Soluble glycosaminoglycanases and methods of preparing and using soluble glycosaminoglycanases

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.

1 . A method for treatment of a hyaluronan-associated disease or condition, comprising:
 administering to a subject a pharmaceutical composition, comprising a PEGylated hyaluronidase in a pharmaceutically acceptable carrier, wherein:   the hyaluronidase contains about three to six PEG moieties per hyaluronidase molecule; and
 the hyaluronidase polypeptide is a human-derived hyaluronidase. 
   
     
     
         2 . The method of  claim 1 , wherein the composition is administered systemically. 
     
     
         3 . The method of  claim 1 , wherein the composition is administered locally for treatment of an ophthalmic condition. 
     
     
         4 . The method of  claim 1 , wherein the composition is administered intravenously. 
     
     
         5 . The method of  claim 1 , wherein the composition is administered intrathecally. 
     
     
         6 . The method of  claim 1 , wherein the composition is administered to the brain. 
     
     
         7 . The method of  claim 1 , wherein the disease or condition is a tumor. 
     
     
         8 . The method of  claim 7 , wherein the composition is administered intravenously. 
     
     
         9 . The method of  claim 1 , wherein the composition is administered for treating myxedema requiring chronic treatment. 
     
     
         10 . The method of  claim 1 , wherein the PEG moieties are branched. 
     
     
         11 . The method of  claim 1 , wherein the PEG moieties result from reaction with a PEG reagent selected from among mPEG-succinimidyl butanoate (mPEG-SBA) (5 kDa), mPEG-SBA (20 kDa), mPEG-SBA (30 kDa), mPEG-succinimidyl α-methylbutanoate (mPEG-SMB) (20 kDa), mPEG-SMB (30 kDa), mPEG-butyraldehyde (30 kDa), mPEG-succinimidyl propionate (mPEG-SPA) (20 kDa), mPEG-SPA (30 kDa), mPEG2-N-Hydroxylsuccinimide (mPEG2-NHS) (10 kDa branched), mPEG2-NHS (20 kDa branched), mPEG2-NHS (40 kD a branched), mPEG2-NHS (60 kD a branched), PEG-NHS-biotin (5 kDa biotinylated), PEG-p-nitrophenyl-carbonate (30 kDa) and PEG-propionaldehyde (30 kDa). 
     
     
         12 . The method of  claim 1 , wherein the PEG moiety results from reaction with mPEG-Succinimidyl Butanoate 30 kD (mPEG-SBA (30 kD)). 
     
     
         13 . The method of  claim 1 , wherein the pharmaceutical composition is formulated for intravenous administration. 
     
     
         14 . The method of  claim 1 , wherein the PEGylated hyaluronidase is soluble and active at neutral pH. 
     
     
         15 . The method of  claim 1 , wherein the pharmaceutical composition comprises a PEGylated hyaluronidase that is active at neutral pH and contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the hyaluronidase polypeptide, wherein:
 the hyaluronidase polypeptide is a C-terminal truncated polypeptide of SEQ ID NO:1; and contains amino acid residues 36-464 as set forth in SEQ ID NO:1 but does not include the complete sequence of amino acids set forth in SEQ ID NO:1 or contains a sequence of amino acid residues that has at least 95% amino acid sequence identity with the sequence of amino acids 36-464 set forth in SEQ ID NO:1.   
     
     
         16 . The method of  claim 1 , wherein:
 the PEGylated hyaluronidase is active at neutral pH and contains at least one sugar moiety that is covalently attached to an asparagine (N) residue of the hyaluronidase polypeptide; and   a) the hyaluronidase consists of the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO:1; or   b) the hyaluronidase contains amino acid substitutions in the sequence of amino acids set forth as amino acids 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 consists of a sequence of amino acids that has at least 95% amino acid sequence identity with the sequence of amino acids set forth as amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482, or 36-483 of SEQ ID NO:1.   
     
     
         17 . The method of  claim 1 , wherein a hyaluronidase in the composition consists of amino acids 36-477, 36-478, 36-479, 36-480, 36-481, 36-482 or 36-483 as set forth in SEQ ID NO:1. 
     
     
         18 . The method of  claim 1 , wherein the hyaluronidase is produced by expression of a nucleic acid molecule that encodes amino acids 1-482 or 36-482 of SEQ ID NO:1. 
     
     
         19 . The method of  claim 1 , wherein the hyaluronidase polypeptide has a sequence of amino acids that has least 95% sequence identity to the sequence set forth in SEQ ID NO:4. 
     
     
         20 . The method of  claim 1 , wherein 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 residues 36-483 in SEQ ID NO:1. 
     
     
         21 . The method of  claim 1 , further comprising administering an anti-cancer agent or treatment, wherein the PEGylated hyaluronidase and the anti-cancer agent or treatment are administered together or sequentially. 
     
     
         22 . The method of  claim 1 , further comprising administering an antibody with the hyaluronidase, wherein the PEGylated hyaluronidase and therapeutic antibody are administered together or sequentially. 
     
     
         23 . The method of  claim 22 , wherein the therapeutic antibody is a monoclonal antibody. 
     
     
         24 . The method of  claim 21 , wherein the anti-cancer agent is an oncolytic virus.