Specificity of human natural antibodies referred to as anti-Tn

To understand the role of human natural IgM known as antibodies against the carbohydrate epitope Tn, the antibodies were isolated using GalNAc alpha-Sepharose affinity chromatography, and their specificity was profiled using microarrays (a glycan array printed with oligosaccharides and bacterial polysaccharides, as well as a glycopeptide array), flow cytometry, and inhibition ELISA. The antibodies bound a restricted number of GalNAc alpha-terminated oligosaccharides better than the parent monosaccharide, e.g., 6-O-Su-GalNAc alpha and GalNAc alpha 1-3Gal beta 1-3(4)GlcNAc beta. The binding with several bacterial polysaccharides that have no structural resemblance to the affinity ligand GalNAc alpha was quite unexpected. Given that GalNAc alpha is considered the key fragment of the Tn antigen, it is surprising that these antibodies bind weakly GalNAc alpha-OSer and do not bind a wide variety of GalNAc alpha-OSer/Thr-containing mucin glycopeptides. At the same time, we have observed specific binding to cells having Tn-positive glycoproteins containing similar glycopeptide motifs in a conformationally rigid macromolecule. Thus, specific recognition of the Tn antigen apparently requires that the naturally occurring "anti-Tn" IgM recognize a complex epitope comprising the GalNAc alpha as an essential component and a fairly long amino acid sequence where the amino acids adjacent to GalNAc alpha do not contact the antibody paratope; i.e., the antibodies recognize a spatial epitope or a molecular pattern rather than a classical continuous sequence. In addition, we have not found any increase in the binding of natural antibodies when GalNAc alpha residues were clustered. These results may help in further development of anticancer vaccines based on synthetic Tn constructs.