Granulocyte colony-stimulating factor (GCSF) stimulates the
proliferation of neutrophils but it has low serum half-life. Therefore,
the present study was done to investigate the effect of XTENylation on
biological activity, pharmacokinetics, and pharmacodynamics of GCSF in a
neutropenic rat model. XTEN tag was genetically fused to the N-terminal
region of GCSF-encoding gene fragment and subcloned into pET28a
expression vector. The cytoplasmic expressed recombinant protein was
characterized through intrinsic fluorescence spectroscopy (IFS), dynamic
light scattering (DLS), and size exclusion chromatography (SEC). In
vitro biological activity of the XTEN-GCSF protein was evaluated on
NFS60 cell line. Hematopoietic properties and pharmacokinetics were also
investigated in a neutropenic rat model. An approximately 140 kDa
recombinant protein was detected on SDS-PAGE. Dynamic light scattering
and size exclusion chromatography confirmed the increase in hydrodynamic
diameter of GCSF molecule after XTENylation. GCSF derivatives showed
efficacy in proliferation of NFS60 cell line among which the XTEN-GCSF
represented the lowest EC50 value (100.6 pg/ml).
Pharmacokinetic studies on neutropenic rats revealed that XTEN polymer
could significantly increase protein serum half-life in comparison with
the commercially available GCSF molecules. PEGylated and XTENylated GCSF
proteins were more effective in stimulation of neutrophils compared to
the GCSF molecule alone. XTENylation of GCSF represented promising
results in in vitro and in vivo studies. This approach can be a
potential alternative to PEGylation strategies for increasing serum
half-life of protein.