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

• US 20090253175A1
• Filed: 06/03/2009
• Published: 10/08/2009
• Est. Priority Date: 03/05/2003
• Status: Active Grant

• Current Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Original Assignee: Halozyme, Inc. (Halozyme Therapeutics, Inc.)
• Inventors: Kundu, Anirbau, Frost, Gregory I., Bookbinder, Louis H.
• Granted Patent: US 8,450,470 B2
• Time in Patent Office: Days
• US Class Current: 435/69.100
• 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

Provided are 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. Sialated and pegylated forms of the sHASEGPs also are provided. Methods of treatment by administering sHASEGPs and modified forms thereof also are provided.

Claims

1. A nucleic acid molecule, comprising a sequence of nucleotides encoding a soluble hyaluronidase polypeptide, wherein:

the encoded soluble hyaluronidase polypeptide is soluble;

the encoded soluble hyaluronidase polypeptide is neutral active; and

the encoded soluble hyaluronidase comprises a sequence of amino acids included in SEQ ID NO;

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

1.

2. The nucleic acid molecule of claim 1, wherein the sequence of nucleotides encodes a soluble hyaluronidase polypeptide truncated in the carboxy terminus of the polypeptide whose sequence is set forth in SEQ ID NO:

1 or a sequence that has at least 91% sequence identity therewith.

3. The nucleic acid molecule of claim 2, wherein the encoded soluble hyaluronidase polypeptide is truncated at amino acid 473, 474, 475, 776, 477, 478, 479, 479, 480, 481, 482 or 483 of SEQ ID NO:

1.

4. The nucleic acid molecule of claim 1, wherein the sequence of nucleotides encodes amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:

1.

5. The nucleic acid molecule of claim 1 that has a sequence of nucleotides set forth in SEQ ID NO:

48.

6. The nucleic acid molecule of claim 1 that has a sequence of nucleotides set forth as nucleotides 106-1446 of SEQ ID NO:

6.

7. The nucleic acid molecule of claim 1, wherein the sequence of nucleotides comprises a signal sequence for secretion of the encoded polypeptide.

8. The nucleic acid molecule of claim 7, wherein the signal sequence is an IgG kappa chain leader peptide.

9. The nucleic acid molecule of claim 8, wherein the IgG kappa chain leader peptide has a sequence of nucleotides set forth in SEQ ID NO:

43.

10. A vector, comprising the nucleic acid molecule of claim 1.

11. A vector of claim 10, comprising a sequence of nucleotides set forth in SEQ ID NO:

51.

12. The vector of claim 10 that is an expression vector.

13. The vector of claim 10 that is a eukaryotic vector.

14. The vector of claim 10 that is a Pichia vector, an E. coli vector, or a viral vector.

15. An isolated cell, comprising the vector of claim 10.

16. The cell of claim 14 that is a prokaryotic cell.

17. The cell of claim 15 that is a eukaryotic cell.

18. The cell of claim 15 selected from among a bacterial cell, a yeast cell, a plant cell, an insect cell and a mammalian cell.

19. The cell of claim 15 that is a Chinese hamster ovary (CHO) cell.

20. A method for producing a substantially purified soluble hyaluronidase polypeptide, comprising:

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

culturing the cells in a suitable growth media under conditions whereby an encoded polypeptide is expressed by the cell; and

recovering the expressed polypeptide(s).

21. The method of claim 20, wherein the cell is a eukaryotic cell.

22. The method of claim 21, wherein the eukaryotic cell is selected from a mammalian cell, an insect cell, a yeast cell and a plant cell.

23. The method of claim 21, wherein the cell is a Chinese hamster ovary (CHO) cell.

24. The method of claim 20, wherein the cells are cultured in the presence of 0.1-1 mM Sodium Butyrate under conditions suitable for production of the polypeptide.

25. The method of claim 23, wherein the cells are cultured in the presence of 0.1-1 mM Sodium Butyrate under conditions suitable for production of the polypeptide.

26. The method of claim 20, wherein the method of recovering the expressed polypeptide(s) comprises:

contacting the media containing the expressed polypeptide(s) under low ionic strength with an anion exchange resin at neutral pH;

eluting the polypeptide with about 400 mM salt;

contacting the polypeptide with a hydrophobic interaction chromatography resin in the presence of about 0.5M ammonium sulfate;

contacting the polypeptide in ammonium sulfate with phenyl boronate resin;

eluting the polypeptide in low salt at neutral pH;

contacting the polypeptide with a Hydroxapatite resin; and

eluting the polypeptide with about 100 mM Na Phosphate, thereby resulting in a polypeptide that is substantially purified.