# US 10,870,691 B2 · Antibodies and fragments thereof that bind hepatitis B virus protein X

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- Number: US 10,870,691 B2
- Document ID: US10870691B2
- Status: Granted
- Kind: B2
- Application number: 16/372,163
- Filed: 2019-04-01
- Published: 2019-10-10
- Granted: 2020-12-22
- Earliest claimed: 2018-04-05
- Assignees: Gilead Sciences, Inc.
- Applicants: Gilead Sciences, Inc.
- Inventors: Simon Paul Fletcher; Christian Alfons Voitenleitner
- Web page: https://patentgrep.com/patent/US10870691B2

## CPC

- C07K 16/082: Hepadnaviridae (F), e.g. hepatitis B virus
- G01N 33/502: {for testing non-proliferative effects}
- G01N 33/56983: {Viruses}
- G01N 33/5761: {Hepatitis B}
- C07K 2317/82: functional in the cytoplasm, the inner aspect of the cell membrane, the nucleus or the mitochondria
- C12N 15/1131: {against viruses}
- C12N 2310/14: interfering nucleic acids [NA]
- C12N 2320/11: for the determination of target sites, i.e. of active nucleic acids
- G01N 2333/02: Hepadnaviridae, e.g. hepatitis B virus
- G01N 2469/10: Detection of antigens from microorganism in sample from host
- G01N 2800/26: Infectious diseases, e.g. generalised sepsis
- 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)
- 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

Provided herein are, inter alia, antibodies, antigen-binding antibody fragments, cells, polynucleotides, compositions, kits, and methods relating to the detection of HBV protein X (HBx), e.g., in vitro and in vivo. Included are antibodies and fragments thereof that bind HBx, as well as kits, cells, and compositions comprising such antibodies and fragments.

## Claims

### Claim 1

An isolated antibody, or an antigen-binding fragment thereof, comprising
(i) a VH-complementarity determining region (CDR) 1 comprising
the amino acid sequence of SEQ ID NO:1, or SEQ ID NO:1 having 1 conservative substitution,
the amino acid sequence of SEQ ID NO:6, or SEQ ID NO:6 having 1 or 2 conservative substitutions,
the amino acid sequence of SEQ ID NO:11, or SEQ ID NO:11 having 1 or 2 conservative substitutions, or
the amino acid sequence of SEQ ID NO:15, or SEQ ID NO:15 having from 1 to 3 conservative substitutions;
(ii) a VH-CDR2 comprising
the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:2 having from 1 to 4 conservative substitutions,
the amino acid sequence of SEQ ID NO:7, or SEQ ID NO:7 having 1 or 2 conservative substitutions,
the amino acid sequence of SEQ ID NO:12, or SEQ ID NO:12 having 1 conservative substitution, or
the amino acid sequence of SEQ ID NO:16, or SEQ ID NO:16 having from 1 to 4 conservative substitutions;
(iii) a VH-CDR3 comprising
the amino acid sequence LAY of SEQ ID NO:74,
the amino acid sequence of SEQ ID NO:8, or SEQ ID NO:8 having 1 conservative substitution,
an alanine residue, or
the amino acid sequence of SEQ ID NO:75;
(iv) a VL-CDR1 comprising
the amino acid sequence of SEQ ID NO:3, or SEQ ID NO:3 having from 1 to 4 conservative substitutions,
the amino acid sequence of SEQ ID NO:9, or SEQ ID NO:9 having 1 or 2 conservative substitutions,
the amino acid sequence of SEQ ID NO:13, or SEQ ID NO:13 having from 1 to 3 conservative substitutions, or
the amino acid sequence of SEQ ID NO:17, or SEQ ID NO:17 having from 1 to 3 conservative substitutions;
(v) a VL-CDR2 comprising
the amino acid sequence of SEQ ID NO:4, or SEQ ID NO:4 having 1 or 2 conservative substitutions,
the amino acid sequence of SEQ ID NO:76, or
the amino acid sequence of SEQ ID NO:18, or SEQ ID NO:18 having from 1 to 3 conservative substitutions; and
(vi) a VL-CDR3 comprising
the amino acid sequence of SEQ ID NO:5, or SEQ ID NO:5 having 1 or 2 conservative substitutions,
the amino acid sequence of SEQ ID NO:10, or SEQ ID NO:10 having 1 or 2 conservative substitutions,
the amino acid sequence of SEQ ID NO:14, or SEQ ID NO:14 having 1 conservative substitution, or
the amino acid sequence of SEQ ID NO:19, or SEQ ID NO:19 having 1 conservative substitution.

### Claim 2 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising:
(i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:1, or SEQ ID NO:1 having 1 conservative substitution;
(ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:2 having from 1 to 4 conservative substitutions;
(iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:74;
(iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:3, or SEQ ID NO:3 having from 1 to 4 conservative substitutions;
(v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:4, or SEQ ID NO:4 having 1 or 2 conservative substitutions; and
(vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:5, or SEQ ID NO:5 having 1 or 2 conservative substitutions.

### Claim 3 (depends on 2)

The antibody or antigen-binding fragment of claim 2, comprising:
(i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:1;
(ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:2;
(iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:74;
(iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:3;
(v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:4; and
(vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:5.

### Claim 4 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising
(i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:6 or SEQ ID NO:6 having 1 or 2 conservative substitutions;
(ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:7, or SEQ ID NO:7 having 1 or 2 conservative substitutions;
(iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:8, or SEQ ID NO:8 having 1 conservative substitution;
(iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:9, or SEQ ID NO:9 having 1 or 2 conservative substitutions;
(v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:76; and
(vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:10, or SEQ ID NO:10 having 1 or 2 conservative substitutions.

### Claim 5 (depends on 4)

The antibody or antigen-binding fragment of claim 4, comprising
(i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:6;
(ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:7;
(iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:8;
(iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:9;
(v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:76; and
(vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:10.

### Claim 6 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising
(i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:11, or SEQ ID NO:11 having 1 or 2 conservative substitutions;
(ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:12, or SEQ ID NO:12 having 1 conservative substitution;
(iii) a VH-CDR3 comprising an alanine residue;
(iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:13, or SEQ ID NO:13 having from 1 to 3 conservative substitutions;
(v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:76; and
(vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:14, or SEQ ID NO:14 having 1 conservative substitution.

### Claim 7 (depends on 6)

The antibody or antigen-binding fragment of claim 6, comprising
(i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:11;
(ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:12;
(iii) a VH-CDR3 comprising an alanine residue;
(iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:13;
(v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:76; and
(vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:14.

### Claim 8 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising
(i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:15, or SEQ ID NO:15 having from 1 to 3 conservative substitutions;
(ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:16, or SEQ ID NO:16 having from 1 to 4 conservative substitutions;
(iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:75;
(iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:17, or SEQ ID NO:17 having from 1 to 3 conservative substitutions;
(v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:18, or SEQ ID NO:18 having from 1 to 3 conservative substitutions; and
(vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:19, or SEQ ID NO:19 having 1 conservative substitution.

### Claim 9 (depends on 8)

The antibody or antigen-binding fragment of claim 8, comprising
(i) a VH-CDR1 comprising the amino acid sequence of SEQ ID NO:15;
(ii) a VH-CDR2 comprising the amino acid sequence of SEQ ID NO:16;
(iii) a VH-CDR3 comprising the amino acid sequence of SEQ ID NO:75;
(iv) a VL-CDR1 comprising the amino acid sequence of SEQ ID NO:17;
(v) a VL-CDR2 comprising the amino acid sequence of SEQ ID NO:18; and
(vi) a VL-CDR3 comprising the amino acid sequence of SEQ ID NO:19.

### Claim 10 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising a VH region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 21 or 22, wherein the amino acid sequence of the VH region has from 0 to 5 amino acid insertions, and/or from 0 to 5 amino acid deletions compared to SEQ ID NO: 21 or 22.

### Claim 11 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising a VL region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 23 or 24, wherein the amino acid sequence of the VL region has from 0 to 5 amino acid insertions, and/or from 0 to 5 amino acid deletions compared to SEQ ID NO: 23 or 24.

### Claim 12 (depends on 10)

The antibody or antigen-binding fragment of claim 10, comprising 1 to 5 amino acid substitutions, wherein at least 1 of the amino acid substitutions is a conservative substitution mutation.

### Claim 13 (depends on 12)

The antibody or antigen-binding fragment of claim 12, wherein all of the amino acid substitutions are conservative substitution mutations.

### Claim 14 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising a heavy chain constant region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 25.

### Claim 15 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising a light chain constant region comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 26.

### Claim 16 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising an IgA, IgD, IgE, IgG, or IgM heavy chain constant region.

### Claim 17 (depends on 16)

The antibody or antigen-binding fragment of claim 16, comprising a mouse, rabbit, goat, rat, or human IgG heavy chain constant region.

### Claim 18 (depends on 1)

The antibody or antigen-binding fragment of claim 1, comprising a kappa light chain or a lambda light chain.

### Claim 19 (depends on 1)

The antibody or antigen-binding fragment of claim 1, which is a linear antibody or a diabody.

### Claim 20 (depends on 1)

The antibody or antigen-binding fragment of claim 1, which is an intrabody.

### Claim 21 (depends on 20)

The antibody or antigen-binding fragment of claim 20, further comprising a nuclear localization signal or a mitochondrial localization signal.

### Claim 22 (depends on 1)

The antibody or antigen-binding fragment of claim 1, which is a monoclonal antibody.

### Claim 23 (depends on 22)

The antibody or antigen-binding fragment of claim 22, which is a humanized antibody or a chimeric antibody.

### Claim 24 (depends on 1)

The antibody or antigen-binding fragment of claim 1, which is a scFv, sc(Fv)2, Fab, F(ab)2, Fab′, F(ab′)2, or Fv fragment.

### Claim 25 (depends on 1)

The antibody or antigen-binding fragment of claim 1, further comprising a cytotoxic agent, a radioisotope, a therapeutic agent, an anti-viral agent, or a detectable label.

### Claim 26 (depends on 1)

A pharmaceutical composition comprising the antibody or antigen-binding fragment of claim 1.

### Claim 27 (depends on 24)

A chimeric antigen receptor (CAR) comprising an antigen-binding fragment according to claim 24.

### Claim 28 (depends on 27)

A CAR T-cell comprising the CAR of claim 27.