Fc fusion proteins of human erythropoietin with high 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 high 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.
Claims20
A recombinant HuEPO-L-vFc fusion protein comprising HuEPO, a peptide linker, and a human IgG Fc variant.
The HuEPO-L-vFc fusion protein of any of the preceding claims exhibits in vitro biological activity similar to or higher than that of rHuEPO on a molar basis.
A CHO-derived cell line producing the HuEPO-L-vFc fusion protein of any of the preceding claims in its growth medium in excess of 10 μg per million cells in a 24 hour period.
A method for making a recombinant fusion protein comprising HuEPO, a flexible peptide linker, and a human IgG Fc variant, which method comprises:
- (a) generating a CHO-derived cell line;
- (b) growing the cell line under conditions the recombinant 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 protein from step (b), wherein the recombinant fusion protein exhibits in vitro biological activity similar to or higher than that of rHuEPO on a molar basis.
A method for making a recombinant fusion protein comprising HuEPO, a flexible peptide linker, and a human IgG Fc variant, which method comprises:
- (a) generating a CHO-derived cell line;
- (b) growing the cell line under conditions the recombinant 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 protein from step (b), wherein the recombinant fusion protein exhibits in vitro biological activity similar to or higher than that of rHuEPO on a molar basis;
- wherein the flexible peptide linker containing about 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 selected from the group consisting of human IgG2 with Pro331Ser mutation as SEQ ID NO:
- 18, human IgG4 with Ser228Pro and Leu235Ala mutations as SEQ ID NO:
- 20, and human IgB1 with Leu234Val, Leu235Ala, and Pro331Ser mutations as SEQ ID NO:
- 22.
Dependencies are inferred from the claim text.