NMR-based structural validation of therapeutic antibody produced in Nicotiana benthamiana
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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.
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.
KeywordsFc Immunoglobulin G Isotope labeling NMR spectroscopy Therapeutic antibody Transgenic tobacco
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.
- 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
- 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
- DeLano WL (2002) The PyMOL Molecular Graphics System. DeLano Scientific, San CarlosGoogle Scholar
- Goddard T, Kneller D (1993) SPARKY 3. University of California, San FranciscoGoogle Scholar
- 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
- 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
- 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
- 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