Producing defucosylated antibodies with enhanced in vitro antibody‐dependent cellular cytotoxicity via <i>FUT8</i> knockout CHO‐S cells

Zong, Huifang; Han, Lei; Ding, Kai; Wang, Jiaxian; Sun, Tao; Zhang, Xinyu; Cagliero, Cédric; Jiang, Hua; Xie, Yueqing; Xu, Jianrong; Zhang, Baohong; Zhu, Jianwei

doi:10.1002/elsc.201600255

Cited by 21 publications

(7 citation statements)

References 35 publications

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“…Repurposing the CRISPR/Cas9 immune system as a fast, efficient, and cheap genome editing tool combined with the sequenced CHO genome as a common biopharmaceutical production host creates new opportunities in the area of targeted genome editing . Several targeted cell line engineering approaches of production cell lines already focused on either KO of disadvantageous acting genes or targeted integration of a gene of interest into the host genome . In this study, we aimed at mimicking a multiplexed genome editing strategy by editing a single miRNA rather than multiple target genes since single miRNAs orchestrate several hundred target mRNAs simultaneously .…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9‐Mediated Knockout of MicroRNA‐744 Improves Antibody Titer of CHO Production Cell Lines

Raab

Mathias

Alt

et al. 2019

Biotechnology Journal

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MicroRNAs (miRNAs) are noncoding RNAs that serve as versatile molecular engineering tools to improve production cells by overexpression or knockdown of miRNAs showing beneficial or adverse effects on cell-culture performance. The genomic knockout (KO) of noncoding RNAs in Chinese hamster ovary (CHO) production cells has not been reported. However, given the significant number of miRNAs showing negative effects on CHO-bioprocess performance and the development of clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas9), genome editing tools facilitate precise optimization of CHO cells via modulation of noncoding RNAs. In a previous high-content miRNA screen, miR-744 was identified as a potential target associated with reduced productivity. Hence, the genomic miR-744 precursor sequence is deleted by two single guide RNA (sgRNA)-Cas9-mediated DNA double-strand breaks (DSB) flanking the miR-744 locus. After fluorescence-activated cell sorting (FACS), clonal miR-744 KO cell lines are recovered and three of them are confirmed as miR-744 KOs. Impacts of CRISPR/Cas9 editing are characterized at the genetic, transcript, and phenotypic levels. During batch cultivation, antibody titers of miR-744 KOs are significantly increased to 190-311 mg L −1 compared to a nontargeting (NT) sgRNA transfected clonal control with 156 mg L −1 , pointing towards the potential of miRNA KO for cell line engineering.

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Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9‐Mediated Knockout of MicroRNA‐744 Improves Antibody Titer of CHO Production Cell Lines

Raab

Mathias

Alt

et al. 2019

Biotechnology Journal

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“…von Horsten et al produced non-fucosylated antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD) in 2010 ( 45 ). In 2017, Zong et al produced defucosylated antibodies with enhanced in vitro ADCC via FUT8 knockout CHO-S cells ( 46 ). In addition, Yuan et al reported a stable cell line expressing defucosylated anti-human epidermal growth factor receptor 2 (anti-HER2) antibody based on an established FUT8 gene knockout CHO-S cell line.…”

Section: Methodsmentioning

confidence: 99%

Combinatory glycoengineering of monoclonal antibodies and its application in cancer therapy: a narrative review

Wang,

Tian,

Tian

et al. 2024

Transl Cancer Res

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Background and Objective The application of immunotherapy, especially in terms of recombinant monoclonal antibodies (mAbs), is a revolution in the pharmaceutical industry field. Glycosylation plays an essential role in the structures and functions of mAbs, which must be carefully monitored and designed throughout their entire lifespan to ensure safety and efficacy. This review aimed to summarize the current status of the glycoengineering of mAbs for providing reference for the pharmaceutical industry. The application of glycoengineering of mAbs in cancer therapy will also be discussed in this article. Methods We searched the PubMed/MEDLINE database to identify studies published between 1970 and 2023 that investigated glycoengineering of recombinant mAbs and its applications. The major findings of these studies were summarized. Key Content and Findings This article reviews the relationship between glycosylation profiles and antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), shelf-life, and other properties of mAbs. Furthermore, the rational design of glycosylation profiles provides solutions to the unmet needs of new drug development and biosimilar manufacturers. This review also emphatically describes various feasible strategies to optimize the glycosylation pattern in biomanufacturing reported in recent years, especially the approaches of combinatory glycoengineering explored in this field. Finally, we share examples of glycoengineering of mAbs applied in cancer therapy. Conclusions Glycan modifications have been achieved through genetic and metabolic glycoengineering. A better understanding of the interplay between cells and the exogenous environment help the combinatorial strategy for glycoengineering in precise control mode. Furthermore, new high-tech approaches for glycoengineering are leading biological engineering and biotherapeutics to a new stage.

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“…13,14 So far, majorities of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8 −/− CHO host cells, which usually have retarded cell growth, less viable cell densities and lower production titers. 15,16 The addition of Fut8 chemical inhibitor 2F-Peracetyl-Fucose into the cell culture medium to generate afucosylated mAbs is another approach. However, the batch-to-batch consistency in both productivity and afucosylation levels in large manufacturing bioreactors need further evaluation.…”

Section: Introductionmentioning

confidence: 99%

“…In the Golgi glycans are fucosylated by fucosyltransferase 8 (Fut8), the only expressed fucose‐transferase in CHO cells 13,14 . So far, majorities of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8 −/− CHO host cells, which usually have retarded cell growth, less viable cell densities and lower production titers 15,16 . The addition of Fut8 chemical inhibitor 2F‐Peracetyl‐Fucose into the cell culture medium to generate afucosylated mAbs is another approach.…”

Section: Introductionmentioning

confidence: 99%

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Generation of FX^−/− and Gmds^−/−CHOZN host cell lines for the production of afucosylated therapeutic antibodies

Liu

Padmashali

Monzon

et al. 2020

Biotechnology Progress

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Antibody-dependent cellular cytotoxicity (ADCC) is the primary mechanism of actions for several marketed therapeutic antibodies (mAbs) and for many more in clinical trials. The ADCC efficacy is highly dependent on the ability of therapeutic mAbs to recruit effector cells such as natural killer cells, which induce the apoptosis of targeted cells. The recruitment of effector cells by mAbs is negatively affected by fucose modification of N-Glycans on the Fc; thus, utilization of afucosylated mAbs has been a trend for enhanced ADCC therapeutics. Most of afucosylated mAbs in clinical or commercial manufacturing were produced from Fut8 −/− Chinese hamster ovary cells (CHO) host cells, generally generating low yields compared to wildtype CHO host. This study details the generation and characterization of two engineered CHOZN® cell lines, in which the enzyme involved in guanosine diphosphate (GDP)fucose synthesis, GDP mannose-4,6-dehydratase (Gmds) and GDP-L-fucose synthase (FX), was knocked out. The top host cell lines for each of the knockouts, FX−/− and Gmds−/−, were selected based on growth robustness, bulk MSX selection tolerance, production titer, fucosylation level, and cell stability. We tested the production of

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Producing defucosylated antibodies with enhanced in vitro antibody‐dependent cellular cytotoxicity via FUT8 knockout CHO‐S cells

Cited by 21 publications

References 35 publications

CRISPR/Cas9‐Mediated Knockout of MicroRNA‐744 Improves Antibody Titer of CHO Production Cell Lines

CRISPR/Cas9‐Mediated Knockout of MicroRNA‐744 Improves Antibody Titer of CHO Production Cell Lines

Combinatory glycoengineering of monoclonal antibodies and its application in cancer therapy: a narrative review

Generation of FX^−/− and Gmds^−/−CHOZN host cell lines for the production of afucosylated therapeutic antibodies

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Producing defucosylated antibodies with enhanced in vitro antibody‐dependent cellular cytotoxicity via FUT8 knockout CHO‐S cells

Cited by 21 publications

References 35 publications

CRISPR/Cas9‐Mediated Knockout of MicroRNA‐744 Improves Antibody Titer of CHO Production Cell Lines

CRISPR/Cas9‐Mediated Knockout of MicroRNA‐744 Improves Antibody Titer of CHO Production Cell Lines

Combinatory glycoengineering of monoclonal antibodies and its application in cancer therapy: a narrative review

Generation of FX−/− and Gmds−/−CHOZN host cell lines for the production of afucosylated therapeutic antibodies

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Generation of FX^−/− and Gmds^−/−CHOZN host cell lines for the production of afucosylated therapeutic antibodies