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A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension Properties.

Unverdorben F, Hutt M, Seifert O, Kontermann RE - PLoS ONE (2015)

Bottom Line: In mice, this affinity-improved mutant (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb).The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity.The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.

ABSTRACT

Background: Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD) from streptococcal protein G (SpGC3) as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions.

Methodology/principal findings: Using site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab) to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpGC3FabRR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics.

Conclusions/significance: The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.

No MeSH data available.


Related in: MedlinePlus

Pharmacokinetics of the fusion proteins in CD1 mice.a) scDb fusion proteins. b) scFv fusion proteins. 25 μg of scDb-SpGC3 (a) or scFv-SpGC3 (b) variants was injected intravenously into female CD1 mice. Serum concentrations were determined by ELISA against CEA and data were normalized considering the maximal concentration at the first time point (3 min).
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pone.0139838.g005: Pharmacokinetics of the fusion proteins in CD1 mice.a) scDb fusion proteins. b) scFv fusion proteins. 25 μg of scDb-SpGC3 (a) or scFv-SpGC3 (b) variants was injected intravenously into female CD1 mice. Serum concentrations were determined by ELISA against CEA and data were normalized considering the maximal concentration at the first time point (3 min).

Mentions: The pharmacokinetic properties of scDb-SpGC3 and scDb-SpGC3Fab were investigated in female CD1 mice after intravenous injection of the fusion proteins. The serum concentration was determined by ELISA to detect CEA-binding molecules. The unmodified scDb showed rapid clearance from the bloodstream with a terminal half-life of 1.3 h (Table 1, Fig 5). Compared to the unmodified scDb, the scDb-SpGC3Fab fusion protein exhibited a strongly augmented terminal half-life (15.1 h), which was also reflected by a strongly increased AUC (Table 1). However, scDb-SpGC3Fab did not reach the half-life (24.3 h) of scDb-SpGC3.


A Fab-Selective Immunoglobulin-Binding Domain from Streptococcal Protein G with Improved Half-Life Extension Properties.

Unverdorben F, Hutt M, Seifert O, Kontermann RE - PLoS ONE (2015)

Pharmacokinetics of the fusion proteins in CD1 mice.a) scDb fusion proteins. b) scFv fusion proteins. 25 μg of scDb-SpGC3 (a) or scFv-SpGC3 (b) variants was injected intravenously into female CD1 mice. Serum concentrations were determined by ELISA against CEA and data were normalized considering the maximal concentration at the first time point (3 min).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4592230&req=5

pone.0139838.g005: Pharmacokinetics of the fusion proteins in CD1 mice.a) scDb fusion proteins. b) scFv fusion proteins. 25 μg of scDb-SpGC3 (a) or scFv-SpGC3 (b) variants was injected intravenously into female CD1 mice. Serum concentrations were determined by ELISA against CEA and data were normalized considering the maximal concentration at the first time point (3 min).
Mentions: The pharmacokinetic properties of scDb-SpGC3 and scDb-SpGC3Fab were investigated in female CD1 mice after intravenous injection of the fusion proteins. The serum concentration was determined by ELISA to detect CEA-binding molecules. The unmodified scDb showed rapid clearance from the bloodstream with a terminal half-life of 1.3 h (Table 1, Fig 5). Compared to the unmodified scDb, the scDb-SpGC3Fab fusion protein exhibited a strongly augmented terminal half-life (15.1 h), which was also reflected by a strongly increased AUC (Table 1). However, scDb-SpGC3Fab did not reach the half-life (24.3 h) of scDb-SpGC3.

Bottom Line: In mice, this affinity-improved mutant (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb).The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity.The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cell Biology and Immunology, University of Stuttgart, Stuttgart, Germany.

ABSTRACT

Background: Half-life extension strategies have gained increasing interest to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. Recently, we established an immunoglobulin-binding domain (IgBD) from streptococcal protein G (SpGC3) as module for half-life extension. SpGC3 is capable of binding to the Fc region as well as the CH1 domain of Fab arms under neutral and acidic conditions.

Methodology/principal findings: Using site-directed mutagenesis, we generated a Fab-selective mutant (SpGC3Fab) to avoid possible interference with the FcRn-mediated recycling process and improved its affinity for mouse and human IgG by site-directed mutagenesis and phage display selections. In mice, this affinity-improved mutant (SpGC3FabRR) conferred prolonged plasma half-lives compared with SpGC3Fab when fused to small recombinant antibody fragments, such as single-chain Fv (scFv) and bispecific single-chain diabody (scDb). Hence, the SpGC3FabRR domain seems to be a suitable fusion partner for the half-life extension of small recombinant therapeutics.

Conclusions/significance: The half-life extension properties of SpGC3 can be retained by restricting binding to the Fab fragment of serum immunoglobulins and can be improved by increasing binding activity. The modified SpGC3 module should be suitable to extend the half-life of therapeutic proteins and, thus to improve therapeutic activity.

No MeSH data available.


Related in: MedlinePlus