Recombinant Granulocyte Colony-Stimulating Factor-Transferrin Fusion Protein as an Oral Myelopoietic Agent

• Published in: Proceedings of the National Academy of Sciences - PNAS 2005-05, Vol.102 (20), p.7292-7296
• Main Authors: Bai, Yun, Ann, David K., Shen, Wei-Chiang, Langer, Robert
• Format: Article
• Language: English
• Subjects: Administration, Oral; Animals; Antibodies; Biological Sciences; Blood; Blotting, Western; Caco 2 cells; Cell Line; Cell Proliferation - drug effects; DNA, Complementary - genetics; Gastrointestinal Tract - metabolism; Gene expression; Genetic engineering; Genetic recombination; Granulocyte Colony-Stimulating Factor - genetics; Granulocyte Colony-Stimulating Factor - metabolism; Granulocyte Colony-Stimulating Factor - pharmacology; HEK293 cells; Humans; Male; Medical treatment; Mice; Myelopoiesis - drug effects; Neutrophils; Oral administration; Placebos; Plasmids - administration & dosage; Plasmids - genetics; Proteins; Receptors; Recombinant fusion proteins; Recombinant Proteins; Time Factors; Transferrin - genetics; Transferrin - metabolism; Transferrin - pharmacology
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0500062102

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.