Advertisement

Plant Cell Reports

, Volume 34, Issue 6, pp 959–968 | Cite as

NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana

  • Hirokazu Yagi
  • Noriho Fukuzawa
  • Yasushi Tasaka
  • Kouki Matsuo
  • Ying Zhang
  • Takumi Yamaguchi
  • Sachiko Kondo
  • Shiori Nakazawa
  • Noritaka Hashii
  • Nana Kawasaki
  • Takeshi Matsumura
  • Koichi Kato
Original Paper

Abstract

Key message

We successfully developed a method for metabolic isotope labeling of recombinant proteins produced in transgenic tobacco. This enabled assessment of structural integrity of plant-derived therapeutic antibodies by NMR analysis.

Abstract

A variety of expression vehicles have been developed for the production of promising biologics, including plants, fungi, bacteria, insects, and mammals. Glycoprotein biologics often experience altered folding and post-translational modifications that are typified by variant glycosylation patterns. These differences can dramatically affect their efficacy, as exemplified by therapeutic antibodies. However, it is generally difficult to validate the structural integrity of biologics produced using different expression vehicles. To address this issue, we have developed and applied a stable-isotope-assisted nuclear magnetic resonance (NMR) spectroscopy method for the conformational characterization of recombinant antibodies produced in plants. Nicotiana benthamiana used as a vehicle for the production of recombinant immunoglobulin G (IgG) was grown in a 15N-enriched plant growth medium. The Fc fragment derived from the 15N-labeled antibody thus prepared was subjected to heteronuclear two-dimensional (2D) NMR measurements. This approach enabled assessment of the structural integrity of the plant-derived therapeutic antibodies by comparing their NMR spectral properties with those of an authentic IgG-Fc derived from mammalian cells.

Keywords

Fc Immunoglobulin G Isotope labeling NMR spectroscopy Therapeutic antibody Transgenic tobacco 

Notes

Acknowledgments

This work was supported, in part, by the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO), and by Grants-in-Aid for Scientific Research (24249002, 25102008, and 25860053) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). This work was partly supported by the Nanotechnology Platform Program (Molecule and Material Synthesis) of MEXT. We gratefully acknowledge Drs. Minoru Tada and Akiko Ishii-Watabe (National Institute of Health Sciences) for providing ADM expression vector for mammalian cells. We also thank Ms. Kiyomi Senda and Ms. Kumiko Hattori (Nagoya City University) for their help in purification of IgG.

Supplementary material

299_2015_1757_MOESM1_ESM.pptx (855 kb)
Supplementary material 1 Supplemental Fig. 1: Detection of glycopeptides in the chromatogram of a tryptic digest of nonlabeled and 15N-labeled IgG expressed in transgenic tobacco.Supplemental Fig. 2: Typical MS spectra of the observed peptides (HC1:VSYLSTASSLDYWGQGTLVTVSSASTK) derived from non-labeled (upper) and 15N-labeled (lower) IgGs produced in transgenic tobacco.Supplemental Fig. 3: Detection of purified Fc fragments derived from transgenic tobacco and CHO cells by Coomassie Brilliant Blue staining of SDS-PAGE gels under non-reducing condition.Supplemental Fig. 4: Superposition of 1H–15N HSQC spectra of the uniformly 15N-labeled IgG-Fc fragments derived from transgenic tobacco (black) and CHO cells (red). The HSQC spectrum of CHO-produced IgG-Fc was adapted from the literature (Yagi et al. 2014). (PPTX 854 kb)

References

  1. Bakker H, Bardor M, Molthoff JW, Gomord V, Elbers I, Stevens LH, Jordi W, Lommen A, Faye L, Lerouge P, Bosch D (2001) Galactose-extended glycans of antibodies produced by transgenic plants. Proc Natl Acad Sci U S A 98(5):2899–2904. doi: 10.1073/pnas.031419998 CrossRefPubMedCentralPubMedGoogle Scholar
  2. Burton DR, Woof JM (1992) Human antibody effector function. Adv Immunol 51:1–84CrossRefPubMedGoogle Scholar
  3. Cabanes-Macheteau M, Fitchette-Laine AC, Loutelier-Bourhis C, Lange C, Vine ND, Ma JK, Lerouge P, Faye L (1999) N-Glycosylation of a mouse IgG expressed in transgenic tobacco plants. Glycobiology 9(4):365–372CrossRefPubMedGoogle Scholar
  4. Cegelski L, Schaefer J (2006) NMR determination of photorespiration in intact leaves using in vivo 13CO2 labeling. J Magn Reson 178(1):1–10. doi: 10.1016/j.jmr.2005.10.010 CrossRefPubMedGoogle Scholar
  5. Chen Q, Santi L, Zhang C (2014) Plant-made biologics. Biomed Res Int 2014:418064. doi: 10.1155/2014/418064 PubMedCentralPubMedGoogle Scholar
  6. Cox KM, Sterling JD, Regan JT, Gasdaska JR, Frantz KK, Peele CG, Black A, Passmore D, Moldovan-Loomis C, Srinivasan M, Cuison S, Cardarelli PM, Dickey LF (2006) Glycan optimization of a human monoclonal antibody in the aquatic plant Lemna minor. Nat Biotechnol 24(12):1591–1597. doi: 10.1038/nbt1260 CrossRefPubMedGoogle Scholar
  7. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMR Pipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6(3):277–293CrossRefPubMedGoogle Scholar
  8. DeLano WL (2002) The PyMOL Molecular Graphics System. DeLano Scientific, San CarlosGoogle Scholar
  9. Dübel S (2007) Recombinant therapeutic antibodies. Appl Microbiol Biotechnol 74(4):723–729. doi: 10.1007/s00253-006-0810-y CrossRefPubMedGoogle Scholar
  10. Goddard T, Kneller D (1993) SPARKY 3. University of California, San FranciscoGoogle Scholar
  11. He J, Lai H, Engle M, Gorlatov S, Gruber C, Steinkellner H, Diamond MS, Chen Q (2014) Generation and analysis of novel plant-derived antibody-based therapeutic molecules against West Nile virus. PLoS One 9(3):e93541. doi: 10.1371/journal.pone.0093541 CrossRefPubMedCentralPubMedGoogle Scholar
  12. Horsch RB, Klee HJ, Stachel S, Winans SC, Nester EW, Rogers SG, Fraley RT (1986) Analysis of Agrobacterium tumefaciens virulence mutants in leaf discs. Proc Natl Acad Sci U S A 83(8):2571–2575CrossRefPubMedCentralPubMedGoogle Scholar
  13. Houde D, Peng Y, Berkowitz SA, Engen JR (2010) Post-translational modifications differentially affect IgG1 conformation and receptor binding. Mol Cell Proteomics 9(8):1716–1728. doi: 10.1074/mcp.M900540-MCP200 CrossRefPubMedCentralPubMedGoogle Scholar
  14. Idusogie EE, Presta LG, Gazzano-Santoro H, Totpal K, Wong PY, Ultsch M, Meng YG, Mulkerrin MG (2000) Mapping of the C1q binding site on rituxan, a chimeric antibody with a human IgG1 Fc. J Immunol 164(8):4178–4184 (ji_v164n8p4178)CrossRefPubMedGoogle Scholar
  15. Ippel JH, Pouvreau L, Kroef T, Gruppen H, Versteeg G, van den Putten P, Struik PC, van Mierlo CP (2004) In vivo uniform 15N-isotope labelling of plants: using the greenhouse for structural proteomics. Proteomics 4(1):226–234. doi: 10.1002/pmic.200300506 CrossRefPubMedGoogle Scholar
  16. Jefferis R, Lund J, Pound JD (1998) IgG-Fc-mediated effector functions: molecular definition of interaction sites for effector ligands and the role of glycosylation. Immunol Rev 163:59–76CrossRefPubMedGoogle Scholar
  17. Kaulfurst-Soboll H, Rips S, Koiwa H, Kajiura H, Fujiyama K, von Schaewen A (2011) Reduced immunogenicity of Arabidopsis hgl1 mutant N-glycans caused by altered accessibility of xylose and core fucose epitopes. J Biol Chem 286(26):22955–22964. doi: 10.1074/jbc.M110.196097 CrossRefPubMedCentralPubMedGoogle Scholar
  18. Loos A, Steinkellner H (2012) IgG-Fc glycoengineering in non-mammalian expression hosts. Arch Biochem Biophys 526(2):167–173. doi: 10.1016/j.abb.2012.05.011 CrossRefPubMedGoogle Scholar
  19. Matsumiya S, Yamaguchi Y, Saito J, Nagano M, Sasakawa H, Otaki S, Satoh M, Shitara K, Kato K (2007) Structural comparison of fucosylated and nonfucosylated Fc fragments of human immunoglobulin G1. J Mol Biol 368(3):767–779. doi: 10.1016/j.jmb.2007.02.034 CrossRefPubMedGoogle Scholar
  20. Matsuo K, Kagaya U, Itchoda N, Tabayashi N, Matsumura T (2014) Deletion of plant-specific sugar residues in plant N-glycans by repression of GDP-d-mannose 4,6-dehydratase and b-1,2-xylosyltransferase genes. J Biosci Bioeng. doi: 10.1016/j.jbiosc.2014.04.005 PubMedGoogle Scholar
  21. Mizushima T, Yagi H, Takemoto E, Shibata-Koyama M, Isoda Y, Iida S, Masuda K, Satoh M, Kato K (2011) Structural basis for improved efficacy of therapeutic antibodies upon defucosylation of their Fc glycans. Genes Cells 16(11):1071–1080. doi: 10.1111/j.1365-2443.2011.01552.x CrossRefPubMedCentralPubMedGoogle Scholar
  22. Mylne JS, Craik DJ (2008) 15N cyclotides by whole plant labeling. Biopolymers 90(4):575–580. doi: 10.1002/bip.21012 CrossRefPubMedGoogle Scholar
  23. Rau R (2002) Adalimumab (a fully human anti-tumour necrosis factor alpha monoclonal antibody) in the treatment of active rheumatoid arthritis: the initial results of five trials. Ann Rheum Dis. doi: 10.1136/ard.61.suppl_2.ii70 PubMedCentralPubMedGoogle Scholar
  24. Schahs M, Strasser R, Stadlmann J, Kunert R, Rademacher T, Steinkellner H (2007) Production of a monoclonal antibody in plants with a humanized N-glycosylation pattern. Plant Biotechnol J 5(5):657–663. doi: 10.1111/j.1467-7652.2007.00273.x CrossRefPubMedGoogle Scholar
  25. Strasser R, Altmann F, Mach L, Glossl J, Steinkellner H (2004) Generation of Arabidopsis thaliana plants with complex N-glycans lacking b1,2-linked xylose and core a1,3-linked fucose. FEBS Lett 561(1–3):132–136. doi: 10.1016/S0014-5793(04)00150-4 CrossRefPubMedGoogle Scholar
  26. Takahashi N, Kato K (2003) GALAXY(glycoanalysis by the three axes of MS and chromatography):a web application that assists structural analyses of N-glycans. Trends Glycosci Glycotech 15(84):235–251CrossRefGoogle Scholar
  27. Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas M, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59(4):687–696CrossRefPubMedGoogle Scholar
  28. Yagi H, Yamamoto M, Yu SY, Takahashi N, Khoo KH, Lee YC, Kato K (2010) N-Glycosylation profiling of turtle egg yolk: expression of galabiose structure. Carbohydr Res 345(3):442–448. doi: 10.1016/j.carres.2009.12.002 CrossRefPubMedGoogle Scholar
  29. Yagi H, Saito T, Yanagisawa M, Yu RK, Kato K (2012) Lewis X-carrying N-glycans regulate the proliferation of mouse embryonic neural stem cells via the Notch signaling pathway. J Biol Chem 287(29):24356–24364. doi: 10.1074/jbc.M112.365643 CrossRefPubMedCentralPubMedGoogle Scholar
  30. Yagi H, Zhang Y, Yagi-Utsumi M, Yamaguchi T, Iida S, Yamaguchi Y, Kato K (2014) Backbone 1H, 13C, and 15N resonance assignments of the Fc fragment of human immunoglobulin G glycoprotein. Biomol NMR assign. doi: 10.1007/s12104-014-9586-7 (in press)Google Scholar
  31. Yamaguchi Y, Kim H, Kato K, Masuda K, Shimada I, Arata Y (1995) Proteolytic fragmentation with high specificity of mouse immunoglobulin G. Mapping of proteolytic cleavage sites in the hinge region. J Immunol Methods 181(2):259–267CrossRefPubMedGoogle Scholar
  32. Yamaguchi Y, Nishimura M, Nagano M, Yagi H, Sasakawa H, Uchida K, Shitara K, Kato K (2006) Glycoform-dependent conformational alteration of the Fc region of human immunoglobulin G1 as revealed by NMR spectroscopy. Biochim Biophys Acta 1760(4):693–700. doi: 10.1016/j.bbagen.2005.10.002 CrossRefPubMedGoogle Scholar
  33. Yamaguchi Y, Takahashi N, Kato K (2007) Molecular interactions: Antibody structures. In: Kamerling JP (ed) Comprehensive glycoscience, vol 3. Elsevier, Oxford, pp 745–763CrossRefGoogle Scholar
  34. Zeitlin L, Pettitt J, Scully C, Bohorova N, Kim D, Pauly M, Hiatt A, Ngo L, Steinkellner H, Whaley KJ, Olinger GG (2011) Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant. Proc Natl Acad Sci U S A 108(51):20690–20694. doi: 10.1073/pnas.1108360108 CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Hirokazu Yagi
    • 1
  • Noriho Fukuzawa
    • 2
  • Yasushi Tasaka
    • 2
  • Kouki Matsuo
    • 2
  • Ying Zhang
    • 1
    • 3
  • Takumi Yamaguchi
    • 1
    • 3
  • Sachiko Kondo
    • 1
    • 4
  • Shiori Nakazawa
    • 5
    • 7
  • Noritaka Hashii
    • 5
  • Nana Kawasaki
    • 5
  • Takeshi Matsumura
    • 2
  • Koichi Kato
    • 1
    • 3
    • 4
    • 6
  1. 1.Faculty and Graduate School of Pharmaceutical SciencesNagoya City UniversityNagoyaJapan
  2. 2.Bioproduction Research InstituteNational Institute of Advanced Industrial Science and Technology (AIST)SapporoJapan
  3. 3.Institute for Molecular Science and Okazaki Institute for Integrative BioscienceNational Institutes of Natural SciencesOkazakiJapan
  4. 4.Medical & Biological Laboratories Co., Ltd.NagoyaJapan
  5. 5.Division of Biological Chemistry and BiologicalsNational Institute of Health SciencesTokyoJapan
  6. 6.The Glycoscience InstituteOchanomizu UniversityTokyoJapan
  7. 7.Sugashima Marine Biological Laboratory, Graduate School of ScienceNagoya UniversityMieJapan

Personalised recommendations