Summary report on the ISOBM TD-4 Workshop: analysis of 56 monoclonal antibodies against the MUC1 mucin. San Diego, Calif., November 17-23, 1996.

Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin-related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRAPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies, highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of antimucin monoclonal antibodies.

Epitope characterization of MUC1 antibodies.

The specificities of the 56 MAbs submitted to the ISOBM TD-4 Workshop were characterized by ELISA assays against MUC1-positive (ZR75-1) and MUC1-negative cell lines (LS174T), binding to a 20-mer peptide corresponding to the tandem repeat region of MUC1 apoprotein, and Pepscan analysis of overlapping 9-mer peptides corresponding to the tandem repeat region of MUC1. The recognition of soluble MUC1 antigen from ZR75-1 cell line was also tested in two-site EIA using the ISOBM TD-4 MAb as catching MAb. The studies indicated that 35 of the submitted antibodies recognized epitopes expressed in the MUC1 tandem repeat region, and that 30 of these recognized epitopes within the immunodominant region (PDTRPAP) of the tandem repeat. Recognition of soluble MUC1 from ZR75-1 cell line indicated that additional 10 MAb recognized epitopes 'outside' the tandem repeat region of MUC1 from ZR75-1 cell line, or that the antibodies recognized epitopes depending on the glycosylation of the MUC1 tandem repeat. Six MAbs reacted only with the MUC1-negative cell line LS174T, and thus most likely did not recognize epitopes specific for MUC1.