Mutant of Granulocyte-Colony Stimulating Factor (G-Csf) and Chemically Conjugated Polypeptide Thereof

• US 20080200657A1
• Filed: 07/19/2006
• Published: 08/21/2008
• Est. Priority Date: 07/20/2005
• Status: Active Grant

• Current Assignee: Mogam Biotechnology Research Institute
• Original Assignee: Mogam Biotechnology Research Institute
• Inventors: Hong, Jeong-Woon, Kang, Kwan-Yub
• Granted Patent: US 8,841,426 B2
• Time in Patent Office: Days
• US Class Current: 530/395
• CPC Class Codes: A61K 47/60 the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
• CPC Class Codes: A61P 37/04 Immunostimulants
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/00 Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF

Abstract

Provided are mutants of human granulocyte-colony stimulating factor (G-CSF) designed for specific chemical conjugation, and chemical conjugates thereof for use as an adjuvant in the treatment of cancer. The present invention provides a mutant of a G-CSF in which a threonine (Thr) residue at position 133 of G-CSF comprising the amino acid sequence identified in SEQ ID NO: 1 is substituted with a cysteine (Cys) residue. In addition, the invention provides a mutant of a G-CSF in which a cysteine (Cys) residue is inserted between a glycine (Gly) residue at position 135 and an alanine (Ala) residue at position 136 of G-CSF. Further, the invention provides a chemically conjugated mutant G-CSF to which biocompatible polymer such as polyethylene glycol (PEG) was attached at the cysteine residue, which was introduced by the substitution or insertion mutation, increasing its in vivo retention time without reducing in vivo biological activity due to the conjugation with the biocompatible polymer, thereby ultimately extending the in vivo biological activity.

Claims

1. A mutant of a human granulocyte-colony stimulating factor (G-CSF), the mutant comprising the amino acid sequence identified in SEQ ID NO:

3, wherein a threonine (Thr) residue at position 133 of G-CSF having the amino acid sequence identified in SEQ ID NO;

1 is substituted with a cysteine (Cys) residue.

2. A conjugated mutant of a human G-CSF, the mutant comprising the amino acid sequence identified in SEQ ID NO:

3, wherein a threonine (Thr) residue at position 133 of G-CSF comprising the amino acid sequence identified in SEQ ID NO;

1 is substituted with a cysteine (Cys) residue, and the substituted Cys residue is attached at a nonprotein chemical compound.

3. The conjugated mutant of claim 2, wherein the mutant is a protein comprising the amino acid sequence identified in SEQ ID NO:

4 having a cysteine (Cys) residue at position 17 substituted with a serine (Ser) residue.

4. The conjugated mutant of claim 2 or 3, wherein the nonprotein chemical compound is a compound of one kind selected from the group comprising of polyethylene glycol (PEG), polyvinyl alcohol (PVA), polycarboxylic acid and polyvinyl pyrrolidone, binding with one kind selected from the group comprising of maleimide, vinyl sulfone, iodacetamide and orthopyridyl disulfide.

5. The conjugated mutant of claim 2 or 3, wherein the nonprotein chemical compound has a molecular weight in the range of about 2 to about 100 kDa.

6. The conjugated mutant of claim 4, wherein the PEG has a molecular weight in the range of about 5 to about 100 kDa.

7. A mutant of a human granulocyte-colony stimulating factor (G-CSF), the mutant comprising the amino acid sequence comprised in SEQ ID NO:

5, wherein a cysteine (Cys) residue is inserted between a glycine (Gly) residue at position 135 and an alanine (Ala) residue at position 136 of the G-CSF comprised in SEQ ID NO;

1.

8. A conjugated mutant of a human granulocyte-colony stimulating factor (G-CSF), the mutant comprising the amino acid sequence comprised in SEQ ID NO:

5, wherein a cysteine (Cys) residue is inserted between a glycine (Gly) residue at position 135 and an alanine (Ala) residue at position 136 of the G-CSF comprising the amino acid sequence comprised in SEQ ID NO;

1, and a nonprotein chemical compound is attached at the inserted cysteine residue.

9. The conjugated mutant of claim 8, wherein the mutant is a protein comprising the amino acid sequence comprised in SEQ ID NO:

6 having a cysteine residue at position 17 substituted with a serine (Ser) residue.

10. The conjugated mutant of claim 8 or 9, wherein the nonprotein chemical compound is a compound of one kind selected from the group comprising of polyethylene glycol (PEG), polyvinyl alcohol (PVA), polycarboxylic acid and polyvinyl pyrrolidone, binding with one kind selected from the group comprising of maleimide, vinyl sulfone, iodacetamide and orthopyridyl disulfide.

11. The conjugated mutant of claim 8 or 9, wherein the nonprotein chemical compound has a molecular weight in the range of about 2 to about 100 kDa.

12. The conjugated mutant of claim 10, wherein the PEG has a molecular weight in the range of about 5 to about 100 kDa.