US2004175824A1PendingUtilityA1

Fc fusion proteins of human erythropoietin with high biological activities

64
Priority: Aug 17, 2001Filed: Jan 21, 2004Published: Sep 9, 2004
Est. expiryAug 17, 2021(expired)· nominal 20-yr term from priority
Inventors:Lee-Hwei K. SunBill N. C. SunCecily Rou-Yun Sun
C07K 2319/30A61K 38/00C07K 14/505C07K 2317/52C07K 2319/00
64
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Claims

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.

Claims

exact text as granted — not AI-modified
1 . A recombinant HuEPO-L-vFc fusion protein comprising HuEPO, a peptide linker, and a human IgG Fc variant.  
     
     
         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 human IgG Fc variant in  claim 1  or  claim 2  comprising a hinge, CH2, and CH3 domains of human IgG2 with Pro331Ser mutation as SEQ ID NO: 18.  
     
     
         4 . The human IgG Fc variant in  claim 1  or  claim 2  comprising a hinge, CH2, and CH3 domains of human IgG4 with Ser228Pro and Leu235Ala mutations as SEQ ID NO: 20.  
     
     
         5 . The human IgG Fc variant in  claim 1  or  claim 2  comprising a hinge, CH2, and CH3 domains of human IgB1 with Leu234Val, Leu235Ala, and Pro331Ser mutations as SEQ ID NO: 22.  
     
     
         6 . 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.  
     
     
         7 . 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.  
     
     
         8 . The CHO-derived cell line producing the HuEPO-L-vFc fusion protein of  claim 7  in its growth medium in excess of 30 μg per million cells in a 24 hour period.  
     
     
         9 . The CHO-derived cell line producing the HuEPO-L-vFc fusion protein of  claim 1 , wherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgG selected from the group consisting of IgB1 as SEQ ID NO: 22, IgG2 as SEQ ID NO: 18, and IgG4 as SEQ ID NO: 20, 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 HuEPO-L-vFc fusion protein exhibits in vitro biological activity similar to or higher than that of rHuEPO on a molar basis.  
     
     
         10 . 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.  
     
     
         11 . The method of  claim 10 , 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.  
     
     
         12 . The method of  claim 10 , wherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgG2 with Pro331Ser mutation.  
     
     
         13 . The method of  claim 10 , wherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgG4 with Ser228Pro and Leu235Ala mutations.  
     
     
         14 . The method of  claim 10 , wherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgG1 with Leu234Val, Leu235Ala, and Pro331Ser mutations as SEQ ID NO: 18.  
     
     
         15 . The method of any claim of claims  10 ,  11 ,  12 ,  13 , and  14 , wherein step (b) is in excess of 30 μg per million cells in a 24 hour period.  
     
     
         16 . The method of  claim 10 , wherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgG4 with Ser228Pro and Leu235Ala mutations as SEQ ID NO: 20.  
     
     
         17 . The method of  claim 16 , wherein step (b) is in excess of 30 μg per million cells in a 24 hour period.  
     
     
         18 . The method of  claim 10 , wherein the human IgG Fc variant comprises a hinge, CH2, and CH3 domains of human IgB1 with Leu234Val, Leu235Ala, and Pro331Ser mutations as SEQ ID NO: 22.  
     
     
         19 . The method of  claim 18 , wherein step (b) is in excess of 30 μg per million cells in a 24 hour period.  
     
     
         20 . 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.

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