Enzymatic removal of αGal antigen in pig kidneys by ex vivo and in vivo administration of endo‐β‐galactosidase C

Abstract

Xenotransplantation using the pig as a donor species is considered to be a promising solution to the serious shortage of organ donors. Both hyperacute and acute vascular rejection (AVR) are believed to be associated with xenoreactive antibody binding to alphaGal epitopes on the pig vascular endothelial cells. Thus, suppression of this antigen-antibody reaction would appear essential for successful long-term xenograft survival. The purpose of this study was to examine the efficacy of ex vivo and in vivo administration of recombinant endo-beta-galactosidase C (EndoGalC which, in previous in vitro studies, has been proven to digest alphaGal antigens completely) on alphaGal epitopes expressed in pig kidneys. Excised pig kidneys were perfused with University of Wisconsin solution containing EndoGalC and preserved for 4 h. After cold storage, the pig kidney was transplanted into another pig. Ex vivo perfusion and cold storage with EndoGalC reduced alphaGal epitope expression on vascular endothelial cells to an undetectable level. However, alphaGal antigens began to be expressed again as early as 1 day after transplantation. The digestion of alphaGal epitopes by EndoGalC did not cause any damage to the kidney graft. EndoGalC was intravenously administered to two pigs (15 kg), without causing any serious adverse effect. Twelve hours later, >98% of alphaGal antigens on pig red blood cells (RBCs) had been digested. Immunohistochemical study revealed almost complete elimination of alphaGal expression on vascular endothelial cells of the kidney graft 4 and 8 h after in vivo administration, but reappearance within 24 h. EndoGalC was administered to a baboon after an interval of 2 months. The second administration did not result in any serious toxicity or reduction in efficacy. These results suggest that ex vivo and in vivo administration of EndoGalC is simple and useful in removing alphaGal epitopes from pig organs. As the effect of EndoGalC is temporary, multiple in vivo administrations of EndoGalC would be required to inhibit the reappearance of alphaGal epitopes. Alternatively, transgenic techniques of introducing the gene for EndoGalC into the donor organ might permanently prevent alphaGal expression.

Identification of a GH110 Subfamily of α1,3-Galactosidases

“…The inverting nature of family GH110 enzymes excludes such undesired reactions. Considerable efforts have been applied to testing the use of an endo-␤-galactosidase with strict specificity for the Gal␣1-3Gal␤1-4GlcNAc structure (35,(37)(38)(39). The action of this enzyme results in exposure of ␤GlcNAc residues rather that the Gal␤1-4GlcNAc structures exposed with exo-␣-galactosidase strategies, and this may result in other immune problems.…”

In vivo gene transfer of endo‐β‐galactosidase C removes αGal antigen on erythrocytes and endothelial cells of the organs

“…Ex vivo treatment of the pig kidney by recombinant EndoGalC resulted in almost complete removal of α Gal antigens [9]. In vivo administration of recombinant EndoGalC did remove α Gal antigens on the pig erythrocytes and on the endothelium of the organ [10,11]. In that study, however, it was difficult to completely rule out low‐level contamination by trace amounts of impure materials of bacterial origin in the final preparation of the recombinant EndoGalC [11].…”

“…In vivo administration of recombinant EndoGalC did remove α Gal antigens on the pig erythrocytes and on the endothelium of the organ [10,11]. In that study, however, it was difficult to completely rule out low‐level contamination by trace amounts of impure materials of bacterial origin in the final preparation of the recombinant EndoGalC [11]. In fact, producing a large amount of recombinant EndoGalC protein with clinical grade purity is costly and laborious.…”

Reduction of α‐galactosyl xenoantigen by expression of endo‐β‐galactosidase C in pig endothelial cells

“…However, as the effect of EndoGalC is temporary, αGal antigens reappear on the cell surface. To inhibit the reappearance of αGal on treated vascular endothelial cells, multiple in vivo administrations of the enzyme might be considered [5]. An alternative approach is to express the enzyme gene in pig endothelial cells.…”