G-CSF polypeptides and 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.

We claim  
     
         1 . A polypeptide conjugate exhibiting G-CSF activity, comprising a polypeptide with 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 with at least one non-polypeptide moiety attached to an attachment group of the polypeptide, the 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.  
     
     
         2 . The conjugate of  claim 1 , having an in vitro bioactivity in the range of about 3-25% of the bioactivity of hG-CSF.  
     
     
         3 . The conjugate of  claim 1 , having an in vitro bioactivity in the range of about 4-20% of the bioactivity of hG-CSF.  
     
     
         4 . The conjugate of  claim 1 , wherein at least a major portion of said conjugate has an apparent molecular weight of at least about 50 kDa as determined by the SDS-PAGE method described herein.  
     
     
         5 . The conjugate of  claim 1 , wherein at least a major portion of said conjugate has an apparent molecular weight of at least about 55 kDa as determined by the SDS-PAGE method described herein.  
     
     
         6 . The conjugate of  claim 1 , wherein at least a major portion of said conjugate has an apparent molecular weight of at least about 60 kDa as determined by the SDS-PAGE method described herein.  
     
     
         7 . The conjugate of  claim 1 , wherein the non-polypeptide moiety is selected from the group consisting of natural and synthetic homopolymers and heteropolymers.  
     
     
         8 . The conjugate of  claim 7 , wherein the polymer is a synthetic homopolymer or heteropolymer selected from the group consisting of linear or branched polyethylene glycol, polyvinyl alcohol (PVA), poly-carboxylic acids and poly-(vinylpyrrolidone).  
     
     
         9 . The conjugate of  claim 8 , wherein the polymer is a linear or branched polyethylene glycol.  
     
     
         10 . The conjugate of  claim 9 , wherein at least one polyethylene glycol moiety is attached to a lysine, cysteine, asparatic acid, glutamic acid or histidine residue.  
     
     
         11 . The conjugate of  claim 10 , wherein at least one native lysine residue has been removed and at least one lysine residue has been introduced compared to SEQ ID NO: 1.  
     
     
         12 . The conjugate of  claim 1 , wherein the polypeptide comprises an amino acid sequence that differs from the amino acid sequence shown in SEQ ID NO: 1 in at least one substitution selected from the group consisting of T1K, P2K, L3K, P5K, A6K, S7K, S8K, L9K, P10K, Q11K, S12K, F13K, L14K, L15K, E19K, Q20K, V21K, Q25K, G26K, D27K, A29K, E33K, A37K, T38K, Y39K, L41K, H43K, P44K, E45K, E46K, V48K, L50K, H52K, S53K, L54K, I56K, P57K, P60K, L61K, S62K, S63K, P65K, S66K, Q67K, A68K, L69K, Q70K, L71K, A72K, G73K, S76K, Q77K, L78K, S80K, F83K, Q86K, G87K, Q90K, E93K, G94K, S96K, P97K, E98K, L99K, G100K, P101K, T102K, D104K, T105K, Q107K, L108K, D109K, A111K, D112K, F113K, T115K, T116K, W118K, Q119K, Q120K, M121K, E122K, E123K, L124K, M126K, A127K, P128K, A129K, L130K, Q131K, P132K, T133K, Q134K, G135K, A136K, M137K, P138K, A139K, A141K, S142K, A143K, F144K, Q145K, S155K, H156K, Q158K, S159K, L161K, E162K, V163K, S164K, Y165K, V167K, L168K, H170K, L171K, A172K, Q173K and P174K.  
     
     
         13 . The conjugate of  claim 12 , wherein the polypeptide comprises at least one substitution selected from the group consisting of Q70K, Q90K, T105K, Q120K, T133K and S159K.  
     
     
         14 . The conjugate of  claim 1 , wherein at least one of amino acid residues K16, K23, K34 and K40 of SEQ ID NO: 1 has been deleted or substituted with another amino acid residue.  
     
     
         15 . The conjugate of  claim 14 , wherein at least one of K16, K23, K34 and K40 has been substituted with an R or Q residue.  
     
     
         16 . The conjugate of  claim 9 , wherein the polyethylene glycol has a molecular weight of about 1000-40,000 Da.  
     
     
         17 . The conjugate of  claim 16 , wherein the polyethylene glycol has a molecular weight of about 2000-20,000 Da.  
     
     
         18 . The conjugate of  claim 17 , wherein the polyethylene glycol has a molecular weight of about 3000-12,000 Da.  
     
     
         19 . The conjugate of  claim 9 , comprising 2-8 polyethylene glycol moieties.  
     
     
         20 . The conjugate of  claim 1 , comprising at least one amino acid alteration, compared to SEQ ID NO: 1, in an 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).  
     
     
         21 . A composition comprising a conjugate according to  claim 1  and a pharmaceutically acceptable carrier or excipient.  
     
     
         22 . A method for treating a mammal having a general haematopoietic disorder, comprising administering to a mammal in need thereof an effective amount of a conjugate according to  claim 1 .  
     
     
         23 . A polypeptide conjugate exhibiting G-CSF activity, comprising a polypeptide with 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 that has 1-8 polyethylene glycol moieties with a molecular weight of about 1000-40,000 Da attached to one or more attachment groups of the polypeptide, wherein the polypeptide conjugate has 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.  
     
     
         24 . A method for preparing a G-CSF conjugate having reduced receptor-mediated clearance compared to hG-CSF, the method comprising preparing a polypeptide with 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 to an attachment group of said polypeptide at least one non-polypeptide moiety 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.  
     
     
         25 . The method of  claim 24 , wherein at least a major portion of the conjugate has an apparent molecular weight of at least about 50 kDa as determined by the SDS-PAGE method described herein.