Hyaluronidase and factor VIII compositions
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.
1. A pharmaceutical composition, comprising in a pharmaceutically acceptable carrier:
a) a hyaluronidase polypeptide, wherein:
the hyaluronidase polypeptide contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide;
the hyaluronidase polypeptide is catalytically active;
the hyaluronidase polypeptide is soluble, and
the hyaluronidase polypeptide is selected from:
i) a polypeptide that consists of a contiguous sequence of amino acids contained within SEQ ID NO:1, whereby the polypeptide comprises amino acids 36-464 of SEQ ID NO:1 and terminates at a residue selected from among 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO:1; or
ii) a polypeptide that contains amino acid substitutions in the sequence of amino acids of the polypeptide of i), whereby the amino acid-substituted polypeptide consists of a sequence of amino acids that has at least about 91% amino acid sequence identity with the polypeptide of i); and
b) a Factor VIII polypeptide.
2. A pharmaceutical composition, comprising, in a pharmaceutically acceptable carrier:
a) a hyaluronidase polypeptide, wherein:
the hyaluronidase polypeptide contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide;
the hyaluronidase polypeptide is catalytically active;
the hyaluronidase polypeptide is soluble; and
the hyaluronidase polypeptide consists of a sequence of amino acids that has at least 98% amino acid sequence identity with the sequence of amino acids set forth as amino acid residues 36-483 of SEQ ID NO:1;
and
b) a Factor VIII polypeptide.
3. The pharmaceutical composition of claim 2 , wherein the hyaluronidase polypeptide consists of amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO: 1.
4. The pharmaceutical composition of claim 1 , wherein the hyaluronidase polypeptide is secreted when produced in CHO cells.
5. The pharmaceutical composition of claim 1 , wherein the hyaluronidase polypeptide is modified with a polymer.
6. The pharmaceutical composition of claim 5 , wherein the polymer is a polyethylene glycol (PEG) or a dextran.
7. The pharmaceutical composition of claim 1 , wherein the hyaluronidase polypeptide is encoded by a nucleic acid molecule that comprises the sequence of nucleotides set forth in SEQ ID NO:48, which encodes amino acids 1-482 of SEQ ID NO:1.
8. The pharmaceutical composition of claim 1 , wherein the hyaluronidase polypeptide is secreted when produced in CHO cells.
9. The pharmaceutical composition of claim 2 , wherein the hyaluronidase polypeptide is modified with a polymer.
10. The pharmaceutical composition of claim 9 , wherein the polymer is a polyethylene glycol (PEG) or a dextran.
11. A method of increasing the delivery of Factor VIII to a subject, comprising administering the pharmaceutical composition of claim 1 , wherein the pharmaceutical composition treats a coagulation disorder.
12. A combination, comprising:
a) a first composition comprising a hyaluronidase polypeptide, wherein:
the hyaluronidase polypeptide contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide;
the hyaluronidase polypeptide is catalytically active;
the hyaluronidase polypeptide is soluble; and
the hyaluronidase polypeptide consists of:
i) a polypeptide that consists of a contiguous sequence of amino acids contained within SEQ ID NO: 1, whereby the polypeptide comprises amino acids 36-464 of SEQ ID NO: 1 and terminates at a residue selected from among 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO: 1; or
ii) a polypeptide that contains amino acid substitutions in the sequence of amino acids of the polypeptide of i), whereby the amino acid-substituted polypeptide consists of a sequence of amino acids that has at least about 91% amino acid sequence identity with the polypeptide of i); and
b) a second composition comprising a Factor VIII polypeptide.
13. The combination of claim 12 , wherein the hyaluronidase polypeptide has a C-terminal amino acid residue selected from among 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO:1.
14. The combination of claim 13 , wherein the hyaluronidase polypeptide consists of amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 of SEQ ID NO: 1.
15. The combination of claim 12 , wherein the hyaluronidase polypeptide is secreted when produced in CHO cells.
16. The combination of claim 12 , wherein the hyaluronidase polypeptide is modified with a polymer.
17. The combination of claim 16 , wherein the polymer is a polyethylene glycol (PEG) or a dextran.
18. A combination, comprising:
a) a first composition comprising a hyaluronidase polypeptide, wherein:
the hyaluronidase polypeptide contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide;
the hyaluronidase polypeptide is C-terminally truncated so that it does not include the full-length of the polypeptide whose sequence is set forth in SEQ ID NO: 1; the hyaluronidase polypeptide is catalytically active; and the hyaluronidase polypeptide consists of:
i) a polypeptide that consists of a contiguous sequence of amino acids contained within SEQ ID NO: 1, whereby the polypeptide comprises amino acids 36-464 of SEQ ID NO: 1 and terminates at a residue selected from among 477, 478, 479, 480, 481, 482 and 483 of SEQ ID NO: 1; or
ii) a polypeptide that contains amino acid substitutions in the sequence of amino acids of the polypeptide of i), whereby the amino acid-substituted polypeptide consists of a sequence of amino acids that has at least about 91% amino acid sequence identity with the polypeptide of i); and
b) a second composition comprising a Factor VIII polypeptide.
19. The combination of claim 18 , wherein the hyaluronidase polypeptide is encoded by a nucleic acid molecule that comprises the sequence of nucleotides 106-1446 of SEQ ID NO: 6.
20. The combination of claim 18 , wherein the hyaluronidase polypeptide is encoded by a nucleic acid molecule that comprises the sequence of nucleotides set forth in SEQ ID NO:48.
21. The combination of claim 18 , wherein the sequence of amino acids is set forth in SEQ ID NO:4 or has at least 91% amino acid sequence identity with the sequence of amino acids set forth as amino acids 1-448 of SEQ ID NO:4.
22. The combination of claim 18 , wherein the hyaluronidase polypeptide is secreted when produced in CHO cells.
23. The combination of claim 18 , wherein the hyaluronidase polypeptide is modified with a polymer.
24. The combination of claim 23 , wherein the polymer is a polyethylene glycol (PEG) or a dextran.
25. A method of increasing the delivery of Factor VIII to a subject, comprising administering the combination of claim 12 .