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Comparison of CD20 Binding Affinities of Rituximab Produced in Nicotiana benthamiana Leaves and Arabidopsis thaliana Callus

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Abstract

Plants are promising drug-production platforms with high economic efficiency, stability, and convenience in mass production. However, studies comparing the equivalency between the original antibodies and those produced in plants are limited. Amino acid sequences that constitute the Fab region of an antibody are diverse, and the post-transcriptional modifications that occur according to these sequences in animals and plants are also highly variable. In this study, rituximab, a blockbuster antibody drug used in the treatment of non-Hodgkin’s lymphoma, was produced in Nicotiana benthamiana leaves and Arabidopsis thaliana callus, and was compared to the original rituximab produced in CHO cells. Interestingly, the epitope recognition and antigen-binding abilities of rituximab from N. benthamiana leaves were almost lost. In the case of rituximab produced in A. thaliana callus, the specific binding ability and CD20 capping activity were maintained, but the binding affinity was less than 50% of that of original rituximab from CHO cells. These results suggest that different plant species exhibit different binding affinities. Accordingly, in addition to the differences in PTMs between mammals and plants, the differences between the species must also be considered in the process of producing antibodies in plants.

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References

  1. Hiatt, A., Cafferkey, R., & Bowdish, K. (1989). Production of antibodies in transgenic plants. Nature, 342, 76–78.

    Article  CAS  Google Scholar 

  2. Ullrich, K. K., Hiss, M., & Rensing, S. A. (2015). Means to optimize protein expression in transgenic plants. Current Opinion in Biotechnology, 32, 61–67.

    Article  CAS  Google Scholar 

  3. Twyman, R. M., Stoger, E., Schillberg, S., Christou, P., & Fischer, R. (2003). Molecular farming in plants: Host systems and expression technology. Trends in Biotechnology, 21, 570–578.

    Article  CAS  Google Scholar 

  4. Lomonossoff, G. P., & D’Aoust, M. A. (2016). Plant-produced biopharmaceuticals: A case of technical developments driving clinical deployment. Science, 353, 1237–1240.

    Article  CAS  Google Scholar 

  5. Shaaltiel, Y., Bartfeld, D., Hashmueli, S., Baum, G., Brill-Almon, E., Galili, G., Dym, O., Boldin-Adamsky, S. A., Silman, I., Sussman, J. L., Futerman, A. H., & Aviezer, D. (2007). Production of glucocerebrosidase with terminal mannose glycans for enzyme replacement therapy of Gaucher’s disease using a plant cell system. Plant Biotechnology Journal, 5, 579–590.

    Article  CAS  Google Scholar 

  6. Fischer, R., & Buyel, J. F. (2020). Molecular farming - The slope of enlightenment. Biotechnology Advances, 40, 107519.

    Article  CAS  Google Scholar 

  7. Gomord, V., & Faye, L. (2004). Posttranslational modification of therapeutic proteins in plants. Current Opinion in Plant Biology, 7, 171–181.

    Article  CAS  Google Scholar 

  8. Murin, C. D., Fusco, M. L., Bornholdt, Z. A., Qiu, X., Olinger, G. G., Zeitlin, L., Kobinger, G. P., Ward, A. B., & Saphire, E. O. (2014). Structures of protective antibodies reveal sites of vulnerability on Ebola virus. Proceedings of the National Academy of Science U S A, 111, 17182–17187.

    Article  CAS  Google Scholar 

  9. Zeitlin, L., Pettitt, J., Scully, C., Bohorova, N., Kim, D., Pauly, M., Hiatt, A., Ngo, L., Steinkellner, H., Whaley, K. J., & Olinger, G. G. (2011). Enhanced potency of a fucose-free monoclonal antibody being developed as an Ebola virus immunoprotectant. Proceedings of the National Academy of Science U S A, 108, 20690–20694.

    Article  CAS  Google Scholar 

  10. Herter, S., Herting, F., Mundigl, O., Waldhauer, I., Weinzierl, T., Fauti, T., Muth, G., Ziegler-Landesberger, D., Van Puijenbroek, E., Lang, S., Duong, M. N., Reslan, L., Gerdes, C. A., Friess, T., Baer, U., Burtscher, H., Weidner, M., Dumontet, C., Umana, P., … Klein, C. (2013). Preclinical activity of the type II CD20 antibody GA101 (obinutuzumab) compared with rituximab and ofatumumab in vitro and in xenograft models. Molecular Cancer Therapeutics, 12, 2031–2042.

    Article  CAS  Google Scholar 

  11. Strasser, R., Altmann, F., & Steinkellner, H. (2014). Controlled glycosylation of plant-produced recombinant proteins. Current Opinion in Biotechnology, 30, 95–100.

    Article  CAS  Google Scholar 

  12. Strasser, R., Stadlmann, J., Schahs, M., Stiegler, G., Quendler, H., Mach, L., Glossl, J., Weterings, K., Pabst, M., & Steinkellner, H. (2008). Generation of glyco-engineered Nicotiana benthamiana for the production of monoclonal antibodies with a homogeneous human-like N-glycan structure. Plant Biotechnology Journal, 6, 392–402.

    Article  CAS  Google Scholar 

  13. Reff, M. E., Carner, K., Chambers, K. S., Chinn, P. C., Leonard, J. E., Raab, R., Newman, R. A., Hanna, N., & Anderson, D. R. (1994). Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood, 83, 435–445.

    Article  CAS  Google Scholar 

  14. Freeman, C. L., & Sehn, L. H. (2018). A tale of two antibodies: Obinutuzumab versus rituximab. British Journal of Haematology, 182, 29–45.

    Article  Google Scholar 

  15. Salles, G., Barrett, M., Foà, R., Maurer, J., O’Brien, S., Valente, N., Wenger, M., & Maloney, D. G. (2017). Rituximab in B-cell hematologic malignancies: A review of 20 years of clinical experience. Advances in Therapy, 34, 2232–2273.

    Article  CAS  Google Scholar 

  16. Smith, M. R. (2003). Rituximab (monoclonal anti-CD20 antibody): Mechanisms of action and resistance. Oncogene, 22, 7359–7368.

    Article  CAS  Google Scholar 

  17. Glennie, M. J., French, R. R., Cragg, M. S., & Taylor, R. P. (2007). Mechanisms of killing by anti-CD20 monoclonal antibodies. Molecular Immunology, 44, 3823–3837.

    Article  CAS  Google Scholar 

  18. Rogers, L. M., Veeramani, S., & Weiner, G. J. (2014). Complement in monoclonal antibody therapy of cancer. Immunologic Research, 59, 203–210.

    Article  CAS  Google Scholar 

  19. Bardor, M., Faveeuw, C., Fitchette, A. C., Gilbert, D., Galas, L., Trottein, F., Faye, L., & Lerouge, P. (2003). Immunoreactivity in mammals of two typical plant glyco-epitopes, core alpha (1,3)-fucose and core xylose. Glycobiology, 13, 427–434.

    Article  CAS  Google Scholar 

  20. Shaaltiel, Y., & Tekoah, Y. (2016). Plant specific N-glycans do not have proven adverse effects in humans. Nature Biotechnology, 34, 706–708.

    Article  CAS  Google Scholar 

  21. Ma, J. K., Drossard, J., Lewis, D., Altmann, F., Boyle, J., Christou, P., Cole, T., Dale, P., van Dolleweerd, C. J., Isitt, V., Katinger, D., Lobedan, M., Mertens, H., Paul, M. J., Rademacher, T., Sack, M., Hundleby, P. A., Stiegler, G., Stoger, E., … Fischer, R. (2015). Regulatory approval and a first-in-human phase I clinical trial of a monoclonal antibody produced in transgenic tobacco plants. Plant Biotechnology Journal, 13, 1106–1120.

    Article  CAS  Google Scholar 

  22. Spoel, S. H. (2018). Orchestrating the proteome with post-translational modifications. Journal of Experimental Botany, 69, 4499–4503.

    Article  CAS  Google Scholar 

  23. Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M., & Dwek, R. A. (2007). The impact of glycosylation on the biological function and structure of human immunoglobulins. Annual Review of Immunology, 25, 21–50.

    Article  CAS  Google Scholar 

  24. Rudd, P. M., Elliott, T., Cresswell, P., Wilson, I. A., & Dwek, R. A. (2001). Glycosylation and the immune system. Science, 291, 2370–2376.

    Article  CAS  Google Scholar 

  25. Castilho, A., Windwarder, M., Gattinger, P., Mach, L., Strasser, R., Altmann, F., & Steinkellner, H. (2014). Proteolytic and N-glycan processing of human α1-antitrypsin expressed in Nicotiana benthamiana. Plant Physiology, 166, 1839–1851.

    Article  Google Scholar 

  26. Walsh, G., & Jefferis, R. (2006). Post-translational modifications in the context of therapeutic proteins. Nature Biotechnology, 24, 1241–1252.

    Article  CAS  Google Scholar 

  27. Strasser, R. (2012). Challenges in O-glycan engineering of plants. Frontiers in Plant Science, 3, 218.

    Article  Google Scholar 

  28. Lee, J. W., Heo, W., Lee, J., Jin, N., Yoon, S. M., Park, K. Y., Kim, E. Y., Kim, W. T., & Kim, J. Y. (2018). The B cell death function of obinutuzumab-HDEL produced in plant (Nicotiana benthamiana L.) is equivalent to obinutuzumab produced in CHO cells. PLoS ONE, 13, e0191075.

    Article  Google Scholar 

  29. Harrison, S. J., Mott, E. K., Parsley, K., Aspinall, S., Gray, J. C., & Cottage, A. (2006). A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation. Plant Methods, 2, 19.

    Article  Google Scholar 

  30. Doelling, J. H., & Pikaard, C. S. (1993). Transient expression in Arabidopsis thaliana protoplasts derived from rapidly established cell suspension cultures. Plant Cell Reports, 12, 241–244.

    Article  CAS  Google Scholar 

  31. Lauber, M. A., Yu, Y.-Q., Brousmiche, D. W., Hua, Z., Koza, S. M., Magnelli, P., Guthrie, E., Taron, C. H., & Fountain, K. J. (2015). Rapid preparation of released N-glycans for HILIC analysis using a labeling reagent that facilitates sensitive fluorescence and ESI-MS detection. Analytical Chemistry, 87, 5401–5409.

    Article  CAS  Google Scholar 

  32. Kommineni, V., Markert, M., Ren, Z., Palle, S., Carrillo, B., Deng, J., Tejeda, A., Nandi, S., McDonald, K. A., Marcel, S., & Holtz, B. (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(1), 194.

    Article  Google Scholar 

  33. Triguero, A., Cabrera, G., Cremata, J. A., Yuen, C. T., Wheeler, J., & Ramirez, N. I. (2005). Plant-derived mouse IgG monoclonal antibody fused to KDEL endoplasmic reticulum-retention signal is N-glycosylated homogeneously throughout the plant with mostly high-mannose-type N-glycans. Plant Biotechnology Journal, 3, 449–457.

    Article  CAS  Google Scholar 

  34. Conrad, U., & Fiedler, U. (1998). Compartment-specific accumulation of recombinant immunoglobulins in plant cells: An essential tool for antibody production and immunomodulation of physiological functions and pathogen activity. Plant Molecular Biology, 38, 101–109.

    Article  CAS  Google Scholar 

  35. Abraham, R., Moller, D., Gabel, D., Senter, P., Hellström, I., & Hellström, K. E. (1991). The influence of periodate oxidation on monoclonal antibody avidity and immunoreactivity. Journal of Immunological Methods, 144, 77–86.

    Article  CAS  Google Scholar 

  36. Bennett, L. D., Yang, Q., Berquist, B. R., Giddens, J. P., Ren, Z., Kommineni, V., Murray, R. P., White, E. L., Holtz, B. R., Wang, L. X., & Marcel, S. (2018). Implementation of glycan remodeling to plant-made therapeutic antibodies. International Journal of Molecular Sciences, 19(2), 421.

    Article  Google Scholar 

  37. Zhao, F., Yu, C. H., & Liu, Y. (2017). Codon usage regulates protein structure and function by affecting translation elongation speed in Drosophila cells. Nucleic Acids Research, 45, 8484–8492.

    Article  CAS  Google Scholar 

  38. Yu, M., Brown, D., Reed, C., Chung, S., Lutman, J., Stefanich, E., Wong, A., Stephan, J. P., & Bayer, R. (2012). Production, characterization, and pharmacokinetic properties of antibodies with N-linked mannose-5 glycans. MAbs, 4, 475–487.

    Article  Google Scholar 

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Acknowledgements

This work was supported by Grants from the National Research Foundation of Korea, Project Nos. NFR-2019R1A2C1086348 to J.Y.K.

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Authors and Affiliations

  1. Department of Pharmacology and Brain Korea 21 Project for Medical Science, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul, 03080, Republic of Korea

    Cho Eun Kang, Woon Heo, Sun Hyung Kwon, JeongRyeol Kim & Joo Young Kim

  2. Department of Systems Biology, College of Life Science and Biotechnology, Yonsei University, Seoul, 03080, Republic of Korea

    Seungeun Lee, Dong Hye Seo & Woo Taek Kim

  3. College of Pharmacy, Yonsei Institute of Pharmaceutical Sciences, Yonsei University, Incheon, 21983, Republic of Korea

    Jinu Lee

  4. Vegetable and Fruit Development Center, Department of Horticultural Sciences, Texas A&M University, College Station, TX77843-2133, USA

    Hisashi Koiwa

  5. Mass Analysis Team, New Drug Development Center, Cheongju, Chungbuk, 28160, Republic of Korea

    Byoung Joon Ko

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  1. Cho Eun Kang
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Kang, C.E., Lee, S., Seo, D.H. et al. Comparison of CD20 Binding Affinities of Rituximab Produced in Nicotiana benthamiana Leaves and Arabidopsis thaliana Callus. Mol Biotechnol 63, 1016–1029 (2021). https://doi.org/10.1007/s12033-021-00360-5

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