Recombinant human G-CSF dimer and use thereof for the treatment of neurological diseases
This invention relates to a recombinant human G-CSF (rhG-CSF) dimer and its use in the treatment of neurological disorder. In particular, upon ischemic neural injury in animal, this invention can be used to protect neurons with the use of rhG-CSF dimer such that function of injured nerves can be restored. Serum half-life of G-CSF dimer of this invention is prolonged and the biological activity thereof is increased.
1. A human granulocyte colony-stimulating factor (G-CSF) dimer of formula (1):
M1-L-M2 (1)
wherein
M1 is a first monomer of G-CSF;
M2 is a second monomer of G-CSF; and
L is a linker connecting said first monomer and said second monomer and disposed therebetween;
wherein said G-CSF dimer retains the biological activity of G-CSF and has a serum half-life of more than twice of that of either said first or said second monomer;
wherein said dimer is produced by two G-CSF-Fc complexes; each of said G-CSF-Fc complex comprises the amino acid sequence of SEQ ID NO: 6.
2. A pharmaceutical composition comprising as its active ingredient, a purified dimer according to claim 1 .
3. The pharmaceutical composition according to claim 2 wherein said dimer has a purity of 90-100%.
4. The dimer according to claim 1 wherein each of said G-CSF-Fc complex comprises a G-CSF monomer, a Fc fragment of human IgG2, and a peptide connecting said G-CSF monomer and said Fc fragment; said dimer is formed through the pairing of two said Fc fragments via a plurality of disulfide bonds disposed therebetween.
5. The dimer according to claim 4 wherein said number of disulfide bonds disposed therebetween is 2 or 4.
6. A method to treat a neurological disorder comprising administering an effective amount of said pharmaceutical composition of claim 2 to a subject in need of the treatment.
7. The method according to claim 6 wherein said effective amount ranges from 0.001-1,000 mg of said dimer per dose.
8. The method according to claim 6 wherein said disease is selected from a group consisting of: stroke, spinal injury, and neurological disorders accompanied with blood brain barrier injury.
9. A method of activating STAT3 in neural cells comprising administering an effective amount of the pharmaceutical composition of claim 2 to a subject in need of the treatment.
10. The method according to claim 9 wherein said effective amount ranges from 0.001-1,000 mg of said dimer per dose.
11. A method for the manufacture of a dimer according to claim 1 comprising the steps of:
a) transforming mammalian cells with an expression vector comprising a DNA sequence comprising the nucleotide sequence of SEQ ID NO:10, encoding a G-CSF-Fc complex;
b) culturing said transformed mammalian cells under conditions sufficient for expressing said G-CSF-Fc complex and said dimer; and
c) isolating and purifying said dimer obtained from step (b);
wherein said dimer comprises two of said G-CSF-Fc complexes; each said G-CSF-Fc complex comprising the amino acid sequence of SEQ ID NO: 6.