Enhanced inhibition of human anti-Gal antibody binding to mammalian cells by synthetic α-Gal epitope polymers

Neoglycopolymers of polyacrylamide backbone conjugated with varying densities of Gal alpha 1-3Gal beta 1-4Glc beta trisaccharide epitopes (alpha-Gal epitopes) were designed and synthesized to study the inhibition of the binding of human natural anti-Gal antibodies to either alpha-Gal-containing glycoproteins or alpha-Gal antigens on the surface of mammalian cells. An inhibition ELISA using mouse laminin and a flow cytometry assay using pig kidney cells (PK15) were established to determine the binding affinity of the synthesized polymers. In comparison to the alpha-Gal monomer (Gal alpha 1-3Gal beta 1-4GlcNHAc beta), the alpha-Gal polymers dramatically enhanced the inhibition of human anti-Gal antibodies (IgG, IgM, and IgA) binding to mouse laminin or mammalian cells. Increases of 7.8 x 10(3)- and 5.0 x 10(4) -fold in inhibitory potential of polymer 7C to IgA and IgM (with IC(50)s of 7.0 and 5.6 nM respectively) were observed over the monomer in inhibition ELISA. The results also indicated that binding enhancement of alpha-Gal polymers is greater for anti-Gal IgA and IgM than for IgG. Such amplified binding differences among the three anti-Gal isotypes can be utilized to selectively inhibit or remove a particular isotype of anti-Gal antibodies. Moreover, it was demonstrated through the flow cytometry assay that certain alpha-Gal polymers are effective in inhibition of anti-Gal antibody (in human serum) binding to pig kidney (PK15) cells. Thus, such synthetic carbohydrate polymers may find practical applications in cell xenotransplantations.