# US 10,246,494 B2 · Polypeptides and antibodies for treating HBV infection and related diseases

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- Number: US 10,246,494 B2
- Document ID: US10246494B2
- Status: Granted
- Kind: B2
- Application number: 15/649,967
- Filed: 2017-07-14
- Published: 2018-01-04
- Granted: 2019-04-02
- Earliest claimed: 2012-06-11
- Assignees: Xiamen University; Yang Sheng Tang Company, Ltd.
- Applicants: Xiamen University; Xiamen Innovax Biotech Co., Ltd.
- Inventors: Quan Yuan; Tianying Zhang; Wenxin Luo; Yixin Chen; Jun Zhang; Ningshao Xia
- Web page: https://patentgrep.com/patent/US10246494B2

## CPC

- C07K 14/005: from viruses
- A61K 39/12: Viral antigens
- A61P 1/16: for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
- A61P 31/20: for DNA viruses
- A61P 37/04: Immunostimulants
- C07K 7/06: having 5 to 11 amino acids
- C07K 16/082: Hepadnaviridae (F), e.g. hepatitis B virus
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- G01N 33/56983: {Viruses}
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- A61K 2039/505: {comprising antibodies}
- A61K 2039/6037: {Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]}
- C07K 2317/24: containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C12N 7/00: Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof (preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00)
- C12N 2710/00034: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- C12N 2730/10122: New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
- C12N 2730/10134: Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
- G01N 2333/02: Hepadnaviridae, e.g. hepatitis B virus
- 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)
- 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
- 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)}
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to epitope peptides (or mutants thereof) for treating hepatitis B virus infection, recombinant proteins comprising such epitope peptides (or mutants thereof) and carrier proteins, and uses of such epitope peptides (or mutants thereof) and recombinant proteins. The present invention also relates to antibodies against such epitope peptides, cell lines producing said antibodies, and uses thereof. Furthermore, the present invention relates to vaccines or pharmaceutical compositions for treating or alleviating one or more symptoms associated with hepatitis B virus infection, which comprise the recombinant proteins or antibodies according to the invention, respectively.

## Claims

### Claim 1

A monoclonal antibody or an antigen binding fragment thereof, wherein the monoclonal antibody specifically binds to positions 119 to 125 of HBsAg protein of HBV, and is selected from one of the following monoclonal antibodies:
1) the monoclonal antibody produced by the hybridoma cell line HBs-E6F6, wherein the hybridoma cell line HBs-E6F6 is deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO. C201270;
2) the monoclonal antibody produced by the hybridoma cell line HBs-E7G11, wherein the hybridoma cell line HBs-E7G11 is deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO. C201271;
3) the monoclonal antibody produced by the hybridoma cell line HBs-G12F5, wherein the hybridoma cell line HBs-G12F5 is deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO. C201272;
4) the monoclonal antibody produced by the monoclonal antibody produced by hybridoma cell line HBs-E13C5, wherein the hybridoma cell line HBs-E13C5 is deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO. C201273 and
5) the monoclonal antibody or an antigen binding fragment selected from a humanized antibody, a chimeric antibody or a single chain antibody made from any of the monoclonal antibodies as described in 1-4 above.

### Claim 2 (depends on 1)

The monoclonal antibody or antigen binding fragment thereof of claim 1, wherein the monoclonal antibody or antigen binding fragment thereof has one or more of the following features:
(1) the antigen binding fragment thereof is selected from a group consisting of Fab, Fab′, F(ab′)2, Fd, Fv, dAb, complementary determining region fragment, or a single chain antibody;
(2) the monoclonal antibody binds to HBsAg protein with a KD of less than about 10−5 M, or less than about 10−6 M, 10−7 M, 10−8 M, 10−9 M, 10−10 M or less;
(3) the monoclonal antibody comprises non-CDR region, and the non-CDR region is from species other than murine species; and
(4) the monoclonal antibody is capable of reducing serum level of HBV DNA and/or HBsAg in a subject.

### Claim 3 (depends on 2)

The monoclonal antibody or antigen binding fragment thereof of claim 2, wherein the single chain antibody is scFv, and/or, the chimeric antibody is a human-mouse chimeric antibody.

### Claim 4 (depends on 2)

The monoclonal antibody or antigen binding fragment thereof of claim 2, wherein the non-CDR region is from human antibody.

### Claim 5 (depends on 1)

An isolated nucleic acid molecule, encoding the monoclonal antibody or antigen binding fragment thereof of claim 1.

### Claim 6 (depends on 5)

A vector, comprising the isolated nucleic acid molecule of claim 5.

### Claim 7 (depends on 5)

A host cell, comprising the isolated nucleic acid molecule according to claim 5 or a vector comprising the isolated nucleic acid molecule.

### Claim 8 (depends on 1)

A method for producing the monoclonal antibody or antigen binding fragment thereof of claim 1, comprising
1) expressing an isolated nucleic acid molecule encoding the monoclonal antibody or antigen binding fragment thereof in a host cell, to produce the monoclonal antibody or antigen binding fragment thereof; and
2) isolating the monoclonal antibody or antigen binding fragment thereof.

### Claim 9

A hybridoma cell line, selected from:
1) hybridoma cell line HBs-E6F6, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO. C201270;
2) hybridoma cell line HBs-E7G11, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO. C201271;
3) hybridoma cell line HBs-G12F5, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO. C201272; and
4) hybridoma cell line HBs-E13C5, deposited in China Center for Type Culture Collection (CCTCC), with a deposition number of CCTCC NO. C201273.

### Claim 10 (depends on 1)

A kit for detecting the presence or level of HBsAg protein in a sample or for diagnosing whether a subject is infected by HBV, comprising the monoclonal antibody or antigen binding fragment thereof of claim 1.

### Claim 11 (depends on 10)

The kit according to claim 10, wherein the monoclonal antibody or antigen binding fragment thereof further comprises a detectable marker.

### Claim 12 (depends on 11)

The kit according to claim 11, wherein the detectable marker comprised in the monoclonal antibody or antigen binding fragment thereof is selected from a radioisotope, a fluorescent substance, a luminescent substance, a chromophoric substance and an enzyme.

### Claim 13 (depends on 10)

The kit according to claim 10, wherein the kit further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen binding fragment thereof; optionally, the second antibody further comprises a detectable marker.

### Claim 14 (depends on 13)

The kit according to claim 13, wherein the detectable marker comprised in the second antibody is selected from a radioisotope, a fluorescent substance, a luminescent substance, a chromophoric substance and an enzyme.

### Claim 15 (depends on 1)

A method for detecting the presence or level of HBsAg protein in a sample, comprising contacting the monoclonal antibody or antigen binding fragment thereof of claim 1 with the sample, and detecting any binding between the monoclonal antibody or antigen binding fragment thereof and HBsAg protein.

### Claim 16 (depends on 1)

A method for diagnosing whether a subject is infected by HBV, comprising (1) contacting the monoclonal antibody or antigen binding fragment thereof of claim 1 with a sample from a subject that is suspected to be infected by HBV, and (2) detecting the presence of HBV in the sample by detecting any binding between the monoclonal antibody or antigen binding fragment thereof and HBsAg protein.

### Claim 17 (depends on 1)

A pharmaceutical composition comprising the monoclonal antibody or antigen binding fragment thereof of claim 1, and a pharmaceutically acceptable carrier and/or excipient.

### Claim 18 (depends on 1)

A method for preventing or treating HBV infection or a disease associated with HBV infection in a subject, comprising administering a prophylactically or therapeutically effective amount of the monoclonal antibody or antigen binding fragment thereof of claim 1 to a subject in need thereof.

### Claim 19 (depends on 18)

The method according to claim 18, wherein the disease associated with HBV infection is hepatitis B.

### Claim 20 (depends on 1)

A method for reducing serum level of HBV DNA and/or HBsAg in a subject, comprising administering to a subject infected with HBV and in need of reducing serum level of HBV DNA and/or HBsAg an effective amount of one of the following:
(1) the monoclonal antibody or antigen binding fragment thereof of claim 1; and
(2) a monoclonal antibody or an antigen binding fragment thereof, capable of blocking the binding of HBsAg protein to the monoclonal antibody or antigen binding fragment thereof as defined in (1) by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%.

### Claim 21 (depends on 1)

The monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein the antibody is a humanized antibody.

### Claim 22 (depends on 1)

The monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein the antibody is a chimeric antibody.

### Claim 23 (depends on 1)

The monoclonal antibody or antigen binding fragment thereof according to claim 1, wherein the antibody is a single chain antibody.