Purification and composition of the human tumor-associated glycoprotein (TAG-72) defined by monoclonal antibodies CC49 and B72.3

Abstract

Human mammary and other carcinoma cells secrete and express on their cell surfaces complex, mucin-like glycoproteins (Mr greater than 10(6] that are recognized as tumor-associated antigens by a variety of monoclonal antibodies (MAbs). One such MAb, B72.3, has been extensively studied as to range of reactivity for a variety of carcinomas versus normal tissues. The B72.3-reactive antigen, designated tumor-associated glycoprotein (TAG)-72, which is a high-molecular-weight mucin, was partially purified and used as immunogen to produce second generation anti-TAG-72 MAbs. One of these second generation MAbs, CC49, was chosen to be used to develop a procedure to yield preparative amounts of purified antigen suitable for analyzing amino acid sequence. One of the reasons for the selection of CC49 for these studies is that it demonstrated a higher Ka for TAG-72 compared with B72.3. Xenografts of LS174T cells (a human colon carcinoma cell line) grown in nude mice were solubilized, extracted with several chaotropic agents and treated with perchloric acid. The acid soluble antigen was subjected to MAb CC49 affinity chromatography, gel filtration, and ion-exchange chromatography using fast protein liquid chromatography and high-performance liquid chromatography methodologies. A double-determinant liquid competition radioimmunoassay for TAG-72 showed greater than a 1000-fold purification. Radiolabeled protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated an apparently homogeneous high molecular weight mucin and a small amount of an additional protein with an apparent Mr of 63,000. Chemical deglycosylation using trifluoromethanesulfonic acid yielded low molecular weight proteins, which could be analyzed for amino acid sequence, and also became susceptible to tryptic digestion. The amino acid composition of the purified TAG-72 mucin was similar to that of other purified mucins.