Progesterone receptor (PgR) is an important clinical marker of endocrine responsiveness and disease prognosis in breast cancer. PgR is also a key regulatory protein in breast cancer cells since it is known to be regulated by estrogens and is the presumed mediator of progestin growth inhibitory effects. We are, therefore, seeking monoclonal antibodies (MAb's) to human PgR for use in development of new clinical assays of PgR in breast tumors and as probes to examine structural and functional properties of receptor not presently accessible by conventional hormone-binding methods of receptor detection. Multiple strategies will be taken to produce a library of MAb's against different functional and structural domains on the receptor molecule. (1) The receptor-hormone complex will be purified from T47D human breast cancer cells by steroid affinity chromatography and used as immunogen to produce MAb's to determinants on the native molecule. (2) To force production of MAb's to the receptor hormone-binding site, anti-idiotypic MAb's will be produced against anti-progesterone antibodies (anti-anti-progesterone). Some of these anti-idiotypes are expected to cross react with the hormone binding site of PgR. (3) Partial proteolytic digestion fragments of PgR will be isolated from SDS-polyacrylamide gels and the individual peptides used as immunogen to produce MAb's to pre-defined structural regions of receptor and to determinants on denatured receptor normally occluded in the native molecule. Selected MA's will be used for ultimate purification of homogeneous human PgR by antibody affinity chromatography. The library of MAb's will be used for structural mapping of the functional domains of receptor polypeptides (i.e., DNA and steroid binding sites) and for critical examination of receptor subunit structure. Finally, MAb's will be used to probe presently inaccessible aspects of receptor dynamics in the cell including, biosynthesis, down regulation, assembly and relationship between A and B-subunits, and mechanism of estrogen regulation. Receptor MAb's will be utilized for the development of an ELISA assay for quantification of PgR in breast tumors and an immunohistochemical procedure for localization of PgR in breast tumor sections. Present methods for clinical assay of PgR often require more tissue than is sometimes available, are expensive and time consuming, are complicated by problems of stability of hormone binding sites, and do not permit histological detection of PgR in the usual heterogeneous tumor specimen. We are seeking, therefore, to develop simpler and more powerful immunological methods for clinical detection of PgR in patient tumors.
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