# US 2014/0154286 A1 · Modified biotin-binding protein, fusion proteins thereof and applications

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- Number: US 2014/0154286 A1
- Document ID: US20140154286A1
- Status: Published application
- Kind: A1
- Application number: 14/116,492
- Filed: 2012-05-11
- Published: 2014-06-05
- Earliest claimed: 2004-09-08
- Assignees: Children's Medical Center Corporation
- Inventors: Richard Malley; Yingjie Lu; Fan Zhang
- Web page: https://patentgrep.com/patent/US20140154286A1

## CPC

- C07K 14/31: from Staphylococcus (G)
- A61K 39/092: {Streptococcus}
- A61K 39/385: Haptens or antigens, bound to carriers
- A61K 47/543: {Lipids, e.g. triglycerides; Polyamines, e.g. spermine or spermidine}
- A61K 47/557: {the modifying agent being biotin}
- A61K 47/61: the organic macromolecular compound being a polysaccharide or a derivative thereof
- A61P 31/04: Antibacterial agents
- A61K 2039/55505: {Inorganic adjuvants}
- A61K 2039/6031: {Proteins}
- A61K 2039/6093: {Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine}
- A61K 2039/625: {binding through the biotin-streptavidin system or similar}
- A61K 2039/627: {characterised by the linker}
- A61K 2039/645: {Dendrimers; Multiple antigen peptides}
- A61K 2039/70: {Multivalent vaccine}
- C07K 2319/00: Fusion polypeptide
- C07K 2319/20: containing a tag with affinity for a non-protein ligand
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The disclosure provides modified biotin-binding proteins which can be expressed in soluble form in high yield in bacteria. Also provided are fusion proteins comprising the modified biotin-binding protein and an antigen. The disclosure further provides non-hemolytic variants of alpha-hemolysin from S. aureus and fusion protein comprising non-hemolytic variant of alpha-hemolysin and a biotin-binding domains. Immunogenic compositions comprising the proteins are also disclosed and use of such immunogenic compositions for inducing an immune response or for vaccinating a subject are also disclosed.

## Claims

### Claim 1

A soluble biotin-binding protein, comprising an amino acid sequence SEQ ID NO: 1 and any functional derivatives thereof.

### Claim at position 2

-67. (canceled)

### Claim 68 (depends on 1)

The biotin-binding of claim 1, wherein the biotin-binding protein is produced in soluble form at a level of at least 10 mg/L of culture media in E. coli.

### Claim 69 (depends on 1)

The biotin-binding protein of claim 1, wherein the biotin-binding protein comprises a bacterial signal sequence at the N-terminus.

### Claim 70 (depends on 69)

The biotin-binding protein of claim 69, wherein the signal sequence is linked to the biotin-protein by a peptide linker.

### Claim 71

A fusion protein comprising a biotin-binding protein and a protein or a peptide.

### Claim 72 (depends on 71)

The fusion protein of claim 71, wherein the protein or peptide is fused to the biotin-binding proteins by a peptide linker.

### Claim 73 (depends on 72)

The fusion protein of claim 72, wherein the peptide linker comprises the amino acid sequence SEQ ID NO: 22.

### Claim 74 (depends on 71)

The fusion protein of claim 71, wherein the protein or peptide is an antigen selected from the group consisting of: pneumococcal antigens, tuberculosis antigens, anthrax antigens, HIV antigens, seasonal or epidemic influenza antigens, Pertussis antigens, Staphylococcus aureus antigens, Meningococcal antigens, Haemophilus antigens, HPV antigens, or combinations thereof.

### Claim 75 (depends on 71)

The fusion protein of claim 71, wherein the protein or peptide is an antigen which is a non-hemolytic variant of S. aureus alpha-hemolysin.

### Claim 76 (depends on 75)

The fusion protein of claim 75, wherein the non-hemolytic variant of S. aureus alpha-hemolysin comprises a mutation at amino acid residue 205, 213 or 209-211 of wild-type S. aureus alpha-hemolysin.

### Claim 77 (depends on 75)

The fusion protein of claim 75, wherein the non-hemolytic variant of S. aureus alpha-hemolysin comprises one of the following mutations in the wild-type S. aureus alpha-hemolysin: (i) residue 205 W to A; (ii) residue 213 W to A; or (iii) residues 209-211 DRD to AAA.

### Claim 78 (depends on 75)

The fusion protein of claim 75, wherein the non-hemolytic variant of S. aureus alpha-hemolysin comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and functional variants, portions, and derivatives thereof.

### Claim 79 (depends on 71)

The fusion protein of claim 71, wherein the fusion protein comprises a bacterial signal sequence at the N-terminus

### Claim 80 (depends on 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79)

The fusion protein of claim 71, wherein the biotin-binding protein is a biotin-binding protein comprising the amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 15 or a functional derivative thereof, or the biotin-binding protein of any of claims 68-79.

### Claim 81 (depends on 71)

The fusion protein of claim 71, wherein the fusion protein comprises the amino acid sequence selected from the group consisting of SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28.

### Claim 82 (depends on 75)

The fusion protein of claim 75, wherein the fusion protein has lower hemolytic activity than an equivalent titer of wild-type alpha-hemolysin (Hla).

### Claim 83 (depends on 75)

The fusion protein of claim 75, wherein the alpha-hemolysin consists of the amino acids sequence of amino acids 27-319 of wild-type alpha-hemolysin of S. aureus.

### Claim 84

A mutant alpha-hemolysin (mHla) protein, comprising a mutation at amino acid residue 205, 213 or 209-211 of wild-type S. aureus alpha-hemolysin, wherein the mutant alpha-hemolysin has lower hemolytic activity than an equivalent titer of wild-type alpha-hemolysin (Hla).

### Claim 85 (depends on 84)

The mutant alpha-hemolysin of claim 84, wherein the mutant alpha-hemolysin comprises one of the following mutations in the wild-type S. aureus alpha-hemolysin: (i) residue 205 W to A; (ii) residue 213 W to A; or (iii) residues 209-211 DRD to AAA.

### Claim 86 (depends on 84)

The mutant alpha-hemolysin any of claim 84, wherein the mutant alpha-hemolysin comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, and functional variants, portions, and derivatives thereof.

### Claim 87 (depends on 1)

A method inducing an immune response in a subject, comprising administering to the subject the biotin-binding protein of claim 1.

### Claim 88 (depends on 87)

The method of claim 87, wherein the immune response is an antibody or B-cell response.

### Claim 89 (depends on 87)

The method of claim 87, wherein the immune response is a CD4+ T-cell response, including Th1, Th2, or Th17 response, or a CD8+ T-cell response or a CD4+/CD8+ T-cell response.