# US 10,245,318 B2 · Flagellin-containing protein nanoparticles as a vaccine platform

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- Number: US 10,245,318 B2
- Document ID: US10245318B2
- Status: Granted
- Kind: B2
- Application number: 15/110,449
- Filed: 2015-01-09
- Published: 2016-11-10
- Granted: 2019-04-02
- Earliest claimed: 2014-01-09
- Assignees: Alpha-O Peptides AG
- Applicants: Alpha-O Peptides AG
- Inventors: Peter Burkhard; Senthil Kumar Raman; Sara Maria Paulillo; Matteo Piazza; Caroline Kulangara; Christian Mittelholzer
- Web page: https://patentgrep.com/patent/US10245318B2

## CPC

- A61K 39/39: characterised by the immunostimulating additives, e.g. chemical adjuvants
- A61K 39/0013: {Therapeutic immunisation against small organic molecules, e.g. cocaine, nicotine}
- A61K 39/015: Hemosporidia antigens, e.g. Plasmodium antigens
- A61P 37/04: Immunostimulants
- C07K 14/195: from bacteria
- A61K 2039/55555: {Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers}
- A61K 2039/6068: {Other bacterial proteins, e.g. OMP}
- A61K 2039/64: {characterised by the architecture of the carrier-antigen complex, e.g. repetition of carrier-antigen units}
- 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
- 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)

## Abstract

The present invention relates to self-assembling protein nanoparticles constructed from suitable oligomerization domains and further incorporating the TLR5 binding protein flagellin as an adjuvant molecule. Furthermore, the invention relates to the use of such nanoparticles for vaccination.

## Claims

### Claim 1

A self-assembling protein nanoparticle consisting of aggregates of a multitude of building blocks of formula (Ia) or (Ib)
X-ND1-L1-ND2-FLA (Ia)
or
FLA-ND1-L1-ND2-X (Ib),
consisting of a continuous chain comprising a protein oligomerization domain ND1, a linker L1, a protein oligomerization domain ND2, FLA, and a further substituent X, wherein
ND1 is a protein that forms oligomers (ND1)m of m subunits ND1,
ND2 is a protein that forms oligomers (ND2)n of n subunits ND2,
m and n each is a figure between 2 and 10, with the proviso that m is not equal to n and not a multiple of n, and n is not a multiple of m,
L1 is a bond or a short flexible linker,
FLA is flagellin, or a derivative of flagellin lacking parts of the flagellin amino acid sequence but at least containing the TLR5 binding domain D1, and wherein the lacking parts are (a) replaced by a flexible linker segment of 1 to 20 amino acids joining two ends of the flagellin amino acid sequence or (b) are replaced by a fully folded protein antigen;
X is absent or a peptide or protein sequence comprising 1 to 1000 amino acids,
optionally co-assembled with a multitude of building blocks of the formula (II)
Y-ND3-L2-ND4-Z (II),
consisting of a continuous chain comprising a protein oligomerization domain ND3, a linker L2, a protein oligomerization domain ND4, and further substituents Y and Z, wherein
ND3 is a protein that forms oligomers (ND3)y of y subunits ND3,
ND4 is a protein that forms oligomers (ND4)z of z subunits ND4,
y and z each is a figure between 2 and 10, with the proviso that y is not equal to z and not a multiple of z, and z is not a multiple of y, and wherein
either ND3 is identical to ND1, or ND4 is identical to ND2 or both ND3 and ND4 are identical to ND1 and ND2, respectively,
L2 is a bond or a short flexible linker that may be different from L1 or identical to L1, and
Y and Z are, independently of each other, absent or a peptide or protein sequence comprising 1 to 1000 amino acids.

### Claim 2 (depends on 1)

The protein nanoparticle according to claim 1 consisting of aggregates of a multitude of building blocks of formula (Ia) or (Ib)
X-ND1-L1-ND2-FLA (Ia)
or
FLA-ND1-L1-ND2-X (Ib),
co-assembled with a multitude of building blocks of the formula (II)
Y-ND3-L2-ND4-Z (II).

### Claim 3 (depends on 1)

The protein nanoparticle according to claim 1 in which at least one of ND1 and ND2 and at least one of ND3 and ND4 is a coiled-coil.

### Claim 4 (depends on 1)

The protein nanoparticle according to claim 1 wherein at least one of X, Y and Z is an antigen of interest.

### Claim 5 (depends on 1)

The protein nanoparticle according to claim 1 wherein the flagellin derivative is lacking the D2 and D3 domains of flagellin.

### Claim 6 (depends on 1)

The protein nanoparticle according to claim 1 wherein the flagellin derivative comprises an antigen of interest.

### Claim 7 (depends on 1)

The protein nanoparticle according to claim 1 wherein n in a building block of formula (Ia) or m in a building block of formula (Ib) is 2.

### Claim 8 (depends on 7)

The protein nanoparticle according of claim 7 wherein FLA is connected at the D2 part of flagellin to the oligomerization domain ND2 in formula (Ia), wherein n is 2, or to the oligomerization domain ND1 in formula (Ib), wherein m is 2.

### Claim 9 (depends on 1)

The protein nanoparticle according to claim 1 wherein at least one of ND1, ND2, ND3 and ND4 is a coiled-coil.

### Claim 10 (depends on 1)

The protein nanoparticle according to claim 1 wherein at least one of ND1, ND2, ND3 and ND4 is the trimerization domain of the bacteriophage T4 protein fibritin.

### Claim 11 (depends on 1)

A composition comprising a protein nanoparticle according to claim 1.

### Claim 12

A monomeric building block of formula (Ia) or (Ib)
X-ND1-L1-ND2-FLA (Ia)
or
FLA-ND1-L1-ND2-X (Ib),
consisting of a continuous chain comprising a protein oligomerization domain ND1, a linker L1, a protein oligomerization domain ND2, FLA, and a further substituent X, wherein
ND1 is a protein that forms oligomers (ND1)m of m subunits ND1,
ND2 is a protein that forms oligomers (ND2)n of n subunits ND2,
m and n each is a figure between 2 and 10, with the proviso that m is not equal to n and not a multiple of n, and n is not a multiple of m,
L1 is a bond or a short flexible linker,
FLA is flagellin, or a derivative of flagellin lacking parts of the flagellin amino acid sequence but at least containing the TLR5 binding domain D1, and wherein the lacking parts are (a) replaced by a flexible linker segment of 1 to 20 amino acids joining two ends of the flagellin amino acid sequence or (b) are replaced by a fully folded protein antigen; and
X is absent or a peptide or protein sequence comprising 1 to 1000 amino acids.

### Claim 13 (depends on 1)

A method of vaccinating a human or non-human animal, which comprises administering an effective amount of a protein nanoparticle according to claim 1 to a subject in need of such vaccination.