# US 12,600,758 B2 · Interleukin-2 muteins for the expansion of T-regulatory cells

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- Number: US 12,600,758 B2
- Document ID: US12600758B2
- Status: Granted
- Kind: B2
- Application number: 17/634,685
- Filed: 2020-08-13
- Published: 2022-09-15
- Granted: 2026-04-14
- Earliest claimed: 2019-08-13
- Assignees: Amgen Inc.
- Applicants: Amgen Inc.
- Inventors: Darren L. Bates; Sue J. Sohn; Hannah Beth Catterall; Zhulun Wang
- Web page: https://patentgrep.com/patent/US12600758B2

## CPC

- C07K 14/55: IL-2
- A61K 38/2013: {IL-2}
- A61P 3/10: for hyperglycaemia, e.g. antidiabetics
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 37/06: Immunosuppressants, e.g. drugs for graft rejection
- 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)
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- 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

Provided herein are IL-2 muteins and IL-2 mutein Fc fusion molecules that preferentially expand and activate cells and are amenable to large scale production. Also provided herein are methods of making and using the compositions of the present invention.

## Claims

### Claim 1

A human interleukin-2 (IL-2) mutein comprising an amino acid sequence that is at least 97% identical to the amino acid sequence set forth in SEQ ID NO: 2, wherein said IL-2 mutein has the V91K and H16E mutations; and wherein said IL-2 mutein preferentially stimulates T regulatory cells relative to other T cells.

### Claim 2 (depends on 1)

The human IL-2 mutein of claim 1, wherein the IL-2 mutein further preferentially stimulates T regulatory cells relative to natural killer (NK) cells.

### Claim 3 (depends on 1)

The human IL-2 mutein of claim 1, wherein the amino acid sequence of said mutein further comprises the C125A mutation.

### Claim 4 (depends on 1)

The human IL-2 mutein of claim 1, wherein the IL-2 mutein has less than 30% phosphorylated Signal Transducer and Activator of Transcription 5 (pSTAT5) activation of T effector cells at 1 nM IL-2 treatment, using a fluorescence-activated cell sorting (FACS)-based peripheral blood mononuclear cell (PBMC) assay.

### Claim 5 (depends on 1)

The human IL-2 mutein of claim 1, wherein the IL-2 mutein has less than 30% pSTAT activation of T effector cells and greater than 40% pSTAT activation of T regulatory cells at 1 nM IL-2 treatment, using a FACS-based PBMC assay.

### Claim 6 (depends on 1)

The human IL-2 mutein of claim 1, wherein the IL-2 mutein has greater than 40% pSTAT activation of T regulatory cells at 1 nM IL-2 treatment, using a FACS-based PBMC assay.

### Claim 7 (depends on 1)

The human IL-2 mutein of claim 1, further wherein the IL-2 mutein has greater than 30% stability at 40° C. after 10 days.

### Claim 8 (depends on 1)

An fragment crystallizable (Fc)-fusion protein comprising an Fc and the human IL-2 mutein of claim 1.

### Claim 9 (depends on 8)

The Fc-fusion protein of claim 8, wherein the Fc is a human IgG1 Fc, wherein the human IgG1 Fc comprises one or more mutations altering effector function of said Fc.

### Claim 10 (depends on 9)

The Fc-fusion protein of claim 9, comprising a substitution or deletion of the C-terminal lysine of said human IgG Fc.

### Claim 11 (depends on 9)

The Fc-fusion protein of claim 9, wherein a linker connects the Fc and human IL-2 mutein portions of said protein.

### Claim 12 (depends on 11)

The Fc-fusion protein of claim 11, wherein the linker is GGGGS (SEQ ID NO: 5), GGNGT (SEQ ID NO: 6), or YGNGT (SEQ ID NO: 7).

### Claim 13 (depends on 9)

The Fc-fusion protein of claim 9, wherein the IL-2 mutein further comprises an amino acid addition, substitution, or deletion altering glycosylation of said Fc-fusion protein when expressed in mammalian cells.

### Claim 14 (depends on 9)

The Fc-fusion protein of claim 9, wherein said Fc-fusion protein comprises an Fc dimer.

### Claim 15 (depends on 14)

The Fc-fusion protein of claim 14, wherein said Fc-fusion protein comprises two IL-2 muteins.

### Claim 16 (depends on 14)

The Fc-fusion protein of claim 14, wherein said Fc-fusion protein comprises a single IL-2 mutein.

### Claim 17 (depends on 8)

An isolated nucleic acid encoding the Fc fusion protein of claim 8.

### Claim 18 (depends on 8)

A method of increasing the ratio of regulatory T cells (Tregs) to non-regulatory T cells within a population of T cells or within peripheral blood of a subject in need thereof, comprising contacting the population of T cells with an effective amount of an Fc-fusion protein of claim 8.

### Claim 19 (depends on 8)

A method of increasing the ratio of regulatory T cells (Tregs) to natural killer (NK) cells within the peripheral blood of a subject in need thereof, comprising administering an effective amount of an Fc-fusion protein of claim 8.

### Claim 20 (depends on 8)

A method of treating a subject with an inflammatory or autoimmune disease, said method comprising administering to said subject a therapeutically effective amount of an Fc-fusion protein of claim 8.

### Claim 21 (depends on 1)

An isolated nucleic acid encoding the human IL-2 mutein of claim 1.

### Claim 22 (depends on 1)

A method of increasing the ratio of regulatory T cells (Tregs) to non-regulatory T cells within a population of T cells or within peripheral blood of a subject in need thereof, comprising contacting the population of T cells with an effective amount of a human IL-2 mutein of claim 1.

### Claim 23 (depends on 1)

A method of increasing the ratio of regulatory T cells (Tregs) to natural killer (NK) cells within the peripheral blood of a subject in need thereof, comprising administering an effective amount of a human IL-2 mutein of claim 1.

### Claim 24 (depends on 1)

A method of treating a subject with an inflammatory or autoimmune disease, said method comprising administering to said subject a therapeutically effective amount of an IL-2 mutein of claim 1.

### Claim 25 (depends on 24)

The method of claim 24, wherein the inflammatory or autoimmune disease is lupus, graft-versus-host disease, hepatitis C-induced vasculitis, type I diabetes, type II diabetes, multiple sclerosis, rheumatoid arthritis, alopecia areata, atherosclerosis, psoriasis, organ transplant rejection, Sjögren's Syndrome, Behcet's disease, spontaneous loss of pregnancy, atopic diseases, asthma, or inflammatory bowel diseases.