Enzymatic remodelling of the carbohydrate surface of a xenogenic cell substantially reduces human antibody binding and complement-mediated cytolysis

Abstract

The major obstacle to successful discordant xenotransplantation is the phenomenon of hyperacute rejection (HAR). In the pig-to-primate discordant transplant setting, HAR results from the deposition of high-titre anti-alpha-galactosyl antibodies and complement activation leading to endothelial cell destruction and rapid graft failure. To overcome HAR, we developed an enzymatic carbohydrate remodelling strategy designed to replace expression of the Gal alpha-1,3-Gal xenoepitope on the surface of porcine cells with the non-antigenic universal donor human blood group O antigen, the alpha-1,2-fucosyl lactosamine moiety (H-epitope). Xenogenic cells expressing the human alpha-1,2-fucosyltransferase expressed high levels of the H-epitope and significantly reduced Gal alpha-1,3-Gal expression. As a result, these cells were shown to be resistant to human natural antibody binding and complement-mediated cytolysis.

Gene therapy in transplantation

“…[156][157][158] In fact, almost all of the human Abs bind to an oligosaccharide pig xenoepitope defined as Gal-␣(1,3)-Gal-␤(1,4)-GlcNAc-protein, also known as the ␣-galactosyl epitope. 159,160 Complement-mediated lysis of the xenograft occurs by the classical pathway culminating in the formation of the membrane attack complex. This hyperacute rejection occurs within minutes.…”

“…This method involves the genetic engineering of xenogeneic cells to express ␣-(1,2)-fucosyltransferase. 159 This enzyme adds fucose residues to a nascent oligosaccharide. In both pig and human, the substrate for the fucosyltransferase is identical, therefore, by overexpressing the enzyme, the amount of xenoantigen would be significantly reduced.…”

“…Indeed, experimental data in vitro demonstrate that as much as 80% of the oligosaccharide is in the fucosylated form in genetically modified cells. 159,160,165,166 Moreover, a transgenic mouse overexpressing the enzyme has been generated. It too shows a diversion to the fucosylated variant to a level as high as 80%.…”

“…It too shows a diversion to the fucosylated variant to a level as high as 80%. 159,165 A potential gene therapy strategy would be to introduce the fucosyltransferase to pig organs, preferably at the time of organ procurement, although recent advances in animal cloning and transgenics may obviate this in the future. 167,168 A recent novel approach to inhibit the production of anti-xenoepitope antibodies involves the establishment of molecular chimerism.…”

Blocking of human anti-pig xenoantibodies by soluble GALα1-3GAL and Galα1-2GAL disaccharides; studies in a pig kidney in vitro perfusion model

“…Second, intravenous infusion of soluble aGa1 terminated oligosaccharides functioning as haptens in baboons transplanted with pig xenografts [30,39]. Third, change of the expression of Galal-3Gal determinants in the graft by al-3galactosyltransferase gene knockout [32,341, diverging the Galal-3Gal biosynthesis by addition of the al,2fucosyltransferase gene forming the blood group H determinant [5,6,28] or removing the antigen determinant by a-Galactosidase [15,21,361. Fourth, blocking complement activation by infusion of human soluble complement receptor 1 [23] or production of pig strains transgenic for human complement regulatory proteins [9,11,14].…”