CHARACTERIZATION OF THE TUMOR-ASSOCIATED CARBOHYDRATE EPITOPE RECOGNIZED BY MONOCLONAL-ANTIBODY 4D3

The tumour-associated epitope recognised by monoclonal antibody (MAb) 4D3 is expressed on a high m.w. mucin glycoprotein preparation known a s small intestinal mucin antigen (SIMA). This epitope is detected in t issue from a high proportion of patients with colorectal cancer, and e levated levels occur in serum from a significant number of such patien ts, highlighting the potential clinical utility of MAb 4D3. In the pre sent study, insight into the composition and structure of the carbohyd rate epitope recognised by MAb 4D3 was gained following characterisati on of 2 glycopeptides that co-purified with SIMA. Sequence analysis of 1 of these glycopeptides revealed that it was identical to the glycop rotein alpha-1-antichymotrypsin. This glycoprotein was subsequently de glycosylated to yield 5 forms corresponding to alpha-1-anti-chymotryps in substituted with 4, 3, 2, 1 or no branched glycans. MAb 4D3 was rea ctive with each of the glycosylated forms, including the form carrying only 1 branched glycan, but did not react with fully deglycosylated a lpha-1-anti-chymotrypsin. MAb 4D3 also reacted to different extents wi th ovine, bovine or porcine submaxillary mucins, each of which has a d ifferent amount of the O-linked sialylated disaccharide known as sialo syl Tn. Of these mucins, MAb 4D3 was most reactive with ovine submaxil lary mucin, in which almost all of the carbohydrate chains are sialosy l Tn. Reactivity of MAb 4D3 towards isolated glycans, sialosyl Tn and related structures led to the conclusion that the preferred MAb 4D3 ep itope involves the sialylated N-acetyl galactosamine disaccharide as w ell as an additional monosaccharide present on a neighbouring carbohyd rate chain, Although the preferred epitope recognised by MAb 4D3 invol ves this sialylated disaccharide, the specificity of MAb 4D3 was diffe rent from that of other MAbs with a reported specificity for sialosyl Tn. (C) 1996 Wiley-Liss, Inc.