# US 11,440,950 B2 · Anti-VEGF protein compositions and methods for producing the same

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- Number: US 11,440,950 B2
- Document ID: US11440950B2
- Status: Granted
- Kind: B2
- Application number: 17/378,362
- Filed: 2021-07-16
- Published: 2022-01-13
- Granted: 2022-09-13
- Earliest claimed: 2019-12-06
- Assignees: Regeneron Pharmaceuticals, Inc.
- Applicants: Regeneron Pharmaceuticals, Inc.
- Inventors: Matthew Franklin
- Web page: https://patentgrep.com/patent/US11440950B2

## CPC

- A61K 38/179: {for growth factors; for growth regulators}
- C07K 14/71: for growth factors; for growth regulators
- A61K 38/1866: {Vascular endothelial growth factor [VEGF]}
- A61P 17/06: Antipsoriatics
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 27/02: Ophthalmic agents
- A61P 35/00: Antineoplastic agents
- C07K 1/06: using protecting groups or activating agents {(C07K1/003, C07K1/006 take precedence)}
- C07K 1/113: without change of the primary structure
- C07K 1/16: by chromatography
- C07K 1/18: Ion-exchange chromatography
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 1/36: by a combination of two or more processes of different types
- C07K 16/22: against growth factors {; against growth regulators}
- C07K 16/283: {against Fc-receptors, e.g. CD16, CD32, CD64 (CD23 C07K16/2851)}
- C12N 5/0018: {Culture media for cell or tissue culture (media for specific animal cell type C12N5/06)}
- C12N 5/0031: {Serum-free culture media}
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12P 21/06: produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00)
- C12Y 304/21004: Trypsin (3.4.21.4)
- G01N 30/80: Fraction collectors
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies
- C12N 2800/10: Plasmid DNA
- G01N 2030/027: {Liquid chromatography}
- 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)
- 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)
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present disclosure pertains to compositions comprising anti-VEGF proteins and methods for producing such compositions.

## Claims

### Claim 1

A recombinant polypeptide having IdeS protease activity comprising the amino acid sequence selected from the group consisting of SEQ ID NO.: 2, SEQ ID NO.: 3, SEQ ID NO.: 4, SEQ ID NO.: 5, SEQ ID NO.: 6, SEQ ID NO.: 7, SEQ ID NO.: 8, SEQ ID NO.: 9, SEQ ID NO.: 10, SEQ ID NO.: 11, SEQ ID NO.: 12, SEQ ID NO.: 13, SEQ ID NO.: 14, SEQ ID NO.: 15 and SEQ ID NO.: 16, wherein said polypeptide has the IdeS protease activity at a basic pH.

### Claim 2 (depends on 1)

The polypeptide of claim 1, wherein said polypeptide is capable of cleaving a target protein having an Fc domain into fragments, wherein at least one fragment comprises said Fc domain.

### Claim 3 (depends on 2)

The polypeptide of claim 2, wherein a cleavage recognition site comprises Glycine-Glycine.

### Claim 4 (depends on 2)

The polypeptide of claim 2, wherein said target protein is aflibercept.

### Claim 5 (depends on 2)

The polypeptide of claim 2, wherein said fragments comprise a Fab fragment and a Fc fragment.

### Claim 6 (depends on 1)

An isolated nucleic acid molecule encoding a polypeptide of claim 1.

### Claim 7 (depends on 6)

A vector comprising said nucleic acid molecule of claim 6.

### Claim 8 (depends on 7)

A vector according to claim 7, wherein said nucleic acid molecule is operatively linked to an expression control sequence capable of directing its expression in a host cell.

### Claim 9 (depends on 7)

A vector according to claim 7, wherein said vector is a plasmid.

### Claim 10 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 2.

### Claim 11 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 3.

### Claim 12 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 4.

### Claim 13 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 5.

### Claim 14 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 6.

### Claim 15 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 7.

### Claim 16 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 8.

### Claim 17 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 8.

### Claim 18 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 10.

### Claim 19 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 11.

### Claim 20 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 12.

### Claim 21 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 13.

### Claim 22 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 14.

### Claim 23 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 15.

### Claim 24 (depends on 1)

The method of claim 1, wherein the amino acid sequence is as set forth in SEQ ID NO.: 16.