Reduced binding of human antibodies to cells from GGTA1/CMAH KO pigs

Abstract

Xenotransplantation using genetically modified pig organs could solve the donor organ shortage problem. Two inactivated genes that make humans unique from pigs are GGTA1 and CMAH, the products of which produce the carbohydrate epitopes, aGal and Neu5Gc that attract preformed human antibody. When the GGTA1 and CMAH genes were deleted in pigs, human antibody binding was reduced in preliminary analysis. We analyzed the binding of human IgM and IgG from 121 healthy human serum samples for binding to GGTA1 KO and GGTA1/CMAH KO peripheral blood mononuclear cells (PBMCs). We analyzed a sub population for reactivity toward genetically modified pig PBMCs as compared to chimpanzee and human PBMCs. Deletion of the GGTA1 and CMAH genes in pigs improved the crossmatch results beyond those observed with chimpanzees. Sorting the 121 human samples tested against the GGTA1/CMAH KO pig PBMCs did not reveal a distinguishing feature such as blood group, age or gender. Modification of genes to make pig carbohydrates more similar to humans has improved the crossmatch with human serum significantly.

Keywords: Antibody-mediated rejection; crossmatch; genetically modified pigs; xenotransplantation.

Figure 1

Scatter plot of human IgM or IgG binding to either PBMC from GGTA1 KO (x-axis) and GGTA1/CMAH KO (y-axis) pigs. Values falling below the trend line indicates less antibody binding to the GGTA1/CMAH KO PBMC than to the GGTA1 KO PBMC.

Figure 2

Comparison of human antibody binding to human, porcine, or chimpanzee cells. PBMC isolated from humans, chimpanzees, and GGTA1/CMAH KO pigs incubated with 25% serum collected from five humans. Levels of IgM or IgG binding were detected using fluorescently labeled anti-human IgM or IgG antibodies followed by flow cytometric analysis. Histogram profiles of human IgM and IgG antibody binding are shown for 3 humans (filled gray), 3 chimpanzees (black) and 3 GGTA1/CMAH KO pigs (red). Histogram profiles of PBMC incubated with fluorescently labeled anti-human IgM or IgG antibodies in the absence of human serum are shown to indicate background fluorescence (2° only).

Figure 3

Human IgM or IgG binding to GGTA1/CMAH KO PBMC sorted by blood types A (n=38), AB (n=22), B (n=30), O (n=31), age defined as young (n=62): 18–30 years or old (n=59): 50–65 years, and gender: female (n=57) and male (n=64). Box and whisker plots are shown with the maximum and minimum values. Statistical significance was determined by unpaired two-tailed T test and indicated with an asterisk (*).