Lawrence D. Papsidero

Immunoaffinity isolation of ductal carcinoma antigen using monoclonal antibody F36/22

Monoclonal antibody (McAb) F36/22, raised against a human breast tumor line, identifies an antigen found in the circulation of cancer patients. Antigen was purified from malignant effusions using McAb-affinity chromatography followed by adsorption-desorption from immobilized wheat germ lectin. Electrophoretic analysis demonstrated the isolation of a single high mol. wt glycoprotein exhibiting an isoionic point near pH 4.2 and a density of approx. 1.45 g/ml. Although highly reactive with wheat germ lectin, a negligible or weak interaction was observed with concanavalin A, lentil and peanut agglutinin. The antigen was immune-precipitable, indicating the occurrence of multiple McAb-binding sites, and was resistant to heat and acid treatments. Antigenicity was not perturbed following protease or neuraminidase treatments, but was affected upon exposure to alkaline conditions. Taken together, these data suggest that McAb F36/22 recognizes a high mol. wt component occurring in circulation as a mucin-like glycoprotein.

Purification and characterization of a human pancreas-specific antigen

A pancreas-specific antigen was identified by immunologic techniques and purified from saline extract of human pancreas. The purified pancreas-specific antigen was shown to be homogeneous by polyacrylamide gel electrophoresis under both denaturing and non-denaturing conditions. It had a molecular weight of 44000 as estimated by gel filtration or sodium dodecyl sulfate-gel electrophoresis, and a sedimentation coefficient of 3.4 S as analyzed by sucrose gradient centrifugation. Pancreas-specific antigen possessed an isoelectric point of 4.9 and migrated to alpha-beta region upon immunoelectrophoresis. By colorimetric assay procedures, pancreas-specific antigen exhibited no enzyme activity, such as amylase, protease, esterase, lipase, acid phosphatase, alkaline phosphatase peroxidase, deoxyribonuclease or ribonuclease. Immunoreactivity of pancreas-specific antigen was sensitive to proteolytic enzymes, perchloric acid and high temperature (70 degrees C, 10 min); but insensitive to neuraminidase or beta-glucosidase. Immunohistochemical staining revealed that pancreas-specific antigen was located in acinar cells of human pancreas. In addition, a higher concentration of pancreas-specific antigen was detected in pancreatic juice than in the saline extract of pancreas. This newly identified pancreas-specific antigen, therefore, may be a useful marker protein in physiological studies of pancreas and pancreatic secretion.

Isoelectric focusing analysis of soluble immune complexes bound to protein A-Sepharose.

Abstract Soluble immune complexes were studied by a newly developed procedure which consisted of precipitation at 2.5% polyethylene glycol, binding to immobilized Protein A, and subjecting to isoelectric focusing (pH 3 to 10). In a model system, bovine serum albumin: anti-bovine serum albumin complexes bound to immobilized Protein A were desorbed from the Protein A matrix and dissociated into antigen and antibody. The antigen and antibody components were separated based upon differences in isoelectric points. By this approach, IgG-type immunoglobulins and putative antigens can be detected from biological fluid of human specimens.

Ductal Carcinoma Antigen: Characteristics, Tissue Distribution and Capacity to Represent a Target for Monoclonal Antibody Therapy

Murine monoclonal antibody F36/22 was generated against the human breast carcinoma cell line MCF-7. The antibody reacts strongly with selected carcinoma cell lines and insignificantly against cell lines of mesenchymal derivation. No reactivity versus normal blood elements, bone marrow components or lymph nodes has been detected. Antigen is highly expressed in primary malignant tumors of ductal lineage to the virtual exclusion of lymphomas, sarcomas and non-ductal carcinomas, hence the designation Ductal Carcinoma Antigen. A small number of normal ductal structures also produce the antigen, although at greatly reduced levels. The antigen is found in the circulation of breast cancer patients where its exists as a high molecular weight glycoprotein. Purified antigen exhibits high density and a significant amount of carbohydrate content, thus resembling a mucin-like structure. The antibody-combining region also represents a carbohydrate component which is released upon alkaline-borohydride cleavage. The antigen behaves as an effective target in vitro for antibody dependent cytotoxicity as mediated both with complement and effector cells. Further, the passive administration of monoclonal antibody F36/22 results in a rapid decrease in the volume of human tumor xenografts as grown on athymic mice. The usefulness of this murine system as a pre-clinical model for human immunotherapies is under study.

Purification of a Human Pancreas-Specific Antigen

Using an antibody reagent, an organ-specific antigen has been purified from human pancreas. Purification was achieved using ammonium sulfate and ethanol precipitations, concanavalin A-Sepharose affinity chromatography, anion-exchange chromatography and gel filtration. The antigen had a molecular weight of approximately 68,000 as determined by gel filtration, was not dissociated into subunits by sodium dodecyl sulfate, and did not bind to immobilized concanavalin A. The biological significance of this antigen in pancreatic cancer is being studied.