mutations into helix D which have begun to define amino acid residues which interact with and transduce signals through the IL7 receptor. One mutant, IL7(W143A) has been shown to bind the IL7 receptor, but was unable to stimulate proliferation of IL7 dependent 2E8 cells in vitro. Thus, IL7(W143A) was the first IL7R antagonist to be reported. Studies proposed in the current application are focused on the further analysis of helix D, as well as the elucidation of amino acid residues in helix A that facilitate binding and signal transduction. They propose to employ BIAcore analysis of native and mutant IL7 binding to both individual and heterodimeric ectodomains of the IL7 receptor. Hydrophobic moment analysis has suggested that helix D of IL7 has the atypical features of presenting a hydrophobic face toward bulk solvent. They will """"""""redesign"""""""" IL7 by circular permutation with the expectation that permuteins with improved properties will be isolated. In addition, permutein variants of IL7 will be used in the construction of diphtheria toxin-related fusion protein toxins that are likely to have improved solubility and folding properties. Finally, they will produce IL7 in sufficient yield and purity to initiate the determination of its X-ray crystal structure. It is envisioned that research proposed in this application will be pivotal in bringing IL7 and/or IL7 variants and DAB389IL7 to clinical trials.
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