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 extracellular epitopes of PSMA. Three of these antibodies--1G9, 3C6, and 4D4--display distinct binding properties consistent with their recognition of conformational epitopes within native PSMA. Results indicate this panel of antibodies binds to native full-length PSMA, but not to fusion proteins containing portions of the linear sequence of the protein. Antibody binding is greatly reduced upon heat denaturation of native PSMA, and these antibodies do not 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 region spanning residues 44-750. Flow cytometric experiments indicate strong specific binding to live LNCaP cells. Antibody inhibition studies demonstrate that these antibodies recognize at least two distinct epitopes. Taken together, the results demonstrate that these antibodies are specific for native protein conformational epitopes within the extracellular domain. Their properties, in particular strong binding to live cancer cells, make them ideal candidates that are clearly superior to linear sequence epitope specific antibodies for in vivo applications.

Prostate-specific membrane antigen (PSMA): current benefits and future value.

We will review the evolution, benefits, and limitations of PSMA testing in the past, as well as its current and future value. Prostate cancer has been the most frequently diagnosed cancer and the second leading cause of cancer death in men in the United States. It has a wide spectrum of biological behavior between latent (indolent) and progressive (aggressive). Further identification of prostate-specific membrane antigen (PSMA) as a prognostic proliferation marker may enhance our understanding of the types of prostate cancer. A review of PSMA testing in the past as well as currently was conducted. Studies were reviewed that deal with detection of PSMA in serum and seminal fluid, reverse transcriptase-polymerase chain reaction (RT-PCR), immunoscintigraphy, and immunohistochemical assays. PSMA is expressed primarily in benign and cancerous prostatic epithelial cells. It is up-regulated in hormone resistant states, and in metastatic situations or other clinical situations where there is tumor recurrence or extension. Based on current results, PSMA detected in the serum by western blotting can assist in the identification, staging, and monitoring of metastatic prostate cancer. In addition, PSMA shows a promising role in directed imaging and therapy of recurrent or metastatic disease.

Isolation and characterization of monoclonal antibodies specific for the extracellular domain of prostate specific membrane antigen.

PURPOSE: Monoclonal antibodies specific for protein epitopes of prostate specific membrane antigen (PSMA) expressed on the external surface of prostatic epithelial cells were prepared to provide material for use in the diagnosis or treatment of prostatic cancer. MATERIALS AND METHODS: Mice were immunized with LNCaP cell membranes followed by purified PSMA before fusion. Hybridomas were screened by reactivity with purified PSMA. Resulting antibodies were characterized by enzyme-linked immunosorbent assay, Western blot and fluorescence-activated cell sorter analyses. RESULTS: Monoclonal antibody producing hybridomas designated 3E11, 3C2, 4E10-1.14, 3C9 and 1G3 were obtained which displayed specificities for differing regions of the extracellular domain of the PSMA protein. These antibodies reacted strongly with PSMA from multiple sources and specifically stained unfixed PSMA expressing cells by flow cytometric analysis. CONCLUSIONS: The antibodies obtained displayed strong reactivity and specificity for extracellular epitopes of PSMA. These antibodies will have value in future diagnostic and therapeutic applications focusing on PSMA as a target antigen.

Measurement of prostate-specific membrane antigen in the serum with a new antibody.

Work to date has identified prostate-specific membrane antigen (PSMA) as a membrane-bound glycoprotein with high specificity for prostatic epithelial cells. PSMA reacts with the monoclonal antibody 7E11.C5, which is present in serum, seminal fluid, and prostatic epithelial cells, and is increased in its expression in the presence of a hormone refractory state associated with prostatic cancer. This report confirms these results and further documents the presence of the monoclonal antibody 3F5.4G6, which reacts with the extracellular domain of PSMA. This region of PSMA is also an element present in a truncated version of the protein, so-called PSM'. Immune precipitation with either 7E11.C5 or 3F5.4G6 yields an isolated protein species that are reactive with the reciprocal antibody in Western blot analysis. Thus, 3F5.4G6 recognizes the same PSMA protein as does 7E11.C5, but at different epitopes on essentially opposite ends of the molecule. These two antibodies are well suited for use in a sandwich immunoassay, either one as a capture or detection antibody. Current work on this is underway. This report also confirms that 7E11.C5 Western blots for PSMA are negative with normal human brain tissue. The monoclonal antibody 9H10 does not react with 3F5.4G6 or with 7E11.C5 in studies conducted herein. Moreover, 3F5.4G6 reacts with PSMA found in the LNCaP cell line, but not DU-145 or PC3, which lack PSMA.

Analysis of glycosylation of prostate-specific membrane antigen derived from LNCaP cells, prostatic carcinoma tumors, and serum from prostate cancer patients.

BACKGROUND: Prostate-specific membrane antigen (PSMA) has been detected in human prostatic cancer tissues, serum, and seminal fluid based on Western blot data with the monoclonal antibody 7E11.C5. The reactive protein is very similar in size to that from human prostatic carcinoma LNCaP cells and corresponds to a protein with a molecular size of about 110,000 daltons. Given that PSMA is known to be a 750 amino acid protein of about 84,000 daltons, a substantial portion, perhaps 20-25% of the native molecular weight, is composed of carbohydrates. METHODS: In this study, we have begun initial analyses of the glycosylation of the PSMA protein from multiple sources using a variety of exo- and endoglycosidase treatments. RESULTS: The results indicate that the carbohydrate is primarily N-linked and in each case the deglycosylated protein has an apparent molecular weight of about 86,000 daltons. The glycan present on in vivo-derived PSMA from tumor tissue or serum was found to be primarily N-linked complex type. A small amount of O-linked glycan also appears to be present. In contrast, only high mannose-type N-linked glycans are present on the PSMA from LNCaP cells. CONCLUSIONS: Oligosaccharides present on PSMA derived from both tissue culture LNCaP cells and in vivo specimens are primarily N-linked and comprise about 20-25% of the native molecular weight. N-linked glycans of PSMA derived from in vivo sources were found to be complex type, lacking polylactosamine structures. In contrast, LNCaP cells express only high mannose-type structures. These results will be useful in our ongoing efforts to develop monoclonal antibodies which are specific for protein epitopes present in the extracellular domain of the protein.