Recombinant granulocyte colony-stimulating factor-transferrin fusion protein as an oral myelopoietic agent

Abstract

An expression construct harboring granulocyte colony-stimulating factor (G-CSF)-transferrin (Tf) fusion protein (G-CSF-Tf) was engineered by fusing human cDNAs encoding G-CSF and Tf to explore the feasibility of using Tf as a carrier moiety for oral delivery of therapeutic proteins. The recombinant protein, G-CSF-Tf, was harvested from protein-free, conditioned medium of transfected HEK293 cells. The in vitro studies demonstrated that the purified G-CSF-Tf fusion protein possesses the activity of both Tf receptor (TfR) binding in Caco-2 cells and G-CSF-dependent stimulation of NFS-60 cell proliferation. Subcutaneous administration of G-CSF-Tf fusion protein to BDF1 mice demonstrated a pharmacological effect comparable to the commercial G-CSF on the increase of absolute neutrophil counts (ANC). However, the fusion protein elicited a significant increase in ANC upon oral administration to BDF1 mice, whereas G-CSF had no effect. This study also showed that orally administered G-CSF-Tf elicits a sustained myelopoietic effect up to 3 days, whereas the s.c. administered G-CSF or G-CSF-Tf lasts only 1 day. Furthermore, coadministration of free Tf abolished the increase of ANC by orally delivered G-CSF-Tf, suggesting that the recombinant protein is absorbed via a TfR-mediated process in the gastrointestinal tract. Taken together, we conclude that the Tf-based recombinant fusion protein technology represents a promising approach for future development of orally effective peptide and protein drugs. myelopoiesis ͉ oral delivery ͉ protein drug

Improving the Oral Efficacy of Recombinant Granulocyte Colony-Stimulating Factor and Transferrin Fusion Protein by Spacer Optimization

“…The method for plasmid construction was similar to that as described in the previous report (20). Human G-CSF cDNA harboring the signal peptide was cloned from human bladder carcinoma 5637 (American Type Culture Collection) by using RT-PCR.…”

“…Furthermore, we have reported oral hypoglycemic activity of an insulin-Tf conjugate in diabetic rats (17,18), and oral myelopoietic activity of a G-CSF and Tf conjugate in BDF-1 mice (19). Most recently, we have produced a fusion protein from recombinant cDNA of G-CSF (20 kD) and Tf (80 kD) that possessed both transferrin receptor (TfR) binding ability in Caco-2 cells and the proliferative activity in NFS-60 cells (20). Furthermore, we have demonstrated that the G-CSF-Tf fusion protein can be absorbed across gastrointestinal epithelia, as indicated by its myelopoietic activity when orally administered in BDF-1 mice (20).…”

“…Most recently, we have produced a fusion protein from recombinant cDNA of G-CSF (20 kD) and Tf (80 kD) that possessed both transferrin receptor (TfR) binding ability in Caco-2 cells and the proliferative activity in NFS-60 cells (20). Furthermore, we have demonstrated that the G-CSF-Tf fusion protein can be absorbed across gastrointestinal epithelia, as indicated by its myelopoietic activity when orally administered in BDF-1 mice (20). However, the fusion protein only retained a small fraction of the in vitro activity of both cell proliferation of G-CSF and TfR-binding of Tf.…”

Preparation of monoPEGylated Cyanovirin-N’s derivative and its anti-influenza A virus bioactivity in vitro and in vivo

“…Meanwhile, the linker peptide facilitated both the independent folding of the SUMO and CVN subunits in the cytoplasm of E. coli and in the downstream purification process of LCVN (17). Direct fusion of functional subunits without a linker may lead to many undesirable outcomes including misfolding of the fusion proteins (18), low yield in protein production (19) or impaired bioactivity (20,21). Usually, PEG chemical conjugation step does not have good production efficiency.…”

Studies on lactoferrin nanoparticles of gambogic acid for oral delivery

“…Furthermore, it has been observed that Lf is relatively resistant to proteolysis in the gastrointestinal tract (Brock et al, 1976;Brines & Brock, 1983). Tf, which has similar properties with Lf, has been investigated for oral delivery of granulocyte colony stimulating factor (Bai et al, 2005), insulin (Shofner et al, 2010) and human growth hormone (Amet et al, 2010). However, Lf has not yet been reported as oral delivery vehicle.…”