# US 11,028,398 B2 · Fusion proteins having mutated immunoglobulin hinge region

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- Number: US 11,028,398 B2
- Document ID: US11028398B2
- Status: Granted
- Kind: B2
- Application number: 13/275,205
- Filed: 2011-10-17
- Published: 2012-04-26
- Granted: 2021-06-08
- Earliest claimed: 2007-07-26
- 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/US11028398B2

## CPC

- C12N 15/62: DNA sequences coding for fusion proteins
- A61K 47/6811: {the drug being a protein or peptide, e.g. transferrin or bleomycin}
- A61K 47/6835: {the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site}
- A61P 37/04: Immunostimulants
- A61P 43/00: Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
- 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- 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)

## Abstract

A fusion protein having a non-immunoglobulin polypeptide having a cysteine residue proximal to the C terminal thereof, and an immunoglobulin component with a mutated hinge region is provided. The mutation comprises a point mutated site corresponding in position to the position in a native hinge region of the cysteine residue located nearest the cysteine residue of the non-Ig component. The distance from the cysteine residue of the non-immunoglobulin polypeptide and any remaining cysteine residues of the mutated hinge region is sufficient to prevent the formation of a disulphide bond therebetween.

## Claims

### Claim 1

A fusion protein comprising from N-terminus to C-terminus:
a non-immunoglobulin polypeptide comprising a cysteine residue nearest to the C-terminus, wherein said polypeptide is selected from the group consisting of human erythropoietin (EPO) and human granulocyte-macrophage colony stimulating factor (GM-CSF); and
an immunoglobulin component comprising a human IgG1 Fc fragment having a mutated IgG1 hinge region, wherein the non-immunoglobulin polypeptide is directly coupled to the immunoglobulin component without an external linker peptide,
wherein said IgG1 Fc fragment comprises said IgG1 mutated hinge region and IgG1 CH2 and CH3 domains;
wherein the amino acid sequence of said mutated IgG1 hinge region is set forth in VEPKSGDKTSTCPPCP (SEQ ID NO: 24) or the amino acid sequence of SEQ ID NO:24, wherein one or more amino acid residues N-terminal to the glycine residue is deleted in SEQ ID NO:24; and
wherein the fusion protein has enhanced biological activity in vivo in comparison to the native non-immunoglobulin polypeptide and in comparison to a fusion protein comprising the native non-immunoglobulin polypeptide and a human IgG1 Fc fragment comprising a native IgG1 hinge region and IgG1 CH2 and CH3 domains.

### Claim 2 (depends on 1)

A fusion protein according to claim 1, wherein the non-immunoglobulin polypeptide is human granulocyte-macrophage colony stimulating factor.

### Claim 3 (depends on 1)

A multimeric protein comprising a plurality of fusion proteins according to claim 1.

### Claim 4 (depends on 3)

The multimeric protein according to claim 3 wherein the multimeric protein is a dimer.

### Claim 5 (depends on 2)

A pharmaceutical composition comprising a fusion protein according to claim 2 and a pharmaceutically acceptable carrier, adjuvant or diluent.

### Claim 6

A fusion protein comprising from N-terminus to C-terminus:
a non-immunoglobulin polypeptide selected from the group consisting of human erythropoietin (EPO) and human granulocyte-macrophage colony stimulating factor (GM-CSF); and
an immunoglobulin component comprising a human IgG1 Fc fragment having a mutated IgG1 hinge region wherein the non-immunoglobulin polypeptide is directly coupled to the immunoglobulin component without an external linker peptide, wherein said IgG1 Fc fragment comprises said mutated IgG1 hinge region and IgG1 CH2 and CH3 domains,
wherein the amino acid sequence of said mutated IgG1 hinge region is set forth in VEPKSGDKTSTCPPCP (SEQ ID NO: 24) or the amino acid sequence of SEQ ID NO:24, wherein one or more amino acid residues N-terminal to the glycine residue is deleted in SEQ ID NO:24; and
wherein the fusion protein has enhanced biological activity in vivo in comparison to the native non-immunoglobulin polypeptide and in comparison to a fusion protein comprising the native non-immunoglobulin polypeptide and a human IgG1 Fc fragment comprising a native IgG1 hinge region and IgG1 CH2 and CH3 domains.

### Claim 7

A fusion protein comprising from N-terminus to C-terminus: (i) a non-immunoglobulin polypeptide selected from the group consisting of human erythropoietin (EPO) and human granulocyte-macrophage colony stimulating factor (GM-CSF) and (ii) a human immunoglobulin component comprising an IgG1 Fc fragment, wherein the fusion protein has a prolonged half-life in vivo in comparison to naturally occurring or recombinant native non-immunoglobulin polypeptide, and wherein the non-immunoglobulin polypeptide is directly coupled to the immunoglobulin component without an external linker peptide,
wherein said IgG1 Fc fragment comprises a mutated IgG1 hinge region and IgG1 CH2 and CH3 domains,
wherein the amino acid sequence of said mutated IgG1 hinge region is set forth in VEPKSGDKTSTCPPCP (SEQ ID NO: 24) or the amino acid sequence of SEQ ID NO:24, wherein one or more amino acid residues N-terminal to the glycine residue is deleted in SEQ ID NO:24; and
wherein the fusion protein has enhanced biological activity in vivo in comparison to the native non-immunoglobulin polypeptide and in comparison to a fusion protein comprising the native non-immunoglobulin polypeptide and a human IgG1 Fc fragment comprising a native IgG1 hinge region and IgG1 CH2 and CH3 domains.

### Claim 8 (depends on 7)

The fusion protein according to claim 7, wherein the half-life of the fusion protein is at least three fold higher than the native non-immunoglobulin polypeptide.

### Claim 9 (depends on 7)

The fusion protein according to claim 7, wherein the half-life of the fusion protein is at least four fold higher than the native non-immunoglobulin polypeptide.

### Claim 10 (depends on 7)

A fusion protein according to claim 7, wherein the non-immunoglobulin polypeptide is a-human granulocyte-macrophage colony stimulating factor.

### Claim 11 (depends on 7)

A multimeric protein comprising a plurality of fusion proteins according to claim 7.

### Claim 12 (depends on 11)

The multimeric protein according to claim 11 wherein the multimeric protein is a dimer.

### Claim 13 (depends on 10)

A pharmaceutical composition comprising a fusion protein according to claim 10 and a pharmaceutically acceptable carrier, adjuvant or diluent.

### Claim 14

A fusion protein comprising the amino acid sequence of SEQ ID NO:6.

### Claim 15 (depends on 1)

The fusion protein as defined in claim 1, wherein the amino acid sequence of said mutated IgG1 hinge region is set forth in VEPKSGDKTSTCPPCP (SEQ ID NO:24).

### Claim 16 (depends on 1)

The fusion protein as defined in claim 1, wherein one amino acid N-terminal to the glycine residue is deleted in SEQ ID NO:24.

### Claim 17

A fusion protein comprising from N-terminus to C-terminus a non-immunoglobulin polypeptide selected from the group consisting of human EPO and human GM-CSF, a mutated human IgG1 hinge region, wherein the amino acid sequence of the mutated human IgG1 hinge region is set forth in SEQ ID NO:24, and human IgG1 CH2 and CH3 domains.

### Claim 18 (depends on 6)

The fusion protein as defined in claim 6, wherein the amino acid sequence of said mutated IgG1 hinge region is set forth in VEPKSGDKTSTCPPCP (SEQ ID NO:24).

### Claim 19 (depends on 6)

The fusion protein as defined in claim 6, wherein one amino acid N-terminal to the glycine residue is deleted in SEQ ID NO:24.

### Claim 20 (depends on 7)

The fusion protein as defined in claim 7, wherein the amino acid sequence of said mutated IgG1 hinge region is set forth in VEPKSGDKTSTCPPCP (SEQ ID NO:24).

### Claim 21 (depends on 7)

The fusion protein as defined in claim 7, wherein one amino acid N-terminal to the glycine residue is deleted in SEQ ID NO:24.

### Claim 22 (depends on 6)

A multimeric protein comprising a plurality of fusion proteins according to claim 6.

### Claim 23 (depends on 22)

The multimeric protein according to claim 22 wherein the multimeric protein is a dimer.

### Claim 24 (depends on 17)

A multimeric protein comprising a plurality of fusion proteins according to claim 17.

### Claim 25 (depends on 24)

The multimeric protein according to claim 24 wherein the multimeric protein is a dimer.