Human Natural Antibodies Recognizing Glycan Galβ1-3GlcNAc (Le©)

The level of human natural antibodies of immunoglobulin M isotype against Le (c) in patients with breast cancer is lower than in healthy women. The epitope specificity of these antibodies has been characterized using a printed glycan array and enzyme-linked immunosorbent assay (ELISA), the antibodies being isolated from donors' blood using Le (c)-Sepharose (Le (c) is Gal beta 1-3GlcNAc beta). The isolated antibodies recognize the disaccharide but do not bind to glycans terminated with Le (c), which implies the impossibility of binding to regular glycoproteins of non-malignant cells. The avidity (as dissociation constant value) of antibodies probed with a multivalent disaccharide is 10(-9)M; the nanomolar level indicates that the concentration is sufficient for physiological binding to the cognate antigen. Testing of several breast cancer cell lines showed the strongest binding to ZR 75-1. Interestingly, only 7% of the cells were positive in a monolayer with a low density, increasing up to 96% at highest density. The enhanced interaction (instead of the expected inhibition) of antibodies with ZR 75-1 cells in the presence of Gal beta 1-3GlcNAc beta disaccharide, indicates that the target epitope of anti-Le (c) antibodies is a molecular pattern with a carbohydrate constituent rather than a glycan.