Pig xenogeneic antigen modification with green coffee bean α‐galactosidase

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Abstract
Green coffee bean a-galactosidase can cleave the terminal a-galactose (αGal) on oligosaccharides that form the major antigen on pig endothelial cells recognized by primate-specific antibodies. Studies have been made of the conditions under which it is functional (e.g. temperature, pH) and of its biochemical and immunologic effects. Pig-to-rhesus monkey vein transplants were studied to identify the efficiency of the enzyme in delaying hyperacute rejection. When a graft became occluded, biopsies were taken for light microscopy (hematoxylin and eosin), scanning electron microscopy (SEM) and immunostaining with Griffonia simplicifolia IB4 lectin (GSIB4), and for IgM, IgG and C3. a-Galactosidase was stable for 72-96 h and was effective at 4°C and pH 6.9 (conditions of human liver graft storage), although better function was obtained at 20°C and pH 6.5. Using the porcine PK15 cell assay, the cytotoxicity of human serum was reduced after treatment of the pig cells with the enzyme. In vitro studies demonstrated that porcine veins treated with a-galactosidase lost endothelial expression of the Gal epitope within 30 min. SEM, however, demonstrated endothelial damage beginning within 2 h, probably caused by the a-galactosidase, as no damage was found in phosphate-buffered saline-treated veins, where the Gal epitope was preserved for > 3 h. No change was found in either group on light microscopy. In vivo studies demonstrated that patency of the a-galactosidase-treated veins (mean 2.5 h) was longer than that of untreated veins (0.23 h) (P < 0.01). Biopsies showed no GSIB4 lectin staining for a-Gal epitopes and much less IgM and C3 deposition in the treated group. Light microscopy and SEM demonstrated more severe endothelial damage, hemorrhage, and fibrin formation in the untreated group. Galactosidase is effective in removing the terminal αGal and delays the onset of hyperacute rejection of pig veins transplanted into monkeys. However, its effect is temporary and, on its own, its use is unlikely to prolong survival of pig organs transplanted into primates sufficiently to be of clinical value.