Enhanced circulating half-life and hematopoietic properties of a human granulocyte colony-stimulating factor/immunoglobulin fusion protein.
OBJECTIVE
The aim of this study was to determine whether fusion proteins comprising human granulocyte colony-stimulating factor (G-CSF) joined to human immunoglobulin G1 and G4 (IgG1 and IgG4) Fc and C(H) domains are biologically active and have improved pharmacokinetic and hematopoietic properties in vivo.


METHODS
Chimeric genes encoding human G-CSF fused to the N-termini of the Fc and C(H) domains of human IgG1 and IgG4 were constructed and used to transfect monkey COS cells. The fusion proteins were purified from the conditioned media by protein A affinity chromatography. Bioactivities of the proteins were measured in a G-CSF-dependent in vitro bioassay. Pharmacokinetic and granulopoietic properties of the G-CSF/IgG1-Fc fusion protein were measured in normal rats.


RESULTS
The G-CSF/IgG-Fc and G-CSF/IgG-C(H) fusion proteins were secreted from transfected COS cells primarily as disulfide-linked homodimers. On a molar basis, the purified G-CSF/IgG-Fc fusion proteins were as active as G-CSF in in vitro bioassays, whereas bioactivities of the purified G-CSF/IgG-C(H) fusion proteins were decreased 3- to 4-fold. The G-CSF/IgG1-Fc fusion protein displayed a slower plasma clearance rate and stimulated greater and longer lasting increases in circulating neutrophils and white blood cells than G-CSF following intravenous and subcutaneous administration to rats.


CONCLUSIONS
Fusion of G-CSF to human IgG domains results in homodimeric fusion proteins possessing high in vitro bioactivities, long circulating half-lives, and enhanced hematopoietic properties in vivo.
Cox
George N
GN
Bolder BioTechnology, Inc., Wheat Ridge, CO 80033, USA. jcox@bolderbio.com
Smith
Darin J
DJ
Carlson
Sharon J
SJ
Bendele
Alison M
AM
Chlipala
Elizabeth A
EA
Doherty
Daniel H
DH
D000818:Animals; D001772:Blood Cell Count; D001856:Bone Marrow Examination; D019556:COS Cells; D016179:Granulocyte Colony-Stimulating Factor / Q000097:blood / Q000235:genetics / Q000494:pharmacology*; D006207:Half-Life; D006410:Hematopoiesis / Q000187:drug effects*; D006801:Humans; D007074:Immunoglobulin G / Q000235:genetics / Q000494:pharmacology*; D007962:Leukocytes / Q000166:cytology / Q000187:drug effects; D008297:Male; D009504:Neutrophils / Q000166:cytology / Q000187:drug effects; D010599:Pharmacokinetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000097:blood / Q000235:genetics / Q000494:pharmacology*; D014162:Transfection
D007074:Immunoglobulin G; D011993:Recombinant Fusion Proteins; D016179:Granulocyte Colony-Stimulating Factor