G-CSF CONJUGATES
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.