Immunological Property of Antibodies against <i>N</i>-Glycolylneuraminic Acid Epitopes in Cytidine Monophospho–<i>N</i>-Acetylneuraminic Acid Hydroxylase-Deficient Mice

Tahara, Hiroyuki; Ide, Kentaro; Basnet, Nabin Bahadur; Tanaka, Yuka; Matsuda, Haruo; Takematsu, Hiromu; Kozutsumi, Yasunori; Ohdan, Hideki

doi:10.4049/jimmunol.0902857

Cited by 39 publications

(48 citation statements)

References 46 publications

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“…Humans, but not nonhuman primates, also make a complex array of antibody to a common mammalian sialic acid modification Neu5GC . This antibody reactivity is widely expected to contribute to xenograft rejection in humans but its significance remains uncertain due to the absence of anti‐Neu5GC antibody in experimental non‐human primate models. Screening strategies based on panels of defined oligosaccharides have failed to detect xenogeneic sensitization to other common oligosaccharides (Forssman antigen and α‐ and β‐lactosamine) .…”

Section: Discussionmentioning

confidence: 99%

Cloning and expression of porcine β1,4 N‐acetylgalactosaminyl transferase encoding a new xenoreactive antigen

Byrne

Stalboerger

et al. 2014

Xenotransplantation

126

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Background Xenograft rejection of pigs organs with an engineered mutation in the GGTA-1 gene (GTKO) remains a predominantly antibody mediated process which is directed to a variety of non-Gal protein and carbohydrate antigens. We previously used an expression library screening strategy to identify six porcine endothelial cell cDNAs which encode pig antigens that bind to IgG induced after pig-to-primate cardiac xenotransplantation. One of these gene products was a glycosyltrasnferase with homology to the bovine β1,4 N-acetylgalactosaminyltransferase (B4GALNT2). We now characterize the porcine B4GALNT2 gene sequence, genomic organization, expression, and functional significance. Methods The porcine B4GALNT2 cDNA was recovered from the original library isolate, subcloned, sequenced and used to identify a bacterial artificial chromosome (BAC) containing the entire B4GALNT2 locus from the Children's Hospital Oakland Research Institute BACPAC Resource Centre (#AC173453). PCR primers were designed to map the intron/exon genomic organization in the BAC clone. A stable human embryonic kidney (HEK) cell line expressing porcine B4GALNT2 (HEK-B4T) was produced. Expression of porcine B4GALNT2 in HEK-B4T cells was characterized by immune staining and siRNA transfection. The effects of B4GALNT2 expression in HEK-B4T cells was measured by flow cytometry and complement mediated lysis. Antibody binding to HEK and HEK-B4T cells was used to detect an induced antibody response to the B4GALNT2 produced glycan and the results were compared to GTKO PAEC specific non-Gal antibody induction. Expression of porcine B4GALNT2 in pig cells and tissues was measured by qualitative and quantitative real time reverse transcriptase PCR and by Dolichos biflorus agglutinin (DBA) tissue staining. Results The porcine B4GALNT2 gene shares a conserved genomic organization and encodes an open reading frame with 76 and 70% amino acid identity to the human and murine B4GALNT2 genes respectively. The B4GALNT2 gene is expressed in porcine endothelial cells and shows a broadly distributed expression pattern. Expression of porcine B4GALNT2 in human HEK cells (HEK-B4T) results in increased binding of antibody to the B4GALNT2 enzyme, and increased reactivity with anti-Sda and DBA. HEK-B4T cells show increased sensitivity to complement mediated lysis when challenged with serum from primates after pig to primate cardiac xenotransplantation. In GTKO and GTKO:CD55 cardiac xenotransplantation recipients there is a significant correlation between the induction of a non-Gal antibody, measured using GTKO PAECs, and the induction of antibodies which preferentially bind to HEK-B4T cells. Conclusion The functional isolation of the porcine B4GALNT2 gene from a PAEC expression library, the pattern of B4GALNT2 gene expression and its sensitization of HEK-B4T cells to antibody binding and complement mediated lysis indicates that the enzymatic activity of porcine B4GALNT2 produces a new immunogenic non-Gal glycan which contributes in part to the non-Gal immune response d...

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Section: Discussionmentioning

confidence: 99%

Cloning and expression of porcine β1,4 N‐acetylgalactosaminyl transferase encoding a new xenoreactive antigen

Byrne

Stalboerger

et al. 2014

Xenotransplantation

126

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“…Inhibition is overcome however with a larger dose of islets and isogenic CMAH +/+ hearts are not rejected by CMAH −/− recipients. (40) Under similar conditions GalT-KO mice reject isogenic GalT + mouse hearts (41) suggesting that the pathogenicity of anti-Neu5Gc antibody may be less then anti-Gal antibody.…”

Section: Human Non-gal Antibodiesmentioning

confidence: 99%

Recent investigations into pig antigen and anti-pig antibody expression

Byrne

McGregor

Breimer

2015

International Journal of Surgery

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Genetic engineering of donor pigs to eliminate expression of the dominant xenogeneic antigen galactose α1,3 galactose (Gal) has created a sea change in the immunobiology of xenograft rejection. Antibody mediated xenograft rejection of GGTA-1 α-galactosyltransferase (GTKO) deficient organs is now directed to a combination of non-Gal pig protein and carbohydrate antigens. Glycan analysis of GTKO tissues identified no new neo-antigens but detected high levels of N-acetylneuraminic acid (Neu5Gc) modified glycoproteins and glycolipids. Humans produce anti-Neu5Gc antibody and in very limited clinical studies sometimes show an induced anti-Neu5Gc antibody response after challenge with pig tissue. The pathogenicity of anti-Neu5Gc antibody in xenotransplantation is not clear however as non-human transplant models, critical for modelling anti-Gal immunity, do not produce anti-Neu5Gc antibody. Antibody induced after xenotransplantation in non-human primates is directed to an array of pig endothelial cells proteins and to a glycan produced by the pig B4GALNT2 gene. We anticipate that immune suppression will significantly affect the T-cell dependent and independent specificity of an induced antibody response and that donor pigs deficient in synthesis of multiple xenogeneic glycans will be important to future studies.

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“…To mimic the human situation, we used mice with a human-like defect in the Cmah gene, which encodes the enzyme that generates activated Neu5Gc (CMP-Neu5Gc) 18 . Such mice can make anti-Neu5Gc antibodies upon immunization with glycosidically-bound but not free Neu5Gc 19,20 . However, these previous studies used whole rodent or chimpanzee cells for immunization, an obviously artificial approach.…”

mentioning

confidence: 99%

“…In this regard, we have also suggested that Neu5Gc contamination of stem cells and other cell types intended for human therapy could pose risks 32,33 . Also, others have recently reported that Cmah null mice can reject Neu5Gc-positive wild-type organ transplants via complement-fixing anti-Neu5Gc antibodies 20 .…”

mentioning

confidence: 99%

Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins

et al. 2010

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Recombinant glycosylated biotherapeutic agents are usually produced in non-human mammalian cell lines, which can synthesize and/or metabolically incorporate the non-human sialic acid N-glycolylneuraminic acid (Neu5Gc). This contamination was previously known but ignored, as normal humans were thought to not react against Neu5Gc. However, recent findings indicate that humans have variable spectra of sometimes high levels of circulating anti-Neu5Gc antibodies. We studied two monoclonal antibodies in clinical use (Cetuximab and Panitumumab), and show covalently-bound Neu5Gc on Cetuximab. Anti-Neu5Gc antibodies from normal humans interact with Cetuximab in a Neu5Gc-specific manner and generate immune complexes in vitro. Mice with a human-like defect in Neu5Gc synthesis generate anti-Neu5Gc antibodies upon injection with Cetuximab. Circulating anti-Neu5Gc antibodies enhance Cetuximab clearance. These findings have potential relevance to half-life, efficacy and immune reactions in patients given such drugs. Finally, we show a method to reduce the Neu5Gc content of cultured cell lines and their secreted glycoproteins.

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Immunological Property of Antibodies against N-Glycolylneuraminic Acid Epitopes in Cytidine Monophospho–N-Acetylneuraminic Acid Hydroxylase-Deficient Mice

Abstract: Material

Cited by 39 publications

References 46 publications

Cloning and expression of porcine β1,4 N‐acetylgalactosaminyl transferase encoding a new xenoreactive antigen

Cloning and expression of porcine β1,4 N‐acetylgalactosaminyl transferase encoding a new xenoreactive antigen

Recent investigations into pig antigen and anti-pig antibody expression

Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins

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