# US 11,299,527 B2 · Long-acting recombinant porcine FSH fusion protein and preparation method and application thereof

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- Number: US 11,299,527 B2
- Document ID: US11299527B2
- Status: Granted
- Kind: B2
- Application number: 16/609,543
- Filed: 2017-11-01
- Published: 2020-07-16
- Granted: 2022-04-12
- Earliest claimed: 2017-09-15
- Assignees: Beijing Vjt Bio Co., Ltd.
- Applicants: Beijing Vjt Bio Co., Ltd.
- Inventors: Haoshu Luo; Lei Shi; Guo Han
- Web page: https://patentgrep.com/patent/US11299527B2

## CPC

- C07K 14/59: Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
- A61K 38/24: Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g. HCG; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
- A61K 47/68: the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61P 15/00: Drugs for genital or sexual disorders (for disorders of sex hormones A61P5/24); Contraceptives
- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/85: for animal cells
- A01K 2227/108: Swine
- A01K 2267/02: Animal zootechnically ameliorated
- 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 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
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## Abstract

Two long-acting recombinant porcine FSH (follicle-stimulating hormone) fusion proteins, comprising pFSH-Fc-1 and pFSH-Fc-2. α subunit/β subunit is directly or indirectly fused on a Fc fragment by means of a linking component; the β subunit/α subunit is combined with the α subunit/β subunit by means of a Van der Waals force or the linking component. The porcine FSH fusion proteins can be prepared by an eukaryotic expression system based on a gene engineering technology. The two porcine FSH fusion proteins have a good medicinal effect and have a longer half-life period as compared with that of a natural porcine FSH; the two porcine FSH fusion proteins do not generate an undesirable effect on animals and can replace pregnant mare serum gonadotropin (PMSG) to be used in animal breeding production. The two porcine FSH fusion proteins can be used for preparation of medicines in the field of animal breeding.

## Claims

### Claim 1

A long-acting recombinant porcine FSH fusion protein pFSH-Fc-1;
wherein the pFSH-Fc-1 comprises two peptide chains and conforms to the equation (pFSHβ:pFSHα-L-Fc)2, the pFSHβ refers to a β subunit of the porcine FSH with a signal peptide removed; the colon represents the relationship that the porcine FSH β subunit and the porcine FSH α subunit are linked by a Van der Waals force; pFSHα refers to an α subunit of the porcine FSH with a signal peptide removed; L represents the linking relationship between the pFSHα subunit and the Fc fragment; Fc refers to an Fc fragment of an immunoglobulin; and the subscript 2 outside of the parenthesis represents that pFSH-Fc-1 is a divalent homodimer, and
the amino acid sequence of the pFSHα-L-Fc is shown in SEQ ID NO: 6, and the amino acid sequence of the pFSHβ is shown in SEQ ID NO: 3.

### Claim 2 (depends on 1)

An expression cassette or an expression vector, comprising a nucleic acid encoding the recombinant porcine FSH fusion protein according to claim 1.

### Claim 3 (depends on 2)

A CHO cell for expressing of a fusion protein, the cell comprising the expression vector of claim 2.

### Claim 4 (depends on 1)

A method for synchronizing estrus and superovulation in an animal comprising the step of administering at least one of recombinant porcine FSH fusion proteins according to claim 1, wherein the animal is a pig, a cow or a goat.

### Claim 5 (depends on 4)

The method of claim 4, wherein the animal is a pig.

### Claim 6 (depends on 1)

A method for treating anestrus in a pig by administering a fusion protein according to claim 1.

### Claim 7 (depends on 1)

A cloning vector comprising a nucleic acid encoding a fusion protein according to claim 1.

### Claim 8

A long-acting recombinant porcine FSH fusion protein pFSH-Fc-2 comprising two peptide chains and conforms to the equation: (pFSHα:pFSHβ-L-Fc)2, wherein the pFSHβ refers to a β subunit of the porcine FSH with a signal peptide removed; the colon represents the relationship that the porcine FSH β subunit and the porcine FSH α subunit are linked by a Van der Waals force; pFSHα refers to an α subunit of the porcine FSH with a signal peptide removed; L represents the linking relationship between the pFSHβ subunit and the Fc fragment; Fc refers to an Fc fragment of an immunoglobulin; and the subscript 2 outside of the parenthesis represents that FSH-Fc-2 is a divalent homodimer, and
the amino acid sequence of the pFSHβ-L-Fc is shown in SEQ ID NO: 8, and the amino acid sequence of the pFSHα is shown in SEQ ID NO:1.

### Claim 9 (depends on 8)

An expression cassette or an expression vector, comprising a nucleic acid encoding the recombinant porcine FSH fusion protein according to claim 8.

### Claim 10 (depends on 9)

A CHO cell for expressing a fusion protein, the cell comprising the expression vector of claim 9.

### Claim 11 (depends on 8)

A cloning vector comprising a nucleic acid encoding a fusion protein according to claim 8.

### Claim 12 (depends on 8)

A method for synchronizing estrus and superovulation in an animal comprising the step of administering at least one of recombinant porcine FSH fusion proteins according to claim 8, wherein the animal is a pig, a cow or a goat.

### Claim 13 (depends on 8)

A method for treating anestrus in a pig by administering a fusion protein according to claim 8.

### Claim 14

A method for preparing a recombinant porcine FSH fusion protein selected from the group consisting of:
(a) a long-acting recombinant porcine FSH fusion protein pFSH-Fc-1; wherein the pFSH-Fc-1 comprises two peptide chains and conforms to the equation (pFSHβ:pFSHα-L-Fc)2, the pFSHβ refers to a β subunit of the porcine FSH with a signal peptide removed; the colon represents the relationship that the porcine FSH β subunit and the porcine FSH α subunit are linked by a Van der Waals force; pFSHα refers to an α subunit of the porcine FSH with a signal peptide removed; L represents the linking relationship between the pFSHα subunit and the Fc fragment; Fc refers to a Fc fragment of an immunoglobulin; and the subscript 2 outside of the parenthesis represents that pFSH-Fc-1 is a divalent homodimer, and
the amino acid sequence of the pFSHα-L-Fc is shown in SEQ ID NO: 6, and the amino acid sequence of the pFSHβ is shown in SEQ ID NO: 3; and
(b) a long-acting recombinant porcine FSH fusion protein pFSH-Fc 2 wherein the pFSH-Fc-2 comprises two peptide chains and conforms to the equation (pFSHα:pFSHβ-L-Fc)2, the pFSHβ refers to a β subunit of the porcine FSH with a signal peptide removed; the colon represents the relationship that the porcine FSH β subunit and the porcine FSH α subunit are linked by a Van der Waals force; pFSHα refers to an α subunit of the porcine FSH with a signal peptide removed; L represents the linking relationship between the pFSHβ subunit and the Fc fragment; Fc refers to an Fc fragment of an immunoglobulin; and the subscript 2 outside of the parenthesis represents that pFSH-Fc-2 is a divalent homodimer, and
the amino acid sequence of the pFSHβ-L-Fc is shown in SEQ ID NO: 8, and the amino acid sequence of the pFSHα is shown in SEQ ID NO: 1, the method comprising:
artificially synthesizing genes encoding pFSHα, pFSHβ, pFSHα-L-Fc and pFSHβ-L-Fc, performing codon optimization, and
cloning the codon optimized genes into eukaryotic expression vectors, respectively;
simultaneously transforming the pFSHα recombinant vector and pFSHβ-L-Fc recombinant vector into eukaryotic cells, and expressing in eukaryotic cells, and isolating and purifying the target protein; or simultaneously transforming the pFSHβ recombinant vector and pFSHα-L-Fc recombinant vector into eukaryotic cells, and expressing in eukaryotic cells, and isolating and purifying the target protein.

### Claim 15 (depends on 14)

The method of claim 14, wherein the eukaryotic expression vector is pcDNA3.1, and the eukaryotic cells include 293 and CHO cells.

### Claim 16 (depends on 15)

A fusion protein produced according to the method of claim 15.

### Claim 17 (depends on 14)

A fusion protein produced according to the method of claim 14.