# US 11,078,300 B2 · Circular tandem repeat proteins

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- Number: US 11,078,300 B2
- Document ID: US11078300B2
- Status: Granted
- Kind: B2
- Application number: 16/802,377
- Filed: 2020-02-26
- Published: 2020-06-18
- Granted: 2021-08-03
- Earliest claimed: 2015-12-02
- Assignees: Fred Hutchinson Cancer Research Center
- Applicants: Fred Hutchinson Cancer Research Center
- Inventors: Philip Bradley; Barry L. Stoddard
- Web page: https://patentgrep.com/patent/US11078300B2

## CPC

- C07K 19/00: Hybrid peptides {, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes}
- C07K 7/08: having 12 to 20 amino acids (gastrins C07K14/595; somatostatins C07K14/655; melanotropins C07K14/68)
- C07K 14/00: Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K 14/001: {by chemical synthesis}
- C07K 14/44: from protozoa
- C07K 14/47: from mammals
- C07K 14/55: IL-2
- C12N 15/62: DNA sequences coding for fusion proteins
- C12N 15/8282: {for fungal resistance}
- 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 2319/00: Fusion polypeptide
- C07K 2319/21: containing a His-tag
- C07K 2319/50: containing protease site
- C07K 2319/70: containing domain for protein-protein interaction
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)

## Abstract

Circular handed alpha-helical repeat proteins are described. The repeat proteins have a number of uses as scaffolds for geometrically precise, arrayed presentation of cell-signaling or immune-related protein and peptide epitopes, as well as numerous other therapeutic, diagnostic, and nanotechnological uses.

## Claims

### Claim 1

A protein having the formula: (a-b-x-y)n wherein
a and x represent linker sequences;
b represents an amino acid sequence that forms an alpha (α) helix;
y represents an amino acid sequence that forms a second α helix;
n=3, 6, 9, 12, or 24;
the protein is handed, and
the N- and C-termini of the protein create a circular architecture.

### Claim 2 (depends on 1)

The protein of claim 1, wherein the linker sequences are flexible linker sequences.

### Claim 3 (depends on 2)

The protein of claim 2, wherein the flexible linker sequences are GBB linker sequences.

### Claim 4 (depends on 3)

The protein of claim 3, wherein the GBB linker sequences are selected from GKS; GIT; GTT; GYS; GDK; GDE; NDK; GDR; GDL; and GIS.

### Claim 5 (depends on 1)

The protein of claim 1, wherein the protein is left-handed.

### Claim 6 (depends on 1)

The protein of claim 1, wherein b and y have 100% sequence identity.

### Claim 7 (depends on 1)

The protein of claim 1, wherein b and y have at least 98% sequence identity.

### Claim 8 (depends on 1)

The protein of claim 1, wherein b and y are selected from the sequences as set forth in SEQ ID NOs. 1-50; 124-129; 139; 140; 146; or 147.

### Claim 9 (depends on 1)

A protein of claim 1, wherein each (a-b-x-y) unit that is not an N-terminal or C-terminal (a-b-x-y) unit has 100% sequence identity with an adjacent (a-b-x-y) unit.

### Claim 10 (depends on 1)

A protein of claim 1, wherein each (a-b-x-y) unit has at least 95% sequence identity with an adjacent (a-b-x-y) unit.

### Claim 11 (depends on 1)

The protein of claim 1, comprising a sequence selected from the sequences as set forth in SEQ ID NOs. 51-70; 117-123; 135-138 or 145.

### Claim 12

A circular, handed protein comprising at least three α-helical structures wherein each α-helical structure comprises an outer α helix and an inner α helix joined by a flexible linker and wherein each α-helical structure has at least 95% sequence identity with an adjacent α-helical structure.

### Claim 13 (depends on 12)

The circular, handed protein of claim 12, wherein the flexible linker is a GBB linker.

### Claim 14 (depends on 13)

The circular, handed protein of claim 13, wherein the GBB linker is a sequence selected from GKS; GIT; GTT; GYS; GDK; GDE; NDK; GDR; GDL; and GIS.

### Claim 15 (depends on 12)

The circular, handed protein of claim 12, further comprising at least two functional domains wherein each functional domain is inserted into the sequence of the protein between an outer α helix of the protein and an adjacent inner α helix of the protein and wherein at least one of the functional domains is selected from a cytokine, a Notch ligand, an immunogenic peptide, a peptide adjuvant, a single-chain class I MHC domain, or a small molecule ligand binding domain.

### Claim 16

A protein having the formula: (d-a-b-x-y)n; (a-d-b-x-y)n; (a-b-d-x-y)n; (a-b-x-d-y)n or (a-b-x-y-d)n wherein
a and x represent linker sequences;
b represents an amino acid sequence that forms an alpha (α) helix;
y represents an amino acid sequence that forms a second α helix;
d represents a functional domain;
n=3, 6, 9, 12, or 24;
the protein is handed, and
the N- and C-termini of the protein create a circular architecture.

### Claim 17 (depends on 16)

The protein of claim 16, wherein the functional domain comprises a cytokine, a Notch ligand, an immunogenic peptide, a peptide adjuvant, a single-chain class I MHC domain, or a small molecule ligand binding domain.

### Claim 18 (depends on 16)

The protein of claim 16, wherein the functional domain comprises SH2, SH3, IL-2, IL-3, IL-17c, single-chain MHC, the extracellular domain of the Delta-1 Notch protein ligand, Protein L, a protein having the sequence set forth in SEQ ID NO: 116, or a protein having the sequence set forth in SEQ ID NO: 115.

### Claim 19 (depends on 16)

The protein of claim 16, wherein the linker sequences are flexible linker sequences.

### Claim 20 (depends on 16)

The protein of claim 16, wherein b and y have at least 98% sequence identity.