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Details for Patent: 8,450,470

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Details for Patent: 8,450,470

Title:Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
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.
Inventor(s): Bookbinder; Louis H. (San Diego, CA), Kundu; Anirbau (San Diego, CA), Frost; Gregory I. (Del Mar, CA)
Assignee: Halozyme, Inc. (San Diego, CA)
Filing Date:Jun 03, 2009
Application Number:12/455,657
Claims:1. A nucleic acid molecule, comprising a sequence of nucleotides encoding a hyaluronidase polypeptide, wherein: the nucleic acid molecule includes a stop codon, whereby the encoded hyaluronidase polypeptide is produced upon expression; and the encoded hyaluronidase polypeptide is selected from among: a polypeptide that consists of a sequence of amino acids selected from among amino acid residues 1-477, 1-478, 1-479, 1-480, 1-481, 1-482, 1-483, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1; and a polypeptide that consists of a sequence of amino acids that contains amino acid substitutions in the sequence of amino acids set forth as 1-477, 1-478, 1-479, 1-480, 1-481, 1-482, 1-483, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1, whereby the amino acid-substituted hyaluronidase glycoprotein consists of a sequence of amino acids that has at least 95% amino acid sequence identity to a sequence of amino acids set forth as amino acid residues 1-477, 1-478, 1-479, 1-480, 1-481, 1-482, 1-483, 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1.

2. The nucleic acid molecule of claim 1, wherein the encoded hyaluronidase polypeptide consists of a sequence of amino acids that contain amino acid substitutions in the sequence of amino acids set forth as 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1, whereby the amino acid-substituted hyaluronidase glycoprotein consists of a sequence of amino acids that has at least 95% amino acid sequence identity to a sequence of amino acids set forth as amino acid residues 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1.

3. 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.

4. The nucleic acid molecule of claim 1, comprising the sequence of nucleotides set forth in SEQ ID NO:48.

5. The nucleic acid molecule of claim 1, comprising the sequence of nucleotides set forth as nucleotides 106-1446 of SEQ ID NO:6.

6. The nucleic acid molecule of claim 1, wherein: the encoded hyaluronidase polypeptide is selected from among: a polypeptide that consists of a sequence of amino acids selected from among amino acid residues 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1; and a polypeptide that consists of a sequence of amino acids that contain amino acid substitutions in the sequence of amino acids set forth as 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1, whereby the amino acid-substituted hyaluronidase glycoprotein consists of a sequence of amino acids that has at least 95% amino acid sequence identity to a sequence of amino acids set forth as amino acid residues 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO:1; and the nucleic acid molecule comprises a sequence of nucleotides encoding a signal sequence for secretion of the encoded polypeptide.

7. The nucleic acid molecule of claim 6, wherein the encoded signal sequence for secretion is an IgG kappa chain leader peptide.

8. The nucleic acid molecule of claim 7, wherein the IgG kappa chain leader peptide is encoded by the sequence of nucleotides set forth in SEQ ID NO:43.

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

10. The vector of claim 9, comprising the sequence of nucleotides set forth in SEQ ID NO:51.

11. The vector of claim 9 that is an expression vector.

12. The vector of claim 9 that is a eukaryotic vector.

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

14. An isolated cell, comprising the vector of claim 9.

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

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

17. The cell of claim 14 selected from among a bacterial cell, a yeast cell, a plant cell, an insect cell and a mammalian cell.

18. The cell of claim 14 that is a Chinese hamster ovary (CHO) cell.

19. A method for producing a hyaluronidase polypeptide, comprising: (a) introducing the nucleic acid of claim 1 operably linked to a promoter into a cell capable of incorporating N-linked sugar moieties into the polypeptide; (b) culturing the cells in a suitable growth medium under conditions whereby an encoded hyaluronidase polypeptide is produced by expression of the introduced nucleic acid and secreted by the cell; and (c) recovering the expressed polypeptide.

20. The method of claim 19, wherein the cell is a eukaryotic cell.

21. The method of claim 20, wherein the eukaryotic cell is selected from a mammalian cell, an insect cell, a yeast cell and a plant cell.

22. The method of claim 20, wherein the cell is a Chinese hamster ovary (CHO) cell.

23. The method of claim 19, wherein the cells are cultured in the presence of 0.1-1 mM Sodium Butyrate under conditions suitable for production of the polypeptide.

24. The method of claim 22, wherein the cells are cultured in the presence of 0.1-1 mM Sodium Butyrate under conditions suitable for production of the polypeptide.

25. A vector, comprising the nucleic acid molecule of claim 4, or degenerates thereof.

26. A vector, comprising the nucleic acid molecule of claim 5, or degenerates thereof.

27. A nucleic acid molecule, comprising a sequence of nucleotides encoding a hyaluronidase polypeptide, wherein: the nucleic acid molecule includes a stop codon, whereby the encoded hyaluronidase polypeptide is produced upon expression; and the encoded hyaluronidase polypeptide consists of a sequence of amino acids that has at least 98% amino acid sequence identity to the sequence of amino acids set forth as amino acid residues 36-483 of SEQ ID NO:1.

28. A vector, comprising the nucleic acid of claim 27.

29. The vector of claim 28 that is a Pichia vector, an E. coli vector, or a viral vector.

30. An isolated cell, comprising the vector of claim 28.
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