ERYTHROPOIETIN ANALOG-IgG FUSION PROTEINS
Erythropoietin analog-human IgG fusion protein (EPOa-IgG) fusion protein and methods of making and using the fusion protein.
1 . An EPOa-IgG fusion protein, wherein at least one amino acid residue of the EPOa moiety of the fusion protein is altered such that a site which serves as a site for glycosylation in EPO does not serve as a site for glycosylation in EPOa.
2 . The EPOa-IgG fusion protein of claim 1 , wherein said fusion protein has the formula:
R1-L-R2; R2-L-R1; or R1-L-R2-L-R1,
wherein R1 is an erythropoietin analog amino acid sequence; L is a peptide linker and R2 is a human IgG immunoglobulin amino acid sequence.
3 - 4 . (canceled)
5 . The EPOa-IgG fusion protein of claim 1 , wherein the at least one amino acid residue of the EPOa moiety which serves as a site for glycosylation has been replaced with an amino acid residue which does not serve as a site for glycosylation.
6 . The EPOa-IgG fusion protein of claim 1 , wherein said at least one amino acid residue is selected from the group consisting of amino acid residues Asn24, Asn38, Asn83 and Ser126 of the EPO moiety.
7 . (canceled)
8 . The EPOa-IgG fusion protein of claim 1 , wherein said glycosylation site is an N-linked glycosylation site and is altered by replacing an amino acid residue Asn of the EPO moiety with Gln.
9 . The EPOa-IgG fusion protein of claim 1 , wherein said glycosylation site is an O-linked glycosylation and is altered by replacing an amino acid residue Ser of the EPO moiety with Gln.
10 . The EPOa-IgG fusion protein of claim 1 , wherein the amino acid residues 24, 38, or 83 of the EPO moiety have been altered.
11 . The EPOa-IgG fusion protein of claim 10 , wherein the amino acid residues 24, 38, or 83 of the EPO moiety have been replaced with Gln.
12 . The EPOa-IgG fusion protein of claim 1 , wherein the amino acid residue 126 of the EPO moiety has been altered.
13 . The EPOa-IgG fusion protein of claim 12 , wherein said amino acid residue 126 of the EPO moiety has been replaced with Ala.
14 . The EPOa-IgG fusion protein of claim 1 , wherein the amino acid residues 24, 38, 83 and 126 of the EPO moiety have been altered such that none of them serves as a glycosylation site.
15 - 19 . (canceled)
20 . The EPOa-IgG fusion protein of claim 14 , wherein the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
21 - 26 . (canceled)
27 . An isolated nucleic acid comprising a nucleotide sequence which encodes an EPOa-IgG fusion protein, wherein at least one amino acid residue of the encoded EPOa-IgG which can serve as a glycosylation site in EPO is altered such that it does not serve as a glycosylation site in EPOa.
28 - 32 . (canceled)
33 . A method of making an EPOa-IgG fusion protein comprising:
providing a transgenic organism which includes a transgene which directs the expression of the EPOa-IgG fusion protein;
allowing the transgene to be expressed; and,
recovering the EPOa-IgG fusion protein.
34 - 39 . (canceled)
40 . A transgenic organism, which includes a transgene which encodes an EPOa-IgG fusion protein.
41 - 45 . (canceled)
46 . A pharmaceutical composition having a therapeutically effective amount of an EPOa-IgG fusion protein.
47 . A method of treating a subject in need of erythropoietin comprising administering a therapeutically effective amount of an EPOa-IgG fusion protein to the subject.
48 - 54 . (canceled)
55 . A method for making an EPOa-IgG fusion protein in a cultured cell comprising supplying a cell which includes a nucleic acid which encodes an EPOa-IgG fusion protein, and expressing the EPOa-IgG fusion protein from the nucleic acid, thereby making the EPOa-IgG fusion protein.
56 . (canceled)
57 . An EPOa-IgG fusion protein, wherein both the EPOa moiety and the human IgG moiety of the fusion protein are altered such that any site that serves as a site for glycosylation is altered such that it cannot serve as a site for glycosylation in the EPOa-IgG fusion protein, making the entire EPOa-IgG fusion protein non-glycosylated.
58 - 60 . (canceled)
61 . The method of claim 55 , wherein the EPOa-IgG fusion protein is made in a mammary gland of a transgenic mammal under the control of a milk specific promoter.