G-csf conjugates

Abstract

The invention relates to polypeptide conjugates comprising a polypeptide exhibiting G-CSF activity and having an amino acid sequence that differs from the amino acid sequence of human G-CSF in at least one specified introduced and/or removed amino acid residue comprising an attachment group for a non-polypeptide moiety, and having at least one non-polypeptide moiety attached to an attachment group of the polypeptide. The attachment group may e.g. be a lysine, cysteine, aspartic acid or glutamic acid residue or a glycosylation site, and the non-polypeptide moiety may e.g. be a polymer such as polyethylene glycol or an oligosaccharide. The conjugate, which has a reduced in vitro bioactivity compared to hG-CSF, has one or more improved properties such as increased biological half-life and increased stimulation of neutrophils.

1 .- 25 . (canceled)  
     
     
         26 . A polypeptide exhibiting G-CSF cell proliferation activity, comprising an amino acid sequence that differs from the hG-CSF sequence shown in SEQ ID NO:1 in no more than 15 amino acid residues and comprises the substitution S159K.  
     
     
         27 . The polypeptide of  claim 26 , further comprising at least one substitution selected from the group consisting of K16R/Q, K34R/Q and K40R/Q.  
     
     
         28 . The polypeptide of  claim 27 , comprising the substitutions K16R, K34R, K40R and S159K.  
     
     
         29 . The polypeptide of  claim 26 , further comprising at least one substitution selected from the group consisting of Q70K, Q90K, T105K, Q120K and T133K.  
     
     
         30 . The polypeptide of  claim 26 , wherein the amino acid sequence differs from SEQ ID NO:1 in no more than 10 amino acid residues.  
     
     
         31 . The polypeptide of  claim 26 , wherein the amino acid sequence differs from SEQ ID NO:1 in no more than 8 amino acid residues.  
     
     
         32 . The polypeptide of  claim 26 , wherein the polypeptide is glycosylated.  
     
     
         33 . The polypeptide of  claim 26 , further comprising at least one non-polypeptide moiety covalently attached to the polypeptide.  
     
     
         34 . The polypeptide of  claim 33 , wherein the non-polypeptide moiety is covalently attached a lysine residue of the polypeptide.  
     
     
         35 . The polypeptide of  claim 33 , wherein the non-polypeptide moiety is a polymer.  
     
     
         36 . The polypeptide of  claim 35 , wherein the polymer is selected from the group consisting of a polyethylene glycol, a polyvinylalcohol (PVA), a poly-carboxylic acid and a poly(vinylpyrrolidone).  
     
     
         37 . A method for treating a mammal having a general haematopoietic disorder, comprising administering to the mammal an effective amount of a composition comprising the polypeptide of  claim 26  and a pharmaceutically acceptable carrier or excipient.  
     
     
         38 . A nucleic acid comprising a nucleotide sequence encoding the polypeptide of  claim 26 .  
     
     
         39 . A method for producing a polypeptide, comprising providing a cell culture comprising a host cell comprising an expression vector comprising the nucleic acid of  claim 38 , cultivating the cell culture under conditions which permit expression of the polypeptide, and recovering the polypeptide.  
     
     
         40 . The method of  claim 39 , wherein the host cell is a glycosylating host cell selected from an  S. cerevisiae  cell, a  Pichia pastoris  cell, a CHO cell, a BHK cell, an HEK 293 cell, and an SF9 cell.  
     
     
         41 . The method of  claim 39 , further comprising covalently attaching at least one non-polypeptide moiety to the polypeptide.  
     
     
         42 . A method for preparing a polypeptide conjugate, the method comprising 
 (i) preparing the polypeptide of  claim 26 , and    (ii) attaching at least one non-polypeptide moiety to a lysine residue of the polypeptide,    wherein the resulting conjugate exhibits G-CSF activity.    
     
     
         43 . The method of  claim 42 , wherein the non-polypeptide moiety is a polymer selected from the group consisting of a polyethylene glycol, a polyvinylalcohol (PVA), a poly-carboxylic acid and a poly-(vinylpyrrolidone).  
     
     
         44 . The method of  claim 42 , wherein the step of preparing the polypeptide comprises: providing a culture comprising a host cell, the host cell comprising an expression vector comprising a nucleic acid comprising a nucleotide sequence which encodes the polypeptide, culturing the culture under conditions which permit expression of the polypeptide, and recovering the polypeptide.  
     
     
         45 . The method of  claim 44 , wherein the host cell is a glycosylating host cell.  
     
     
         46 . The method of  claim 44 , wherein the host cell is a bacterial host cell.  
     
     
         47 . An expression vector comprising the nucleic acid of  claim 38 .  
     
     
         48 . A host cell comprising the nucleic acid of  claim 38 .  
     
     
         49 . The host cell of  claim 48 , wherein the host cell is a bacterial host cell.  
     
     
         50 . The host cell of  claim 48 , wherein the host cell is a glycosylating host cell.  
     
     
         51 . The host cell of  claim 50 , selected from an  S. cerevisiae  cell, a  Pichia pastoris  cell, a CHO cell, a BHK cell, an HEK 293 cell, and an SF9 cell.