Summary Report on the ISOBM TD-4 Workshop: Analysis of 56 Monoclonal Antibodies against the MUC1 Mucin

Abstract

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 PDTRPAP. 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 anti-mucin monoclonal antibodies.

Mice with Spontaneous Pancreatic Cancer Naturally Develop MUC-1-Specific CTLs That Eradicate Tumors When Adoptively Transferred

“…2, C and D), HMFG-2 and SM-3 (not shown). Although BC2 has been described as glycosylation insensitive in the mammary gland, which exhibits a lower level of glycosylation than the pancreas, this clearly is not the case in the pancreas (22). Normal pancreas in humans and mice do not exhibit epitopes for BC2.…”

“…MUC1 Abs used were CT1, a rabbit polyclonal Ab that recognizes mouse and human cytoplasmic tail region of MUC1 (21), HMFG-2, BC2, and SM-3, which have epitopes in the TR domain of MUC1. The TR epitope of HMFG-2 has been mapped to DTR, that of BC2 to APDTR, and that of SM-3 to PDTRP (22). All TR Abs are specific for human MUC1 and do not cross-react with mouse Muc1.…”

Characterization of the MUC1.Tg/MIN transgenic mouse as a model for studying antigen-specific immunotherapy of adenomas

“…Cell lysates were resolved on 5% denaturing polyacrylamide gels, transferred to polyvinylidene difluoride membranes electrophoretically and blocked in 5% dry milk in PBST (PBS, 0.01% Tween 20) overnight. The membrane was then incubated (1 h, 25°C) with either isotype control antibody or the B27.29 antibody that recognizes the human MUC1 tandem repeat sequences but is not reactive with the mouse Muc1 protein [23]. Both antibodies were diluted at 2 µg/mL in blocking solution.…”

Correlation study of Carcino Embryonic Antigen & Cancer Antigen 15.3 in pretreated female breast cancer patients

“…The DF3 antibody was produced against a membrane preparation from a breast carcinoma metastasis. The 115D8 antibody was prepared against milk fat globulin membrane and may be directed to a carbohydrate epitope (6).…”