Impacts of fast production of afucosylated antibodies and Fc mutants in ExpiCHO-S™ for enhancing FcγRIIIa binding and NK cell activation
This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only pa...
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| Published in: | Journal of biotechnology Vol. 360; pp. 79 - 91 |
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| Main Authors: | , , , , , , , , , , , , , , , , , , , , , , , , , |
| Format: | Journal Article |
| Language: | English |
| Published: |
Netherlands
Elsevier B.V
10-12-2022
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| Subjects: | |
| Online Access: | Get full text |
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| Summary: | This study has employed mammalian transient expression systems to generate afucosylated antibodies and antibody Fc mutants for rapid candidate screening in discovery and early development. While chemical treatment with the fucose analogue 2-fluoro-peracetyl-fucose during transient expression only partially produced antibodies with afucosylated N-glycans, the genetic inactivation of the FUT8 gene in ExpiCHO-S™ by CRISPR/Cas9 enabled the transient production of fully afucosylated antibodies. Human IgG1 and murine IgG2a generated by the ExpiCHOfut8KO cell line possessed a 8-to-11-fold enhanced FcγRIIIa binding activity in comparison with those produced by ExpiCHO-S™. The Fc mutant S239D/S298A/I332E produced by ExpiCHO-S™ had an approximate 2-fold higher FcγRIIIa affinity than that of the afucosylated wildtype molecule, although it displayed significantly lower thermal-stability. When the Fc mutant was produced in the ExpiCHOfut8KO cell line, the resulting afucosylated Fc mutant antibody had an additional approximate 6-fold increase in FcγRIIIa binding affinity. This synergistic effect between afucosylation and the Fc mutations was further verified by a natural killer (NK) cell activation assay. Together, these results have not only established an efficient large-scale transient CHO system for rapid production of afucosylated antibodies, but also confirmed a cooperative impact between afucosylation and Fc mutations on FcγRIIIa binding and NK cell activation.
●Our manuscript describes the use of the industrially relevant transient expression host ExpiCHO-S™ for expression of antibody molecules having enhanced ADCC activity.●We first demonstrate that treatment of cell culture with the chemical additive 2 F-paracetyl-fucose is inefficient in a transient setting for production of molecules having afucosylated N-glycans.●We then describe the creation of an ExpiCHO-S™ cell host lacking activity of the FUT8 gene, resulting in the ability to produce molecules having completely afucosylated N-glycans.●Using both this host as well as the wild-type ExpiCHO-S™ cell line, we produce a series of molecules with either (a) afucosylated N-glycans, (b) Fc mutations aimed at enhancing ADCC activity, or (c) molecules having both afucosylated N-glycans and Fc mutations and compare the ability of these molecules to bind to FcγRIIIa as well as the ability to activate NK cells.●Finally, we demonstrate that the Fc mutation approach does lead to thermal instability of the molecule, relative to both the wild-type antibody and the wild type antibody with afucosylated N-glycans. |
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| Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
| ISSN: | 0168-1656 1873-4863 |
| DOI: | 10.1016/j.jbiotec.2022.10.016 |