Production of chimeric monoclonal antibodies by genetically manipulated chickens

Kamihira, Masamichi; Kawabe, Yoshinori; Shindo, Takuya; Ono, Kosuke; Esaka, Kazuhisa; Yamashita, Takashi; Nishijima, Ken ichi; Iijima, Shinji

doi:10.1016/j.jbiotec.2009.02.022

Cited by 39 publications

(46 citation statements)

References 45 publications

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“…We previously reported that the molecular mass of serum-derived TNFR/Fc was higher than that of yolk, and that this difference disappeared after the removal of N-and O-glycans (Kyogoku et al 2008). In this study, we showed that the extent of sialylation of scFv/Fc obtained from the serum of genetically manipulated chickens (Kamihira et al 2009) was different from that of the yolk (Fig. 1).…”

Section: Discussionmentioning

confidence: 46%

“…1). Yolk-derived scFv/ Fc did not contain the disialyl forms, which were detected in serum-derived scFv/Fc (Kamihira et al 2009). These results indicate that scFv/Fc recovered from the yolk was partly sialylated.…”

Section: Scfv/fc Produced In the Yolk Is Partly Sialylatedmentioning

confidence: 92%

“…We previously analyzed the glycan structure of serumderived scFv/Fc and found that more than 10 % of glycan had sialic acid, including more than 7 % of the disialyl form (Kamihira et al 2009). However, the sialylation of scFv/Fc in the yolk was reduced as shown in Fig.…”

Section: Scfv/fc Produced In the Yolk Is Partly Sialylatedmentioning

confidence: 99%

“…This is in contrast to glycans in mammalian species other than humans: N-glycolylneuraminic acid alone or both Nglycolyl-and N-acetylneuraminic acids have been added as the terminal residue of N-glycans. However, exogenously expressed antibodies and erythropoietin in the egg white of genetically manipulated chickens were shown to mainly possess the truncated forms of N-glycans without galactose and sialic acid (Kamihira et al 2009;Kodama et al 2008;Zhu et al 2005), which was partly improved by the genetic introduction of the galactosyltransferase gene (Mizutani et al 2012).…”

Section: Introductionmentioning

confidence: 99%

“…Due to this transport system, several recombinant proteins with the human Fc domain accumulated in the yolk of genetically manipulated chickens (Kamihira et al 2005(Kamihira et al , 2009Kyogoku et al 2008;Penno et al 2010): the single-chained Fv fragment fused to Fc (scFv/Fc), the extracellular domain of the TNF receptor fused to Fc (TNFR/Fc), and erythropoietin fused to Fc (EPO/Fc), as well as humanized antibody, were deposited in the yolk when they were expressed under the control of a ubiquitous promoter. Structural analysis of N-glycan on scFv/Fc purified from the serum revealed the considerable proportions of sialylated glycans (Kamihira et al 2009), which implies the potential to obtain sialylated Fc-fused proteins from the egg yolk of genetically manipulated chickens. In this study, we examined the sialylation of yolk-derived Fc-fused proteins.…”

Section: Introductionmentioning

confidence: 99%

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Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan

et al. 2013

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The transgenic chicken is a candidate for the production of biopharmaceutical proteins with several economic superiorities. In general, the addition of sialic acid at the terminal of N-glycan is important for the bioactivity of biopharmaceuticals including plasma half-life; however, sialic acid has not been detected in the N-glycan of proteins produced in the egg white of genetically manipulated chickens. In this study, the extracellular domain of the TNF receptor and single chain Fv fused to Fc (referred to as TNFR/Fc and scFv/Fc, respectively) were purified from the egg yolk of genetically manipulated chickens and their sialylation in N-glycan was examined. In contrast to the glycan in egg white, yolk-derived proteins were partly sialylated. Lectin blot showed the existence of a2,6-sialic acid on TNFR/Fc, which disappeared with the removal of N-glycan by PNGase. In scFv/Fc, up to 7 % of N-glycan contained sialic acid. Disialyl glycans, which were detected in serumderived scFv/Fc in a previous study, were not found in the yolk sample. Ovarian follicular tissue, which surrounds growing yolk, expressed several neuraminidases, suggesting the partial truncation of glycan during the yolk transfer process from the blood.

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Section: Discussionmentioning

confidence: 46%

Section: Scfv/fc Produced In the Yolk Is Partly Sialylatedmentioning

confidence: 92%

Section: Scfv/fc Produced In the Yolk Is Partly Sialylatedmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan

et al. 2013

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Inducible transgene expression in CHO cells using an artificial transcriptional activator with estrogen‐binding domain

Rahman,

Kawabe,

Suzuki

et al. 2023

Biotechnology Journal

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Biopharmaceuticals, including therapeutic antibodies, are rapidly growing products in the pharmaceutical market. Mammalian cells, such as Chinese hamster ovary (CHO) cells, are widely used as production hosts because recombinant antibodies require complex three‐dimensional structures modified with sugar chains. Recombinant protein production using mammalian cells is generally performed with cell growth. In this study, we developed a technology that controls cell growth and recombinant protein production to induce recombinant protein production with predetermined timing. Expression of green fluorescent protein (GFP) gene and a single‐chain antibody fused with the Fc‐region of the human IgG1 (scFv‐Fc) gene can be induced and mediated by the estrogen receptor‐based artificial transcription factor Gal4‐ERT2‐VP16 and corresponding inducer drugs. We generated CHO cells using an artificial gene expression system. The addition of various concentrations of inducer drugs to the culture medium allowed control of proliferation and transgene expression of the engineered CHO cells. Use of 4‐hydroxytamoxifen, an antagonist of estrogen, as an inducing agent yielded high gene expression at a concentration more than 10‐fold lower than that of β‐estradiol. When scFv‐Fc was produced under inducing conditions, continuous production was possible for more than 2 weeks while maintaining high specific productivity (57 pg cell−1 day−1). This artificial gene expression control system that utilizes the estrogen response of estrogen receptors can be an effective method for inducible production of biopharmaceuticals.

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Cre recombinase‐mediated site‐specific modification of a cellular genome using an integrase‐defective retroviral vector

Huang

Kawabe

Ito

et al. 2010

Biotech & Bioengineering

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Retroviral integrase is an enzyme responsible for the integration of retroviruses. A single mutation in the integrase core domain can severely compromise its integration ability, leading to the accumulation of circular retroviral cDNA in the nuclei of infected cells. We therefore attempted to use those cDNA as substrates for Cre recombinase to perform a recombinase-mediated cassette exchange (RMCE), thereby targeting retroviral vectors to a predetermined site. An expression unit containing a promoter, an ATG codon and marker genes (hygromycin resistance gene and red fluorescent protein gene) flanked by wild-type and mutant loxP sites was first introduced into cellular chromosome to build founder cell lines. We then constructed another plasmid for the production of integrase-defective retroviral vectors (IDRV), which contains an ATG-deficient neomycin resistance gene and green fluorescent protein gene, flanked by a compatible pair of loxPs. After providing founder cells with Cre and infecting with IDRV later, effective RMCE occurred, resulting in the appearance of G418-resistant colonies and a change in the color of fluorescence from red to green. Southern blot and PCR analyses on selected clones further confirmed site-specific recombination. The successful substitution of the original viral integration machinery with a non-viral mechanism could expand the application of retroviral vectors.

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Production of chimeric monoclonal antibodies by genetically manipulated chickens

Cited by 39 publications

References 45 publications

Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan

Recombinant proteins produced into yolk of genetically manipulated chickens are partly sialylated in N-glycan

Inducible transgene expression in CHO cells using an artificial transcriptional activator with estrogen‐binding domain

Cre recombinase‐mediated site‐specific modification of a cellular genome using an integrase‐defective retroviral vector

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