# US 7,611,700 B2 · Protease resistant modified interferon alpha polypeptides

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- Number: US 7,611,700 B2
- Document ID: US7611700B2
- Status: Granted
- Kind: B2
- Application number: 10/658,834
- Filed: 2003-09-08
- Published: 2004-07-08
- Granted: 2009-11-03
- Earliest claimed: 2002-09-09
- Assignees: Hanall Pharmaceuticals, Co., Ltd.
- Inventors: Rene Gantier; Manuel Vega; Lila Drittanti; Thierry Guyon
- Web page: https://patentgrep.com/patent/US7611700B2

## CPC

- C07K 14/475: Growth factors; Growth regulators
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 3/00: Drugs for disorders of the metabolism (of the blood or the extracellular fluid A61P7/00)
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 7/00: Drugs for disorders of the blood or the extracellular fluid
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 11/00: Drugs for disorders of the respiratory system
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 25/16: Anti-Parkinson drugs
- A61P 25/28: for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 35/00: Antineoplastic agents
- A61P 37/00: Drugs for immunological or allergic disorders
- A61P 37/08: Antiallergic agents (antiasthmatic agents A61P11/06; ophthalmic antiallergics A61P27/14)
- C07K 14/505: Erythropoietin [EPO]
- C07K 14/52: Cytokines; Lymphokines; Interferons
- C07K 14/53: Colony-stimulating factor [CSF]
- C07K 14/535: Granulocyte CSF; Granulocyte-macrophage CSF
- C07K 14/54: Interleukins [IL]
- C07K 14/555: Interferons [IFN]
- C07K 14/575: Hormones (derived from pro-opiomelanocortin, pro-enkephalin or pro-dynorphin C07K14/665, e.g. corticotropin C07K14/695)
- C07K 14/61: Growth hormone [GH], i.e. somatotropin
- 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: 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
- 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)

## Abstract

Processes and systems for the high throughput directed evolution of peptides and proteins, particularly cytokines that act in complex biological settings, are provided. Also provided is a rational method for generating protein variants and the resulting variants.

## Claims

### Claim 1

An isolated interferon (IFN) alpha cytokine, comprising an amino acid replacement in its sequence of amino acids, wherein:
an amino acid replacement is E41 Q, whereby the interferon alpha cytokine exhibits increased resistance to proteolysis so that it can be administered orally compared to the unmodified interferon alpha cytokine that does not comprise the amino acid replacement; and
in the interferon-alpha cytokine,
the interferon is not glycosylated at E41Q; and
the unmodified interferon alpha cytokine is selected from among an interferon α-2b (IFNα-2b), an interferon α-2a (IFNα-2a), an interferon α-2c (IFNα-2c) and consensus interferon whose sequences are set forth in SEQ ID Nos. 1, 182, 185 and 232, respectively.

### Claim 2

An interferon alpha cytokine selected from among IFNα-2b, IFNα-2a, IFNα-2c, consensus interferon, IFNα-c, IFNα-d, IFNα-5, IFNα-6, IFNα-4, IFNα-4b, IFNα-I, IFNα-J, IFNα-H, IFNα-F and IFNα-8, each comprising the modification E41Q, wherein:
the sequence of a human wild-type interferon alpha for each of IFNα-2b, IFNα-2a, IFNα-2c, consensus interferon, IFNα-c, IFNα-d, IFNα-5, IFNα-6, IFNα-4, IFNα-4b, IFNα-I, IFNα-J, IFNα-H, IFNα-F and IFNα-8; is set forth in SEQ ID Nos. 1, 182, 185, 232, 183 and 186-195, respectively; the modification at E41Q increases protease resistance of the interferon alpha cytokine so that it can be administered orally.
the interferon alpha cytokine is not glycosylated at E41Q; and
residue 1 in each SEQ ID corresponds to residue 1 of the mature interferon-alpha.

### Claim 3 (depends on 2)

An interferon alpha cytokine of claim 2, comprising two, three, four or five amino acid replacements in its sequence of amino acids.

### Claim 4 (depends on 3)

The interferon alpha cytokine of claim 3 that is a modified IFNα-2b cytokine.

### Claim 5 (depends on 1)

A pharmaceutical composition, comprising a cytokine of claim 1 in a pharmaceutically acceptable carrier.

### Claim 6 (depends on 1)

An interferon alpha cytokine of claim 1 that is an IFNα-2b.

### Claim 7 (depends on 1)

The interferon alpha cytokine of claim 1 that contains only a single amino acid replacement compared to the interferon cytokine that does not include the E41Q replacement.

### Claim 8 (depends on 5)

The pharmaceutical composition of claim 5, formulated for oral administration.

### Claim 9

An interferon alpha cytokine, comprising the sequence of amino acids set forth in SEQ ID NO:87.

### Claim 10 (depends on 1)

The interferon alpha cytokine of claim 1, comprising two amino acid replacements.

### Claim 11 (depends on 2)

A modified interferon (IFN) alpha cytokine of claim 2, comprising one or two amino acid replacements.

### Claim 12 (depends on 11)

The modified IFN alpha cytokine of claim 11 that is an IFN-α2b cytokine.

### Claim 13 (depends on 11)

A pharmaceutical composition, comprising a cytokine of claim 11 in a pharmaceutically acceptable carrier.

### Claim 14 (depends on 12)

A pharmaceutical composition, comprising a cytokine of claim 12 in a pharmaceutically acceptable carrier.

### Claim 15 (depends on 6)

A pharmaceutical composition, comprising a cytokine of claim 6 in a pharmaceutically acceptable carrier.

### Claim 16 (depends on 15)

The pharmaceutical composition of claim 15 that is formulated as a tablet or capsule for oral administration.

### Claim 17 (depends on 9)

A pharmaceutical composition, comprising a cytokine of claim 9 in a pharmaceutically acceptable carrier.

### Claim 18 (depends on 4)

The modified interferon alpha cytokine of claim 4, comprising amino acid replacements corresponding to E41Q and D94G.

### Claim 19 (depends on 2)

A pharmaceutical composition, comprising a modified interferon alpha cytokine of claim 2.

### Claim 20 (depends on 17)

The pharmaceutical composition of claim 17 that is formulated for oral administration.

### Claim 21 (depends on 20)

The pharmaceutical composition of claim 20 that is formulated as tablet or capsule.

### Claim 22 (depends on 2)

The interferon alpha cytokine of claim 2 that contains only a single amino acid replacement compared to the interferon cytokine that does not include the E41Q replacement.

### Claim 23 (depends on 19)

The pharmaceutical composition of claim 19 that is formulated for oral administration.

### Claim 24 (depends on 23)

The pharmaceutical composition of claim 23 that is formulated as a tablet or capsule.