# US 2015/0329598 A1 · Stabilized hepatitis b core polypeptides

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- Number: US 2015/0329598 A1
- Document ID: US20150329598A1
- Status: Published application
- Kind: A1
- Application number: 14/807,787
- Filed: 2015-07-23
- Published: 2015-11-19
- Earliest claimed: 2013-01-23
- Assignees: The Board of Trustees of the Leland Stanford Junior University
- Applicants: The Board of Trustees of the Leland Stanford Junior University
- Inventors: Yuan Lu; James Robert Swartz
- Web page: https://patentgrep.com/patent/US20150329598A1

## CPC

- C07K 14/005: from viruses
- C07K 14/02: Hepadnaviridae, e.g. hepatitis B virus
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- A61K 39/29: Hepatitis virus
- C12N 2730/10122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2730/10123: Virus like particles [VLP]
- C12N 2770/32423: Virus like particles [VLP]
- C12N 2770/32434: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- 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)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Genetically modified HBc polypeptides are provided.

## Claims

### Claim at position 1

1-18. (canceled)

### Claim 19

An HBc polypeptide comprising the amino acid sequence of any one of SEQ ID NO:3-31, 34-52.

### Claim at position 3

20-23. (canceled)

### Claim 24 (depends on 19)

A method of producing a polypeptide of claim 19, comprising introducing a polynucleotide sequence encoding the fusion protein operably linked to a promoter, into a cell or cell free protein synthesis reaction mix and incubating for a period of time sufficient to synthesize the protein.

### Claim at position 5

25-26. (canceled)

### Claim 27

A stabilized, chimeric virus like particle (VLP) having an artificial disulfide bond network comprising multiple HBc polypeptides wherein:
a. at least two amino acids of each of the HBc polypeptides are substituted with cysteine residues that can form interdimer disulfide bonds after the HBc is assembled into the VLP thereby forming the artificial disulfide bond network that stabilizes the VLP;
b. wherein each of the HBc polypeptides comprises a transplanted spike polypeptide sequence of an HBc polypeptide which is different from the spike polypeptides sequence of the wildtype HBc polypeptide of (a); and
c. wherein two HBc polypeptides of (b) dimerized to form a spike.

### Claim at position 7

28-29. (canceled)

### Claim 30 (depends on 27)

The stabilized VLP of claim 27, wherein the spike sequence of the HBc polypeptide is a sequence from amino-to-carboxyl end of about (a) twenty four amino acids that form a long alpha helix 3 (α3) with 6.4 alpha helical turns followed by (b) five amino acids that loop back joined to (c) thirty-two amino acids that form alpha helix 4 (α4) with a kink that breaks the alpha helix 4 at the thirteenth amino acid separating α4 into about (i) twelve amino acids that form three alpha helical turns of alpha helix 4a (α4a) and (ii) nineteen amino acids that form five alpha helical turns of alpha helix 4b (α4b), which forms a hairpin structure and (b) participates in a 4-helix bundle in the HBc dimer of claim 27(c) or 28(c), or portion thereof.

### Claim 31 (depends on 30)

The stabilized VLP of claim 30, wherein the sequence forming alpha helix 3 (α3) to end of alpha helix 4b (α4b) is any of a sequence starting with amino acid residue, proline, at position 50 and ending with amino acid residue, phenylalanine, at position 110 of SEQ ID NO: 1 or UniProt accession number P03147 (FIG. 4A) or equivalent thereof.

### Claim 32 (depends on 31)

The stabilized VLP of claim 31, wherein the amino acid residues equivalent to position 50 to 110 of SEQ ID NO: 1 or UniProt accession number P03147 (FIG. 4A) are any of: the top six aligned HBV sequences shown in FIG. 14A beginning with about amino acid residue, alanine or proline, at position 50 and ending with about amino acid residue, phenylalanine, at position 110.

### Claim 33 (depends on 27)

The stabilized VLP of claim 27, wherein the spike of (c) has a hydrophobic pocket or portion thereof and a spike tip.

### Claim 34 (depends on 33)

The stabilized VLP of claim 33, wherein the sequence of the hydrophobic pocket is a sequence selected from a group consisting of:
i. amino acid residue, isoleucine, at about position 59 and ends with amino acid residue, phenylalanine, at about position 97 of SEQ ID NO: 1 or UniProt accession number P03147 (FIG. 4A) or equivalent thereof, or ii. amino acid residue, valine, at about position 88 and ending with amino acid residue, isoleucine, at about position 126 of UniProt accession number Q8B6N7 (FIG. 14A-B) or equivalent thereof, and
iii. an about 39-amino acid sequence of SS1(HP) as underlined and shown in Table S2 beginning with valine (V) and ending with isoleucine (I).

### Claim 35 (depends on 34)

The stabilized VLP of claim 34, wherein the amino acid residues equivalent to about positions 59 to 97 of SEQ ID NO: 1 or UniProt accession number P03147 of FIG. 4A are shown in FIG. 14A beginning with isoleucine or valine at about amino acid 59 and ending with leucine, phenylalanine or isoleucine at about amino acid 97 for the top six aligned HBV sequences; or aligned HBV sequences or portions thereof corresponding to about position 59 to 97 of SEQ ID NO: 1 or UniProt accession number P03147.

### Claim 36 (depends on 34)

The stabilized VLP of claim 34, wherein amino acid residues equivalent to about amino acid position 88 and ending at amino acid position 126 of UniProt accession number Q8B6N7 are shown in FIG. 14A beginning with valine or isoleucine at about amino acid position 88 and ending with isoleucine at amino acid position 126 for the bottom two aligned HBV sequences or additional aligned HBV sequences or portions thereof corresponding to position 88 to 126 of UniProt accession number Q8B6N7 of FIG. 14A-B.

### Claim 37 (depends on 34)

The stabilized VLP of claim 34, wherein the spike tip comprises an 8-amino acid sequence of SS1(ST) as shown underlined in Table S2 having:
i. a sequence starting with amino acid residue, threonine, at about position 74 and ending with amino acid residue, serine, at about position 81 of SEQ ID NO: 1 or UniProt accession number P03147 of FIG. 4A or equivalent thereof; or
ii. a sequence starting with amino acid residue, asparagine, at about position 103 and ending with amino acid residue, alanine, at about position 110 of UniProt accession number Q8B6N7 of FIG. 14A-B or equivalent thereof.

### Claim at position 16

38-39. (canceled)

### Claim 40 (depends on 27)

The stabilized VLP of claim 27, wherein at least two amino acids of the HBc polypeptide of (a) are located in proximity between HBc dimer forming both 5-fold units and 6-fold units of the VLP.

### Claim 41 (depends on 40)

The stabilized VLP of claim 40, wherein the amino acids of the HBC polypeptide so substituted are D29 and R127 as shown in SEQ ID NO:1 or SEQ ID NO:2 .

### Claim 42 (depends on 29)

The stabilized VLP of claim 29, wherein the two amino acids of the HBC polypeptide so substituted are any of P134-P135, P134-N136, P134-A137, or P135-N136 as shown in SEQ ID NO:1 or SEQ ID NO:2.

### Claim 43 (depends on 1)

The HBc polypeptide of claim 1 further comprising an internal sequence from a second different HBc polypeptide, wherein the internal sequence of the second, different HBc polypeptide comprises a spike tip with a lower number of net negative charges at neutral pH and is used to substitute a corresponding portion of the HBc polypeptide thereby resulting in a chimeric HBc polypeptide having lower net negative charges at the spike tip than two net negative charges at neutral pH at the spike tip of an unsubstituted HBc polypeptide or amino acid residues 74-81 of SEQ ID NO:1.

### Claim 44

(canceled)

### Claim 45 (depends on 43)

The HBc polypeptide of claim 43, wherein the internal sequence is a sequence selected from a group consisting of an HBc spike tip, an HBc hydrophobic pocket, and an HBc spike or a portion thereof.

### Claim at position 23

46-47. (canceled)

### Claim 48 (depends on 18)

The HBc polypeptide of claim 18, wherein the additional moiety so conjugated is any one or more of an antibody or a fragment or derivative thereof, a cytokine, a bacterial protein and a tumor antigen.

### Claim 49

(canceled)

### Claim 50 (depends on 27)

A method for making a VLP of claim 27 having a transplanted spike sequence or portion thereof comprising:
a. aligning amino acid sequences of VLP coat or capsid proteins;
b. identifying amino acid sequence with a desired spike sequence or portion thereof from the aligned sequences;
c. replacing nucleic acid sequence encoding a spike sequence or portion thereof of a VLP coat or capsid protein with nucleic acid sequence encoding the desired spike sequence or portion thereof so as to produce a nucleic acid sequence encoding a chimeric VLP coat or capsid protein with a transplanted spike sequence or portion thereof;
d. producing the chimeric VLP coat or capsid protein with a transplanted spike sequence or portion thereof from the nucleic acid sequence of (c),
e. permitting the chimeric VLP coat or capsid protein of (d) to undergo self-assembly to form VLP, thereby making a chimeric VLP having a transplanted spike sequence or portion thereof.

### Claim 51 (depends on 50)

A chimeric VLP produced by the method of claim 50.

### Claim at position 28

52-56. (canceled)

### Claim 57

A kit for the display of an antigen on the surface of VLP comprising a chimeric VLP with a transplanted spike or portion thereof stabilized by artificial disulfide bond network and comprised of an non-natural amino acid, and an informational insert.