G-CSF conjugates

Abstract

Polypeptide conjugates with G-CSF activity comprising a polypeptide having at least one introduced lysine residue and at least one removed lysine residue compared to the sequence of human G-CSF, and which are conjugated to 2-6 polyethylene glycol moieties. The conjugates have a low in vitro bioactivity, a long in vivo half-life, a reduced receptor-mediated clearance, and provide a more rapid stimulation of production of white blood cells and neutrophils than non-conjugated recombinant human G-CSF.

1 . A polypeptide conjugate exhibiting G-CSF activity, comprising a polypeptide comprising at least one substitution selected from the group consisting of K16R/Q, K34R/Q and K40R/Q, and at least one substitution selected from the group consisting of T105K and S159K, relative to the amino acid sequence of hG-CSF shown in SEQ. ID NO:1 or in a corresponding position relative to an amino acid sequence having at least 80% sequence identity with SEQ ID NO:1, the conjugate having 2-6 polyethylene glycol moieties with a molecular weight of about 1000-10,000 Da attached to attachment groups of the polypeptide.  
     
     
         2 . The polypeptide conjugate of  claim 1 , comprising three, four or five of said substitutions.  
     
     
         3 . The polypeptide conjugate of  claim 1 , further comprising the substitution H170K, H1170Q or H170R.  
     
     
         4 . The polypeptide conjugate of  claim 1 , comprising substitutions selected from the group consisting of: Q70K+S 159K, Q70K+H170K, Q90K+S 159K, Q90K+H170K, T105K+S159K, T105K+H170K, Q120K+S159K, Q120K+H170K, T133K+S159K, T133K+H170K, S159K+H170K, Q70K+Q90K+S159K, Q70K+Q90K+H170K, Q70K+T105K+S159K, Q70K+T105K+H170K, Q70K+Q120K+S159K, Q70K+Q120K+H170K, Q70K+T133K+S159K, Q70K+T133K+H170K, Q70K+S159K+H170K, Q90K+T105K+S159K, Q90K+T105K+H170K, Q90K+Q120K+S159K, Q90K+Q120K+H170K, Q90K+T133K+S159K, Q90K+T133K+H170K, Q90+S159K+H170K, T 05K+Q120K+S159K, T105K+Q120K+H170K, T105K+T133K+S159K, T105K+T133K+H170K, T105K+S159K+H170K, Q120K+T133K+S159K, Q120K+T133K+H170K, Q120K+S159K+H170K, T133K+S159K+H170K, Q70K+Q90K+T105K+S159K, Q70K+Q90K+T105K+H170K, Q70K+Q90K+Q120K+S159K, Q70K+Q90K+Q120K+H170K, Q70K+Q90K+T133K+S159K, Q70K+Q90K+T133K+H170K, Q70K+Q90K+S159K+H170K, Q70K+T105K+Q120K+S159K, Q70K+T105K+Q120K+H170K, Q70K+T105K+T133K+S159K, Q70K+T105K+T133K+H170K, Q70K+T105K+S159K+H170K, Q70K+Q120K+T133K+S159K, Q70K+Q120K+T133K+H170K, Q70K+T133K+S159K+H170K, Q90K+T105K+Q120K+S159K, Q90K+T105K+Q120K+H170K, Q90K+T105+T133K+S159K, Q90K+T105+T133K+H170K, Q90K+T105+S159K+H170K, Q90K+Q120K+T133K+S159K, Q90K+Q120K+T133K+H170K, Q90K+Q120K+S159K+H170K, Q90K+T133K+S159K+H170K, T105K+Q120K+T133K+S159K, T105K+Q120K+T133K+H170K, T105K+Q120K+S159K+H170K, T105K+T133K+S159K+H170K and Q120K+T133K+S159K+H170K.  
     
     
         5 . The polypeptide conjugate of  claim 1 , comprising substitutions selected from the group consisting of K16R+K23R, K16R+K34R, K16R+K40R, K23R+K34R, K23R+K40R, K34R+K40R, K16R+K23R+K34R, K16R+K23R+K40R, K23R+K34R+K40R, K16R+K34R+K40R and K16R+K23R+K34R+K40R.  
     
     
         6 . The polypeptide conjugate of  claim 5 , comprising the substitutions K16R+K34R+K40R, and comprising a lysine residue in position 23.  
     
     
         7 . The polypeptide conjugate of  claim 1 , wherein said substitutions are in a corresponding position relative to an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:1.  
     
     
         8 . The polypeptide conjugate of  claim 7 , wherein said substitutions are in a corresponding position relative to an amino acid sequence having at least about 95% sequence identity with SEQ ID NO:1  
     
     
         9 . The polypeptide conjugate of  claim 1 , wherein the polyethylene glycol moieties are attached to at least one lysine residue and optionally also to the N-terminal amino group.  
     
     
         10 . The polypeptide conjugate of  claim 1 , comprising a polypeptide comprising the substitutions K16R, K34R, K40R, T105K and S159K relative to the amino acid sequence of hG-CSF shown in SEQ ID NO:1 and having 2-6 polyethylene glycol moieties with a molecular weight of about 1000-10,000 Da attached to attachment groups of the polypeptide.  
     
     
         11 . The polypeptide conjugate of  claim 1 , which is glycosylated at T133.  
     
     
         12 . The polypeptide conjugate of  claim 1 , having 3-5, 4-6, 3-4, 4-5 or 5-6 attached PEG moieties.  
     
     
         13 . The polypeptide conjugate of  claim 12 , having 3-6 polyethylene glycol moieties with a molecular weight of about 5000-6000 Da attached.  
     
     
         14 . The polypeptide conjugate of  claim 13 , having 4 PEG moieties of about 5 kDa attached.  
     
     
         15 . The polypeptide conjugate of  claim 13 , having 5 PEG moieties of about 5 kDa attached.  
     
     
         16 . The polypeptide conjugate of  claim 1 , having an in vitro bioactivity in the range of about 2-30% of the bioactivity of non-conjugated hG-CSF as determined by the luciferase assay described herein.  
     
     
         17 . A method for preparing a G-CSF conjugate which provides a reduced duration of neutropenia compared to hG-CSF, the method comprising preparing a polypeptide comprising an amino acid sequence that differs in at least one amino acid residue from the amino acid sequence of hG-CSF shown in SEQ ID NO:1, and attaching a non-polypeptide moiety to each of at least two attachment groups of said polypeptide to result in a conjugate having an in vitro bioactivity in the range of about 2-30% of the bioactivity of non-conjugated hG-CSF as determined by the luciferase assay described herein.  
     
     
         18 . The method of  claim 17 , wherein a desired in vitro bioactivity is obtained by alteration of at least one amino acid residue selected from amino acid position 11-41 (helix A), 71-95 (helix B), 102-125 (helix C), and 145-170 (helix D), relative to the amino acid sequence of SEQ ID NO:1, and by conjugation of at least one non-polypeptide moiety to the at least one altered amino acid residue.  
     
     
         19 . A composition comprising the polypeptide conjugate of  claim 1  and at least one pharmaceutically acceptable carrier or excipient.  
     
     
         20 . A method for treating a mammal suffering from an insufficient neutrophil level, comprising administering to a mammal in need thereof a therapeutically effective amount of the polypeptide conjugate of  claim 1.