Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof

• US 20090181032A1
• Filed: 02/20/2009
• Published: 07/16/2009
• Est. Priority Date: 03/05/2003
• Status: Active Grant

• Current Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Original Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Inventors: Frost, Gregory I., Kundu, Anirban, Bookbinder, Louis H.
• Granted Patent: US 8,431,380 B2
• Time in Patent Office: Days
• US Class Current: 424/141.100
• CPC Class Codes: A61K 2039/505 comprising antibodies
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/47 acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• CPC Class Codes: A61K 39/395 Antibodies agglutinins A61K38/36 ; as drug carriers A61K47/50; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
• CPC Class Codes: A61K 9/0048 Eye, e.g. artificial tears
• CPC Class Codes: A61P 17/00 Drugs for dermatological disorders
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 25/16 Anti-Parkinson drugs
• CPC Class Codes: A61P 25/24 Antidepressants
• 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 31/00 Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: A61P 31/10 Antimycotics
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 33/00 Antiparasitic agents
• CPC Class Codes: A61P 33/04 Amoebicides
• CPC Class Codes: A61P 33/06 : Antimalarials
• CPC Class Codes: A61P 35/00 : Antineoplastic agents
• CPC Class Codes: A61P 43/00 : Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 : Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: A61P 7/10 : Antioedematous agents; Diuretics
• CPC Class Codes: A61P 9/00 : Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 : Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 : for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: A61P 9/12 : Antihypertensives
• CPC Class Codes: C12N 9/2474 : Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
• CPC Class Codes: C12Y 302/01035 : Hyaluronoglucosaminidase (3.2.1.35), i.e. hyaluronidase
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention relates to the discovery of novel soluble neutral active Hyaluronidase Glycoproteins (sHASEGP'"'"'s), methods of manufacture, and their use to facilitate administration of other molecules or to alleviate glycosaminoglycan associated pathologies. Minimally active polypeptide domains of the soluble, neutral active sHASEGP domains are described that include asparagine-linked sugar moieties required for a functional neutral active hyaluronidase domain. Included are modified amino-terminal leader peptides that enhance secretion of sHASEGP. The invention further comprises sialated and pegylated forms of a recombinant sHASEGP to enhance stability and serum pharmacokinetics over naturally occurring slaughterhouse enzymes. Further described are suitable formulations of a substantially purified recombinant sHASEGP glycoprotein derived from a eukaryotic cell that generate the proper glycosylation required for its optimal activity.

Claims

1. A substantially purified hyaluronidase polypeptide, comprising at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide, wherein:

the substantially purified hyaluronidase polypeptide is soluble;

the soluble hyaluronidase polypeptide is a neutral active glycoprotein; and

the substantially purified hyaluronidase comprises a sequence of amino acids included in SEQ ID NO;

1 or a sequence that has at least about 91% amino acid sequence identity with a sequence of amino acids included in SEQ ID NO;

1.

2. A substantially purified hyaluronidase polypeptide of claim 1, wherein the polypeptide is encoded by a nucleic acid molecule that encodes amino acids 36-482 of SEQ ID NO:

1.

3. The substantially purified hyaluronidase polypeptide of claim 2, wherein the nucleic acid molecule has a sequence of nucleotides set forth in SEQ ID NO:

48.

4. The substantially purified hyaluronidase polypeptide of claim 1, wherein the polypeptide includes a sequence of amino acids set forth in SEQ ID NO:

1 that is truncated at an amino acid residue that is or is between amino acid residues 467 to 483.

5. The substantially purified hyaluronidase polypeptide of claim 1, wherein the polypeptide includes a sequence of amino acids set forth in SEQ ID NO:

1 that is truncated at an amino acid residue selected from among 467, 477, 478, 479, 480, 481, 482 and 483.

6. The substantially purified hyaluronidase polypeptide of claim 5, wherein the polypeptide is secreted in CHO cells.

7. The substantially purified hyaluronidase polypeptide of claim 1, wherein the polypeptide has the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO:

1.

8. A substantially purified hyaluronidase polypeptide of claim 1, wherein the polypeptide is modified with a polymer.

9. A substantially purified hyaluronidase polypeptide of claim 8, wherein the polymer is a polyethylene glycol (PEG) or a dextran.

10. A nucleic acid molecule encoding a polypeptide of claim 1.

11. A vector, comprising the nucleic acid molecule of claim 10.

12. A vector of claim 11 comprising a sequence of nucleotides set forth in SEQ ID NO:

51.

13. The vector of claim 11 that is an expression vector.

14. The vector of claim 11 that is a eukaryotic vector.

15. The vector of claim 11 that is a Pichia vector, an E. coli vector, or a viral vector.

16. An isolated cell, comprising the vector of claim 11.

17. The cell of claim 16 that is a prokaryotic cell.

18. The cell of claim 16 that is a eukaryotic cell.

19. The cell of claim 16 selected from among a bacterial cell, a yeast cell, a plant cell, an insect cell and a mammalian cell.

20. The cell of claim 16 that is a CHO cell.

21. A method for producing a substantially purified hyaluronidase polypeptide of claim 1, comprising:

introducing a nucleic acid that encodes a polypeptide of claim 1 operably linked to a suitable promoter into a cell capable of incorporating N-linked sugar moieties into the polypeptide;

culturing the cell under conditions whereby an encoded polypeptide is expressed by the cell; and

recovering the expressed polypeptide(s).

22. The method of claim 21, wherein the cell is a eukaryotic cell.

23. The method of claim 22, wherein the eukaryotic cell is selected from a mammalian cell, an insect cell, a yeast cell and a plant cell.

25. A pharmaceutical composition, comprising a substantially purified hyaluronidase polypeptide of claim 1.

26. The pharmaceutical composition of claim 25, wherein the polypeptide is produced by expression in mammalian cells of a nucleic acid molecule that encodes amino acids 36-482 of SEQ ID NO:

1.

27. The pharmaceutical composition of claim 26, wherein the mammalian cell is a CHO cell.

28. The pharmaceutical composition of claim 25, further comprising a pharmaceutically active agent.

29. The pharmaceutical composition of claim 28, wherein the pharmaceutically active agent is selected from among a small molecule, a nucleic acid and a vehicle comprising a nucleic acid.

30. The pharmaceutical composition of claim 29, wherein the nucleic acid or the vehicle comprising the nucleic acid molecule is selected from among a retrovirus, an adenovirus, an adeno-associated virus and a DNA complex.

31. The pharmaceutical composition of claim 28, wherein the pharmaceutically active agent is selected from among a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amoebicidal agent, a trichomonocidal agent, an anti-parkinson agent, an anti-malarial agent, an anticonvulsant agent, an anti-depressant agent, and antiarthritics agent, an anti-fungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasite agent, an antihistamine agent, an alpha-adrenargic agonist agent, an alpha blocker agent, an anesthetic agent, a bronchial dialator agent, a biocide agent, a bactericide agent, a bacteriostat agent, a beta adrenergic blocker agent, a calcium channel blocker agent, a cardiovascular drug agent, a contraceptive agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent, a electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contractant agent, an ophthalmic agent, a parasympathomimetic agent, a psychic energizer agent, a sedative agent, a sympathomimetic agent, a tranquilizer agent, an urinary agent, a vaginal agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent, a polypeptide, a protein, a nucleic acid, a drug, a organic molecule and a sleep inducer.

32. The pharmaceutical composition of claim 31, wherein the chemotherapeutic agent is a toxin or a tumor necrosis factor.

33. The pharmaceutical composition of claim 31, wherein the anesthetic agent is lidocaine or bupivacaine.

34. The pharmaceutical composition of claim 28, further comprising a hormonal agent.

35. The pharmaceutical composition of claim 34, wherein the hormonal agent is epinephrine.

36. The pharmaceutical composition of claim 28, wherein the pharmaceutically active agent is selected from among insulin, a cytokine, an antibody and a monoclonal antibody.

37. A conjugate, comprising a soluble glycoprotein of claim 1 or comprising a domain of a soluble glycoprotein of claim 1.

38. A combination, comprising:

a substantially purified hyaluronidase polypeptide of claim 1; and

a pharmaceutically active agent.

39. The combination of claim 38, wherein the pharmaceutically active agent is selected from among a small molecule, a nucleic acid and a vehicle comprising a nucleic acid.

40. The combination of claim 39, wherein the nucleic acid or the vehicle comprising the nucleic acid molecule is selected from among a retrovirus, an adenovirus, an adeno-associated virus and a DNA complex.

41. The combination of claim 38, wherein the pharmaceutically active agent is selected from among a chemotherapeutic agent, an analgesic agent, an anti-inflammatory agent, an antimicrobial agent, an amoebicidal agent, a trichomonocidal agent, an anti-parkinson agent, an anti-malarial agent, an anticonvulsant agent, an anti-depressant agent, and antiarthritics agent, an anti-fungal agent, an antihypertensive agent, an antipyretic agent, an anti-parasite agent, an antihistamine agent, an alpha-adrenargic agonist agent, an alpha blocker agent, an anesthetic agent, a bronchial dilator agent, a biocide agent, a bactericide agent, a bacteriostat agent, a beta adrenergic blocker agent, a calcium channel blocker agent, a cardiovascular drug agent, a contraceptive agent, a decongestant agent, a diuretic agent, a depressant agent, a diagnostic agent, a electrolyte agent, a hypnotic agent, a hormone agent, a hyperglycemic agent, a muscle relaxant agent, a muscle contractant agent, an ophthalmic agent, a parasympathomimetic agent, a psychic energizer agent, a sedative agent, a sympathomimetic agent, a tranquilizer agent, an urinary agent, a vaginal agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent, a polypeptide, a protein, a nucleic acid, a drug, a organic molecule and a sleep inducer.

42. The combination of claim 38, wherein the pharmaceutically active agent is selected from among insulin, a cytokine, an antibody and a monoclonal antibody.

24. The method of claim 24, wherein the cell is a CHO cell.