Removal of xenoreactive human anti-pig antibodies by absorption on recombinant mucin-containing glycoproteins carrying the Gal alpha1,3Gal epitope

Abstract

Background: The hyperacute rejection caused by preformed natural antibodies in the recipient species reacting with donor species endothelial antigens is one of the major obstacles preventing routine use of clinical xenotransplantation. Based on the known structure and biosynthetic pathway of the major porcine xenoantigen, Gal alpha1,3Gal, we developed a novel strategy aimed at specific removal of human, natural anti-pig antibodies.

Methods: Cotransfection of COS cells with expression plasmids encoding a secreted mucin/immunoglobulin chimera and the porcine alpha1,3-galactosyltransferase facilitated simple immunoaffinity purification of a highly Gal alpha1,3Gal-substituted mucin/immunoglobulin fusion protein from transfected cell supernatants.

Results: Cotransfection of COS cells resulted in a mucin/Ig concentration in the supernatants ranging from 150 to 200 ng/ml. Approximately 300 ng of mucin/Ig chimeras absorbed onto 50 microl of packed anti-mouse IgG agarose beads could completely remove cytotoxic human anti-pig antibodies from 1 ml of human AB serum, as estimated in porcine endothelial cell cytotoxicity assays. Purified human IgG, IgM, and IgA all bound porcine endothelium, but only IgG and IgM were cytotoxic in the presence of rabbit complement. When the cytotoxicity of human IgG at 8 mg/ml and IgM at 1 mg/ml was completely removed by absorption on the mucin/Ig chimera, the binding to porcine endothelium was only partly reduced. Antibodies mediating antibody-dependent cellular cytotoxicity against porcine endothelium were also absorbed, indicating the importance of Gal alpha1,3Gal epitopes for this effect.

Conclusions: We describe the construction and production of a new and effective Gal alpha1,3Gal-substituted, mucin domain-containing absorber that can be used in a pretransplant extracorporeal immunoabsorption setting to remove anti-pig antibodies involved in antibody-dependent, complement- and cell-mediated cytotoxicity of pig endothelial cells.