Isolation and characterization of monoclonal antibodies specific for protein conformational epitopes present in prostate-specific membrane antigen (PSMA)

Prostate-specific membrane antigen (PSMA) is a 750-amino acid glycoprotein highly expressed in malignant prostate tissues. PSMA reacts with the murine monoclonal antibody 7E11.C5, whose binding epitope has been mapped to the N-terminal of the protein distributed on the cytoplasmic side of the plasma membrane. We have developed murine monoclonal antibodies specific for extr acellular epitopes of PSMA, Three of these antibodies-1G9 3C6, and 4D4-disp lay distinct binding properties consistent with their recognition of confor mational epitopes within native PSMA, Results indicate this panel of antibo dies binds to native full-length PSMA, but not to fusion proteins containin g portions of the linear sequence of the protein. Antibody binding is great ly reduced upon heat denaturation of native PSMA, and these antibodies do n ot detect PSMA by Western blot. Immunoprecipitation experiments demonstrate the ability of each to bind to full-length PSMA as well as PSM', a form of the protein missing the first 57 amino acids. These results indicate each antibody is specific for an epitope within the extracellular domain, a regi on spanning residues 44-750. Flow cytometric experiments indicate strong sp ecific binding to live LNCaP cells. Antibody inhibition studies demonstrate that these antibodies recognize at least two distinct epitopes, Taken toge ther, the results demonstrate that these antibodies are specific for native protein conformational epitopes within the extracellular domain. Their pro perties, in particular strong binding to live cancer cells, make them ideal candidates that are clearly superior to linear sequence epitope specific a ntibodies for in vivo applications.