Branched Peg Remodeling and Glycosylation of Glucagon-Like Peptides-1 [Glp-1]

Abstract

The present invention provides polypeptides that include an O-linked glycoconjugate in which a species such as a water-soluble polymer, a therapeutic agent of a biomolecule is covalently linked through an intact O-linked glycosyl residue to the polypeptide. The polypeptides of the invention include wild-type peptides and mutant peptides that include an O-linked glycosylation site that is not present in the wild-type peptide. Also provided are methods of making the peptides of the invention and methods, pharmaceutical compositions containing the peptides and methods of treating, ameliorating or preventing diseased in mammals by administering an amount of a peptide of the invention sufficient to achieve the desired response.

1 . A peptide having a formula selected from: 
       
         
           
           
               
               
           
         
         in which AA is an amino acid with a side chain that comprises a hydroxyl moiety; and
 X a modifying group or it is a saccharyl moiety. 
 
       
     
     
         2 . The peptide according to  claim 1 , wherein AA is introduced into said peptide via mutation of wild-type peptide. 
     
     
         3 . The peptide according to  claim 1 , wherein X comprises a group selected from sialyl, galactosyl and Gal-Sia moieties, wherein at least one of said sialyl, galactosyl and Gal-Sia comprises a modifying group. 
     
     
         4 . The peptide according to  claim 1 , wherein X comprises poly(ethylene glycol). 
     
     
         5 . The peptide according to  claim 1 , wherein X comprises monomethoxy-poly(ethylene glycol). 
     
     
         6 . The peptide according to  claim 5 , wherein X comprises the structure: 
       
         
           
           
               
               
           
         
         in which L is a substituted or unsubstituted alkyl or substituted or unsubstituted heteroalkyl group; and n is selected from the integers from 0 to about 500. 
       
     
     
         7 . The peptide according to  claim 5 , wherein X comprises the structure: 
       
         
           
           
               
               
           
         
         in which s is selected from the integers from 0 to 20. 
       
     
     
         8 . An isolated nucleic acid comprising a polynucleotide sequence encoding a mutant polypeptide, wherein the mutant polypeptide comprises an O-linked glycosylation site that does not exist in the corresponding wild-type polypeptide. 
     
     
         9 . The nucleic acid of  claim 8 , wherein the polypeptide is a GLP-1 polypeptide. 
     
     
         10 . An expression cassette comprising the nucleic acid of  claim 8 . 
     
     
         11 . A cell comprising the nucleic acid of  claim 8 . 
     
     
         12 . A method for making a glycoconjugate of a mutant polypeptide, which comprises an O-linked glycosylation that does not exist in the corresponding wild-type polypeptide, comprising the steps of:
 (a) recombinantly producing the mutant polypeptide, and   (b) enzymatically glycosylating the mutant polypeptide with a modified sugar at said O-linked glycosylation site.   
     
     
         13 . The method of  claim 12 , wherein the corresponding mutant polypeptide has an amino acid sequence selected from the group consisting of: 
       
         
           
                 
               
                     
                 
                   GLP-1 Glycopeptides 
                 
                   
                     
                       
                       
                           
                           
                       
                     
                   
                 
                     
                 
                     
                 
                 
                 
               
                     
                   Ac-X-HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2   
                 
                 
                 
               
                     
                   H-X-EGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HA-X-GTFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAE-X-TFTSDVSSYLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEG-X-FTSDVSSYLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGT-X-TSDVSSYLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTF-X-SDVSSYLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFT-X-DVSSYLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTS-X-VSSYLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSD-X-SSYLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFVSDV-X-SYLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVS-X-YLEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSS-X-LEGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSY-X-EGQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYL-X-GQAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLE-X-QAAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEG-X-AAKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQ-X-AKEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQA-X-KEFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAA-X-EFIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAK-X-FIAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAKE-X-IAWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAKEF-X-AWLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAKEFI-X-WLVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAKEFIA-X-LVKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAKEFIAW-X-VKGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAKEFIAWL-X-KGR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAKEFIAWLV-X-GR-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAKEFIAWLVK-X-R-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAKEFIAWLVKG-X-NH 2   
                 
                     
                   HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-X-NH 2   
                 
                     
                     
                 
             
                
                
                
                
               
               
                
               
            
             
                
               
            
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         14 . A pharmaceutical composition of a Glucagon-Like Peptide-1 comprising an effective amount of a mutant polypeptide, which comprises an O-linked glycosylation site that does not exist in the corresponding wild-type Glucagon-Like Peptide-1, wherein said peptide is glycoconjugated with a modified sugar. 
     
     
         15 . The pharmaceutical composition according to  claim 13 , wherein said modified sugar is modified with a member selected from poly(ethylene glycol) and m-poly(ethylene glycol). 
     
     
         16 . A method of providing Glucagon-Like Peptide-1 therapy to a subject in need of said therapy, said method comprising, administering to said subject an amount of an O-linked glyco-PEG-ylated Glucagon-Like Peptide-1 sufficient to provide a therapeutic effect. 
     
     
         17 . The method according to  claim 16 , wherein said O-linked glyco-PEG-ylated Glucagon-Like Peptide-1 is glyco-PEG-ylated on an amino acid residue not present in wild type Glucagon-Like Peptide-1.