Erythropoietin analog-human serum albumin fusion

Details Erythropoietin analog-human serum albumin fusion Erythropoietin analog-human serum albumin fusion

1999-06-15

2003-04-15

Crouch, Deborah (Department: 1632)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S070100, C435S325000

Reexamination Certificate

active

06548653

ABSTRACT:

BACKGROUND OF THE INVENTION
The invention relates to erythropoiein analog-human serum albumin (EPOa-hSA) fusion proteins, nucleic acids which encode EPOa-hSA fusion proteins, and methods of making and using EPOa-hSA fusion proteins and nucleic acids.
SUMMARY OF THE INVENTION
In general, the invention features, an EPOa-hSA 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 erythropoietin (EPO) does not serve as a site for glycosylation in the EPOa, e.g., an EPOa-hSA fusion protein in which at least one amino acid residue which can serve as a glycosylation site in erythropoietin is altered, e.g., by substitution or deletion, such that it does not serve as a glycosylation site.
In a preferred embodiment, the EPOa-hSA fusion protein has the formula: R1-L-R2; R2-L-R1; or R1-L-R2-L-R1, wherein R1 is an EPOa amino acid sequence, L is a peptide linker and R2 is human serum albumin amino acid sequence. Preferably, R1 and R2 are covalently linked via the peptide linker.
In a preferred embodiment: an amino acid residue of EPO which serves as an attachment point for glycosylation has been deleted; an amino acid residue of EPO which serves as a site for glycosylation has been replaced with an amino acid residue which does not serve as a site for glycosylation; the amino acid residue which is altered is selected from the group consisting of amino acid residues Asn24, Asn38, Asn83 and Ser126; the glycosylation site at amino acid residue Ser126 and at least one additional N-linked glycosylation site selected from the group consisting of Asn24, Asn38 and Asn83 are altered; a glycosylation site which provides for N-linked glycosylation is altered by replacing an Asn residue with an amino acid residue other than it, e.g., Gln; a glycosylation site which provides for O-linked glycosylation is altered by replacing a Ser residue with an amino acid residue other than it, e.g., Ala.
In preferred embodiments, the EPOa-hSA fusion protein is made in a mammary gland of a transgenic mammal, e.g., a ruminant, e.g., a goat.
In preferred embodiments, the EPOa-hSA fusion protein is secreted into the milk of a transgenic mammal, e.g., a ruminant, e.g., a goat.
In preferred embodiments, the EPOa-hSA fusion protein is made, in a transgenic animal, under the control of a mammary gland specific promoter, e.g., a milk specific promoter, e.g., a milk serum protein or casein promoter. The milk specific promoter can be a casein promoter, beta lactoglobulin promoter, whey acid protein promoter, or lactalbumin promoter. Preferably, the promoter is a goat &bgr; casein promoter.
In preferred embodiments, the EPOa-hSA fusion protein, in a transgenic animal, and is secreted into the milk of a transgenic mammal at concentrations of at least about 0.2 mg/ml, 0.5 mg/ml, 0.75 mg/ml, 1 mg/ml, 2 mg/ml, 3 mg/ml or higher.
In a preferred embodiment, amino acid residue Asn24 has been altered, e.g., substituted or deleted. Preferably, the amino acid residue Asn24 has been replaced with Gln.
In a preferred embodiment, amino acid residue Asn38 has been altered, e.g., substituted or deleted. Preferably, amino acid residue Asn38 has been replaced with Gln.
In a preferred embodiment, amino acid residue Asn83 has been altered, e.g., substituted or deleted. Preferably, the amino acid residue Asn83 has been replaced with Gln.
In yet another embodiment, amino acid residue Ser126 has been altered, e.g., substituted or deleted. Preferably, the amino acid residue Ser126 has been replaced with Ala.
In a preferred embodiment: each of amino acid residue Asn24, Asn38, Asn83 and Ser126 has been altered, e.g., substituted or deleted, such that it does not serve as a glycosylation site; each of the amino acid residues Asn24, Asn28, Asn83 and Ser126 has, respectively, been replaced with Gln, Gln, Gln, and Ala.
In a preferred embodiment, the fusion protein includes a peptide linker and the peptide linker has one or more of the following characteristics: a) it allows for the rotation of the erythropoietin analog amino acid sequence and the human serum albumin amino acid sequence relative to each other; b) it is resistant to digestion by proteases; and c) it does not interact with the erythropoietin analog or the human serum albumin.
In a preferred embodiment: the fusion protein includes a peptide linker and the peptide linker is 5 to 60, more preferably, 10 to 30, amino acids in length; the peptide linker is 20 amino acids in length; the peptide linker is 17 amino acids in length; each of the amino acids in the peptide linker is selected from the group consisting of Gly, Ser, Asn, Thr and Ala; the peptide linker includes a Gly-Ser element.
In a preferred embodiment, the fusion protein includes a peptide linker and the peptide linker includes a sequence having the formula (Ser-Gly-Gly-Gly-Gly)
y
wherein y is 1, 2, 3, 4, 5, 6, 7, or 8. Preferably, the peptide linker includes a sequence having the formula (Ser-Gly-Gly-Gly-Gly)
3
. Preferably, the peptide linker includes a sequence having the formula ((Ser-Gly-Gly-Gly-Gly)
3
-Ser-Pro).
In a preferred embodiment, the fusion protein includes a peptide linker and the peptide linker includes a sequence having the formula (Ser-Ser-Ser-Ser-Gly)
y
wherein y is 1, 2, 3, 4, 5, 6, 7, or 8. Preferably, the peptide linker includes a sequence having the formula ((Ser-Ser-Ser-Ser-Gly)
3
-Ser-Pro).
In another aspect, the invention features, an EPOa-hSA fusion protein wherein the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO (i.e., only amino acids 24, 38, 83, and 126 differ from wild type).
In another aspect, the invention features, an EPOa-hSA fusion protein which includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)
3
-Ser-Pro), and human serum albumin.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment the fusion protein is from left to right, Gln24, Gln38, Gln83, Ala126 EPO, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)
3
-Ser-Pro), and human serum albumin.
In another aspect, the invention features, an EPOa-hSA fusion protein which includes, from left to right, human serum albumin, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)
3
-Ser-Pro), and an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In a preferred embodiment the fusion protein is from left to right, human serum albumin, a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)
3
-Ser-Pro), and Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, an isolated nucleic acid having a nucleotide sequence which encodes an EPOa-hSA fusion protein described herein, e.g., an EPOa-hSA fusion protein wherein at least one amino acid residue is altered such that a site which serves as a site for glycosylation in EPO does not serve as a site for glycosylation in the EPOa, e.g., an EPOa-hSA fusion protein in which at least one amino acid residue of the encoded EPOa-hSA which can serve as a glycosylation site in erythropoietin is altered, e.g., by substitution or deletion, such that it does not serve as a glycosylation site.
In another aspect, the invention features, a nucleic acid which encodes an EPOa-hSA fusion protein wherein the EPOa includes amino acid residues Gln24, Gln38, Gln83 and Ala126.
In a preferred embodiment the EPOa is Gln24, Gln38, Gln83, Ala126 EPO.
In another aspect, the invention features, a nucleic acid which encodes an EPOa-hSA fusion protein which includes from left to right, an EPOa which includes amino acid residues Gln24, Gln38, Gln83 and Ala126, a peptide linker, e.g., a peptide linker having the formula ((Ser-Gly-Gly-Gly-Gly)
3
-Ser-Pro), and human serum albumin.
In a preferred embodiment the EPOa i

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