Porcine organs are rapidly rejected after transplantation into primate recipients due to the presence of preexisting immunoglobulins that bind to terminal galactose alpha1,3 galactose residues (alpha-galactosyl) present on porcine glycoproteins and glycolipids. Currently available immunosuppressive reagents have been largely ineffective at controlling the synthesis of these anti-Gal antibodies. Nonantigenic hapten polymers have been shown to be effective materials for blocking humoral immune responses in various model systems. We have developed a series of alpha-galactosyl glycoconjugate polymers and tested their ability to block anti-Gal antibody binding in vitro and in vivo. A galactose alpha1,3 galactose beta 1,4 GlcNAc trisaccharide free acid (TRFA) with a hexanoic acid spacer, containing five methylene groups and a carboxylic acid, was produced and coupled to a variety of polymeric backbones including dextran, branched poly(ethylene glycol) (PEG), and poly-L-lysine. The ability of monomeric TRFA and the alpha-galactosyl conjugates to block anti-Gal IgG and IgM binding was determined using a competition ELISA assay on defined HSA-Gal glycoconjugates and porcine microvascular endothelial cell substrates. We show that branched PEG carriers, with a TRFA sugar attached to each branch, exhibit enhanced antibody blocking ability compared to TRFA, but at higher target antigen densities these simple PEG conjugates are no more effective then an equivalent amount of TRFA in blocking anti-Gal IgM antibody interactions. In contrast, polymers of the branched PEG conjugates and linear conjugates made using dextran and poly-L-lysine were 2000 to 70000-fold more effective inhibitors of anti-Gal antibodies. In a study using nonhuman primates, a single dose infusion of polymeric PEG or dextran glycoconjugates dramatically reduced the level of circulating anti-Gal antibodies in cynomologus monkeys for at least 72 h. Glycoconjugates similar to these might be useful both to block anti-Gal interactions in vivo and to specifically control the induced anti-Gal immune response.

Unlike natural antibodies, single-chain Fv (sFv) proteins normally lack asparagine-linked glycosylation. Many designed immunoconjugates and other therapeutics currently employ the advantageous conjugation chemistry or targeting properties provided by the glycoprotein oligosaccharide domain. sFv proteins with engineered N-glycan designs were evaluated in Pichia pastoris for glycosylation efficiency, expression level, oligosaccharide chain length and composition, and affinity. In contrast to nearly all natural glycoproteins, the engineered attachment of N-glycans conveniently near the polypeptide C-terminus was found to produce the optimal results. Furthermore, the percentage modification and chain length of the attached mannose chains were controllable by the use of tandem and overlapping Asn-X-Thr tripeptide sites. The glycosylated sFv mannose chains could be effectively conjugated to polyethylene glycol and the resulting conjugate displayed a 10-fold increased circulating life in mice. The potential to control polymer:sFv or drug:sFv molar ratios by site-specific conjugation may substantially improve the therapeutic efficacy of these minimal antigen-binding molecules.