Skip to main content
SpringerLink
Log in

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

  • Original Article
  • Published:
Glycoconjugate Journal Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

BY2:

Nicotiana tabacum L. cv. Bright Yellow 2

CHO:

Chinese hamster ovary

CTLA4:

Cytotoxic T-lymphocyte antigen-4

ELISA:

Enzyme-linked immunosorbent assay

Fuc:

Fucose

Gal:

Galactose

GlcNAc:

N-acetylglucosamine

GU:

Glucose unit

hCTLA4Ig:

Human cytotoxic T-lymphocyte antigen 4-immunoglobulin

hCTLA4IgM :

CHO cell-derived human cytotoxic T-lymphocyte antigen 4-immunoglobulin

hCTLA4IgP :

Rice cell-derived human cytotoxic T-lymphocyte antigen 4-immunoglobulin

hCTLA4IgP-Gal:

β1,4-galactose-extended rice cell-derived human cytotoxic T-lymphocyte antigen 4-immunoglobulin

hGalT:

Human β1,4-galactosyltransferase

HPLC:

High-performance liquid chromatography

HRP:

Horseradish peroxidase

HygR :

Hygromycin selection marker

MAA:

Maackia amurensis agglutinin

Man:

Mannose

ODS:

Octadecylsilyl

PA:

Pyridylamino

RCA:

Ricinus communis agglutinin RCA120

RT-PCR:

Reverse transcription-polymerase chain reaction

UTR:

Untranslated region

Xyl:

Xylose

References

  1. Saint-Jore-Dupas, C., Faye, L., Gomord, V.: From planta to pharma with glycosylation in the toolbox. Trends Biotechnol. 25, 317–323 (2007)

    Article  CAS  PubMed  Google Scholar 

  2. Bosch, D., Castilho, A., Loos, A., Schots, A., Steinkellner, H.: N-glycosylation of plant-produced recombinant proteins. Curr. Pharm. Des. 19, 5503–5512 (2013)

    Article  CAS  PubMed  Google Scholar 

  3. Jenkins, N., Curling, E.M.: Glycosylation of recombinant proteins: problems and prospects. Enzym. Microb. Technol. 16, 354–364 (1994)

    Article  CAS  Google Scholar 

  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 

  5. Gomord, V., Fitchette, A.C., Menu-Bouaouiche, L., Saint-Jore-Dupas, C., Plasson, C., Michaud, D., Faye, L.: Plant-specific glycosylation patterns in the context of therapeutic protein production. Plant Biotechnol. J. 8, 564–587 (2010)

    Article  CAS  PubMed  Google Scholar 

  6. Lerouge, P., Bardor, M., Pagny, S., Gomord, V., Faye, L.: N-glycosylation of recombinant pharmaceutical glycoproteins produced in transgenic plants: towards an humanisation of plant N-glycans. Curr. Pharm. Biotechnol. 1, 347–354 (2000)

    Article  CAS  PubMed  Google Scholar 

  7. Palacpac, N.Q., Kimura, Y., Fujiyama, K., Yoshida, T., Seki, T.: Structures of N-linked oligosaccharides of glycoproteins from tobacco BY2 suspension cultured cells. Biosci. Biotechnol. Biochem. 63, 35–39 (1999)

    Article  CAS  PubMed  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)

    Article  CAS  PubMed  Google Scholar 

  9. Palacpac, N.Q., Yoshida, S., Sakai, H., Kimura, Y., Fujiyama, K., Yoshida, T., Seki, T.: Stable expression of human beta1,4-galactosyltransferase in plant cells modifies N-linked glycosylation patterns. Proc. Natl. Acad. Sci. U. S. A. 96, 4692–4697 (1999)

    Article  PubMed Central  CAS  PubMed  Google 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)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  11. Robert, J.P., Peter, S.L.: CTLA4Ig: a novel immunoglobulin chimera with immunosuppressive properties. Methods 8, 116–123 (1995)

    Article  Google Scholar 

  12. Lee, S.J., Park, C.I., Park, M.Y., Jung, H.S., Ryu, W.S., Lim, S.M., Tan, H.K., Kwon, T.H., Yang, M.S., Kim, D.I.: Production and characterization of human CTLA4Ig expressed in transgenic rice cell suspension cultures. Protein Expr. Purif. 51, 293–302 (2007)

    Article  CAS  PubMed  Google 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)

    Article  CAS  PubMed  Google Scholar 

  14. Krapp, S., Mimura, Y., Jefferis, R., Huber, R., Sondermann, P.: Structural analysis of human IgG-Fc glycoforms reveals a correlation between glycosylation and structural integrity. J. Mol. Biol. 325, 979–989 (2003)

    Article  CAS  PubMed  Google Scholar 

  15. Deisenhofer, J.: Crystallographic refinement and atomic models of a human Fc fragment and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry 20, 2361–2370 (1981)

    Article  CAS  PubMed  Google Scholar 

  16. Matsumiya, S., Yamaguchi, Y., Saito, J., Nagano, M., Sasakawa, H., Otaki, S., Satoh, M., Shitara, K., Kato, K.: Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1. J. Mol. Biol. 368, 767–779 (2007)

    Article  CAS  PubMed  Google Scholar 

  17. Mizushima, T., Yagi, H., Takemoto, E., Shibata-Koyama, M., Isoda, Y., Iida, S., Masuda, K., Satoh, M., Kato, K.: Structural basis for improved efficacy of therapeutic antibodies upon defucosylation of their Fc glycans. Genes Cells 16, 1071–1080 (2011)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Boyd, P.N., Lines, A.C., Patel, A.K.: The effect of the removal of sialic acid, galactose and total carbohydrate on the functional activity of Campath-1H. Mol. Immunol. 32, 1311–1318 (1995)

    Article  CAS  PubMed  Google Scholar 

  19. Wright, A., Morrison, S.L.: Effect of glycosylation on antibody function: implications for genetic engineering. Trends Biotechnol. 15, 26–32 (1997)

    Article  CAS  PubMed  Google Scholar 

  20. Watzele, G., Berger, E.G.: Near identity of HeLa cell galactosyltransferase with the human placental enzyme. Nucleic Acids Res. 18, 7174 (1990)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  21. Wise, A.A., Liu, Z., Binns, A.N.: Three methods for the introduction of foreign DNA into Agrobacterium. Methods Mol. Biol. 343, 43–53 (2006)

    CAS  PubMed  Google Scholar 

  22. Terada, R., Asao, H., Iida, S.: A large-scale Agrobacterium-mediated transformation procedure with a strong positive-negative selection for gene targeting in rice (Oryza sativa L.). Plant Cell Rep. 22, 653–659 (2004)

    Article  CAS  PubMed  Google Scholar 

  23. Thompson, J.A., Abdullah, R., Cocking, E.C.: Protoplast culture of rice (Oryza sativa L.) using media solidified with agarose. Plant Sci. 47, 123–133 (1986)

    Article  Google 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)

    Article  CAS  PubMed  Google Scholar 

  25. Takahashi, N., Kato, K.: GALAXY (glycoanalysis by the three axes of MS and chromatography): a web application that assists structural analyses of N-glycans. Trends Glycosci. Glycotechnol. 15, 235–251 (2003)

    Article  CAS  Google Scholar 

  26. Hwang, H.S., Kim, B.S., Park, H., Park, H.Y., Choi, H.D., Kim, H.H.: Type and branched pattern of N-glycans and their structural effect on the chicken egg allergen ovotransferrin: a comparison with ovomucoid. Glycoconj. J. 31, 41–50 (2014)

    Article  CAS  PubMed  Google 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)

    Article  CAS  PubMed  Google 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)

    Article  CAS  PubMed  Google Scholar 

  29. Narimatsu, H.: Recent progress in molecular cloning of glycosyltransferase genes of eukaryotes. Microbiol. Immunol. 38, 489–504 (1994)

    Article  CAS  PubMed  Google Scholar 

  30. Raju, T.S.: Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr. Opin. Immunol. 20, 471–478 (2008)

    Article  CAS  PubMed  Google Scholar 

  31. Byrne, B., Donohoe, G.G., O’Kennedy, R.: Sialic acids: carbohydrate moieties that influence the biological and physical properties of biopharmaceutical proteins and living cells. Drug Discov. Today 12, 319–326 (2007)

    Article  CAS  PubMed  Google Scholar 

  32. Fujiyama, K., Palacpac, N.Q., Sakai, H., Kimura, Y., Shinmyo, A., Yoshida, T., Seki, T.: In vivo conversion of a glycan to human compatible type by transformed tobacco cells. Biochem. Biophys. Res. Commun. 289, 553–557 (2001)

    Article  CAS  PubMed  Google Scholar 

  33. Schähs, M., Strasser, R., Stadlmann, J., Kunert, R., Rademacher, T., Steinkellner, H.: Production of a monoclonal antibody in plants with a humanized N-glycosylation pattern. Plant Biotechnol. J. 5, 657–663 (2007)

    Article  PubMed  Google Scholar 

  34. Vézina, L.P., Faye, L., Lerouge, P., D’Aoust, M.A., Marquet-Blouin, E., Burel, C., Lavoie, P.O., Bardor, M., Gomord, V.: Transient co-expression for fast and high-yield production of antibodies with human-like N-glycans in plants. Plant Biotechnol. J. 7, 442–455 (2009)

    Article  PubMed  Google Scholar 

  35. Johnson, K.D., Chrispeels, M.J.: Substrate specificities of N-acetylglucosaminyl-, fucosyl-, and xylosyltransferases that modify glycoproteins in the Golgi apparatus of bean cotyledons. Plant Physiol. 84, 1301–1308 (1987)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  36. Chen, M., Liu, X., Wang, Z., Song, J., Qi, Q., Wang, P.G.: Modification of plant N-glycans processing: the future of producing therapeutic protein by transgenic plants. Med. Res. Rev. 25, 343–360 (2005)

    Article  CAS  PubMed  Google 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)

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  38. Dong, X., Storkus, W.J., Salter, R.D.: Binding and uptake of agalactosyl IgG by mannose receptor on macrophages and dendritic cells. J. Immunol. 163, 5427–5434 (1999)

    CAS  PubMed  Google Scholar 

  39. Wright, A., Morrison, S.L.: Effect of C2-associated carbohydrate structure on Ig effector function: studies with chimeric mouse-human IgG1 antibodies in glycosylation mutants of Chinese hamster ovary cells. J. Immunol. 160, 3393–3402 (1998)

    CAS  PubMed  Google Scholar 

  40. Lerouge, P., Cabanes-Macheteau, M., Rayon, C., Fischette-Laine, A.C., Gomord, V., Faye, L.: N-glycoprotein biosynthesis in plants: recent developments and future trends. Plant Mol. Biol. 38, 31–48 (1998)

    Article  CAS  PubMed  Google Scholar 

  41. Goetze, A.M., Liu, Y.D., Zhang, Z., Shah, B., Lee, E., Bondarenko, P.V., Flynn, G.C.: High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans. Glycobiology 21, 949–959 (2011)

    Article  CAS  PubMed  Google 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)

    Article  CAS  PubMed  Google Scholar 

  43. Gawlitzek, M., Ryll, T., Lofgren, J., Sliwkowski, M.B.: Ammonium alters N-glycan structures of recombinant TNFR-IgG: degradative versus biosynthetic mechanisms. Biotechnol. Bioeng. 68, 637–646 (2000)

    Article  CAS  PubMed  Google Scholar 

  44. Becerra-Arteaga, A., Shuler, M.L.: Influence of culture medium supplementation of tobacco NT1 cell suspension cultures on the N-glycosylation of human secreted alkaline phosphatase. Biotechnol. Bioeng. 97, 1585–1593 (2007)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

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.

Author information

Authors and Affiliations

  1. Boryung Central Research Institute, Boryung Pharmaceutical Co. Ltd., 1122-3, Shingil-dong, Danwon-gu, Ansan-si, Kyungki-do, 425-839, Republic of Korea

    Seung Hoon Kang, Hahn Sun Jung, Song Jae Lee, Cheon Ik Park & Sang Min Lim

  2. Biotherapeutics and Glycomics Laboratory, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul, 156-756, Republic of Korea

    Heajin Park, Byung Sun Kim, Kwang Heum Na, Gyeong Jin Han, Jae Woo Bae, Hyun Joo Park, Keuk Chan Bang, Byung Tae Park, Hye Seong Hwang & Ha Hyung Kim

  3. College of Life Sciences and Biotechnology, Korea University, 5ga Anam-dong, Sungbuk-ku, Seoul, 136-701, Republic of Korea

    In-Soo Jung

  4. School of Life Sciences, Gwangju Institute of Science and Technology, Oryong-dong, Buk-gu, Gwangju, 500-712, Republic of Korea

    Jae Il Kim

  5. Korea Research Institute of Bioscience & Biotechnology (KRIBB), 125 Gwahakro, Yuseong-gu, Daejeon, 305-806, Republic of Korea

    Doo Byung Oh

  6. Department of Biological Engineering, Inha University, 100 Inha-ro, Nam-gu, Incheon, 4022-751, Republic of Korea

    Dong Il Kim

  7. Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, 467-8603, Japan

    Hirokazu Yagi & Koichi Kato

  8. Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi, 444-8787, Japan

    Koichi Kato

  9. Department of Environmental & Health Chemistry, College of Pharmacy, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul, 156-756, Republic of Korea

    Dae Kyong Kim

Authors
  1. Seung Hoon Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hahn Sun Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Song Jae Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cheon Ik Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sang Min Lim
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Heajin Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Byung Sun Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Kwang Heum Na
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Gyeong Jin Han
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Jae Woo Bae
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Hyun Joo Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Keuk Chan Bang
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Byung Tae Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Hye Seong Hwang
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. In-Soo Jung
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Jae Il Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Doo Byung Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Dong Il Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Hirokazu Yagi
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Koichi Kato
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Dae Kyong Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Ha Hyung Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Dae Kyong Kim or Ha Hyung Kim.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kang, S.H., Jung, H.S., Lee, S.J. et al. 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. Glycoconj J 32, 161–172 (2015). https://doi.org/10.1007/s10719-015-9590-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10719-015-9590-x

Keywords

Search

Navigation