Biochemical and Immunological Properties of the Human Carcinoma-associated CAR-3 Epitope Defined by the Monoclonal Antibody AR-3

The monoclonal antibody AR-3 reacts with an epitope (CAR-3) carried on a high-molecular-weight glycoprotein associated with carcinomas of the pancreas, stomach, colon, uterus, and ovary. This study reports the partial purification and characterization of CAR-3-bearing molecule. The antigen was quantified by a double determinant immunoradiometric assay. CAR-3 antigen was purified by a three-step procedure, consisting of perchloric add extraction, molecular sieving on Sepharose CL-4B, and affinity chromatography on AR-3 antibodies coupled to Sepharose 4B. Following this procedure CAR-3 antigen was purified about 400-fold with a 36% yield. Treatment of the CAR-3 antigen with 16 dim meta-periodate or with 1 n NaOH resulted in complete loss of activity. Antigenicity survived enzymatic treatments known to destroy proteins. The epitope was found to be carried on a molecule with a molecular weight of 400,000 with a density of 1.45 g/ml, metabolically labeled with [35S]isulfate, [3H]glucosamine, and [methionine. It is concluded that CAR-3 epitope is expressed on a carbohydrate moiety linked to a sulfo-mucin-like molecule via an Oglycosidic bond. Cross-competition experiments showed that CAR-3 epitope is strictly related or in close topografie proximity to Lewis2 and Lewisb antigens. Cross-double determinant immunoradiometric assay experiments indicated that the same mucin carrying CAR-3 bears also CA 19-9, CA 125, and BW 494 epitopes.