Fc fusion proteins of human erythropoietin with increased biological activities
View Patent ↗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.
1. 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 IgG2 with Pro331Ser mutation as SEQ ID NO: 18.
2. The peptide linker in claim 1 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.
3. The HuEPO-L-vFc fusion protein of claim 1 which exhibits an enhanced in vitro biological activity of at least 2 fold relative to that of rHuEPO on a molar basis.
4. A CHO cell line transfected with DNA encoding the recombinant HuEPO-L-vFc fusion protein of claim 1 in its growth medium in excess of 10 μg per million cells in a 24 hour period.
5. The CHO cell line transfected with DNA encoding the recombinant HuEPO-L-vFc fusion protein of claim 4 in its growth medium in excess of 30 μg per million cells in a 24 hour period.
6. The CHO cell line transfected with DNA encoding the recombinant HuEPO-L-vFc fusion protein of claim 1 , wherein the human lgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgG2 with Pro331Ser mutation as SEQ ID NQ:18, the IgG Fc contains amino acid mutations to attenuate effector functions, a flexible peptide linker containing about 20 or fewer amino acids is present between HuEPO and human IgG Fc variant, and the HuEPQ-L-vFc fusion protein exhibits an enhanced in vitro biological activity of at least 2 fold relative to that of rHuEPO on a molar basis.
7. 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 domains of human IgG2 with Pro331Ser mutation as SEQ ID NO: 18, which 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 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 an enhanced in vitro biological activity of at least 2 fold relative to that of rHuEPO on a molar basis.
8. The method of claim 7 , wherein step (b) is in excess of 30 μg per million cells in a 24 hour period.
9. The method of claim 7 , 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.
10. The method of claim 9 , wherein step (b) is in excess of 30 μg per million cells in a 24 hour period.
11. The method of claim 7 , wherein step (b) is in excess of 30 μg per million cells in a 24 hour period.
12. A method for making a recombinant fusion protein consisting of HuEPO, a flexible peptide linker, and a human IgG Fc variant, which method comprises: (a) generating a CHO cell line transfected with DNA encoding the recombinant HuEPQ-L-vFc fusion protein; (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 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 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 of human IgG2 with Pro331Ser mutation as SEQ ID NO:18.