Recombinant mucin-immunoglobulin chimeras as xenoreactive anti-pig antibody absorbers

Abstract

Human allotransplantation is nowadays the generally accepted treatment of choice for several illnesses leading to organ failure. A major problem facing clinical organ transplantation is the lack of human donor organs and tissues. Xenotransplantation from pig to man is considered a plausible solution to this problem. However, several hurdles, the hyperacute rejection (HAR) being the most immediate, need to be overcome before xenotransplantation will become a clinical reality. HAR is initiated by complement, activated by naturally occuring antibodies in man binding the Galalpha1,3Gal (cc-Gal) carbohydrate epitope on pig tissues. Although cellular xenografts are not lost in a HAR, antibodies have been shown to decrease graft survival in pig to mouse models of neural and islet cell xenotransplantation. Various methods, including plasmapheresis and extracorporeal immunoabsorption, have been used in order to remove anti-Gal antibodies from human serum.

The main aims of this thesis have been to: (i) construct, produce and characterize a novel anti-pig antibody absorber based on an alpha-Gal-substituted mucin-like immunoglobulin chimera, (ii) compare its anti-pig antibody absorption efficacy with that of porcine thyroglobulin and Galalpha1,3Gal disaccharides, and to (iii) analyze the relationship between the expression of alpha-Gal epitopes in individual pig embryonic brain cells and their susceptibility to the cytotoxicity of human blood group AB serum depleted of anti-alpha-Gal antibodies by absorption on the a-Gal substituted chimera.

The secreted P-selectin glycoprotein ligand-1/mouse IgG2b (PSGL-17mIgG2b) fusion protein had an apparent MW under reducing conditions of around 145 kDa with a wide size distribution characteristic of a heavily glycosylated protein. Following coexpression of the porcine alpha1,3 galactosyltransferase, the mucin/Ig stained strongly with the alpha-Gal specific lectin Griffonia simplicifolia 1 IB4 in Western blot experiments. The carbohydrate portion of the mucin/lg constituted 43 % of its molecular weight and the majority of the a-Gal epitopes were O-linked. The fusion protein carried approximately 140 mol of terming alpha-linked galactose per mol protein, and was on a carbohydrate molar basis a much more efficient anti-pig antibody absorber than pig thyroglobulin and Galalpha1,3Gal disaccharides. CHO-K1, 293T, COS7m6, Sp2/0 and High-Five cells stably expressing PSGL-1/mIgG2b with and without the porcine alpha1,3 galactosyltransferase were established. The alpha-Gal epitope density on PSGL-1/mIgG2b was dependent on the host cell used for its production, and was highest on the mucin/lg made in COS and 293T cells and lowest on CHO cells.

The anti-pig antibody absorption efficacy correlated to the cc-Gal epitope density. Transfection of CHO cells producing alpha-Gal-substituted mucin/lgs with plasmids encoding a core 2 beta1,6GIcNAc transferase led to the production of a mucin chimera with higher alpha-Gal epitope density and anti-pig antibody absorption capacity. The level of cc-Gal expression on neurons and astrocytes of porcine embryonic brain tissue was much lower than the level of expression on brain microvascular endothelial cells and microglia. Both non-depleted and anti-alpha-Gal- depleted human AB serum was cytotoxic to pig embryonic brain cells, suggesting that also non- alpha-Galspecific antibodies may contribute to the rejection of pig brain cell xenografts.

In conclusion, we have described the construction, production and characterization of a novel type of anti-pig antibody absorber based on a recombinant mucin-type fusion protein with rich Galalpha1,3Gal substitution on O-linked. glycans. Provided production costs can be kept low, this fusion protein may be used in extracorporeal absorption columns aimed at anti- pig antibody removal prior to future pigto-human organ transplantations.