ANTI-GAL ANTIBODIES FROM HUMANS AND 1,3??-GALACTOSYLTRANSFERASE KNOCKOUT MICE.

Abstract

54 All mammals, except Old World monkeys including humans, express the enzyme 1,3α-galactosyltransferase that couples terminal αGAL epitopes to glycoproteins and glycolipids. As a result of the lack of αGAL epitopes human serum contains a high level of natural antibodies recognizing αGAL. These anti-GAL antibodies have been identified as a major obstacle for the successful transplantation of GAL+ organs into primates. Mice have been generated in which the 1,3α-galactosyltransferase gene is knocked-out by homologous recombination (GAL/TKO mice). In young GAL/TKO mice however, the levels of anti-GAL antibodies were below detection limit. In order to increase IgG and IgM anti-GAL antibody titers in GAL/TKO mice, an immunization protocol was developed in which rabbit red blood cells were used as a source of GAL epitopes. Repeated immunizations were necessary to increase anti-GAL antibody titers. Following immunization. the observed titers varied from 1:100 to 1:1000. Anti-GAL antibodies from mice and humans were compared in binding studies. Direct binding assays of human or mouse anti-GAL IgG or IgM to BSA/GAL showed that antibody titers in immunized mice were higher than those in humans (1:1000 vs 1:40). The affinity of anti-GAL antibodies was measured in competition assays using soluble α1-3,β1-4-Dgalactotriose. Antibody binding to BSA/GAL was measured in the presence of increasing concentrations of galactotriose. The concentration of galactotriose inhibiting antibody binding by 50% was 20 uM for human and mouse anti-GAL IgG antibodies. In contrast, IgM anti-GAL antibodies had a substantially lower affinity (800 uM for human IgM Vs 100 uM for mouse IgM). When hyperimmunized mice were used as recipients of GAL+ heart transplants, hyperacute rejection of these grafts (<2 hrs) was observed. In non-immunized GAL/TKO mice GAL+ hearts remained functional for up to 6 days. Immunohistological examination of the rejected GAL+ hears revealed massive deposition of IgM on endothelial cells of the graft with a concomitant deposition of complement. Therefore, our studies demonstrate that anti-GAL antibodies expressed in immunized GAL/TKO mice bind αGAL with similar affinity as human anti-GAL antibodies and cause hyperacute rejection of GAL+ heart transplants.