Analysis of the Role of Type 1 Core O-Glycans in the Binding of Anti-MUC1 Antibodies by Cytofluorometry and Synthetic Peptide/Glycopeptide Binding Inhibition Studies

Abstract

A panel of 56 MAbs submitted to the ISOBM TD-4 (MUC1) Workshop were analysed in two systems. These systems were designed to screen for peptide type 1 core O-glycan-related reactivities. Using synthetic MUC1 mucin-related peptides and glycopeptides, the panel of MAbs were tested for relative binding affinities to type 1 core O-glycan-substituted MUC1 structures. These studies utilized a competitive binding format with a native human adenocarcinoma-derived mucin as a solid phase. This system allows for analysis of the type 1 core glycoform subspecificity of each MAb. The second approach taken in parallel, utilized MCF-7 (BrCa) and OVCAR (OVCa) cell lines which were grown in the presence or absence of phenyl-N-acetylgalactosaminide (p-gal), a blocker of mucin O-linked glycosylation. These cells were analysed by FACS to examine the role these same glycan substitutions play with regard to either the diagnostic or therapeutic application of these MAbs. By FACS analysis there was a consistent increased ‘epitope exposure’ for peptide-specific MAbs binding in the presence of p-gal. In addition, a single MAb (TD-4 #150) is interpreted to react with a type 1 core O-glycan, probably with Tn, TF or STn specificity.

Galectin-3 regulates MUC1 and EGFR cellular distribution and EGFR downstream pathways in pancreatic cancer cells

“…However, since the influence of glycosylation on MUC1 immunodetection is not understood fully (Reddish et al, 1998;Reis et al, 1998), the possibility that changes of glycosylation may explain changes in MUC1 immunoreactivity could not be excluded. Anyway, our data strengthen the clinical relevance of studying the impact of galectin-3 on MUC1 localization.…”

Characterization of an Anti-MUC1 Monoclonal Antibody with Potential as a Cancer Vaccine

“…The MUC1 TD-4 workshop has compared 56 anti-MUC1 antibodies for the influence of carbohydrate moieties on antibody binding. Studies included various approaches like comparative binding to MUC1 peptides with and without o-glycan substitution, (24) binding to oligosaccharides, (25) binding after desialylation of MUC1 from ZR75-1 cells, (26) binding to nonglycosylated peptides, GalNAc-substituted peptides and fully glycosylated MUC1, 27,28 various non-MUC1 mucins, (29) or comparison of binding to fully, under-or nonglycosylated full-length MUC1. (30) Whereas a few antibodies clearly bind to a carbohydrate epitope (SH1, DH-1, M26, 3E1.2, 115D8, Ma695, HH6, 43, CC2, FH6, BW835, 27.1, KC4, 5F4, BCRU-G7, 7539MR), the majority of the 56 anti-MUC1 antibodies were directed against the MUC1 core protein, in particular the variable numbers of tandem repeat (VNTR) region.…”

Characterization of MUC1 glycoprotein on prostate cancer for selection of targeting molecules

“…With the exception of BrE-3, all of these MAbs were characterized extensively in the 1998 Workshop on MUC1 MAbs (42); in vitro and in vivo binding of BrE-3 has been described elsewhere (19,22,23,27,(43)(44)(45). MAb characterization included comparison for core peptide epitopes (46), VNTR affinity (47), effect of glycosylation on binding to VNTR (48), ability to bind to highly glycosylated (49) and hypoglycosylated cell lines (50) and binding to normal MUC1-expressing tissues (breast, small intestine, colon) (51). In addition to their MUC1 epitope diversity, these MAbs were also selected based on published information reflecting their usefulness for imaging, therapy and immunopathology correlations with cancer stage and prognosis.…”