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Glycoconjugate Journal

, Volume 32, Issue 3–4, pp 161–172 | Cite as

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

  • Seung Hoon Kang
  • Hahn Sun Jung
  • Song Jae Lee
  • Cheon Ik Park
  • Sang Min Lim
  • 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
  • In-Soo Jung
  • Jae Il Kim
  • Doo Byung Oh
  • Dong Il Kim
  • Hirokazu Yagi
  • Koichi Kato
  • Dae Kyong Kim
  • Ha Hyung Kim
Original Article

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.

Keywords

Plant cell culture CTLA4Ig Human β1,4-galactosyltransferase N-glycan Half-life Clearance 

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

Notes

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.

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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Seung Hoon Kang
    • 1
  • Hahn Sun Jung
    • 1
  • Song Jae Lee
    • 1
  • Cheon Ik Park
    • 1
  • Sang Min Lim
    • 1
  • Heajin Park
    • 2
  • Byung Sun Kim
    • 2
  • Kwang Heum Na
    • 2
  • Gyeong Jin Han
    • 2
  • Jae Woo Bae
    • 2
  • Hyun Joo Park
    • 2
  • Keuk Chan Bang
    • 2
  • Byung Tae Park
    • 2
  • Hye Seong Hwang
    • 2
  • In-Soo Jung
    • 3
  • Jae Il Kim
    • 4
  • Doo Byung Oh
    • 5
  • Dong Il Kim
    • 6
  • Hirokazu Yagi
    • 7
  • Koichi Kato
    • 7
    • 8
  • Dae Kyong Kim
    • 9
  • Ha Hyung Kim
    • 2
  1. 1.Boryung Central Research InstituteBoryung Pharmaceutical Co. Ltd.Ansan-siRepublic of Korea
  2. 2.Biotherapeutics and Glycomics Laboratory, College of PharmacyChung-Ang UniversitySeoulRepublic of Korea
  3. 3.College of Life Sciences and BiotechnologyKorea UniversitySeoulRepublic of Korea
  4. 4.School of Life SciencesGwangju Institute of Science and TechnologyGwangjuRepublic of Korea
  5. 5.Korea Research Institute of Bioscience & Biotechnology (KRIBB)DaejeonRepublic of Korea
  6. 6.Department of Biological EngineeringInha UniversityIncheonRepublic of Korea
  7. 7.Graduate School of Pharmaceutical SciencesNagoya City UniversityNagoyaJapan
  8. 8.Okazaki Institute for Integrative Bioscience and Institute for Molecular ScienceNational Institutes of Natural SciencesOkazakiJapan
  9. 9.Department of Environmental & Health Chemistry, College of PharmacyChung-Ang UniversitySeoulRepublic of Korea

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