We transplanted kidneys from alpha1,3-galactosyltransferase knockout (GalT-KO) pigs into six baboons using two different immunosuppressive regimens, but most of the baboons died from severe acute humoral xenograft rejection. Circulating induced antibodies to non-Gal antigens were markedly elevated at rejection, which mediated strong complement-dependent cytotoxicity against GalT-KO porcine target cells. These data suggest that antibodies to non-Gal antigens will present an additional barrier to transplantation of organs from GalT-KO pigs to humans.

In a pig-to-baboon kidney transplant model, both preformed and induced anti-non-Gal antibodies are strongly associated with the pathogenesis of AHXR when anti-Gal antibodies are neutralized.

Thus, pigs transgenic for multiple transgenes provide a greater degree of human complement regulation and hence might be more suitable for xenotransplantation.

Anti-pig antibodies (APA) were analysed in serum from 28 naïve wild-caught baboons (originating from Kenya) and 31 naïve captive-bred cynomolgus monkeys (13 from the Philippines and 18 from Mauritius), using a haemolytic assay with pig erythrocytes (APA), flow cytometry on the porcine lymphoma T-cell cell line L35, and enzyme linked immunosorbent assay (ELISA) using alpha-Gal type II and type VI antigen. This was extended in baboon samples by the evaluation in two laboratories (Imutran, Cambridge, UK and Immerge, Boston, USA), and by antibody absorption using either immobilized alpha-Gal type II or alpha-Gal type VI. Anti-porcine antibodies were demonstrated in all assays with substantial variability within and between the three non-human primate groups. Immunoglobulin (Ig)M antibody levels tended to be similar to or higher than those in a pooled normal human standard serum while IgG levels tended to be lower. Highest antibody levels were recorded in Mauritius cynomolgus monkeys. There were statistically significant correlations between assays for IgM or IgG class anti-Gal antibodies using either alpha-Gal type II or alpha-Gal type VI as antigen, both for different assays and two laboratories involved. Also, significant correlations were observed between the anti-Gal and L35 binding assays. Baboon sera before and after absorption to immobilized alpha-Gal type II or type VI were analysed for anti-Gal type VI or type II antibody: levels were almost undetectable indicating that most anti-Gal antibodies react to epitopes shared between alpha-Gal type II and type VI oligosaccharides. Finally, the relation between APA and outcome of porcine heart xenotransplantation in cynomolgus monkeys and baboons showed no apparent relation between pre-transplant APA levels and the occurrence of hyperacute rejection (HAR) when compared with non-immunological cause of organ/recipient dysfunction or acute humoral xenograft rejection during the first 4 days post-transplantation or survival exceeding 4 days post-transplantation.

It is recommended to use multiple assays (preferably hemolytic, anti-Gal, and anti-endothelial cell) to be able to fully monitor the peripheral antibody responses in pig-to-primate xenograft recipients.