CHARACTERIZATION OF MONOCLONAL-ANTIBODIES DIRECTED TO THE AMINO-TERMINUS OF THE WT1, WILMS-TUMOR SUPPRESSOR PROTEIN

We have produced and characterized three monoclonal antibodies (MAbs) directed to the amino terminus of the WT1 Wilms' tumor suppressor tran scription factor and compared their properties to rabbit polyclonaI se ra raised to the same immunogen, A recombinant protein consisting of a mino acids 1-181 of human WT1 was overexpressed in E. coli, purified, and used as the immunogen, Three MAbs designated 6F-H2, 6F-H7, and 6F- HC-17-all of the IgG1 subclass-were selected and further characterized . Each recognized all isoforms of the full-length WT1 protein in Weste rn blot assays and immunoprecipitated WT1 in both physiologic buffers and under high detergent/high salt (RIPA) conditions. Preliminary epit ope mapping suggests that all three MAbs recognize a region in the ami no terminal 84 amino acids of WT1 and that the MAbs do not recognize t he polyglycine or polyproline regions of the protein. The WT1 antibodi es do not recognize the structurally and functionally related early gr owth response (EGR)1, EGR2, EGR3, or EGR4 proteins. All WT1 MAbs recog nize the murine WT1 protein and immunohistochemical staining of murine embryonic and newborn kidney sections show strong staining of condens ing metanephric mesenchyme and primitive podocytes in developing glome ruli, These WT1-specific MAbs should be useful in characterizing the b iochemical and developmental roles of WT1 and in defining the emerging role of WT1 as a diagnostic and/or prognostic marker in mesothelioma, leukemias, and breast cancer.