Soluble hyaluronidase glycoprotein (sHASEGP), process for preparing the same, uses and pharmaceutical compositions comprising thereof
First Claim
1. A substantially purified glycoprotein, comprising a neutral active soluble hyaluronidase polypeptide and at least one N-linked sugar moiety, wherein the N-linked sugar moiety is covalently attached to an asparagine residue of the polypeptide.
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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.
197 Citations
161 Claims
- 1. A substantially purified glycoprotein, comprising a neutral active soluble hyaluronidase polypeptide and at least one N-linked sugar moiety, wherein the N-linked sugar moiety is covalently attached to an asparagine residue of the polypeptide.
- 13. A polypeptide, consisting essentially of a hyaluronidase domain or a catalytically active portion thereof of a human hyaluronidase protein.
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29. An isolated substantially pure polypeptide that consists essentially of the hyaluronidase domain of sHASEGP.
- 31. An isolated soluble human hyaluronidase glycoprotein (sHASEGP), wherein the potency of the sHASEGP is greater than 40,000 USP Units/mg protein.
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39. A nucleic acid molecule, comprising a sequence selected from:
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(a) a sequence as set forth in SEQ ID NO. 6;
(b) a sequence that hybridizes under high stringency to at least about 70% of the full-length to the sequence of nucleotides set forth in SEQ ID NO. 6;
(c) a sequence of nucleotides that encodes the polypeptide set forth in SEQ ID No. 1, 3, 4, 5 and 50;
(d) a sequence that is a splice variant of (a), (b) or (c);
(e) a sequence that encodes the hyaluronidase domain or a catalytically active portion thereof that includes a sequence of nucleotides having at least about 60% sequence identity with the sequence set forth in SEQ ID No. 6 or 50; and
(f) a sequence comprising degenerate codons of (a), (b), (c), (d) or (e).
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40. A vector having a sequence as set forth in SEQ ID NO:
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54. A method for producing a soluble polypeptide that contains sHASEGP, comprising:
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introducing a nucleic acid as set forth in SEQ ID NO. 6 operably linked to a promoter into a cell that incorporates N-linked sugar moieties into sHASEGP;
culturing the cell under conditions whereby the encoded polypeptide is expressed by the cell; and
recovering the expressed polypeptide. - View Dependent Claims (56)
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67. A composition, comprising a substantially purified sHASEGP glycoprotein polypeptide and a suitable pharmaceutical carrier.
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75. A method for increasing the diffusion of a therapeutic substance smaller than about 0.5 microns in diameter in a subject, comprising:
administering to a subject a sHASEGP polypeptide in an amount sufficient to open or to form channels smaller than about 0.5 microns in diameter and a therapeutic substance, whereby the diffusion of the therapeutic substance is increased. - View Dependent Claims (76, 77)
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78. A kit, comprising:
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(a) a sHASEGP polypeptide at a dose between 0.1 and 1500 Units/ml in an acceptable carrier;
(b) at least one therapeutic substance in an acceptable carrier; and
(c) optionally, instructions for delivering therapeutic substances. - View Dependent Claims (79)
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140. A method for the treatment of a pathologic accumulation of glycosaminoglycans, comprising:
administration of a recombinant sHASEGP, wherein said sHASEGP is free of bovine, ovine or bacterialenzymes, in an amount sufficient to ameliorate or reduce the accumulated glycosaminoglycan. - View Dependent Claims (141)
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142. A method of facilitating revascularization of necrotic tissue, comprising administration of a recombinant sHASEGP in an amount sufficient to induce the growth of neovasculature into said necrotic tissue.
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143. A method for removal of glycosaminoglucans from the vitreous humor comprising;
administration of a sHASEGP.
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154. A process for producing a substantially purified neutral active soluble hyaluronidase polypeptide and at least one N-linked sugar moiety, wherein the N-linked sugar moiety is covalently attached to an asparagine residue of the polypeptide, comprising growing Chinese Hamster Ovary cells in a suitable growth media and in the presence of 0.1-1 mM Sodium Butyrate under conditions suitable for production of the polypeptide.
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155. A process for purifying a sHASEGP, said process comprising;
- contacting sHASEGP containing media under low ionic strength with an anion exchange resin at neutral pH, and eluting said sHASEGP with about 400 mM of a salt;
contact of said sHASEGP with hydrophobic interaction chromatography resin in the presence of about 0.5M ammonium sulfate;
contact of said sHASEGP in ammonium sulfate with phenyl boronate resin;
eluting said sHASEGP in low salt at neutral pH;
contact said sHASEGP with Hydroxapatite resin, and elution of said sHASEGP with about 100 mM Na Posphate, resulting in a sHASEGP wherein said sHASEGP is substantially purified.
- contacting sHASEGP containing media under low ionic strength with an anion exchange resin at neutral pH, and eluting said sHASEGP with about 400 mM of a salt;
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159. A method of reducing intraocular pressure in the eye of a subject comprising administering to a subject in need thereof, human sHASEGP characterized as
being substantially free of bovine, ovine or bacterial enzymes; -
being greater than about 40,000 USP Units/mg protein; and
thereby reducing intraocular pressure in the subject. - View Dependent Claims (160, 161)
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Specification