Peptide extended glycosylated polypeptides
Glycosylated polypeptides comprising the primary structure NH 2 -X-Pp-COOH, wherein X is a peptide addition comprising or contributing to a glycosylation site, and Pp is a polypeptide of interest or comprising the primary structure NH 2 -P x -X-P y -COOH, wherein P x is an N-terminal part of a polypeptide Pp of interest, P y is a C-terminal part of said polypeptide Pp, and X is a peptide addition comprising or contributing to a glycosylation site are provided. The glycosylated polypeptides possess improved properties as compared to the polypeptide of interest.
1 .- 57 . (canceled)
58 . A method for increasing the serum half-life of a mature form of a polypeptide of interest, the method comprising:
a) preparing a nucleic acid comprising a nucleotide sequence encoding a peptide-extended polypeptide with the primary structure NH 2 -X-Pp-COOH, wherein
NH 2 and COOH represent the N-terminus and the C-terminus of the peptide-extended polypeptide, respectively;
X is a peptide addition 1-30 consecutive amino acids in a linear chain, wherein X comprises or contributes to an in vivo N-glycosylation site; and
Pp is the sequence of the mature form of the polypeptide of interest, wherein X and Pp are linked by a peptide linkage; and
b) expressing the nucleic acid in a mammalian host cell to provide a peptide-extended glycosylated polypeptide;
wherein the peptide-extended glycosylated polypeptide exhibits an altered glycosylation pattern and an increased serum half-life compared to that of the mature form of the polypeptide of interest when expressed under the same conditions.
59 . The method of claim 58 , further comprising:
recovering the peptide-extended glycosylated polypeptide.
60 . The method of claim 58 , wherein the polypeptide of interest is a cytokine or a hormone.
61 . The method of claim 58 , wherein X is of the formula:
X 1 ′-N-X 2 -[T/S]/C-Z wherein
X 1 ′ is absent or comprises at least one amino acid;
X 2 is any one amino acid except proline;
Z is absent or comprises at least one amino acid;
N is asparagine; and
[T/S]/C is threonine, serine, or cysteine.
62 . The method of claim 61 , wherein X is of the formula:
X 1 -(N-X 2 -[T/S])-X 3 -(N-X 2 -[T/S]) n -X 4 wherein
X 1 is absent, or is any 1, 2, 3, or 4 amino acids;
X 2 is any one amino acid except proline;
X 3 is absent, or is any 1, 2, 3, or 4 amino acids;
X 4 is absent, or is any 1, 2, 3, or 4 amino acids;
n is an integer between 0 and 6; N is asparagine; and [T/S] is threonine or serine.
63 . The method of claim 61 , wherein
X 2 is isoleucine, alanine, glycine, valine, or serine.
64 . The method of claim 58 , wherein the mammalian host cell is selected from a hamster cell line, a monkey cell line, a mouse cell line, a hamster cell line, and a human cell line.
65 . The method of claim 64 , wherein the mammalian host cell is a CHO cell.
66 . The method of claim 64 , wherein the mammalian host cell is selected from CHO-K1, COS 1, COS 7, NS/O, BHK, and HEK 293.
67 . The method of claim 59 , further comprising:
incubating the peptide-extended glycosylated polypeptide with a non-peptide moiety which differs from an oligosaccharide moiety, under conditions suitable to covalently attach said non-peptide moiety to an attachment group of the polypeptide.