In vivo Neutralization of naturally Existing Antibodies against Linear (1,3)-Galactosidic Carbohydrate Epitopes by Multivalent Antigen Presentation : a Solution for the First Hurdle of Pig-to-Human Xenotransplantation

Pig-to-human xenotransplantation of islet cells or of vascularized organs would offer a welcome treatment alternative for the ever-increasing number of patients with end-stage organ failure who are waiting for a suitable allograph. The main hurdle are preexisting antibodies, most of which are specific for 'Linear-B', carbohydrate epitopes terminated by the unbranched Gal-α(1,3)Gal disaccharide. These antibodies are responsible for the 'hyper-acute rejection' of the xenograft by complement mediated hemorrhage. For depletion of such antibodies we have developed an artificial injectable antigen, a glycopolymer (GAS914) with a charge neutral poly-lysine backbone (degree of polymerization n = 1000) and 25% of its side chains coupled to Linear-B-trisaccharide. With an average molecular weight of 400 to 500 kD, presenting 250 trisaccharide epitopes per molecule, this multivalent array binds anti-αGal antibodies with at least three orders of magnitude higher avidity on a per-saccharide basis than the monomeric epitope. In vivo experiments with non-human primates documented that rather low doses – 1 to 5 mg/kg of GAS914 injected i.v. – efficiently reduce the load of anti-Linear-B antibodies quickly by at least 80%. This treatment can be repeated without any sensitization to GAS914. Interestingly, although the antibody levels start raising 12 h after injection, they do not reach pretreatment levels. The polymer is degraded and excreted within hours, with a minute fraction remaining in lymphoid tissue of anti-αGal producing animals only, probably binding to and inhibiting antibody-producing B-cells. The results of pig-to-non-human primate xenotransplantations established GAS914 as a relevant therapeutic option for pig-to-human transplantations as well. The synthesis of GAS914 was successfully scaled up to kg amounts needed for first clinical studies. Key was the use of galactosyl transferases and UDP-galactose for the synthesis of the trisaccharide

Removal of anti-Gal\u3b11,3Gal xenoantibodies with an injectable polymer

Preformed and elicited Ab's against the Gal\u3b11,3Gal terminating carbohydrate chains (\u3b1Gal Ab's) are the primary cause of hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation. \u3b1Gal Ab's are produced by long-lived Ab-producing cells that are not susceptible to pharmacological immunosuppression. We reasoned that antigen-specific elimination of \u3b1Gal Ab's might be achieved in vivo by systemic administration of nonimmunogenic polyvalent \u3b1Gal structures with high avidity for \u3b1Gal Ab's. We devised GAS914, a soluble trisaccharide-polylysine conjugate of approximately 500 kDa that effectively competes for \u3b1Gal binding by \u3b1Gal IgM (IC50, 43 nM) and IgG (IC50, 28 nM) in vitro. Injections of GAS914 in cynomolgus monkeys, at the dose of 1 mg/kg, resulted in the immediate decrease of more than 90% of circulating \u3b1Gal Ab's and serum anti-pig cytotoxicity. In baboons, repeated injections of GAS914 effectively reduced both circulating \u3b1Gal Ab's and cytotoxicity over several months. Studies with [14C]GAS914 in rhesus monkeys and Gal-/- mice indicate that GAS914 binds to circulating \u3b1Gal Ab's and that the complex is quickly metabolized by the liver and excreted by the kidney. Remarkably, posttreatment \u3b1Gal Ab titers never exceeded pretreatment levels and no sensitization to either \u3b1Gal or the polylysine backbone has been observed. Furthermore there was no apparent acute or chronic toxicity associated with GAS914 treatment in primates. We conclude that GAS914 may be used therapeutically for the specific removal of \u3b1Gal Ab's