Inhibition of anti-Gal antibody capture and complement lysis of porcine endothelial cells by soluble Gal-bearing molecules
Introduction The xenotransplantation of organs from pigs to primates, as a model for pig to human transplantation, leads to an immediate complement-mediated hyperacute rejection (HAR) of the organ, which is principally due to the presence of natural anti-pig antibodies circulating in the recipient. Studies have shown that these antibodies are mainly directed against the Galα1-3Gal (Gal) epitope expressed on the porcine endothelium. Strategies to reduce HAR have included removal or neutralization of the anti-Gal antibodies, reduction of Gal expression in the donor organ, and complement inhibition. The aim of this study was to test certain soluble sugar preparations for their ability to inhibit the coupling of anti-Gal antibodies to pig endothelial cells in vitro.
Materials and methods Six Gal-bearing molecules were tested, Galα1-3Gal, trisaccharide, pentasaccharide, tripolymer, BSA-Gal and an oligosaccharide bound to polyacrilamide (Bdi-PAA) (kind gifts from Synthesome, Germany/Russia and Glycorex, Sweden). The pig aortic endothelium cell line C7 was a kind gift from Mike Parkhouse. The methods used to assess the sugars were as follows: (a) Inhibition of cell lysis by human AB serum containing complement. Cell viability was measured with the chromogen MTS. (b) Inhibition of anti-Gal capture (human serum or purified anti-Gal IgG) by cells fixed in microwell plates. Antibody capture was revealed with peroxidase-conjugated goat antihuman IgG antibody, with TMB as chromogen. (c) Inhibition of anti-Gal binding to C7 cells assessed by cytometry using goat antihuman IgG-FITC. The sugars were preincubated for 4 h with the serum or purified anti-Gal IgG prior to addition to the cells.
Results The greatest inhibition (up to 60%) was obtained with the pentasaccharide, tripolymer, BSA-Gal and Bdi-PAA, at the highest concentration (30 mg/ml), whereas the disaccharide and trisaccharide were much less effective (up to 16% inhibition).
Conclusions It is evident that the smaller saccharides are less effective competitors for anti-Gal antibodies than the more complex molecules. Larger molecules with several Gal epitopes spaced apart on an inert backbone should improve the avidity of the antibody-sugar reaction, and therefore be more effective in the neutralization or removal of the natural anti-Gal antibodies present in human blood.