Fc fusion proteins of human erythropoietin with increased biological activities
OwnerLee-Hwei K. Sun, Bill N. C. Sun +1inventors
InventorsLee-Hwei K. Sun, Bill N. C. Sun, Cecily R. Y. Sun
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Abstract
Fc fusion proteins of human EPO with increased biological activities relative to rHuEPO on a molar basis are disclosed. The HuEPO-L-vFc fusion protein comprises HuEPO, a flexible peptide linker of about 20 or fewer amino acids, and a human IgG Fc variant. The Fc variant is of a non-lytic nature and shows minimal undesirable Fc-mediated side effects. A method is also disclosed to make or produce such fusion proteins at high expression levels. Such HuEPO-L-vFc fusion proteins exhibit extended serum half-life and increased biological activities, leading to improved pharmacokinetics and pharmacodynamics, thus fewer injections will be needed within a period of time.
Claims12
A recombinant HuEPO-L-vFc fusion protein consisting of HuEPO, a peptide linker, and a human IgG Fc variant, wherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgG4 with Ser228Pro and Leu235Ala mutations of SEQ ID NO:
- 20.
A method for making a recombinant fusion protein consisting of HuEPO, a flexible peptide linker, and a human IgG Fc variant, wherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgG4 with Ser228Pro and Leu235Ala mutations of SEQ ID NO:
- 20, wherein said method comprises:
- (a) generating a CHO cell line transfected with DNA encoding the recombinant HuEPO-L-vFc fusion protein;
- (b) growing the cell line under conditions wherein the recombinant fusion protein is expressed in its growth medium in excess of 10 μg per million cells in a 24 hour period;
- and (c) purifying the expressed recombinant fusion protein from step (b), wherein the recombinant fusion protein exhibits an enhanced in vitro biological activity of at least 2 fold relative to that of rHuEPO on a molar basis.
A method for making a recombinant fusion protein consisting of HuEPO, a flexible peptide linker, and a human IgG Fc variant, wherein said method comprises:
- (a) generating a CHO cell line transfected with DNA encoding the recombinant HuEPO-L-vFc fusion protein;
- (b) growing the cell line under conditions, wherein the recombinant fusion protein is expressed in its growth medium in excess of 10 μg per million cells in a 24 hour period;
- and (c) purifying the expressed recombinant fusion protein from step (b), wherein the recombinant fusion protein exhibits an enhanced in vitro biological activity of at least 2 fold relative to that of rHuEPO on a molar basis;
- wherein the flexible peptide linker contains 20 or fewer amino acids is present between HuEPO and the human IgG Fc variant;
- and the peptide linker comprises two or more amino acids selected from the group consisting of glycine, serine, alanine, and threonine;
- wherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgG4 with Ser228Pro and Leu235Ala mutations of SEQ ID NO:
- 20.
Dependencies are inferred from the claim text.