The main objective of this research is to generate human-human hybridomas which secrete human monoclonal antibodies specific for prostate tumor antigens. It is thought that such human monoclonal antibodies will be superior to mouse monoclonal antibodies as diagnostic and therapeutic reagents, since after repeated injections of the mouse antibodies humans may recognize these antibodies as foreign. Also, by generating human monoclonal antibodies, we will determine the optimal conditions for generating human-human hybridomas and the type of immune response humans generate in response to their own tumor antigens. Lymphocytes isolated from regional draining lymph nodes of patients with prostate tumors will be fused with UC729-6, a 6-thioguanine-resistant human lymphoblastoid B cell line we have characterized in our laboratory. This cell line was chosen for its effectiveness in fusing with immunoglobulin-secreting human lymphocytes, resulting in human-human hybridomas. Supernatants from the immunoglobulin-secreting human hybridomas will be screened by an enzyme-linked immunosorbent assay against a panel of cells, both normal and neoplastic, to establish the specificity of the generated human monoclonal antibodies. The human hybridomas secreting anti-tumor monoclonal antibodies will be expanded, cloned, and further tested. We will use these human anti-tumor monoclonal antibodies to biochemically characterize the recognized antigens, specifically: (1) to define individual antigens associated with prostate tumors and which tumor types express the recognized antigen(s); (2) to isolate these antigens to determine their molecular profile; and (3) to determine the mechanisms regulating their biosynthesis, expression in the membrane, and shedding. The production and use of human monoclonal antibodies with predefined specificities should greatly aid our understanding of man's immune response and may lead to new immunotherapeutic approaches without fear of sensitization as a foreign protein (e.g., murine monoclonal antibodies). (A)