Soluble Glycosaminoglycanases and Methods of Preparing and Using Soluble Glycosaminoglycanases
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 (sHASEGPs), 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.
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Citations
157 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.
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2-12. -12. (canceled)
- 13. A polypeptide, consisting essentially of a hyaluronidase domain or a catalytically active portion thereof of a human hyaluronidase protein.
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14-79. -79. (canceled)
- 80. A method of promoting delivery of a sHASEGP or other glycosaminoglycanase (GAG Enzyme) into a first tissue within a mammal, comprising introducing a volume of liquid comprising the GAG Enzyme into the first tissue and allowing the volume of liquid and catalytic activity of the GAG Enzyme to promote delivery of the GAG Enzyme into the tissue.
- 94. A method of increasing the permeability of a target tissue of a mammal, comprising introducing a sHASEGP into the tissue.
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119. A method of ameliorating the consequences of stroke or other CNS injury in a mammal, comprising introducing a sHASEGP into a mammal following said stroke or CNS injury.
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124-127. -127. (canceled)
- 128. A PEGylated hyaluronidase, wherein the hyaluronidase comprises at least about three PEG moieties per polypeptide.
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129-133. -133. (canceled)
- 134. A method of preparing a PEGylated hyaluronidase, comprising reacting a hyaluronidase with a molar excess of a PEG reagent selected from the group consisting of a PEG-succinimidyl butanoate, a PEG-succinimidyl methylbutanoate, a PEG-succinimidyl proprionate, a PEG-N-hydroxysuccinimide, a PEG-aldehyde, a PEG-carbonate, a PEG-propionaldehyde and a PEG-maleimide.
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142-143. -143. (canceled)
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145-153. -153. (canceled)
Specification