Effect of Carbohydrates on the Pharmacokinetics of Human Interferon-γ

Sareneva, Timo; Cantell, Kari; Pyhälä, Liisa; Pirhonen, Jaana; Julkunen, Ilkka

doi:10.1089/jir.1993.13.267

Cited by 52 publications

(29 citation statements)

References 4 publications

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“…Thus, an engineered N-glycosylated scFv containing high-mannose carbohydrate chains at a C-terminal extension was more rapidly cleared from circulation than the unmodified scFv (32). Similar observations were made for interferon-␥ (29). We found that the three N-glycosylated scDb variants all contained N-glycans of the complex type (bi-, tri-, and tetra-antennary).…”

Section: Discussionsupporting

confidence: 76%

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

Stork

Zettlitz

Müller

et al. 2008

Journal of Biological Chemistry

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The therapeutic efficacy of recombinant antibodies such as single-chain Fv fragments and small bispecific or bifunctional molecules is often limited by rapid elimination from the circulation because of their small size. Here, we have investigated the effects of N-glycosylation on the activity and pharmacokinetics of a small bispecific single-chain diabody (scDb CEACD3) developed for the retargeting of cytotoxic T cells to CEA-expressing tumor cells. We could show that the introduction of N-glycosylation sequons into the flanking linker and a C-terminal extension results in the production of N-glycosylated molecules after expression in transfected HEK293 cells. N-Glycosylated scDb variants possessing 3, 6, or 9 N-glycosylation sites, respectively, retained antigen binding activity and bispecificity for target and effector cells as shown in a target cell-dependent IL-2 release assay, although activity was reduced ϳ3-5-fold compared with the unmodified scDb. All N-glycosylated scDb variants exhibited a prolonged circulation time compared with scDb, leading to a 2-3-fold increase of the area under curve (AUC). In comparison, conjugation of a branched 40-kDa PEG chain increased AUC by a factor of 10.6, while a chimeric anti-CEA IgG1 molecule had the longest circulation time with a 17-fold increase in AUC. Thus, N-glycosylation complements the repertoire of strategies to modulate pharmacokinetics of small recombinant antibody molecules by an approach that moderately prolongs circulation time.Whole antibodies, especially chimeric, humanized or fully human IgG molecules, exhibit a long circulation time in the human body that can reach a half-life of 27 days (1, 2). In contrast, antibody fragments, e.g. Fab fragments, or recombinant formats, such as single-chain Fv fragments (scFv) 3 or bispecific derivatives thereof (tandem scFv, diabodies, single-chain diabodies), are rapidly cleared from circulation (2-4). This is mainly due to the small size leading to rapid renal clearance and the lack of recycling processes mediated by the neonatal Fc receptor (FcRn). Thus, repeated injections or infusions are required to maintain a therapeutically effective dose in the body (5). Several strategies to improve pharmacokinetic properties and thus dosing and therapeutic efficacy of recombinant antibodies have been developed in recent years. These strategies can be divided into: 1) those based on reducing renal clearance by increasing the apparent size of the therapeutic molecule, and 2) those that in addition implement FcRn-mediated recycling processes (6 -8).PEGylation of proteins is a well-established strategy to improve pharmacokinetic properties by increasing the molecular mass and the hydrodynamic radius (9 -11). Several PEGylated proteins are in clinical use, e.g. PEGylated interferon ␣-2a (PegIntron, PEGASYS) and PEGylated granulocyte-colony stimulating factor (Neulasta), or are under clinical development, e.g. a PEGylated anti-TNF Fab fragment (certolizumab pegol) (9, 12) Furthermore, direct fusion to albumin or an albumin bin...

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Section: Discussionsupporting

confidence: 76%

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

Stork

Zettlitz

Müller

et al. 2008

Journal of Biological Chemistry

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“…Kanda et al (2007) also demonstrated faster clearance in the b-phase of an anti-CD 20 antibody with Man5 glycans after intravenous injection of 20 mg antibody per mouse. Although a 1.5-to 3-fold lower half-life in serum was observed for oligomannose containing antibodies compared to the control antibody in these studies, the 4.6-day (Kanda et al, 2007) and 2.8-to 4-day (Wright et al, 1994(Wright et al, , 1998 half-lives of the oligomannose containing antibodies are still much longer than many non-antibody like glycoproteins with oligomannose residues, which have half-lives of a few minutes (Kogerlberg et al, 2007;Sareneva et al, 1993;Van Patten et al, 2007). The difference in half-lives between antibody and nonantibody like glycoprotein with oligomannose residues prompted us to compare their binding to mannose receptor (Jones et al, 2007;Su et al, 2005).…”

Section: Discussionmentioning

confidence: 68%

Development of a simple and rapid method for producing non‐fucosylated oligomannose containing antibodies with increased effector function

Zhou

Shankara

Roy

et al. 2007

Biotech & Bioengineering

167

170

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Glycosylation in the Fc region of antibodies has been shown to play an important role in antibody function. In the current study, glycosylation of human monoclonal antibodies was metabolically modulated using a potent alpha-mannosidase I inhibitor, kifunensine, resulting in the production of antibodies with oligomannose-type N-glycans. Growing Chinese hamster ovary cells for 11 days in batch culture with a single treatment of kifunensine was sufficient to elicit this effect without any significant impact on cell viability or antibody production. Antibodies expressed in the presence of kifunensine at a concentration as low as 60 ng/mL contained mainly oligomannose-type glycans and demonstrated increased ADCC activity and affinity for FcgammaRIIIA, but reduced C1q binding. Although the kifunensine-mediated shift to oligomannose-type glycans could, in theory, result in rapid clearance of the antibody through increased mannose receptor binding, the serum levels of antibody in mice were not significantly altered up to 168 h following injection. The use of kifunensine provides a simple and rapid method for the production of antibodies with increased ADCC without the time-consuming need to re-engineer either the antibody molecule or the host cell line.

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“…Tunicamycin treatment resulted in a 4-5-fold decrease in the level of synthesized IFN-y protein, and the amount of secreted protein was about 30% of the (Fig. 3 B), is probably due to the C-terminal protein processing found earlier by us and others both in natural and recombinant IFN-y [8,[25][26][27].…”

Section: Role Of N-glycosylation In the Synthesis And Secretion Of Ifmentioning

confidence: 58%

Biosynthesis and N‐glycosylation of Human Interferon‐γ

Sareneva

Mørtz²,

Tölö

et al. 1996

European Journal of Biochemistry

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Interferon-y (IFN-y) is a secretory glycoprotein produced by T cells in response to antigenic or mitogenic stimuli. We studied the kinetics of the synthesis, N-glycosylation, and secretion of IFN-y in human CD8' T lymphocytes stimulated via T-cell receptor. Highly elevated IFN-y mRNA levels were found as early as 1 h after stimulation. Maximal IFN-y protein synthesis was observed 2-4 h after induction and appeared to correlate to steady-state IFN-y mRNA levels. As analyzed by pulse/chase experiments, the secretion of IFN-y from T cells was very rapid, the secretion half-time being approximately 20-25 min. Inhibition of N-glycosylation by tunicamycin dramatically reduced the expression of IFN-y, but did not block its secretion. Natural IFN-y is heterogeneously glycosylated and doubly, singly, and unglycosylated forms exist. Experiments performed in a cell-free translation/glycosylation system with mutated IFN-y constructs lacking either one of the potential glycosylation sites suggested that Am25 is more efficiently glycosylated than Asn97. Site-specific oligosaccharide analysis of natural IFN-y by glycosidase treatment followed by matrix-assisted-laser-desorption-ionization mass spectrometry revealed considerable variation in the carbohydrate structures, with more than 30 different forms. The glycans at Am25 consisted of fucosylated, mainly complex-type oligosaccharides, whereas the glycans at Asn97 were more heterogeneous, with hybrid and high-mannose structures. Our results emphasize the essential role of N-linked glycans in the biology of IFN-y and show that there is a considerable heterogeneity in the individual sugar chains of this important human cytokine.

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Effect of Carbohydrates on the Pharmacokinetics of Human Interferon-γ

Cited by 52 publications

References 4 publications

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

N-Glycosylation as Novel Strategy to Improve Pharmacokinetic Properties of Bispecific Single-chain Diabodies

Development of a simple and rapid method for producing non‐fucosylated oligomannose containing antibodies with increased effector function

Biosynthesis and N‐glycosylation of Human Interferon‐γ

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