Effect of desialylation on binding, affinity, and specificity of 56 monoclonal antibodies against MUC1 mucin

We evaluated 56 monoclonal antibodies (MAbs), submitted to the ISOBM TD-4 Workshop, for changes in binding following desialylation of the MUC1 molecule and for epitope specificity. Antibody binding of MAbs was assayed by an ELISA method using microtiter plates coated with the MUC1 mucin obtained from supernatants of the ZR75-1 cell line. The MUC1 mucin was desialylated directly on the plate by treatment with neuraminidase. For each MAb, binding to untreated mucin was compared over a range of antibody concentrations. The concentration at which binding was half-maximal (K-50) was determined for all antibodies whose binding reached saturation in the assay. Results showed that K-50 values for MAb binding to untreated MUC1 mucin varied from 10(-10) to 10(-6) M. These data suggest that MAbs to MUC1 mucin bind with a broad range of intrinsic affinities, Desialylation was found to have variable effects on antibody binding, in that binding was either increased, decreased, or unchanged. No relationship was found between the apparent affinities for untreated mucin and changes in binding following desialylation. Among the 56 Workshop MAbs, 33 were found reactive with synthetic peptides which mimic the MUC1 tandem repeat. We determined the epitope specificity of the 33 MAbs by competitive binding using IO amino acid peptides corresponding to various regions of the 20-amino acid tandem repeat domain of MUC1. All antibodies which recognized epitopes in the 1-10 amino acid region of the tandem repeat showed increased binding to desialylated mucin. Antibodies to other peptide epitopes showed no consistent pattern of change in binding following desialylation. Our results suggest that sialic acid residues on the MUC1 mucin may contribute either positively or negatively to antibody binding. In addition, our results suggest that improved antibody selection methods could provide MAbs with improved selectivity for cancer-derived mucin compared with mucin from normal tissues. This could form the basis of improved biomarker assays for breast cancer.