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Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/?4GalNT2 genes.

ABSTRACT: Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The ?4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes.The Cas9 endonuclease and gRNA were used to create pigs lacking GGTA1, GGTA1/CMAH, or GGTA1/CMAH/?4GalNT2 genes. Peripheral blood mononuclear cells were isolated from these animals and examined for binding to IgM and IgG from humans, rhesus macaques, and baboons.Cells from GGTA1/CMAH/?4GalNT2 deficient pigs exhibited reduced human IgM and IgG binding compared to cells lacking both GGTA1 and CMAH. Non-human primate antibody reactivity with cells from the various pigs exhibited a slightly different pattern of reactivity than that seen in humans. Simultaneous inactivation of the GGTA1 and CMAH genes increased non-human primate antibody binding compared to cells lacking either GGTA1 only or to those deficient in GGTA1/CMAH/?4GalNT2.Inactivation of the ?4GalNT2 gene reduces human and non-human primate antibody binding resulting in diminished porcine xenoantigenicity. The increased humoral immunity of non-human primates toward GGTA1-/CMAH-deficient cells compared to pigs lacking either GGTA1 or GGTA1/CMAH/?4GalNT2 highlights the complexities of carbohydrate xenoantigens and suggests potential limitations of the non-human primate model for examining some genetic modifications. The progressive reduction of swine xenoantigens recognized by human immunoglobulin through inactivation of pig GGTA1/CMAH/?4GalNT2 genes demonstrates that the antibody barrier to xenotransplantation can be minimized by genetic engineering.

SUBMITTER: Estrada JL

PROVIDER: S-EPMC4464961 | biostudies-literature | 2015 May-Jun

REPOSITORIES: biostudies-literature

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Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes.

Estrada Jose L JL Martens Greg G Li Ping P Adams Andrew A Newell Kenneth A KA Ford Mandy L ML Butler James R JR Sidner Richard R Tector Matt M Tector Joseph J

Xenotransplantation 20150301 3

<h4>Background</h4>Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan-based species incompatibilities, we examined human and non-human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate-modifying genes.<h4>Methods</h4>The Cas9 endonuclease and gRNA were used to create pigs lacking ...[more]

PMID: 25728481

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Project description:BACKGROUND:Islet transplantation is an effective therapy for selected patients with type 1 diabetes with labile glycemic control and hypoglycemic unawareness, but donor organs are limited. Islet xenotransplantation using porcine islets will potentially solve this problem. Although successful proof of concept studies using clinically inapplicable anti-CD154 monoclonal antibody (mAb) in pig-to-non-human primate (NHP) islet xenotransplantation has been demonstrated by several groups worldwide, potentially clinically applicable anti-CD40 (2C10R4) mAb-based studies have not been reported. METHODS:Nine streptozotocin (STZ)-induced diabetic rhesus monkeys were transplanted with adult porcine islets isolated from designated pathogen-free (DPF) miniature pigs. They were treated with anti-CD40 mAb-based immunosuppressive regimen and were divided into 3 groups: anti-CD40 only group (n = 2), belatacept group (anti-CD40 mAb+belatacept, n = 2), and tacrolimus group (anti-CD40 mAb+tacrolimus, n = 5). All monkeys received anti-thymocyte globulin (ATG), cobra venom factor (CVF), adalimumab, and sirolimus. Blood glucose levels (BGL) and serum porcine C-peptide concentrations were measured. Humoral and cellular immune responses were assessed by ELISA and ELISPOT, respectively. Liver biopsy and subsequent immunohistochemistry were conducted. RESULTS:All animals restored normoglycemia immediately after porcine islet transplantation and finished the follow-up without any severe adverse effects except for one animal (R092). Most animals maintained their body weight. Median survival, as defined by a serum porcine C-peptide concentration of >0.15 ng/mL, was 31, 27, and 60 days for anti-CD40 only, belatacept, and tacrolimus groups, respectively. Anti-?Gal IgG levels in serum and the number of interferon-? secreting T cells in peripheral blood mononuclear cells did not increase in most animals. CONCLUSION:These results showed that anti-CD40 mAb combined with tacrolimus was effective in prolonging porcine islet graft survival, but anti-CD40 mAb was not as effective as anti-CD154 mAb in terms of preventing early islet loss.

Dataset Information

Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/?4GalNT2 genes.

Publications

Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes.

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