# US 10,117,949 B2 · Recombinant human EPO-Fc fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo

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- Number: US 10,117,949 B2
- Document ID: US10117949B2
- Status: Granted
- Kind: B2
- Application number: 12/162,320
- Filed: 2007-01-25
- Published: 2009-12-03
- Granted: 2018-11-06
- Earliest claimed: 2006-01-27
- Assignees: Novagen Holding Corporation
- Applicants: Haitao Wang; Yong Du; Rui Zhang; Jing Xu; Longbin Liu
- Inventors: Haitao Wang; Yong Du; Rui Zhang; Jing Xu; Longbin Liu
- Web page: https://patentgrep.com/patent/US10117949B2

## CPC

- C07K 14/505: Erythropoietin [EPO]
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 47/6811: {the drug being a protein or peptide, e.g. transferrin or bleomycin}
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 7/06: Antianaemics
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C07K 2317/53: Hinge
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS

## Abstract

A recombinant fusion protein comprising a human erythropoietin peptide portion linked to an immunoglobulin peptide portion is described. The fusion protein has a prolonged half-life in vivo in comparison to naturally occurring or recombinant native human erythropoietin. In one embodiment of the invention the protein has a half-life in vivo at least three fold higher than native human erythropoietin. The fusion protein also exhibits enhanced erythropoietic bioactivity in comparison to native human erythropoietin. In one embodiment, the fusion protein comprises the complete peptide sequence of a human erythropoietin (EPO) molecule and the peptide sequence of an Fc fragment of human immunoglobulin IgG1. The Fc fragment in the fusion protein includes the hinge region, CH2 and CH3 domains of human immunoglobulin IgG1. The EPO molecule may be linked directly to the Fc fragment to avoid extraneous peptide linkers and lessen the risk of an immunogenic response when administered in vivo. In one embodiment the hinge region is a human Fc fragment variant having a non-cysteine residue at amino acid 6. The invention also relates to nucleic acid and amino acid sequences encoding the fusion protein and transfected cell lines and methods for producing the fusion protein. The invention further includes pharmaceutical compositions comprising the fusion protein and methods of using the fusion protein and/or the pharmaceutical compositions, for example to stimulate erythropoiesis in subjects in need of therapy.

## Claims

### Claim 1

An isolated multimeric protein comprising a plurality of polypeptides each having the amino acid sequence present in SEQ ID NO:2 or a sequence having at least 90% sequence identity thereto, wherein:
(i) each polypeptide comprises an erythropoietin molecule and an immunoglobulin Fc fragment having a mutated IgG1 hinge region,
(ii) the C-terminal of said erythropoietin molecule is directly linked to the N-terminal of said Fc fragment such that no external peptide linkers are interposed therebetween,
(iii) the amino acid sequence of said mutated IgG1 hinge region is set forth in SEQ ID NO:17 (VEPKSGDKTSTCPPCP), or a sequence having at least 90% sequence identity thereto, provided that said mutated IgG1 hinge region does not have a cysteine at the sixth amino acid of SEQ ID NO:17, and
(iv) said multimeric protein has an in vivo half-life and erythropoietic potency at least equal to native human erythropoietin.

### Claim 2 (depends on 1)

The protein as defined in claim 1, wherein said protein is a dimer.

### Claim 3 (depends on 2)

The protein as defined in claim 2, wherein said dimer comprises disulfide bonds between said mutated IgG1 hinge regions of said polypeptides.

### Claim 4 (depends on 2)

The protein as defined in claim 2 wherein said dimer has a molecular mass of about 180 kDa.

### Claim 5 (depends on 1)

The protein as defined in claim 1, wherein each of said polypeptides has a molecular mass of about 75 kDa.

### Claim 6

An isolated fusion protein comprising:
(a) an erythropoietin molecule having a cysteine residue near the C-terminal thereof; and
(b) a Fc fragment comprising a mutated IgG1 hinge region, wherein the N-terminal of said Fc fragment is directly linked to said C-terminal of said erythropoietin molecule such that no external peptide linkers are interposed therebetween, and wherein the amino acid sequence of said mutated IgG1 hinge region is set forth in SEQ ID NO:17 (VEPKSGDKTSTCPPCP), or a sequence having at least 90% sequence identity thereto, provided that said mutated IgG1 hinge region does not have a cysteine at the sixth amino acid of SEQ ID NO:17;
wherein said fusion protein comprises the amino acid sequence present in SEQ ID NO:2 or a sequence having at least 90% sequence identity thereto, and
wherein said fusion protein has an in vivo half-life and erythropoietic potency at least equal to native human erythropoietin.

### Claim 7 (depends on 6)

The fusion protein as defined in claim 6, wherein the half-life of said fusion protein is at least three fold higher than said native human erythropoietin.

### Claim 8 (depends on 7)

The fusion protein as defined in claim 7, wherein said half-life of said fusion protein is at least four fold higher than said native human erythropoietin.

### Claim 9 (depends on 7)

The fusion protein as defined in claim 7, wherein said fusion protein has enhanced erythropoietic potency in comparison to said native human erythropoietin.

### Claim 10 (depends on 6)

The fusion protein as defined in claim 6, wherein said Fc fragment is a human IgG1 Fc fragment comprising a mutated human IgG1 hinge and human IgG1 CH2 and CH3 domains.

### Claim 11 (depends on 6)

An isolated multimeric protein construct comprising a plurality of fusion proteins as defined in claim 6.

### Claim 12 (depends on 11)

The multimeric protein construct of claim 11, wherein said construct is a dimer.

### Claim 13 (depends on 6)

A pharmaceutical composition comprising the fusion protein as defined in claim 6 together with a pharmaceutically acceptable carrier, adjuvant or diluent.

### Claim 14 (depends on 13)

A method of stimulating erythropoiesis in a mammal comprising administering to said mammal a pharmaceutical composition as defined in claim 13.

### Claim 15 (depends on 14)

The method as defined in claim 14, wherein said mammal is a primate.

### Claim 16 (depends on 15)

The method as defined in claim 15, wherein said primate is a human.

### Claim 17

An isolated fusion protein comprising:
(a) an erythropoietin molecule having a cysteine residue near the C terminal thereof; and
(b) a Fc fragment comprising a mutated IgG1 hinge region, wherein the N-terminal of said Fc fragment is directly linked to said C-terminal of said erythropoietin molecule such that no external peptide linkers are interposed therebetween, and wherein the amino acid sequence of said mutated IgG1 hinge region is set forth in SEQ ID NO:17 (VEPKSGDKTSTCPPCP), or a sequence having at least 90% sequence identity thereto, provided that said mutated IgG1 hinge region does not have a cysteine at the sixth amino acid of SEQ ID NO:17, wherein the first cysteine residue of said mutated IgG1 hinge region located nearest said N-terminal is spaced at least 12 amino acids apart from said cysteine residue of said erythropoietin molecule,
wherein said fusion protein comprises the amino acid sequence present in SEQ ID NO:2 or a sequence having at least 90% sequence identity thereto, and
wherein said fusion protein has an in vivo half-life and erythropoietic potency at least equal to native human erythropoietin.

### Claim 18 (depends on 17)

The fusion protein as defined in claim 17, wherein said Fc fragment is a human IgG1 Fc fragment comprising a mutated human IgG1 hinge and human IgG1 CH2 and CH3 domains.

### Claim 19 (depends on 17)

The fusion protein as defined in claim 17, wherein the half-life of said fusion protein when administered to a mammal is at least three fold higher than native human erythropoietin administered to said mammal by the same means.

### Claim 20 (depends on 19)

The fusion protein as defined in claim 19, wherein the half-life of said fusion protein when administered to a mammal is at least four fold higher than native human erythropoietin administered to said mammal by the same means.

### Claim 21 (depends on 19)

The fusion protein as defined in claim 19, wherein said mammal is a human.

### Claim 22 (depends on 17)

An isolated dimer comprising first and second fusion proteins each as defined in claim 17, wherein said IgG1 hinge region of said first fusion protein is linked to said IgG1 hinge region of said second fusion protein by disulfide bonds.

### Claim 23 (depends on 17)

An isolated multimeric protein construct comprising a plurality of linked fusion proteins each as defined in claim 17.

### Claim 24

An isolated fusion protein comprising:
(a) an erythropoietin molecule having a cysteine residue near the C terminal thereof; and
(b) a Fc fragment comprising a mutated IgG1 hinge region, wherein the N-terminal of said Fc fragment is directly linked to said C-terminal of said erythropoietin molecule such that no external peptide linkers are interposed therebetween, provided that said mutated IgG1 hinge region has a mutation whereby a cysteine residue nearest to said N-terminal of said Fc fragment is replaced with a non-cysteine residue such that the first cysteine residue of said mutated IgG1 hinge region located nearest said N-terminal is spaced at least 12 amino acids apart from said cysteine residue of said erythropoietin molecule,
wherein said fusion protein has the amino acid sequence present in SEQ ID NO:2 or a sequence having at least 98% sequence identity thereto, and
wherein said fusion protein has an in vivo half-life and erythropoietic potency at least equal to native human erythropoietin.

### Claim 25

An isolated fusion protein comprising:
(a) an erythropoietin molecule having a cysteine residue near the C-terminal thereof; and
(b) a Fc fragment comprising a mutated IgG1 hinge region, wherein the N-terminal of said Fc fragment is directly linked to said C-terminal of said erythropoietin molecule such that no external peptide linkers are interposed therebetween, provided that said mutated IgG1 hinge region has a mutation whereby a cysteine residue nearest to said N-terminal of said Fc fragment is replaced with a non-cysteine residue,
wherein said fusion protein has the amino acid sequence present in SEQ ID NO:2 or a sequence having at least 98% sequence identity thereto, and
wherein said fusion protein has an in vivo half-life and erythropoietic potency at least equal to native human erythropoietin.

### Claim 26

A fusion protein having a prolonged half-life in vivo in comparison to naturally occurring or recombinant native human erythropoietin comprising:
(a) a naturally occurring human erythropoietin molecule having a cysteine residue near the C-terminal thereof; and
(b) a human IgG1 Fc fragment comprising a mutated human IgG1 hinge region, and human IgG1 CH2 and CH3 domains, wherein the N-terminal of the human IgG1 Fc fragment is directly linked to said C-terminal of said erythropoietin molecule such that no external peptide linkers are interposed therebetween, and wherein the amino acid sequence of said mutated human IgG1 hinge region is set forth in SEQ ID NO:17 (VEPKSGDKTSTCPPCP), or a sequence having at least 90% sequence identity thereto, provided that the mutated human IgG1 hinge region does not have a cysteine residue at the sixth amino acid of SEQ ID NO:17, whereby the first cysteine residue of said mutated human IgG1 hinge region located nearest said N-terminal is spaced at least 12 amino acids apart from said cysteine residue of said erythropoietin molecule.

### Claim 27

A fusion protein having a prolonged half-life in vivo in comparison to naturally occurring or recombinant native human erythropoietin comprising:
(a) an erythropoietin molecule having a cysteine residue near the C-terminal thereof, wherein the C-terminal of said erythropoietin molecule is directly linked without the interposition of an external peptide linker to
(b) the N-terminal of a human IgG1 Fc fragment comprising a mutated human IgG1 hinge region and human IgG1 CH2 and CH3 domains, wherein the amino acid sequence of said mutated human IgG1 hinge region is
(i) SEQ ID NO:17 (VEPKSGDKTSTCPPCP) or
(ii) a sequence having at least 90% sequence identity thereto, provided that the mutated human IgG1 hinge region does not have a cysteine residue at the sixth amino acid of SEQ ID NO:17,
whereby the cysteine residue of said mutated human IgG1 hinge region located nearest said erythropoietin molecule is spaced at least 12 amino acids apart from said cysteine residue of said erythropoietin molecule.