In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants
Abstract
:1. Introduction
2. Results
2.1. Kifunensine Treatment of N. benthamiana did not Impede Rituximab Expression
2.2. A Concentration of 0.375 μM of Kifunensine is Sufficient to Produce Rituximab with Afucosylated, Oligomannose-Type Glycoforms Exclusively
2.3. Rituximab Decorated with Oligomannose Residues Binds Target CD20 as Efficiently as Rituxan®
2.4. Effect of Kifunensine on Biological Activity of Rituximab Produced in N. benthamiana
3. Discussion
3.1. Kifunensine Treatment Is Well Tolerated During Expression of Rituximab in N. benthamiana
3.2. Kifunensine Has a Strong Effect on the Plant N-glycosylation Machinery, even at Low Concentration
3.3. Kifunensine Represents a Cost-Effective Method to Produced Biobetter Anti-Cancer Antibodies
4. Materials and Methods
4.1. Construction of Heavy Chain and Light Chain Expression Vectors
4.2. Expression and Extraction
4.3. Purification and SDS-PAGE Analysis
4.4. Intact Protein Analysis
4.5. Glycopeptide Profiling
4.6. Cell Lines and Cell Culture Conditions
4.7. CD20 Binding Assay
4.8. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) Assay
5. Conclusions
Author Contributions
Acknowledgments
Conflicts of Interest
Abbreviations
mAb | Monoclonal antibody |
ADCC | antibody-dependent cell-mediated cytotoxicity |
Man8 | mannose-8 glycan |
Man9 | mannose-9 glycan |
EC50 | half maximal effective concentration |
PGC | polypropylene glycol |
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Kommineni, V.; Markert, M.; Ren, Z.; Palle, S.; Carrillo, B.; Deng, J.; Tejeda, A.; Nandi, S.; McDonald, K.A.; Marcel, S.; et al. In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants. Int. J. Mol. Sci. 2019, 20, 194. https://doi.org/10.3390/ijms20010194
Kommineni V, Markert M, Ren Z, Palle S, Carrillo B, Deng J, Tejeda A, Nandi S, McDonald KA, Marcel S, et al. In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants. International Journal of Molecular Sciences. 2019; 20(1):194. https://doi.org/10.3390/ijms20010194
Chicago/Turabian StyleKommineni, Vally, Matthew Markert, Zhongjie Ren, Sreenath Palle, Berenice Carrillo, Jasmine Deng, Armando Tejeda, Somen Nandi, Karen A. McDonald, Sylvain Marcel, and et al. 2019. "In Vivo Glycan Engineering via the Mannosidase I Inhibitor (Kifunensine) Improves Efficacy of Rituximab Manufactured in Nicotiana benthamiana Plants" International Journal of Molecular Sciences 20, no. 1: 194. https://doi.org/10.3390/ijms20010194