O-linked glycosylation of peptides

• US 8,361,961 B2
• Filed: 01/10/2005
• Issued: 01/29/2013
• Est. Priority Date: 01/08/2004
• Status: Expired due to Fees

• Current Assignee: Ratiopharm GmbH (Teva Pharmaceutical Industries Limited)
• Original Assignee: Biogenerix Ag (Teva Pharmaceutical Industries Limited)
• Inventors: DeFrees, Shawn, Zopf, David A., Wang, Zhi-Guang, Clausen, Henrik
• Primary Examiner(s): Bradley, Christina
• Application Number:
  US10/585,385
• Publication Number:
  US 20090169509A1
• Time in Patent Office: 2,941 Days
• Field of Search: None
• US Class Current: 514/7.6
• CPC Class Codes: A61K 38/193 Colony stimulating factors [CSF]
• CPC Class Codes: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 1/04 for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 11/02 Nasal agents, e.g. decongestants
• CPC Class Codes: A61P 11/06 Antiasthmatics
• CPC Class Codes: A61P 13/00 Drugs for disorders of the urinary system
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 15/08 for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: A61P 19/10 for osteoporosis
• CPC Class Codes: A61P 21/00 Drugs for disorders of the muscular or neuromuscular system
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/14 for treating abnormal movements, e.g. chorea, dyskinesia
• CPC Class Codes: A61P 25/20 Hypnotics; Sedatives
• CPC Class Codes: A61P 25/28 for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• CPC Class Codes: A61P 27/02 Ophthalmic agents
• CPC Class Codes: A61P 29/00 : Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 : Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/10 : for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 31/12 : Antivirals
• CPC Class Codes: A61P 31/14 : for RNA viruses
• CPC Class Codes: A61P 31/18 : for HIV
• CPC Class Codes: A61P 31/20 : for DNA viruses
• CPC Class Codes: A61P 31/22 : for herpes viruses
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 35/02 : specific for leukemia
• CPC Class Codes: A61P 37/06 : Immunosuppressants, e.g. drugs for graft rejection
• CPC Class Codes: A61P 37/08 : Antiallergic agents antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 5/00 : Drugs for disorders of the endocrine system
• CPC Class Codes: A61P 5/06 : of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• CPC Class Codes: A61P 5/10 : of the posterior pituitary hormones, e.g. oxytocin, ADH
• CPC Class Codes: A61P 7/00 : Drugs for disorders of the blood or the extracellular fluid
• CPC Class Codes: A61P 7/06 : Antianaemics
• CPC Class Codes: C07K 1/1077 : by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 14/535 : Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C07K 9/00 : Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
• CPC Class Codes: C07K 9/001 : the peptide sequence having less than 12 amino acids and not being part of a ring structure

Abstract

The present invention provides polypeptides that include an O-linked glycosylation site that is not present in the wild-type peptide. The polypeptides of the invention include glycoconjugates in which a species such as a water-soluble polymer, a therapeutic agent of a biomolecule is covalently linked through an intact O-linked glycosyl residue to the polypeptide. Also provided are methods of making the peptides of the invention and methods, pharmaceutical compositions containing the peptides and methods of treating, ameliorating or preventing diseased in mammals by administering an amount of a peptide of the invention sufficient to achieve the desired response.

Claims

1. An isolated mutant G-CSF polypeptide comprising the wild-type G-CSF sequence SEQ ID NO:

143 wherein the sequence M¹TPLGP (SEQ ID NO;

     190) in SEQ ID NO;

143 is replaced with a mutant peptide sequence, wherein the mutant peptide sequence encodes an O-linked glycosylation site that does not exist in the wild-type G-CSF polypeptide, and wherein the mutant peptide sequence is selected from the group consisting of;

MVTPLGP (SEQ ID NO;

     1), MQTPLGP (SEQ ID NO;

     2), MIATPLGP (SEQ ID NO;

     3), MPTQGAMPLGP (SEQ ID NO;

     5), MVQTPLGP (SEQ ID NO;

     6), MQSTPLGP (SEQ ID NO;

     7), MGQTPLGP (SEQ ID NO;

     8), MAPTSSSPLGP (SEQ ID NO;

     9), MAPTPLGPA (SEQ ID NO;

     10), MTPTLGP (SEQ ID NO;

     11), MTPTQLGP (SEQ ID NO;

     12), MTPTSLGP (SEQ ID NO;

     13), MTPTQGP (SEQ ID NO;

     14), MTPTSSP (SEQ ID NO;

     15), MTPQTP (SEQ ID NO;

     16), MTPTGP (SEQ ID NO;

     17), MTPLTP (SEQ ID NO;

     18), MTPNTGP (SEQ ID NO;

     19), MTPVTP (SEQ ID NO;

     20), MTPMVTP (SEQ ID NO;

     21), and MTPTQGLGPAS (SEQ ID NO;

     22).

2. The mutant G-CSF polypeptide of claim 1, wherein the mutant peptide sequence is selected from the group consisting of:

MVTPLGP (SEQ ID NO;

     1), MQTPLGP (SEQ ID NO;

     2), MIATPLGP (SEQ ID NO;

     3), MPTQGAMPLGP (SEQ ID NO;

     5), MVQTPLGP (SEQ ID NO;

     6), MQSTPLGP (SEQ ID NO;

     7), MGQTPLGP (SEQ ID NO;

     8), MAPTSSSPLGP (SEQ ID NO;

     9), MAPTPLGPA (SEQ ID NO;

     10).

3. The mutant G-CSF polypeptide of claim 1, wherein the mutant peptide sequence is selected from the group consisting of:

MTPTLGP (SEQ ID NO;

     11), MTPTQLGP (SEQ ID NO;

     12), MTPTSLGP (SEQ ID NO;

     13), MTPTQGP (SEQ ID NO;

     14), MTPTSSP (SEQ ID NO;

     15), MTPQTP (SEQ ID NO;

     16), MTPTGP (SEQ ID NO;

     17), MTPLTP (SEQ ID NO;

     18), MTPNTGP (SEQ ID NO;

     19), MTPVTP (SEQ ID NO;

     20), MTPMVTP (SEQ ID NO;

     21), and MTPTQGLGPAS (SEQ ID NO;

     22).

4. An isolated nucleic acid encoding the mutant G-CSF polypeptide of claim 1.

5. An expression cassette comprising the nucleic acid of claim 4.

6. A cell comprising the nucleic acid of claim 4.

7. The mutant G-CSF polypeptide of claim 1, further comprising a formula selected from:

8. The polypeptide according to claim 7, wherein X comprises a group selected from sialyl, galactosyl and Gal-Sia moieties, wherein at least one of said sialyl, galactosyl and Gal-Sia comprises a modifying group.

9. The polypeptide according to claim 7, wherein X comprises the moiety:

10. The polypeptide according to claim 7, wherein X comprises the structure:

11. The polypeptide according to claim 7, wherein X comprises the structure:

12. A method for making a glycoconjugate of the mutant G-CSF polypeptide of claim 1, comprising the steps of:

(a) recombinantly producing the mutant G-CSF polypeptide of claim 1, and

(b) enzymatically glycosylating the mutant G-CSF polypeptide with a modified sugar at said O-linked glycosylation site.

13. A pharmaceutical composition of a granulocyte colony stimulating factor (G-CSF) comprising:

an effective amount of the mutant G-CSF polypeptide of claim 1, wherein said mutant G-CSF polypeptide is glycoconjugated with a modified sugar.

14. The pharmaceutical composition according to claim 13, wherein said modified sugar is modified with a member selected from poly(ethylene glycol) and methoxy-poly(ethylene glycol) (m-PEG).

15. A method of providing G-CSF therapy to a subject in need of said therapy, said method comprising, administering to said subject an effective amount the pharmaceutical composition of claim 13.