The epitope recognized by the unique anti-MUC1 monoclonal antibody MY.1E12 involves sialylα2-3galactosylβ1-3N-acetylgalactosaminide linked to a distinct threonine residue in the MUC1 tandem repeat

The specificity of the MY.1E12 mAb that was generated by immunizing mice with human milk fat globule (HMFG) was investigated. Fluorescein isothiocyanate (FITC)-conjugated peptides corresponding to a portion of the MUC1 tandem repeat were enzymatically glycosylated with N-acetylgalactosamine, galactose, and then sialic acid. The MY.1E12 mAb was examined for its affinity to the resulting glycopeptides by fluorescence polarization. Its affinity for the peptide whose Thr within the VTS sequence bears a Neu5Acalpha2-3Galbeta1-3GalNAc trisaccharide (K-d = 1.4 x 10(-7) M) was significantly higher than for the same peptide whose Thr bears an unsialylated disaccharide (K-d = 3.9 x 10(-6) M). The MY.1E12 mAb also bound strongly to a purified recombinant MUC1 fusion protein with six tandem repeats that was expressed by transfected MCF-7 breast cancer cells. The removal of sialic acids from the fusion protein significantly decreased MY. I E 12 mAb reactivity, much more so than the MUC1-specific 115138 antibody, whose epitope is known to be destroyed by desialylation. Thus, the attachment of the sialy1alpha2-3Galbeta1-3beta1-3GalNAc trisaccharide onto the Thr within the VTS motif significantly increases the binding of the MY 1 E 12 antibody to the MUC1 repeat sequence. (C) 2002 Elsevier Science B.V. All rights reserved.