Production of afucosylated antibodies in CHO cells by coexpression of an anti-FUT8 intrabody.
ADCC
IgG
antibody
cell culture
flow cytometry
fucosylation
glycoengineering
glycosylation
intrabody
Journal
Biotechnology and bioengineering
ISSN: 1097-0290
Titre abrégé: Biotechnol Bioeng
Pays: United States
ID NLM: 7502021
Informations de publication
Date de publication:
08 2022
08 2022
Historique:
revised:
17
04
2022
received:
27
01
2022
accepted:
21
04
2022
pubmed:
6
5
2022
medline:
14
7
2022
entrez:
5
5
2022
Statut:
ppublish
Résumé
Some effector functions prompted by immunoglobulin G (IgG) antibodies, such as antibody-dependent cell-mediated cytotoxicity (ADCC), strongly depend on the N-glycans linked to asparagine 297 of the Fc region of the protein. A single α-(1,6)-fucosyltransferase (FUT8) is responsible for catalyzing the addition of an α-1,6-linked fucose residue to the first GlcNAc residue of the N-linked glycans. Antibodies missing this core fucose show a significantly enhanced ADCC and increased antitumor activity, which could help reduce therapeutic dose requirement, potentially translating into reduced safety concerns and manufacturing costs. Several approaches have been developed to modify glycans and improve the biological functions of antibodies. Here, we demonstrate that expression of a membrane-associated anti-FUT8 intrabody engineered to reside in the endoplasmic reticulum and Golgi apparatus can efficiently reduce FUT8 activity and therefore the core-fucosylation of the Fc N-glycan of an antibody. IgG1-producing CHO cells expressing the intrabody secrete antibodies with reduced core fucosylation as demonstrated by lectin blot analysis and UPLC-HILIC glycan analysis. Cells engineered to inhibit directly and specifically alpha-(1,6)-fucosyltransferase activity allows for the production of g/L levels of IgGs with strongly enhanced ADCC effector function, for which the level of fucosylation can be selected. The quick and efficient method described here should have broad practical applicability for the development of next-generation therapeutic antibodies with enhanced effector functions.
Substances chimiques
Antibodies, Monoclonal
0
Immunoglobulin G
0
Polysaccharides
0
Fucose
28RYY2IV3F
Fucosyltransferases
EC 2.4.1.-
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
2206-2220Informations de copyright
© 2022 National Research Council Canada. Biotechnology and Bioengineering published by Wiley Periodicals LLC. Reproduced with the permission of the Minister of Human Health Therapeutics Research Center.
Références
Agostinetto, R., Rossi, M., Dawson, J., Lim, A., Simoneau, M. H., Boucher, C., Valldorf, B., Ross-Gillespie, A., Jardine, J. G., Sok, D., Burton, D. R., Hassell, T., Broly, H., Palinsky, W., Dupraz, P., Feinberg, M., & Dey, A. K. (2022). Rapid cGMP manufacturing of COVID-19 monoclonal antibody using stable CHO cell pools. Biotechnology and Bioengineering, 119(2), 663-666. https://doi.org/10.1002/bit.27995
Allen, J. G., Mujacic, M., Frohn, M. J., Pickrell, A. J., Kodama, P., Bagal, D., San Miguel, T., Sickmier, E. A., Osgood, S., Swietlow, A., Li, V., Jordan, J. B., Kim, K. W., Rousseau, A. C., Kim, Y. J., Caille, S., Achmatowicz, M., Thiel, O., Fotsch, C. H., … McCarter, J. D. (2016). Facile modulation of antibody fucosylation with small molecule fucostatin inhibitors and cocrystal structure with GDP-mannose 4,6-dehydratase. ACS Chemical Biology, 11(10), 2734-2743. https://doi.org/10.1021/acschembio.6b00460
Bigge, J. C., Patel, T. P., Bruce, J. A., Goulding, P. N., Charles, S. M., & Parekh, R. B. (1995). Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid. Analytical Biochemistry, 230(2), 229-238. https://doi.org/10.1006/abio.1995.1468
Cambay, F., Henry, O., Durocher, Y., & De Crescenzo, G. (2019). Impact of N-glycosylation on Fcγ receptor/IgG interactions: Unravelling differences with an enhanced surface plasmon resonance biosensor assay based on coiled-coil interactions. mAbs, 11(3), 435-452. https://doi.org/10.1080/19420862.2019.1581017
Chang, M. M., Gaidukov, L., Jung, G., Tseng, W. A., Scarcelli, J. J., Cornell, R., Marshall, J. K., Lyles, J. L., Sakorafas, P., Chu, A. A., Cote, K., Tzvetkova, B., Dolatshahi, S., Sumit, M., Mulukutla, B. C., Lauffenburger, D. A., Figueroa, B., Jr., Summers, N. M., Lu, T. K., & Weiss, R. (2019). Small-molecule control of antibody N-glycosylation in engineered mammalian cells. Nature Chemical Biology, 15(7), 730-736. https://doi.org/10.1038/s41589-019-0288-4
Cobb, B. A. (2020). The history of IgG glycosylation and where we are now. Glycobiology, 30(4), 202-213. https://doi.org/10.1093/glycob/cwz065
Daboussi, F., Zaslavskiy, M., Poirot, L., Loperfido, M., Gouble, A., Guyot, V., Leduc, S., Galetto, R., Grizot, S., Oficjalska, D., Perez, C., Delacôte, F., Dupuy, A., Chion-Sotinel, I., Le Clerre, D., Lebuhotel, C., Danos, O., Lemaire, F., Oussedik, K., … Pâques, F. (2012). Chromosomal context and epigenetic mechanisms control the efficacy of genome editing by rare-cutting designer endonucleases. Nucleic Acids Research, 40(13), 6367-6379. https://doi.org/10.1093/nar/gks268
Derer, S., Glorius, P., Schlaeth, M., Lohse, S., Klausz, K., Muchhal, U., Desjarlais, J. R., Humpe, A., Valerius, T., & Peipp, M. (2014). Increasing FcγRIIa affinity of an FcγRIII-optimized anti-EGFR antibody restores neutrophil-mediated cytotoxicity. mAbs, 6(2), 409-421. https://doi.org/10.4161/mabs.27457
Dorion-Thibaudeau, J., Raymond, C., Lattova, E., Perreault, H., Durocher, Y., & De Crescenzo, G. (2014). Towards the development of a surface plasmon resonance assay to evaluate the glycosylation pattern of monoclonal antibodies using the extracellular domains of CD16a and CD64. Journal of Immunological Methods, 408, 24-34. https://doi.org/10.1016/j.jim.2014.04.010
Durocher, Y., Perret, S., & Kamen, A. (2002). High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Research, 30(2), E9. https://doi.org/10.1093/nar/30.2.e9
Ferrara, C., Brünker, P., Suter, T., Moser, S., Püntener, U., & Umaña, P. (2006). Modulation of therapeutic antibody effector functions by glycosylation engineering: Influence of Golgi enzyme localization domain and co-expression of heterologous β1, 4-N-acetylglucosaminyltransferase III and Golgi α-mannosidase II. Biotechnology and Bioengineering, 93(5), 851-861. https://doi.org/10.1002/bit.20777
Garber, K. (2018). No added sugar: Antibody makers find an upside to ‘no fucose’. Nature Biotechnology, 36(11), 1025-1027. https://doi.org/10.1038/nbt1118-1025
Grav, L. M., Lee, J. S., Gerling, S., Kallehauge, T. B., Hansen, A. H., Kol, S., Lee, G. M., Pedersen, L. E., & Kildegaard, H. F. (2015). One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment. Biotechnology Journal, 10(9), 1446-1456. https://doi.org/10.1002/biot.201500027
Hossler, P., Chumsae, C., Racicot, C., Ouellette, D., Ibraghimov, A., Serna, D., Mora, A., McDermott, S., Labkovsky, B., Scesney, S., Grinnell, C., Preston, G., Bose, S., & Carrillo, R. (2017). Arabinosylation of recombinant human immunoglobulin-based protein therapeutics. mAbs, 9(4), 715-734. https://doi.org/10.1080/19420862.2017.1294295
Imai-Nishiya, H., Mori, K., Inoue, M., Wakitani, M., Iida, S., Shitara, K., & Satoh, M. (2007). Double knockdown of α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in antibody-producing cells: A new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC. BMC Biotechnology, 7(1), 84. https://doi.org/10.1186/1472-6750-7-84
Jefferis, R. (2009). Glycosylation as a strategy to improve antibody-based therapeutics. Nature Reviews Drug Discovery, 8(3), 226-234. https://doi.org/10.1038/nrd2804
Jennewein, M. F., & Alter, G. (2017). The immunoregulatory roles of antibody glycosylation. Trends in Immunology, 38(5), 358-372. https://doi.org/10.1016/j.it.2017.02.004
Kanda, Y., Imai-Nishiya, H., Kuni-Kamochi, R., Mori, K., Inoue, M., Kitajima-Miyama, K., Okazaki, A., Iida, S., Shitara, K., & Satoh, M. (2007). Establishment of a GDP-mannose 4,6-dehydratase (GMD) knockout host cell line: A new strategy for generating completely non-fucosylated recombinant therapeutics. Journal of Biotechnology, 130(3), 300-310. https://doi.org/10.1016/j.jbiotec.2007.04.025
Kellner, C., Otte, A., Cappuzzello, E., Klausz, K., & Peipp, M. (2017). Modulating cytotoxic effector functions by Fc engineering to improve cancer therapy. Transfusion Medicine and Hemotherapy, 44(5), 327-336. https://doi.org/10.1159/000479980
Kelly, R. M., Kowle, R. L., Lian, Z., Strifler, B. A., Witcher, D. R., Parekh, B. S., Wang, T., & Frye, C. C. (2018). Modulation of IgG1 immunoeffector function by glycoengineering of the GDP-fucose biosynthesis pathway. Biotechnology and Bioengineering, 115(3), 705-718. https://doi.org/10.1002/bit.26496
Kubota, T., Niwa, R., Satoh, M., Akinaga, S., Shitara, K., & Hanai, N. (2009). Engineered therapeutic antibodies with improved effector functions. Cancer Science, 100(9), 1566-1572. https://doi.org/10.1111/j.1349-7006.2009.01222.x
Liu, W., Padmashali, R., Monzon, O. Q., Lundberg, D., Jin, S., Dwyer, B., Lee, Y. J., Korde, A., Park, S., Pan, C., & Zhang, B. (2020). Generation of FX −/− and Gmds −/− CHOZN host cell lines for the production of afucosylated therapeutic antibodies. Biotechnology Progress, 37(1), 3061. https://doi.org/10.1002/btpr.3061
Malphettes, L., Freyvert, Y., Chang, J., Liu, P. -Q., Chan, E., Miller, J. C., Zhou, Z., Nguyen, T., Tsai, C., Snowden, A. W., Collingwood, T. N., Gregory, P. D., & Cost, G. J. (2010). Highly efficient deletion of FUT8 in CHO cell lines using zinc-finger nucleases yields cells that produce completely nonfucosylated antibodies. Biotechnology and Bioengineering, 106(5), 774-783. https://doi.org/10.1002/bit.22751
Mellor, J. D., Brown, M. P., Irving, H. R., Zalcberg, J. R., & Dobrovic, A. (2013). A critical review of the role of Fc gamma receptor polymorphisms in the response to monoclonal antibodies in cancer. Journal of Hematology & Oncology, 6, 1. https://doi.org/10.1186/1756-8722-6-1
Mori, K., Kuni-Kamochi, R., Yamane-Ohnuki, N., Wakitani, M., Yamano, K., Imai, H., Kanda, Y., Niwa, R., Iida, S., Uchida, K., Shitara, K., & Satoh, M. (2004). Engineering Chinese hamster ovary cells to maximize effector function of produced antibodies using FUT8 siRNA. Biotechnology and Bioengineering, 88(7), 901-908. https://doi.org/10.1002/bit.20326
Munro, T. P., Le, K., Le, H., Zhang, L., Stevens, J., Soice, N., Benchaar, S. A., Hong, R. W., & Goudar, C. T. (2017). Accelerating patient access to novel biologics using stable pool-derived product for non-clinical studies and single clone-derived product for clinical studies. Biotechnology Progress, 33(6), 1476-1482. https://doi.org/10.1002/btpr.2572
Okeley, N. M., Alley, S. C., Anderson, M. E., Boursalian, T. E., Burke, P. J., Emmerton, K. M., Jeffrey, S. C., Klussman, K., Law, C. L., Sussman, D., Toki, B. E., Westendorf, L., Zeng, W., Zhang, X., Benjamin, D. R., & Senter, P. D. (2013). Development of orally active inhibitors of protein and cellular fucosylation. Proceedings of the National Academy of Sciences of the United States of America, 110(14), 5404-5409. https://doi.org/10.1073/pnas.1222263110
Peipp, M., Lammerts van Bueren, J. J., Schneider-Merck, T., Bleeker, W. W., Dechant, M., Beyer, T., Repp, R., van Berkel, P. H., Vink, T., van de Winkel, J. G., Parren, P. W., & Valerius, T. (2008). Antibody fucosylation differentially impacts cytotoxicity mediated by NK and PMN effector cells. Blood, 112(6), 2390-2399. https://doi.org/10.1182/blood-2008-03-144600
Pereira, N. A., Chan, K. F., Lin, P. C., & Song, Z. (2018). The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity. mAbs, 10(5), 693-711. https://doi.org/10.1080/19420862.2018.1466767
Popp, O., Moser, S., Zielonka, J., Rüger, P., Hansen, S., & Plöttner, O. (2017). Development of a pre-glycoengineered CHO-K1 host cell line for the expression of antibodies with enhanced Fc mediated effector function. mAbs, 10(2), 290-303. https://doi.org/10.1080/19420862.2017.1405203
Poulain, A., Perret, S., Malenfant, F., Mullick, A., Massie, B., & Durocher, Y. (2017). Rapid protein production from stable CHO cell pools using plasmid vector and the cumate gene-switch. Journal of Biotechnology, 255, 16-27. https://doi.org/10.1016/j.jbiotec.2017.06.009
Rajendra, Y., Balasubramanian, S., McCracken, N. A., Norris, D. L., Lian, Z., Schmitt, M. G., Frye, C. C., & Barnard, G. C. (2017). Evaluation of piggyBac-mediated CHO pools to enable material generation to support GLP toxicology studies. Biotechnology Progress, 33(6), 1436-1448. https://doi.org/10.1002/btpr.2495
Ronda, C., Pedersen, L. E., Hansen, H. G., Kallehauge, T. B., Betenbaugh, M. J., Nielsen, A. T., & Kildegaard, H. F. (2014). Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool. Biotechnology and Bioengineering, 111(8), 1604-1616. https://doi.org/10.1002/bit.25233
Roy, G., Martin, T., Barnes, A., Wang, J., Jimenez, R. B., Rice, M., Li, L., Feng, H., Zhang, S., Chaerkady, R., Wu, H., Marelli, M., Hatton, D., Zhu, J., & Bowen, M. A. (2018). A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies. mAbs, 10(3), 416-430. https://doi.org/10.1080/19420862.2018.1433975
Russell, A., Adua, E., Ugrina, I., Laws, S., & Wang, W. (2018). Unravelling immunoglobulin G Fc N-Glycosylation: A dynamic marker potentiating predictive, preventive and personalised medicine. International Journal of Molecular Sciences, 19(2). https://doi.org/10.3390/ijms19020390
Shi, C., Shin, Y. O., Hanson, J., Cass, B., Loewen, M. C., & Durocher, Y. (2005). Purification and characterization of a recombinant G-protein-coupled receptor, Saccharomyces cerevisiae Ste2p, transiently expressed in HEK293 EBNA1 cells. Biochemistry, 44(48), 15705-15714. https://doi.org/10.1021/bi051292p
Shields, R. L., Lai, J., Keck, R., O'Connell, L. Y., Hong, K., Meng, Y. G., Weikert, S. H., & Presta, L. G. (2002). Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcγRIII and antibody-dependent cellular toxicity. Journal of Biological Chemistry, 277(30), 26733-26740. https://doi.org/10.1074/jbc.M202069200
Stuible, M., Burlacu, A., Perret, S., Brochu, D., Paul-Roc, B., Baardsnes, J., Loignon, M., Grazzini, E., & Durocher, Y. (2018). Optimization of a high-cell-density polyethylenimine transfection method for rapid protein production in CHO-EBNA1 cells. Journal of Biotechnology, 281, 39-47. https://doi.org/10.1016/j.jbiotec.2018.06.307
Stuible, M., van Lier, F., Croughan, M. S., & Durocher, Y. (2018). Beyond preclinical research: Production of CHO-derived biotherapeutics for toxicology and early-phase trials by transient gene expression or stable pools. Current Opinion in Chemical Engineering, 22, 145-151. https://doi.org/10.1016/j.coche.2018.09.010
Sun, Y., Gadoury, C., Hirakawa, M. P., Bennett, R. J., Harcus, D., Marcil, A., & Whiteway, M. (2016). Deletion of a Yci1 domain protein of Candida albicans allows homothallic mating in MTL heterozygous cells. mBio, 7(2), 00465-16. https://doi.org/10.1128/mBio.00465-16
Takahashi, M., Kuroki, Y., Ohtsubo, K., & Taniguchi, N. (2009). Core fucose and bisecting GlcNAc, the direct modifiers of the N-glycan core: Their functions and target proteins. Carbohydrate Research, 344(12), 1387-1390. https://doi.org/10.1016/j.carres.2009.04.031
Treffers, L. W., van Houdt, M., Bruggeman, C. W., Heineke, M. H., Zhao, X. W., van der Heijden, J., Nagelkerke, S. Q., Verkuijlen, P., Geissler, J., Lissenberg-Thunnissen, S., Valerius, T., Peipp, M., Franke, K., van Bruggen, R., Kuijpers, T. W., van Egmond, M., Vidarsson, G., Matlung, H. L., & van den Berg, T. K. (2018). FcγRIIIb restricts antibody-dependent destruction of cancer cells by human neutrophils. Frontiers in Immunology, 9, 3124. https://doi.org/10.3389/fimmu.2018.03124
Umana, P., Jean-Mairet, J., Moudry, R., Amstutz, H., & Bailey, J. E. (1999). Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nature Biotechnology, 17(2), 176-180. https://doi.org/10.1038/6179
von Horsten, H. H., Ogorek, C., Blanchard, V., Demmler, C., Giese, C., Winkler, K., Kaup, M., Berger, M., Jordan, I., & Sandig, V. (2010). Production of non-fucosylated antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase. Glycobiology, 20(12), 1607-1618. https://doi.org/10.1093/glycob/cwq109
Wang, Q., Chung, C.-Y., Chough, S., & Betenbaugh, M. J. (2018). Antibody glycoengineering strategies in mammalian cells. Biotechnology and Bioengineering, 115(6), 1378-1393. https://doi.org/10.1002/bit.26567
Yamane-Ohnuki, N., Kinoshita, S., Inoue-Urakubo, M., Kusunoki, M., Iida, S., Nakano, R., Wakitani, M., Niwa, R., Sakurada, M., Uchida, K., Shitara, K., & Satoh, M. (2004). Establishment ofFUT8 knockout Chinese hamster ovary cells: An ideal host cell line for producing completely defucosylated antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnology and Bioengineering, 87(5), 614-622. https://doi.org/10.1002/bit.20151
Zimmermann, M., Ehret, J., Kolmar, H., & Zimmer, A. (2019). Impact of acetylated and non-acetylated fucose analogues on IgG glycosylation. Antibodies, 8(1). https://doi.org/10.3390/antib8010009