Stabilized hepatitis B core polypeptides

Patent metadata

Field	Value
Publication number	US-9896483-B2
Application number	US-201514807787-A
Country	US
Kind code	B2
Filing date	Jul 23, 2015
Priority date	Jan 23, 2013
Publication date	Feb 20, 2018
Grant date	Feb 20, 2018

Abstract

Genetically modified HBc polypeptides are provided.

First claim

What is claimed is:

1. A stabilized, chimeric virus like particle (VLP) having an artificial disulfide bond network comprising multiple HBc polypeptides:
a. wherein 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 spike sequence—which has been modified to have a lower negative charge or neutral charge compared to the spike sequence of the wild-type HBc polypeptide at neutral pH; and
c. wherein two HBc polypeptides of (b) are dimerized to form a spike, and wherein the at least two amino acids are selected from the group consisting of P134-P135, P134-N136, P134-A137, and P135-N136 as provided in SEQ ID NO:1 or SEQ ID NO:2 or amino acid pairs at corresponding positions in aligned sequences of HBC polypeptides.

2. The stabilized VLP of claim 1 , wherein the spike sequence of the HBc polypeptide is a sequence from amino-to-carboxyl end of (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 (i) twelve amino acids that form three alpha helical turns of alpha helix4a (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 1 (c), or portion thereof.

3. The stabilized VLP of claim 2 , wherein the sequence forming alpha helix 3 (α3) to the end of alpha helix 4b (α4b) is 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 SEQ ID NO: 32 or corresponding residues thereto.

4. The stabilized VLP of claim 3 , wherein the amino acid residues corresponding to position 50 to 110 of SEQ ID NO: 1 or SEQ ID NO: 32 are in any of: SEQ ID NOS: 111-116 beginning with amino acid residue, alanine or proline, at position 50 and ending with amino acid residue, phenylalanine, at position 110.

5. The stabilized VLP of claim 1 , wherein the spike of (c) has a hydrophobic pocket or portion thereof and a spike tip.

6. The stabilized VLP of claim 5 , wherein the sequence of the hydrophobic pocket is a sequence selected from the group consisting of:
i. amino acid residue, isoleucine, at position 59 and ends with amino acid residue, phenylalanine, at position 97 of SEQ ID NO: 1 or SEQ ID NO: 32 or corresponding residues thereto, or
ii. amino acid residue, valine, at position 88 and ending with amino acid residue, isoleucine, at position 126 of SEQ ID NO: 117 or corresponding residues thereto, and
iii. an 39-amino acid sequence of SS1(HP) as shown in 2 SEQ ID NO: 110 beginning with valine (V) at position 59 and ending with isoleucine (I) at position 97.

7. The stabilized VLP of claim 6 , wherein the amino acid residues corresponding to positions 59 to 97 of SEQ ID NO: 1 or SEQ ID NO: 32 begin with isoleucine or valine at amino acid 59 and end with leucine, phenylalanine or isoleucine at amino acid 97 ; or aligned HBV sequences or portions thereof corresponding to position 59 to 97 of SEQ ID NO: 1 or SEQ ID NO: 32.

8. The stabilized VLP of claim 6 , wherein the spike tip comprises an 8-amino acid sequence of SS1(ST) having:
i. a sequence starting with amino acid residue, threonine, at position 74 and ending with amino acid residue, serine, at position 81 of SEQ ID NO: 1 or SEQ ID NO: 32 or corresponding residues thereto; or
ii. a sequence starting with amino acid residue, asparagine, at position 103 and ending with amino acid residue, alanine, at position 110 of SEQ ID NO: 117 or corresponding residues thereto.

9. A stabilized, chimeric virus like particle (VLP) having an artificial disulfide bond network comprising multiple HBc polypeptides wherein:
a. wherein 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 spike sequence of an HBc polypeptide which has been modified to have a lower negative charge or neutral charge compared to the spike sequence of the wild-type HBc polypeptide at neutral pH; and
c. wherein two HBc polypeptides of (b) are dimerized to form a spike and wherein the at least 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.

10. The polypeptide of claim 1 , additionally comprising substituting a cysteine at amino acid position 61 of SEQ ID NO: 1 or SEQ ID NO: 2 with a non-cysteine amino acid so as to further stabilize a VLP or reduce the possibility of self-assembly to a VLP with T=3 icosahedral symmetry.

Assignees

• Univ Leland Stanford Junior

Inventors

• Lu Yuan
• Swartz James Robert

Classifications

• C12P21/02 Primary

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C07K14/005 Primary

  from viruses · CPC title

• C12N2770/32423

  Virus like particles [VLP] · CPC title

• C12N2730/10123

  Virus like particles [VLP] · CPC title

• C07K14/02

  Hepadnaviridae, e.g. hepatitis B virus · CPC title