Gamma interferon (IFN gamma) is an important lymphokine that Plays a pivotal role in the regulation of essential immune functions in host defense against cancer. Among these are: (1) modulation of expression of products of the major histocompatibility complex on the cell membrane, particularly the upregulation of expression of Ia antigens or macrophages; (2) priming/activation of macrophages for tumor cell killing; (3) enhancement of cytoxicity of lymphocytes, including that of antigen stimulated T lymphocytes and that of natural killer cells against tumor cells: (4) maturation of B cells for immunoglobulin secretion which, depending on the stage of clonal expansion of the cells, is manifested as either enhancement or suppression of antibody production. We propose to further examine the structure/function basis for the biological effects of IFN gamma using the synthetic peptide approach with monoclonal and polyclonal antibodies to IFN gamma and IFN gamma synthetic peptides. It is planed that the objective be achieved through the following approach: (1) generate monoclonal antibodies to mouse (and human) IFN gamma and determine their ability to block function and/or binding of IFN gamma to membrane receptors; (2) map epitope specificity of monoclonal antibodies using synthetic peptides that correspond to regions of the IFN gamma molecule that are located on the surface; initial mapping will involve longer peptides, followed by use of shorter peptides for possibly more precise mapping; (3) use synthetic peptides to produce monoclonal and polyclonal antibodies and determine their effect on function and receptor binding by IFN gamma; (4) determine the steric relationships of epitopes by competitive binding between monoclonal antibodies (and their Fab fragments) of defined epitope specificities with the IFN gamma molecule; (5) perform competitive receptor binding and functional experiments between synthetic peptides and IFN gamma, and determine the ability of appropriate peptides to bind to IFN gamma receptor; particular emphasis will placed on the use of longer peptides; (6) modify peptides that are important in epitope mapping and receptor competition by systematic removal and/or substitution of amino acids in order to more precisely identify sequences or regions of IFN gamma that are involved in function; (7) synthesize hybrid molecules to help identify discontinuous regions of the IFN gamma molecule that may act cooperatively to form structures that are critical for IFN gamma function. The proposed studies are important because they will provide information on the structural basis for the action of IFN gamma in regulation of immune functions and neoplasia.
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