Phosphorylated polypeptide antigen vaccine, preparation method therefor and application thereof

Patent metadata

Field	Value
Publication number	US-12414987-B2
Application number	US-201917059665-A
Country	US
Kind code	B2
Filing date	Mar 26, 2019
Priority date	May 31, 2018
Publication date	Sep 16, 2025
Grant date	Sep 16, 2025

Abstract

The present invention discloses a phosphorylated polypeptide antigen vaccine, comprising at least two polypeptide fragments or conservatively modified variants thereof from human full-length Tau protein, wherein the polypeptide fragments or conservatively modified variants thereof contain phosphorylation sites. The present invention also discloses a complex vaccine formed by coupling a phosphorylated polypeptide antigen vaccine with a carrier. The polypeptide antigen vaccine and the complex vaccine can be used for preventing and/or treating tauopathy comprising Alzheimer's disease (AD).

First claim

The invention claimed is:

1. A phosphorylated polypeptide antigen vaccine, which comprises at least two polypeptide fragments or conservatively modified variants thereof from human full-length Tau protein, wherein the polypeptide fragments or conservatively modified variants thereof contain phosphorylation sites, wherein the polypeptide fragments are connected directly by peptide bonds or connected by amino acid linkers.

2. The phosphorylated polypeptide antigen vaccine of claim 1 , wherein the polypeptide fragments are connected directly by peptide bonds.

3. The phosphorylated polypeptide antigen vaccine of claim 1 , wherein the polypeptide fragments are derived from phosphorylation modification site-rich regions of human full-length Tau protein.

4. The phosphorylated polypeptide antigen vaccine of claim 3 , wherein the phosphorylation sites include two or more of phosphorylated amino acid sites corresponding to positions 18, 202, 205, 212, 214, 231, 235, 238, 262, 396 and 404 of the amino acid sequence of human full-length Tau protein, namely, 18(P-Tyr 18 ), 202(P-Ser 202 ), 205(P-Thr 205 ), 212(P-Thr 212 ), 214(P-Ser 214 ), 231(P-Thr 231 ), 235(P-Ser 235 ), 238(P-Ser 238 ), 262(P-Ser 262 ), 396(P-Ser 396 ) and 404(P-Ser 404 ).

5. The phosphorylated polypeptide antigen vaccine of claim 1 , wherein the conservatively modified variant of the polypeptide fragment is a variant obtained by conservatively substitution of one or more amino acids of the polypeptide fragment with functionally similar amino acids.

6. The phosphorylated polypeptide antigen vaccine of claim 1 , which has an amino acid sequence as represented by any one of SEQ ID NOs: 1-1331 and the amino acid sequence contains two or more phosphorylation sites selected from 18(P-Tyr 18 ), 202(P-Ser 202 ), 205(P-Thr 205 ), 212(P-Thr 212 ), 214(P-Ser 214 ), 231(P-Thr 231 ), 235(P-Ser 235 ), 238(P-Ser 238 ), 262(P-Ser 262 ), 396(P-Ser 396 ) and 404(P-Ser 404 ).

7. A complex vaccine formed by coupling the phosphorylated polypeptide antigen vaccine of claim 1 with a carrier.

8. The complex vaccine of claim 7 , wherein the carrier is selected from the group consisting of human serum albumin, keyhole limpet hemocyanin, bacterium-like particles (BLP), immunoglobulin molecules, thyroglobulin, ovalbumin, bovine serum albumin component V, influenza hemagglutinin, PAN-DR binding peptide (PADRE polypeptide), malaria circumsporozoite (CS) protein, hepatitis B surface antigen (HB S Ag 19-28 ), Heat Shock Protein (HSP) 65, Bacille Calmette-Guérin (BCG), cholera toxin, attenuated cholera toxin variants, diphtheria toxin, norovirus capsid P protein, recombinant Streptococcus C5a peptidase, Streptococcus pyogenes ORF1224, Streptococcus pyogenes ORF1664, Streptococcus pyogenes ORF2452, pneumolysin, attenuated pneumolysin toxicity variants, Chlamydia pneumoniae ORFT367, Chlamydia pneumoniae ORFT858, Tetanus toxoid, HIV gp120T1, microbial surface components recognizing adhesive matrix molecules (MSCRAMMS), growth factor/hormone and/or chemokines.

9. A method for preparing the complex vaccine of claim 7 , comprising the following steps:
1) artificially synthesizing the phosphorylated polypeptide antigen vaccine of claim 1 ;
2) preparing a carrier to be coupled to the phosphorylated polypeptide antigen vaccine;

3) mixing the phosphorylated polypeptide antigen vaccine with the carrier to perform coupling reaction; and
4) separating and purifying the conjugate obtained in 3), thereby obtaining a complex vaccine.

10. The method of claim 9 , wherein the carrier in step 2) is a norovirus capsid P protein, preferably PP-3C or PP-3K, and step 2) specifically comprises:
i) obtaining an expression vector comprising a nucleic acid encoding a PP-3C or PP-3K protein;
ii) transferring the expression vector into a receptor cell;
iii) expressing the PP-3C or PP-3K protein, and allowing it to self-assemble into a recombinant P particle in the receptor cell;
preferably, step 2) also comprises isolation and purification steps; and more preferably, ion exchange chromatography and/or hydrophobic chromatography are used for purification.

11. The method of claim 9 , wherein in step 3), PP-3C is used as a vaccine carrier for coupling, a preferred buffer system is an ammonium bicarbonate buffer system, and a preferred pH ranges from 7.5 to 8.8; preferably, the phosphorylated polypeptide antigen vaccine and the carrier are mixed in a ratio of 10:1 to 100:1, and a preferred reaction temperature ranges from 2° C. to 10° C.; or in step 3), PP-3K is used as a vaccine carrier for coupling, and a preferred buffer system is a phosphate buffer system, and a preferred pH ranges from 7.0 to 8.5; preferably, the phosphorylated polypeptide antigen vaccine and the carrier are mixed in a ratio of 10:1 to 100:1, and a preferred reaction temperature ranges from 2° C. to 25° C.

12. The method of claim 9 , wherein the carrier in step 2) is bacterium-like particles (BLP), and step 3) specifically comprises:
i) obtaining a purified protein adaptor—C-PA protein with a sequence as represented by SEQ ID NO: 1364; and
ii) connecting the carrier—bacterium-like particles (BLP)—obtained in step 2) with the C-PA protein to obtain C-PA-BLP; and iii) preforming coupling reaction of C-PA-BLP with the phosphorylated polypeptide antigen vaccine;
wherein a Tris buffer system is used as a buffer system and a preferred pH ranges from 7.2 to 8.8; a preferred C-PA protein concentration is 0.1 mg/mL to 1.5 mg/mL, and a preferred reaction temperature ranges from 2° C. to 30° C.; or an ammonium bicarbonate buffer system is used as a buffer system, and a preferred pH ranges from 7.5 to 8.8; preferably, the phosphorylated polypeptide antigen vaccine and the C-PA-BLP are mixed in a ratio of 10:1 to 100:1, and a preferred reaction temperature ranges from 2° C. to 10° C.

13. The method of claim 9 , wherein step 4) comprises removing coupling agent and polypeptide antigens which are not successfully connected by methods including desalting chromatography, dialysis and ultrafiltration.

14. A vaccine composition, wherein the vaccine composition comprises the phosphorylated polypeptide antigen vaccine of claim 1 or a complex vaccine; preferably, the vaccine composition further comprises a pharmaceutically acceptable adjuvant; more preferably, the pharmaceutically acceptable adjuvant is selected from one or more of CpG, MF59, AS02, AS03, Freund's complete adjuvant and Freund's incomplete adjuvant;
wherein the complex vaccine is formed by coupling the phosphorylated polypeptide antigen vaccine of claim 1 with a carrier.

15. Use of the phosphorylated polypeptide antigen vaccine of claim 1 or a complex vaccine or a vaccine composition for preparing a medicament for prevention and/or treatment of neurodegenerative disorders; and/or for preparing a medicament for maintaining or improving, preferably recovering, and more preferably completely recovering the cognitive memory of mammals, especially human beings;
wherein the complex vaccine is formed by coupling the phosphorylated polypeptide antigen vaccine of claim 1 with a carrier;
wherein the vaccine composition comprises the phosphorylated polypeptide antigen vaccine of claim 1 or the complex vaccine; preferably, the vaccine composition further comprises a pharmaceutically acceptable adjuvant; more preferably, the pharmaceutically acceptable adjuvant is selected from one or more of CpG, MF59, AS02, AS03, Freund's complete adjuvant and Freund's incomplete adjuvant.

16. Use of claim 15 , wherein the neurodegenerative disorders are selected from one or more of AD, Creu

Assignees

• Changchun Bcht Biotechnology Co
• Univ Jilin

Inventors

• Kong Wei
• Wu Hui
• Sun Yao
• Guo Yongqing

Classifications

• A61K2039/627

  characterised by the linker · CPC title

• A61K2039/6081

  Albumin; Keyhole limpet haemocyanin [KLH] · CPC title

• A61K2039/6075

  Viral proteins · CPC title

• A61K2039/6068

  Other bacterial proteins, e.g. OMP · CPC title

• A61K2039/6037

  Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT] · CPC title