Glycan structure and serum half-life of recombinant CTLA4Ig, an immunosuppressive agent, expressed in suspension-cultured rice cells with coexpression of human β1,4-galactosyltransferase and human CTLA4Ig
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Human cytotoxic T-lymphocyte antigen 4-immunoglobulin (hCTLA4Ig) is an immunosuppressive therapeutic, and recently produced rice cell-derived hCTLA4Ig (hCTLA4IgP) reportedly exhibits in vitro immunosuppressive activities equivalent to those of Chinese hamster ovary cell-derived hCTLA4Ig (hCTLA4IgM). However, limitations of hCTLA4IgP include shortened in vivo half-life as well as the presence of nonhuman N-glycans containing (β1-2)-xylose and α1,3-fucose, which cause immunogenic reactions in humans. In the present study, human β1,4-galactose-extended hCTLA4IgP (hCTLA4IgP-Gal) was expressed through the coexpression of human β1,4-galactosyltransferase (hGalT) and hCTLA4Ig in an attempt to overcome these unfavorable effects. The results indicated that both encoding hGalT and hCTLA4Ig were successfully coexpressed, and the analysis of N-glycan and its relative abundance in purified hCTLA4IgP-Gal indicated that not only were the two glycans containing (β1-4)-galactose newly extended, but also glycans containing both β1,2-xylose and α1,3-fucose were markedly reduced and high-mannose-type glycans were increased compared to those of hCTLA4IgP, respectively. Unlike hCTLA4IgP, hCTLA4IgP-Gal was effective as an acceptor via (β1-4)-galactose for in vitro sialylation. Additionally, the serum half-life of intravenously injected hCTLA4IgP-Gal in Sprague–Dawley rats was 1.9 times longer than that of hCTLA4IgP, and the clearance pattern of hCTLA4IgP-Gal was close to that for hCTLA4IgM. These results indicate that the coexpression with hGalT and hCTLA4IgP is useful for both reducing glycan immunogens and increasing in vivo stability. This is the first report of hCTLA4Ig as an effective therapeutics candidate in glycoengineered rice cells.
KeywordsPlant cell culture CTLA4Ig Human β1,4-galactosyltransferase N-glycan Half-life Clearance
Nicotiana tabacum L. cv. Bright Yellow 2
Chinese hamster ovary
Cytotoxic T-lymphocyte antigen-4
Enzyme-linked immunosorbent assay
Human cytotoxic T-lymphocyte antigen 4-immunoglobulin
CHO cell-derived human cytotoxic T-lymphocyte antigen 4-immunoglobulin
Rice cell-derived human cytotoxic T-lymphocyte antigen 4-immunoglobulin
β1,4-galactose-extended rice cell-derived human cytotoxic T-lymphocyte antigen 4-immunoglobulin
High-performance liquid chromatography
Hygromycin selection marker
Maackia amurensis agglutinin
Ricinus communis agglutinin RCA120
Reverse transcription-polymerase chain reaction
This study was supported by a grant (no. 2013M3A9B6075896) from the National Research Foundation of Korea, Republic of Korea. This work was also supported, in part, by the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), Japan, and by Grants-in-Aid for Scientific Research (A) (no. 24249002) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.
Conflict of interest
The authors declare that they have no conflict of interest.
Compliance with Ethical Standards
The animal experiments were performed in accordance with approval granted by the Institutional Animal Care and Use Committee at Boryung Pharmaceutical Co. Ltd.
- 4.Chrispeels, M.J., Faye, L.: The production of recombinant glycomproteins with defiined non-immunogenic glycans. In: Owen, M.R.L., Pen, J. (eds.) Transgenic Plants : A Production System for Industrial and Pharmaceutical Proteins, pp. 99–113. Wiley, New York (1996)Google Scholar
- 8.Fujiyama, K., Furukawa, A., Katsura, A., Misaki, R., Omasa, T., Seki, T.: Production of mouse monoclonal antibody with galactose-extended sugar chain by suspension cultured tobacco BY2 cells expressing human beta(1,4)-galactosyltransferase. Biochem. Biophys. Res. Commun. 358, 85–91 (2007)CrossRefPubMedGoogle Scholar
- 10.Bakker, H., Bardor, M., Molthoff, J.W., Gomord, V., Elbers, I., Stevens, L.H., Jordi, W., Lommen, A., Faye, L., Lerouge, P., Bosch, D.: Galactose-extended glycans of antibodies produced by transgenic plants. Proc. Natl. Acad. Sci. U. S. A. 98, 2899–2904 (2001)CrossRefPubMedCentralPubMedGoogle Scholar
- 13.Metzler, W.J., Bajorath, J., Fenderson, W., Shaw, S.Y., Constantine, K.L., Naemura, J., Leytze, G., Peach, R.J., Lavoie, T.B., Mueller, L., Linsley, P.S.: Solution structure of human CTLA-4 and delineation of a CD80/CD86 binding site conserved in CD28. Nat. Struct. Biol. 4, 527–531 (1997)CrossRefPubMedGoogle Scholar
- 24.Nakagawa, H., Kawamura, Y., Kato, K., Shimada, I., Arata, Y., Takahashi, N.: Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography. Anal. Biochem. 226, 130–138 (1995)CrossRefPubMedGoogle Scholar
- 27.Jung, H.S., Koo, J.K., Lee, S.J., Park, C.I., Shin, J.Y., Kim, M.H., Tan, H.K., Lim, S.M., Kim, D.I.: Characterization of human cytotoxic T lymphocyte-associated antigen 4-immunoglobulin (hCTLA4Ig) expressed in transgenic rice cell suspension cultures. Biotechnol. Lett. 28, 2039–2048 (2006)CrossRefPubMedGoogle Scholar
- 28.Wilson, I.B., Harthill, J.E., Mullin, N.P., Ashford, D.A., Altmann, F.: Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts. Glycobiology 8, 651–661 (1998)CrossRefPubMedGoogle Scholar
- 37.Ko, K., Tekoah, Y., Rudd, P.M., Harvey, D.J., Dwek, R.A., Spitsin, S., Hanlon, C.A., Rupprecht, C., Dietzschold, B., Golovkin, M., Koprowski, H.: Function and glycosylation of plant-derived antiviral monoclonal antibody. Proc. Natl. Acad. Sci. U. S. A. 100, 8013–8018 (2003)CrossRefPubMedCentralPubMedGoogle Scholar
- 42.Li, H., Sethuraman, N., Stadheim, T.A., Zha, D., Prinz, B., Ballew, N., Bobrowicz, P., Choi, B.K., Cook, W.J., Cukan, M., Houston-Cummings, N.R., Davidson, R., Gong, B., Hamilton, S.R., Hoopes, J.P., Jiang, Y., Kim, N., Mansfield, R., Nett, J.H., Rios, S., Strawbridge, R., Wildt, S., Gerngross, T.U.: Optimization of humanized IgGs in glycoengineered Pichia pastoris. Nat. Biotechnol. 24, 210–215 (2006)CrossRefPubMedGoogle Scholar