# US 12,180,258 B2 · T-cell modulatory multimeric polypeptides and methods of use thereof

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- Number: US 12,180,258 B2
- Document ID: US12180258B2
- Status: Granted
- Kind: B2
- Application number: 18/391,486
- Filed: 2023-12-20
- Published: 2024-06-20
- Granted: 2024-12-31
- Earliest claimed: 2016-12-22
- Assignees: Cue Biopharma, Inc.
- Applicants: Cue Biopharma, Inc.
- Inventors: Ronald D. Seidel, III; Rodolfo J. Chaparro
- Web page: https://patentgrep.com/patent/US12180258B2

## CPC

- A61K 39/12: Viral antigens
- C07K 14/55: IL-2
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- A61K 35/17: Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes (when activated by a specific antigen A61K39/00)
- A61P 31/20: for DNA viruses
- A61P 35/00: Antineoplastic agents
- A61P 37/04: Immunostimulants
- C07K 14/005: from viruses
- C07K 14/4748: {Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE}
- C07K 14/70539: {MHC-molecules, e.g. HLA-molecules}
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- 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)
- A61K 2039/505: {comprising antibodies}
- A61K 2039/55533: {IL-2}
- C07K 2317/34: Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
- C07K 2319/30: Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
- C12N 2710/20034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- 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
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present disclosure provides variant immunomodulatory polypeptides, and fusion polypeptides comprising the variant immunomodulatory peptides. The present disclosure provides T-cell modulatory multimeric polypeptides, and compositions comprising same, where the T-cell modulatory multimeric polypeptides comprise a variant immunomodulatory polypeptide of the present disclosure. The present disclosure provides nucleic acids comprising nucleotide sequences encoding the T-cell modulatory multimeric polypeptides, and host cells comprising the nucleic acids. The present disclosure provides methods of modulating the activity of a T cell; the methods comprise contacting the T cell with a T-cell modulatory multimeric polypeptide of the present disclosure.

## Claims

### Claim 1

A fusion polypeptide comprising:
a) one or more variant IL-2 polypeptides, wherein each of the one or more variant IL-2 polypeptides comprises up to four amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO:1, wherein amino acid 42 is an amino acid other than a phenylalanine, wherein amino acid 16 is an amino acid other than a histidine, and wherein each of the one or more variant IL2 polypeptides exhibit reduced binding affinity to an IL-2 receptor (IL2R) comprising alpha, beta, and gamma polypeptides having amino acid sequences set forth in SEQ ID NOs: 18-20, compared to the binding affinity of the IL-2 amino acid sequence set forth in SEQ ID NO: 1 for the IL2R; and
b) a heterologous fusion partner comprising an antibody Fc region, wherein the antibody Fc region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO:57, wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST,
wherein the fusion polypeptide does not comprise a major histocompatibility complex (MHC) polypeptide.

### Claim 2 (depends on 1)

The fusion polypeptide of claim 1, wherein the antibody Fc region comprises one or more substitutions selected from an L14A substitution, an L15A substitution, an N77A substitution, a P111S substitution based on the amino acid numbering of SEQ ID NO:57.

### Claim 3 (depends on 1)

The fusion polypeptide of claim 1, wherein the fusion polypeptide comprises from 400 to 600 amino acids.

### Claim 4 (depends on 1)

The fusion polypeptide of claim 1, wherein the fusion polypeptide comprises from 500 to 700 amino acids.

### Claim 5 (depends on 1)

The fusion polypeptide of claim 1, wherein the heterologous fusion partner comprises an antibody Fc region and an antigen-binding region of an antibody.

### Claim 6 (depends on 5)

The fusion polypeptide of claim 5, wherein the fusion polypeptide comprises from 400 to 700 amino acids.

### Claim 7 (depends on 5)

The fusion polypeptide of claim 5, wherein the fusion polypeptide comprises from 700 to 900 amino acids.

### Claim 8 (depends on 1)

The fusion polypeptide of claim 1, wherein amino acid 16 of each of the one or more variant IL-2 polypeptides is Ala, Thr, Asp, or Glu; and wherein amino acid 42 of each of the one or more variant IL-2 polypeptides is Ala or Thr.

### Claim 9 (depends on 6)

The fusion polypeptide of claim 6, wherein amino acid 16 of each of the one or more variant IL-2 polypeptides is Ala, Thr, Asp, or Glu; and wherein amino acid 42 of each of the one or more variant IL-2 polypeptides is Ala or Thr.

### Claim 10 (depends on 7)

The fusion polypeptide of claim 7, wherein amino acid 16 of each of the one or more variant IL-2 polypeptides is Ala, Thr, Asp, or Glu; and wherein amino acid 42 of each of the one or more variant IL-2 polypeptides is Ala or Thr.

### Claim 11

A fusion polypeptide comprising:
a) one or more variant IL-2 polypeptides, wherein each of the one or more variant IL-2 polypeptides comprises up to three amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO:1, wherein amino acid 42 is an amino acid other than a phenylalanine, wherein amino acid 16 is an amino acid other than a histidine, and wherein each of the one or more variant IL2 polypeptides exhibit reduced binding affinity to an IL-2 receptor (IL2R) comprising alpha, beta, and gamma polypeptides having amino acid sequences set forth in SEQ ID NOs: 18-20, compared to the binding affinity of the IL-2 amino acid sequence set forth in SEQ ID NO: 1 for the IL2R; and
b) a heterologous fusion partner comprising an antibody Fc region, wherein the antibody Fc region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57, wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST,
wherein the fusion polypeptide does not comprise a major histocompatibility complex (MHC) polypeptide.

### Claim 12 (depends on 11)

The fusion polypeptide of claim 11, wherein the antibody Fc region comprises one or more substitutions selected from an L14A substitution, an L15A substitution, an N77A substitution, a P111S substitution based on the amino acid numbering of SEQ ID NO:57.

### Claim 13 (depends on 11)

The fusion polypeptide of claim 11, wherein the fusion polypeptide comprises from 400 to 600 amino acids.

### Claim 14 (depends on 11)

The fusion polypeptide of claim 11, wherein the fusion polypeptide comprises from 500 to 700 amino acids.

### Claim 15 (depends on 11)

The fusion polypeptide of claim 11, wherein the heterologous fusion partner comprises an antibody Fc region and an antigen-binding region of an antibody.

### Claim 16 (depends on 15)

The fusion polypeptide of claim 15, wherein the fusion polypeptide comprises from 400 to 700 amino acids.

### Claim 17 (depends on 15)

The fusion polypeptide of claim 15, wherein the fusion polypeptide comprises from 700 to 900 amino acids.

### Claim 18 (depends on 11)

The fusion polypeptide of claim 11, wherein amino acid 16 of each of the one or more variant IL-2 polypeptides is Ala, Thr, Asp, or Glu; and wherein amino acid 42 of each of the one or more variant IL-2 polypeptides is Ala or Thr.

### Claim 19 (depends on 16)

The fusion polypeptide of claim 16, wherein amino acid 16 of each of the one or more variant IL-2 polypeptides is Ala, Thr, Asp, or Glu; and wherein amino acid 42 of each of the one or more variant IL-2 polypeptides is Ala or Thr.

### Claim 20 (depends on 17)

The fusion polypeptide of claim 17, wherein amino acid 16 of each of the one or more variant IL-2 polypeptides is Ala, Thr, Asp, or Glu; and wherein amino acid 42 of each of the one or more variant IL-2 polypeptides is Ala or Thr.

### Claim 21

A fusion polypeptide comprising:
a) one or more variant IL-2 polypeptides, wherein each of the one or more variant IL-2 polypeptides comprises two amino acid substitutions relative to the amino acid sequence set forth in SEQ ID NO:1, wherein amino acid 42 is an amino acid other than a phenylalanine, wherein amino acid 16 is an amino acid other than a histidine, and wherein each of the one or more variant IL2 polypeptides exhibit reduced binding affinity to an IL-2 receptor (IL2R) comprising alpha, beta, and gamma polypeptides having amino acid sequences set forth in SEQ ID NOs: 18-20, compared to the binding affinity of the IL-2 amino acid sequence set forth in SEQ ID NO: 1 for the IL2R; and
b) a heterologous fusion partner comprising an antibody Fc region, wherein the antibody Fc region comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 57, wherein the percent sequence identity is determinable by a sequence alignment performed using BLAST,
wherein the fusion polypeptide does not comprise a major histocompatibility complex (MHC) polypeptide.

### Claim 22 (depends on 21)

The fusion polypeptide of claim 21, wherein the antibody Fc region comprises one or more substitutions selected from an L14A substitution, an L15A substitution, an N77A substitution, a P111S substitution based on the amino acid numbering of SEQ ID NO:57.

### Claim 23 (depends on 21)

The fusion polypeptide of claim 21, wherein the fusion polypeptide comprises from 400 to 600 amino acids.

### Claim 24 (depends on 21)

The fusion polypeptide of claim 21, wherein the fusion polypeptide comprises from 500 to 700 amino acids.

### Claim 25 (depends on 21)

The fusion polypeptide of claim 21, wherein the heterologous fusion partner comprises an antibody Fc region and an antigen-binding region of an antibody.

### Claim 26 (depends on 25)

The fusion polypeptide of claim 25, wherein the fusion polypeptide comprises from 400 to 700 amino acids.

### Claim 27 (depends on 25)

The fusion polypeptide of claim 25, wherein the fusion polypeptide comprises from 700 to 900 amino acids.

### Claim 28 (depends on 21)

The fusion polypeptide of claim 21, wherein amino acid 16 of each of the one or more variant IL-2 polypeptides is Ala, Thr, Asp, or Glu; and wherein amino acid 42 of each of the one or more variant IL-2 polypeptides is Ala or Thr.

### Claim 29 (depends on 26)

The fusion polypeptide of claim 26, wherein amino acid 16 of each of the one or more variant IL-2 polypeptides is Ala, Thr, Asp, or Glu; and wherein amino acid 42 of each of the one or more variant IL-2 polypeptides is Ala or Thr.

### Claim 30 (depends on 27)

The fusion polypeptide of claim 27, wherein amino acid 16 of each of the one or more variant IL-2 polypeptides is Ala, Thr, Asp, or Glu; and wherein amino acid 42 of each of the one or more variant IL-2 polypeptides is Ala or Thr.