Polypeptide carrier for presenting target polypeptide and uses thereof

Patent metadata

Field	Value
Publication number	US-10548973-B2
Application number	US-201615754766-A
Country	US
Kind code	B2
Filing date	Aug 24, 2016
Priority date	Aug 25, 2015
Publication date	Feb 4, 2020
Grant date	Feb 4, 2020

Abstract

The invention relates to a polypeptide carrier for presenting a target polypeptide, and use thereof. In particular, the invention relates to a nucleic acid molecule, comprising a nucleotide sequence encoding a polypeptide carrier, and being used for insertion of a nucleotide sequence encoding a target polypeptide. In addition, the invention further relates to a recombinant protein comprising the polypeptide carrier and a target polypeptide. Furthermore, the invention further relates to use of the nucleic acid molecule and the recombinant protein. In addition, the invention further relates to a vaccine or a pharmaceutical composition useful for preventing, alleviating or treating HBV infection or a disease associated with HBV infection (e.g., hepatitis B), comprising a recombinant protein comprising the polypeptide carrier of the invention and an epitope from HBV.

First claim

The invention claimed is:

1. A nucleic acid molecule, comprising:
a nucleotide sequence encoding a polypeptide carrier, or a variant thereof,
wherein the variant has at least 90% identity to the nucleotide sequence, or is capable of hybridizing to the nucleotide sequence under a stringent condition or a high stringent condition, and
wherein the polypeptide carrier is selected from the group consisting of:
(1) RBHBcAg carrier, which differs from a core antigen protein from roundleaf bat HBV (RBHBcAg protein) in that (a) 1 to 6 amino acid residues corresponding to the amino acid residues at positions 78-83 of SEQ ID NO: 1 are deleted or substituted with a linker, and (b) optionally, 1-40 amino acid residues at C-terminus of the RBHBcAg protein are deleted;
(2) TBHBcAg carrier, which differs from a core antigen protein from tent-making bat HBV (TBHBcAg protein) in that (a) 1 to 5 amino acid residues corresponding to the amino acid residues at positions 80-84 of SEQ ID NO: 2 are deleted or substituted with a linker, and (b) optionally, 1-35 amino acid residues at C-terminus of the TBHBcAg protein are deleted; and
(3) HBHBcAg carrier, which differs from a core antigen protein from horseshoe bat HBV (HBHBcAg protein) in that (a) 1 to 6 amino acid residues corresponding to the amino acid residues at positions 78-83 of SEQ ID NO: 3 are deleted or substituted with a linker, and (b) optionally, 1-40 amino acid residues at C-terminus of the HBHBcAg protein are deleted.

2. A vector, comprising the nucleic acid molecule of claim 1 .

3. A host cell, comprising the nucleic acid molecule of claim 1 or a vector comprising the nucleic acid molecule.

4. A method for presenting a target polypeptide, comprising:
(1) inserting a nucleotide sequence encoding the target polypeptide into the nucleic acid molecule of claim 1 to obtain a nucleic acid molecule encoding a recombinant protein, wherein the nucleotide sequence encoding the target polypeptide is inserted at a position of nucleotides encoding the deleted amino acid residues, inserted in the nucleotide sequence encoding the linker, or inserted at one or both termini of the nucleotide sequence encoding the linker; and
(2) expressing the nucleic acid molecule encoding the recombinant protein to produce the recombinant protein.

5. A recombinant protein, comprising:
a polypeptide carrier; and
a target polypeptide,
wherein the polypeptide carrier is selected from the group consisting of:
(1) RBHBcAg carrier, which differs from a core antigen protein from roundleaf bat HBV (RBHBcAg protein) in that (a) 1 to 6 amino acid residues corresponding to the amino acid residues at positions 78-83 of SEQ ID NO: 1 are deleted or substituted with a linker, and (b) optionally, 1-40 amino acid residues at C-terminus of the RBHBcAg protein are deleted;
(2) TBHBcAg carrier, which differs from a core antigen protein from tent-making bat HBV (TBHBcAg protein) in that (a) 1 to 5 amino acid residues corresponding to the amino acid residues at positions 80-84 of SEQ ID NO: 2 are deleted or substituted with a linker, and (b) optionally, 1-35 amino acid residues at C-terminus of the TBHBcAg protein are deleted; and
(3) HBHBcAg carrier, which differs from a core antigen protein from horseshoe bat HBV (HBHBcAg protein) in that (a) 1 to 6 amino acid residues corresponding to the amino acid residues at positions 78-83 of SEQ ID NO: 3 are deleted or substituted with a linker, and (b) optionally, 1-40 amino acid residues at C-terminus of the HBHBcAg protein are deleted, and
wherein the target polypeptide is inserted at a position of the deleted amino acid residues, inserted in the linker, or inserted at one or both of the termini of the linker.

6. A virus-like particle, comprising the recombinant protein of claim 5 .

7. A pharmaceutical composition, comprising:
the recombinant protein of claim 5 or a virus-like particle comprising the recombinant protein; and
at least one pharmaceutically acceptable vehicle or excipient.

8. A polynucleotide, encoding the recombinant protein of claim 5 .

9. A vector, comprising the polynucleotide of claim 8 .

10. A host cell, comprising the polynucleotide of claim 8 or a vector comprising the polynucleotide.

11. A method for preparing the recombinant protein of claim 5 , comprising:
culturing a host cell comprising a polynucleotide encoding the recombinant protein under a condition suitable for expressing the recombinant protein; and
recovering the recombinant protein.

12. The nucleic acid molecule of claim 1 , wherein at least one of (i) to (xi) is satisfied:
(i) the RBHBcAg protein has the amino acid sequence of SEQ ID NO: 1;
(ii) the RBHBcAg carrier differs from the RBHBcAg protein in that (a) amino acid residues corresponding to the amino acid residues at positions 78-82 of SEQ ID NO: 1 are deleted or substituted with a linker, and (b) optionally, 5-35 amino acid residues at C-terminus of the RBHBcAg protein are deleted;
(iii) the TBHBcAg protein has the amino acid sequence of SEQ ID NO: 2;
(iv) the TBHBcAg carrier differs from the TBHBcAg protein in that (a) amino acid residues corresponding to the amino acid residues at positions 80-83 of SEQ ID NO: 2 are deleted or substituted with a linker, and (b) optionally, 5-30 amino acid residues at C-terminus of the TBHBcAg protein are deleted;
(v) the HBHBcAg protein has the amino acid sequence of SEQ ID NO: 3;
(vi) the HBHBcAg carrier differs from the HBHBcAg protein in that (a) amino acid residues corresponding to the amino acid residues at positions 78-82 of SEQ ID NO: 3 are deleted or substituted with a linker, and (b) optionally, 5-35 amino acid residues at C-terminus of the HBHBcAg protein are deleted;
(vii) the linker is a flexible linker;
(viii) a restriction enzyme cleavage site is introduced at a position of nucleotides encoding the amino acid residues that are deleted;
(ix) a restriction enzyme cleavage site is introduced in the nucleotide sequence encoding the linker, and/or at either or both of the termini thereof;
(x) the nucleic acid molecule is used to insert a nucleotide sequence encoding a target polypeptide; and
(xi) the nucleic acid molecule further comprises a nucleotide sequence encoding a target polypeptide, wherein the target polypeptide is heterologous relative to the polypeptide carrier, and the nucleotide sequence encoding the target polypeptide is inserted at a position of nucleotides encoding the deleted amino acid residues, inserted in the nucleotide sequence encoding the linker, or inserted at one or both termini of the nucleotide sequence encoding the linker.

13. The nucleic acid molecule of claim 1 , wherein the polypeptide carrier has the amino acid sequence of SEQ ID NO: 4, 5, 6, 7, 8 or 9, or the nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 12, 13, 14, 15, 16 or 17.

14. The method of claim 4 , wherein the target polypeptide is an epitope peptide, and optionally,
the epitope peptide satisfies at least one of (i) to (iii):
(i) the epitope peptide comprises an epitope of HBsAg from human HBV, or an epitope of HIV GP120 protein, or an epitope of human PD-L1;
(ii) the epitope peptide comprises amino acids corresponding to the amino acids at positions 113-135 of SEQ ID NO: 44 (HBsAg protein), the amino acids at positions 361-375 of an amino acid sequence of HIV GP120 protein, or the amino acids at positions 147-160 of an amino acid sequence of human PD-L1 protein; and
(iii) the epitope peptide has the amino acid sequence of SEQ ID NO: 20, 21, 22, 60, 61, or 62.

15. The recombinant protein of claim 5 , wherein at least one of (i) to (x) i

Assignees

• Univ Xiamen
• Yang Sheng Tang Co Ltd

Inventors

• Zhang Tianying
• Yuan Quan
• Guo Xueran
• Zhang Ying
• Zhao Qinjian
• Zhang Jun
• Xia Ningshao

Classifications

• C07K14/705

  Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} · CPC title

• A61P35/00

  Antineoplastic agents · CPC title

• C12N2730/10133

  Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• A61K39/385

  Haptens or antigens, bound to carriers · CPC title