Background: Clinical xenotransplantation is not possible because humans possess antibodies that recognize antigens on the surface of pig cells. and N-glycolylneuraminic acid (Neu5Gc) are two known xenoantigens. Methods: We report the homozygous disruption of the a1, 3-galactosyltransferase (GGTA1) and the cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) genes in liver-derived female pig cells using zinc-finger nucleases (ZFNs). Somatic cell nuclear transfer (SCNT) was used to produce healthy cloned piglets from the genetically modified liver cells. Antibody-binding and antibody-mediated complement-dependent cytotoxicity assays were used to examine the immunoreactivity of pig cells deficient in Neu5Gc and Gal. Results: This approach enabled rapid production of a pig strain deficient in multiple genes without extensive breeding protocols. Immune recognition studies showed that pigs lacking both CMAH and GGTA1 gene activities reduce the humoral barrier to xenotransplantation, further than pigs lacking only GGTA1. Conclusions: This technology will accelerate the development of pigs for xenotransplantation research.

Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end-stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig-to-primate kidney xenotransplantation has met limited success. Preformed anti-pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti-pig antibody levels. We then selected animals with both low and high titers of anti-pig antibodies to proceed with kidney transplant from galactose-α1,3-galactose knockout/CD55 transgenic pig donors. All animals received T-cell depletion followed by maintenance therapy with costimulation blockade (either anti-CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti-pig antibody rejected the kidney xenograft within the first week. Low-titer animals treated with anti-CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21 days, respectively). Long-term surviving animals treated with the anti-CD154-based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig-to-non-human primate kidney xenotransplantation, now >125 days, provides promise for further study and potential clinical translation.

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 (PBMC). We analyzed a sub population for reactivity towards genetically modified pig PBMC as compared to chimpanzee and human PBMC. 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 PBMC 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 cross match with human serum significantly.

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Pigs are currently the preferred species for future organ xenotransplantation. With advances in the development of genetically modified pigs, clinical xenotransplantation is becoming closer to reality. In preclinical studies (pig-to-nonhuman primate), the xenotransplantation of livers from pigs transgenic for human CD55 or from α1,3-galactosyltransferase gene-knockout pigs+/− transgenic for human CD46, is associated with survival of approximately 7–9 days. Although hepatic function, including coagulation, has proved to be satisfactory, the immediate development of thrombocytopenia is very limiting for pig liver xenotransplantation even as a ‘bridge’ to allotransplantation. Current studies are directed to understand the immunobiology of platelet activation, aggregation and phagocytosis, in particular the interaction between platelets and liver sinusoidal endothelial cells, hepatocytes and Kupffer cells, toward identifying interventions that may enable clinical application.

Burlak C, Wang ZY, Chihara RK, Lutz AJ, Wang Y, Estrada JL, Tector AJ. Identification of human preformed antibody targets in GTKO pigs. Xenotransplantation 2012; 19: 92–101. © 2012 John Wiley & Sons A/S. Abstract: Background:  Human preformed antibodies continue to recognize porcine xenografts, despite the advent of α‐galactosyltransferase knockout (GTKO) pigs. This study examined the potential reactivity of human preformed IgG and IgM antibodies toward antigens in the GTKO pig liver. Methods:  Human serum was analyzed for the concentration of IgG, IgM, anti‐αgal antibody, anti‐non‐αgal antibody and cytotoxicity toward domestic and GTKO fibroblasts and liver sinusoidal endothelial cells (LSEC). We detected preformed antibodies in human serum directed toward GTKO pig liver cells and tissue samples using advanced proteomic techniques. The targets of preformed antibodies were identified by MALDI TOF TOF mass spectrometry and validated by confocal microscopy, immunoblot, and immunoprecipitation. Results:  Human serum used in this study contained 2.06 μg/ml IgG and 0.013 μg/ml IgM directed toward GTKO fibroblasts. Human IgG and IgM bound to GTKO LSEC in a dose‐dependent manner and were cytotoxic. We detected 357 protein spots recognized by human IgG and 233 by human IgM. Two hundred and nineteen proteins were common to both human IgG and IgM. Mass spectrometry identified numerous immunoreactive proteins, of which 19 were membrane proteins on liver cells. The most significant to this study were α‐enolase, CFTR, and E‐cadherin, which were abundant in GTKO pig tissues and expressed on the surface of GTKO LSEC. Human IgG captured α‐enolase, CFTR, and E‐cadherin by immunoprecipitation validating the proteomic identification. Conclusion:  These experiments indicate that several membrane antigens in GTKO pigs could be recognized directly by human IgG or IgM. Further studies on the contribution of these antigens to antibody‐mediated xenograft rejection are necessary.

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The development of anti-human growth hormone (anti-hGH) and anti-host-cell protein antibodies to recombinant hGH (rhGH) of mammalian cell origin was determined in 395 children (304 GH deficiency; 91 Turner syndrome) undergoing long-term treatment (up to 54 months) for growth disorders. In all patients, blood samples were obtained prior to and every 2-3 months during treatment, and analyzed at a central laboratory for anti-hGH antibodies by RIA and antibodies to host-cell antigens by ELISA. During the first 24 months of treatment, 9 (3%) of the 304 patients with GH deficiency developed antibodies to rhGH for longer than 3 months. However, persistent antibodies were seen in only 2 patients, both of whom had proven hGH-N gene defects. In the remaining 7 (2%) anti-rhGH-antibody-positive patients, antibody concentrations showed a tendency to increase for 3-12 months, irrespective of the time of onset of measurable concentrations, and declined thereafter. In these patients, binding capacities were between 0.01 and 0.1 mg/l, and binding affinities were between 7 × 10⁸ and 8 × 10⁹1/mol. Height velocity was unaffected in these children. None of the 91 patients with Turner syndrome developed persistent anti-hGH antibodies. Further, no child developed antibodies to host-cell antigens during treatment with rhGH of mammalian cell origin.