Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
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 substantially purified hyaluronidase polypeptide, wherein:
the polypeptide contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide;
the polypeptide is catalytically active;
the polypeptide is secreted; and
the 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.
2. The hyaluronidase polypeptide of claim 1 that has at least 99% amino acid sequence identity with the sequence of amino acids set forth as amino acid residues 36-483 of SEQ ID NO:1.
3. The substantially purified hyaluronidase polypeptide of claim 1 that consists of the sequence of amino acids set forth as amino acids 36-485 of SEQ ID NO:1.
4. A substantially purified hyaluronidase polypeptide that consists of the sequence of amino acids set forth as amino acids 36-494 of SEQ ID NO:1, wherein:
the polypeptide contains at least one sugar moiety that is covalently attached to an asparagine residue of the hyaluronidase polypeptide;
the polypeptide is catalytically active; and
the polypeptide is secreted.
5. The substantially purified hyaluronidase polypeptide of claim 1 , wherein the polypeptide is secreted when produced in CHO cells.
6. The substantially purified hyaluronidase polypeptide of claim 1 , wherein the polypeptide is conjugated to a polymer.
7. The substantially purified hyaluronidase polypeptide of claim 6 , wherein the polymer is a polyethylene glycol (PEG) or a dextran.
8. A pharmaceutical composition, comprising the substantially purified hyaluronidase polypeptide of claim 1 .
9. The pharmaceutical composition of claim 8 , further comprising a pharmaceutically active agent.
10. The pharmaceutical composition of claim 9 , wherein the pharmaceutically active agent is a small molecule.
11. The pharmaceutical composition of claim 9 , 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 anti-convulsant 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, an 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 sedative agent, a sympathomimetic agent, a tranquilizer agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent and a sleep inducer.
12. The pharmaceutical composition of claim 11 , wherein the agent is a toxin or a tumor necrosis factor.
13. The pharmaceutical composition of claim 11 , wherein the agent is an anesthetic agent that is lidocaine or bupivacaine.
14. The pharmaceutical composition of claim 9 , further comprising a hormonal agent.
15. The pharmaceutical composition of claim 14 , wherein the hormonal agent is epinephrine.
16. The pharmaceutical composition of claim 9 , wherein the pharmaceutically active agent is selected from among an insulin, a cytokine and an antibody.
17. A conjugate comprising:
the polypeptide of claim 1 , a linker and a targeting agent, wherein the linker is attached to the targeting agent, and wherein the linker is attached directly or indirectly to the polypeptide.
18. A conjugate comprising:
the polypeptide of claim 6 , a linker and a targeting agent, wherein the linker is attached to the targeting agent, and wherein the linker is attached directly or indirectly to the polypeptide.
19. A combination, comprising:
a first composition and a second composition, wherein:
the first composition comprises the hyaluronidase polypeptide of claim 1 ;
the second composition comprises a pharmaceutically active agent; and
the first and second compositions are separate or combined to form a single composition.
20. The combination of claim 19 wherein the pharmaceutically active agent is selected from among a small molecule and an antibody.
21. The combination of claim 19 , 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, an 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 sedative agent, a sympathomimetic agent, a tranquilizer agent, a viricide agent, a vitamin agent, a non-steroidal anti-inflammatory agent, an angiotensin converting enzyme inhibitor agent and a sleep inducer.
22. The combination of claim 19 , wherein the pharmaceutically active agent is selected from among insulin, a cytokine and an antibody.
23. The pharmaceutical composition of claim 9 , wherein the pharmaceutically active agent is a monoclonal antibody.
24. The combination of claim 19 , wherein the pharmaceutically active agent is a monoclonal antibody.