Fc-erythropoietin fusion protein with improved pharmacokinetics

Abstract

The present invention provides Fc-erythropoietin (“Fc-EPO”) fusion proteins with improved pharmacokinetics. Nucleic acids, cells, and methods relating to the production and practice of the invention are also provided.

1 . A population of purified Fc-EPO fusion proteins suitable for administration to a mammal, the Fc-EPO fusion proteins comprising an Fc portion towards the N-terminus of the Fc-EPO fusion proteins and an erythropoietin portion towards the C-terminus of the Fc-EPO fusion proteins, said population having an average of 11-28 sialic acid residues per purified Fc-EPO fusion protein. 
     
     
         2 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the population has an average of 15-28 sialic acid residues per purified Fc-EPO fusion protein. 
     
     
         3 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the population has an average of 19-28 sialic acid residues per purified Fc-EPO fusion protein. 
     
     
         4 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the population has an average of 20-22 sialic acid residues per purified Fc-EPO fusion protein. 
     
     
         5 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the purified Fc-EPO fusion proteins are synthesized in a BHK cell. 
     
     
         6 . The population of purified Fc-EPO fusion proteins of  claim 5 , wherein the BHK cell is adapted for growth in suspension. 
     
     
         7 . The population of purified Fc-EPO fusion proteins of  claim 5 , wherein the BHK cell is adapted for growth in a protein-free medium. 
     
     
         8 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the population has a longer serum half-life compared to a population of corresponding Fc-EPO fusion proteins synthesized in NS/0, PerC6, or 293 cells. 
     
     
         9 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the Fc-EPO fusion proteins do not include a variable region of an immunoglobulin. 
     
     
         10 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the Fc portion comprises a CH2 domain. 
     
     
         11 . The population of purified Fc-EPO fusion proteins of  claim 10 , wherein the Fc portion further comprises a CH3 domain. 
     
     
         12 . The population of purified Fc-EPO fusion proteins of  claim 10 , wherein the CH2 domain is derived from a human IgG2 heavy chain. 
     
     
         13 . The population of purified Fc-EPO fusion proteins of  claim 12 , wherein the CH2 domain comprises a mutation of the glycosylation site within the CH2 domain of the IgG2 heavy chain. 
     
     
         14 - 17 . (canceled) 
     
     
         18 . The population of purified Fc-EPO fusion proteins of  claim 10 , wherein the Fc portion further comprises a hinge region. 
     
     
         19 . The population of purified Fc-EPO fusion proteins of  claim 18 , wherein the hinge region is derived from a human IgG1 heavy chain. 
     
     
         20 . The population of purified Fc-EPO fusion proteins of  claim 18 , wherein the CH2 domain is from a first antibody isotype and the hinge region is from a second antibody isotype. 
     
     
         21 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the Fc portion is derived from an IgG sequence in which the Leu-Ser-Leu-Ser (SEQ ID NO:20) amino acid sequence near the C-terminus of the Fc portion is replaced with an Ala-Thr-Ala-Thr (SEQ ID NO:16) amino acid sequence. 
     
     
         22 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the Fc portion is derived from an IgG sequence in which the C-terminal lysine residue is replaced. 
     
     
         23 . The population of purified Fc-EPO fusion proteins of  claim 22 , wherein the C-terminal lysine residue is replaced with alanine. 
     
     
         24 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the Fc-EPO fusion proteins further comprise a linker between the Fc portion and the erythropoietin portion. 
     
     
         25 . The population of purified Fc-EPO fusion proteins of  claim 24 , wherein the linker comprises between 1 and 25 amino acids. 
     
     
         26 . The population of purified Fc-EPO fusion proteins of  claim 24 , wherein the linker comprises a glycosylation site. 
     
     
         27 . The population of purified Fc-EPO fusion proteins of  claim 26 , wherein the linker comprises an Asn-Ala-Thr amino acid sequence. 
     
     
         28 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the erythropoietin portion is derived from human erythropoietin. 
     
     
         29 . The population of purified Fc-EPO fusion proteins of  claim 28 , wherein the erythropoietin portion comprises at least one of the following mutations: Arg 131 ->Glu and Arg 139 ->Glu. 
     
     
         30 . The population of purified Fc-EPO fusion proteins of  claim 28 , wherein the erythropoietin portion comprises at least one of the following mutations: His 32 ->Gly, Ser 34 ->Arg, and Pro 90 ->Ala. 
     
     
         31 . The population of purified Fc-EPO fusion proteins of  claim 1 , wherein the erythropoietin portion comprises a pattern of disulfide bonding distinct from human erythropoietin. 
     
     
         32 . The population of purified Fc-EPO fusion proteins of  claim 31 , wherein the erythropoietin portion comprises at least one of the following amino acid substitutions: a non-cysteine residue at position 29, a non-cysteine residue at position 33, a cysteine residue at position 88, and a cysteine residue at position 139. 
     
     
         33 . A pharmaceutical composition comprising the population of Fc-EPO fusion proteins of  claim 1  and a pharmaceutically-acceptable carrier. 
     
     
         34 . A method of treating a mammal comprising administering to the mammal the pharmaceutical composition of  claim 33 . 
     
     
         35 . The method of  claim 34 , wherein the mammal has a hematopoietic disorder or deficiency. 
     
     
         36 . The method of  claim 34 , wherein the pharmaceutical composition is administered fewer than three times per week. 
     
     
         37 . A method of selecting a BHK cell stably maintaining a nucleic acid sequence encoding an Fc-EPO fusion protein comprising an Fc portion and an erythropoietin portion, the method comprising the steps of:
 (a) introducing into a BHK cell a nucleic acid sequence encoding hygromycin B and a nucleic acid sequence encoding the Fc-EPO fusion protein; and   (b) culturing the BHK cell in the presence of hygromycin B.   
     
     
         38 . The method of  claim 37 , wherein the nucleic acid sequence encoding hygromycin B and the nucleic acid sequence encoding the Fc-EPO fusion protein are present in a single nucleic acid. 
     
     
         39 . A BHK cell selected by the method of  claim 37 . 
     
     
         40 . The BHK cell of  claim 39 , wherein the cell is adapted for growth in suspension. 
     
     
         41 . The BHK cell of  claim 39 , wherein the cell is adapted for growth in a protein-free medium. 
     
     
         42 . A method of producing an Fc-EPO fusion protein comprising:
 (a) maintaining the BHK cell of  claim 39  under conditions suitable for expression of the Fc-EPO fusion protein; and   (b) recovering the expressed Fc-EPO fusion protein.   
     
     
         43 . The method of  claim 42 , wherein the conditions comprise culturing the BHK cell in a protein-free medium. 
     
     
         44 . The method of  claim 42 , wherein the conditions comprise culturing the BHK cell in suspension. 
     
     
         45 . An Fc-EPO fusion protein produced by the method of  claim 42 , wherein the Fc-EPO fusion protein has a longer serum half-life than a corresponding Fc-EPO protein synthesized in NS/0, PerC6, or 293 cells. 
     
     
         46 . A BHK cell adapted for growth in a protein-free medium comprising a nucleic acid sequence encoding an Fc-erythropoietin (Fc-EPO) fusion protein, the Fc-EPO protein comprising an Fc portion towards the N-terminus of the Fc-EPO fusion protein and an erythropoietin portion towards the C-terminus of the Fc-EPO fusion protein. 
     
     
         47 . The BHK cell of  claim 46 , wherein the cell is adapted for growth in suspension. 
     
     
         48 . The BHK cell of  claim 46 , wherein the nucleic acid sequence encoding an Fc-EPO fusion protein is stably maintained in the BHK cell. 
     
     
         49 . A method of producing an Fc-EPO fusion protein comprising:
 (a) maintaining the BHK cell of  claim 46  under conditions suitable for expression of the Fc-EPO fusion protein; and   (b) recovering the expressed Fc-EPO fusion protein.   
     
     
         50 . An Fc-EPO fusion protein produced by the method of  claim 49 . 
     
     
         51 . A pharmaceutical composition comprising a pharmaceutically-acceptable carrier and an Fc-EPO fusion protein synthesized in a BHK cell stably maintaining a nucleic acid encoding the Fc-EPO fusion protein, the Fc-EPO fusion protein comprising an Fc portion towards the N-terminus of the Fc-EPO fusion protein and an erythropoietin portion towards the C-terminus of the Fc-EPO fusion protein. 
     
     
         52 . A method of treating a mammal, the method comprising the step of administering to the mammal the pharmaceutical composition of  claim 51 . 
     
     
         53 . The pharmaceutical composition of  claim 51 , wherein the Fc-EPO fusion protein has not been treated to remove sialic acid residues. 
     
     
         54 . A method of treating a mammal, the method comprising the step of administering to the mammal the pharmaceutical composition of  claim 53 . 
     
     
         55 . The method of  claim 54 , wherein the pharmaceutical composition is administered fewer than three times per week.