# US 2016/0355602 A1 · Novel bispecific antibody binding to human tlr2 and human tlr4

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2016/0355602 A1
- Document ID: US20160355602A1
- Status: Published application
- Kind: A1
- Application number: 15/121,567
- Filed: 2015-02-27
- Published: 2016-12-08
- Earliest claimed: 2014-02-28
- Assignees: Astellas Pharma Inc.
- Applicants: Astellas Pharma Inc.
- Inventors: Satoshi TAKEUCHI; Masahito SATO; Jotarou SUZUKI; Shinji SOGA; Jun TAKASAKI; Koji AOYAMA; Chie OKUYAMA
- Web page: https://patentgrep.com/patent/US20160355602A1

## CPC

- C07K 16/2896: {against molecules with a "CD"-designation, not provided for elsewhere}
- A61P 1/18: for pancreatic disorders, e.g. pancreatic enzymes
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 31/04: Antibacterial agents
- A61P 37/02: Immunomodulators
- C12N 5/12: Fused cells, e.g. hybridomas
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- C07K 2317/31: multispecific
- C07K 2317/40: characterized by post-translational modification
- C07K 2317/515: Complete light chain, i.e. VL + CL
- C07K 2317/52: Constant or Fc region; Isotype
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2317/64: comprising a combination of variable region and constant region components
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- C07K 2319/00: Fusion polypeptide
- C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

[Problem] To provide a bispecific antibody for preventing or treating an immune inflammatory disease by acting on both human TLR2 and human TLR4 to inhibit human TLR2- and human TLR4-mediated immune inflammatory effects. [Means for Solution] Provided is a bispecific antibody for human TLR2 and human TLR4, including a heavy chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 4; a light chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 6; a heavy chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 491 to 609 of SEQ ID NO: 4; and a light chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 625 to 732 of SEQ ID NO: 4.

## Claims

### Claim 1

A bispecific antibody binding to human TLR2 and human TLR4, which is selected from the following (1) or (2):
(1) a bispecific antibody binding to human TLR2 and human TLR4, comprising
1) a heavy chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 4, and a light chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 6, and
2) a heavy chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 491 to 609 of SEQ ID NO: 4, and a light chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 625 to 732 of SEQ ID NO: 4; or
(2) a bispecific antibody binding to human TLR2 and human TLR4, which is a bispecific antibody derived from post-translational modification of the bispecific antibody of (1).

### Claim 2 (depends on 1)

The bispecific antibody according to claim 1, which is selected from the following (1) or (2):
(1) a bispecific antibody comprising an anti-human TLR2 antibody and an anti-human TLR4 antibody fragment, wherein
the anti-human TLR2 antibody is an IgG antibody and comprises a heavy chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 4, and a light chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 6, and
the anti-human TLR4 antibody fragment is a single chain variable region fragment (scFv) and comprises a heavy chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 491 to 609 of SEQ ID NO: 4, and a light chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 625 to 732 of SEQ ID NO: 4; or
(2) a bispecific antibody binding to human TLR2 and human TLR4, which is a bispecific antibody derived from post-translational modification of the bispecific antibody of (1).

### Claim 3 (depends on 2)

The bispecific antibody according to claim 2, wherein the bispecific antibody comprises an anti-human TLR2 antibody and an anti-human TLR4 antibody fragment,
the anti-human TLR2 antibody is an IgG antibody and comprises a heavy chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 4, and a light chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 6, and
the anti-human TLR4 antibody fragment is an scFv and comprises a heavy chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 491 to 609 of SEQ ID NO: 4, and a light chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 625 to 732 of SEQ ID NO: 4.

### Claim 4 (depends on 2)

The bispecific antibody according to claim 2, wherein the post-translational modification is pyroglutamylation at the amino terminal (N terminal) of the heavy chain variable region of the anti-human TLR2 antibody, deletion of lysine at the heavy chain carboxy terminal (C terminal) of the anti-human TLR2 antibody, and/or pyroglutamylation at the N terminal of the heavy chain variable region of the anti-human TLR4 antibody fragment.

### Claim 5 (depends on 2, 3, 4)

The bispecific antibody according to any one of claims 2 to 4, wherein the anti-human TLR2 antibody comprises a heavy chain constant region which is a human Igγ1 constant region.

### Claim 6 (depends on 5)

The bispecific antibody according to claim 5, wherein the human Igγ1 constant region is a human Igγ1 constant region having amino acid mutations of L234A and L235A or amino acid mutations of L234A, L235A and I253A.

### Claim 7 (depends on 2, 3, 4)

The bispecific antibody according to any one of claims 2 to 4, wherein the anti-human TLR2 antibody comprises a light chain constant region which is a human Igκ constant region.

### Claim 8 (depends on 2, 3, 4)

The bispecific antibody according to any one of claims 2 to 4, wherein the anti-human TLR2 antibody comprises a heavy chain constant region which is a human Igγ1 constant region and a light chain constant region which is a human Igκ constant region.

### Claim 9 (depends on 8)

The bispecific antibody according to claim 8, wherein the human Igγ1 constant region is a human Igγ1 constant region having amino acid mutations of L234A and L235A or amino acid mutations of L234A, L235A and I253A.

### Claim 10 (depends on 2, 3)

The bispecific antibody according to claim 2 or 3, wherein the anti-human TLR2 antibody comprises a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 450 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6.

### Claim 11 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10)

The bispecific antibody according to any one of claims 2 to 10, wherein the anti-human TLR4 antibody fragment is an scFv having a structure where the N terminal of the light chain variable region is connected via a linker to the C terminal of the heavy chain variable region.

### Claim 12 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The bispecific antibody according to any one of claims 2 to 11, wherein the anti-human TLR4 antibody fragment is an scFv consisting of the amino acid sequence of amino acid numbers 491 to 732 of SEQ ID NO: 4.

### Claim 13 (depends on 2, 3)

The bispecific antibody according to claim 2 or 3, wherein the anti-human TLR2 antibody comprises a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 450 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6, and the anti-human TLR4 antibody fragment is an scFv consisting of the amino acid sequence of amino acid numbers 491 to 732 of SEQ ID NO: 4.

### Claim 14 (depends on 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The bispecific antibody according to any one of claims 2 to 13, wherein the N terminal of the anti-human TLR4 antibody fragment is connected via a linker to the C terminal of each heavy chain of the anti-human TLR2 antibody.

### Claim 15 (depends on 14)

The bispecific antibody according to claim 14, wherein the linker connecting the heavy chain of the anti-human TLR2 antibody and the anti-human TLR4 antibody fragment consists of a GS linker.

### Claim 16 (depends on 15)

The bispecific antibody according to claim 15, wherein the GS linker consists of the amino acid sequence of SEQ ID NO: 9.

### Claim 17 (depends on 2, 3)

The bispecific antibody according to claim 2 or 3, wherein the anti-human TLR2 antibody comprises a heavy chain consisting of the amino acid sequence of amino acid numbers 1 to 450 of SEQ ID NO: 4 and a light chain consisting of the amino acid sequence of SEQ ID NO: 6, the anti-human TLR4 antibody fragment is an scFv consisting of the amino acid sequence of amino acid numbers 491 to 732 of SEQ ID NO: 4, and the N terminal of the anti-human TLR4 antibody fragment is connected via a linker to the C terminal of each heavy chain of the anti-human TLR2 antibody.

### Claim 18 (depends on 17)

The bispecific antibody according to claim 17, wherein the linker connecting the heavy chain of the anti-human TLR2 antibody and the anti-human TLR4 antibody fragment consists of a GS linker.

### Claim 19 (depends on 18)

The bispecific antibody according to claim 18, wherein the GS linker consists of the amino acid sequence of SEQ ID NO: 9.

### Claim 20 (depends on 17, 18, 19)

A bispecific antibody derived from post-translational modification of the bispecific antibody according to any one of claims 17 to 19.

### Claim 21 (depends on 1)

A polynucleotide comprising a base sequence encoding the heavy chain variable region of the anti-human TLR2 antibody, the light chain variable region of the anti-human TLR2 antibody, the heavy chain variable region of the anti-human TLR4 antibody and/or the light chain variable region of the anti-human TLR4 antibody of the bispecific antibody according to claim 1.

### Claim 22 (depends on 17, 18, 19)

A polynucleotide comprising a base sequence encoding a fusion where the N terminal of the anti-human TLR4 antibody fragment is connected via a linker to the C terminal of the heavy chain of the anti-human TLR2 antibody of the bispecific antibody according to any one of claims 17 to 19.

### Claim 23 (depends on 22)

The polynucleotide according to claim 22, comprising a base sequence encoding the fusion consisting of the amino acid sequence of SEQ ID NO: 4.

### Claim 24 (depends on 17, 18, 19)

A polynucleotide comprising a base sequence encoding the light chain of the anti-human TLR2 antibody of the bispecific antibody according to any one of claims 17 to 19.

### Claim 25 (depends on 21)

An expression vector comprising the polynucleotide according to claim 21.

### Claim 26 (depends on 22, 23)

An expression vector comprising the polynucleotide according to claim 22, 23 and/or 24.

### Claim 27 (depends on 25)

A host cell transformed with the expression vector according to claim 25, selected from the group consisting of the following (a) to (d):
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 4, a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 491 to 609 of SEQ ID NO: 4, and a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 625 to 732 of SEQ ID NO: 4, and an expression vector comprising a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 6;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 4, a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 491 to 609 of SEQ ID NO: 4, a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 625 to 732 of SEQ ID NO: 4, and a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 6;
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 4, a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 491 to 609 of SEQ ID NO: 4, and a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 625 to 732 of SEQ ID NO: 4; and
(d) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 6.

### Claim 28 (depends on 22, 23, 24, 25)

A host cell transformed with the expression vector according to claim 25, selected from the group consisting of the following (a) to (d):
(a) a host cell transformed with an expression vector comprising the polynucleotide according to claim 22 or 23 and an expression vector comprising the polynucleotide according to claim 24;
(b) a host cell transformed with an expression vector comprising the polynucleotide according to claim 22 or 23 and the polynucleotide according to claim 24;
(c) a host cell transformed with an expression vector comprising the polynucleotide according to claim 22 or 23; and
(d) a host cell transformed with an expression vector comprising the polynucleotide according to claim 24.

### Claim 29

A method for producing a bispecific antibody binding to human TLR2 and human TLR4, comprising culturing host cell(s) selected from the group consisting of the following (a) to (c) to express the bispecific antibody binding to human TLR2 and human TLR4:
(a) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 4, a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 491 to 609 of SEQ ID NO: 4, and a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 625 to 732 of SEQ ID NO: 4, and an expression vector comprising a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 6;
(b) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 4, a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 491 to 609 of SEQ ID NO: 4, a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 625 to 732 of SEQ ID NO: 4, and a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 6; and
(c) a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 120 of SEQ ID NO: 4, a polynucleotide comprising a base sequence encoding a heavy chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 491 to 609 of SEQ ID NO: 4, and a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR4 antibody consisting of the amino acid sequence of amino acid numbers 625 to 732 of SEQ ID NO: 4, and a host cell transformed with an expression vector comprising a polynucleotide comprising a base sequence encoding a light chain variable region of an anti-human TLR2 antibody consisting of the amino acid sequence of amino acid numbers 1 to 113 of SEQ ID NO: 6.

### Claim 30 (depends on 22, 23, 24)

A method for producing a bispecific antibody binding to anti-human TLR2 and human TLR4, comprising culturing host cell(s) selected from the group consisting of the following (a) to (c) to express a bispecific antibody binding to human TLR2 and human TLR4:
(a) a host cell transformed with an expression vector comprising the polynucleotide according to claim 22 or 23 and an expression vector comprising the polynucleotide according to claim 24;
(b) a host cell transformed with an expression vector comprising the polynucleotide according to claim 22 or 23 and the polynucleotide according to claim 24; and
(c) a host cell transformed with an expression vector comprising the polynucleotide according to claim 22 or 23, and a host cell transformed with an expression vector comprising the polynucleotide according to claim 24.

### Claim 31 (depends on 29)

A bispecific antibody binding to human TLR2 and human TLR4, produced by the method according to claim 29.

### Claim 32 (depends on 30)

A bispecific antibody binding to human TLR2 and human TLR4, produced by the method according to claim 30.

### Claim 33 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 31, 32)

A pharmaceutical composition comprising the bispecific antibody according to any one of claims 1 to 20, 31 and 32, and a pharmaceutically acceptable excipient.

### Claim 34 (depends on 33)

The pharmaceutical composition according to claim 33, which is a pharmaceutical composition for preventing or treating an immune inflammatory disease.

### Claim 35 (depends on 34)

The pharmaceutical composition according to claim 34, wherein the immune inflammatory disease is sepsis or acute pancreatitis.

### Claim 36 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 31, 32)

A method for preventing or treating an immune inflammatory disease, comprising administering a therapeutically effective amount of the bispecific antibody according to any one of claims 1 to 20, 31 and 32.

### Claim 37 (depends on 36)

The method for preventing or treating according to claim 36, wherein the immune inflammatory disease is sepsis or acute pancreatitis.

### Claim 38 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 31, 32)

The bispecific antibody according to any one of claims 1 to 20, 31 and 32 for use in preventing or treating an immune inflammatory disease.

### Claim 39 (depends on 38)

The bispecific antibody according to claim 38, wherein the immune inflammatory disease is sepsis or acute pancreatitis.

### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 31, 32)

Use of the bispecific antibody according to any one of claims 1 to 20, 31 and 32 in the manufacture of a pharmaceutical composition for preventing or treating an immune inflammatory disease.

### Claim 41 (depends on 40)

The use of the bispecific antibody according to claim 40, wherein the immune inflammatory disease is sepsis or acute pancreatitis.

### Claim 42

A bispecific antibody binding to human TLR2 and human TLR4, comprising:
1) a heavy chain variable region of an anti-human TLR2 antibody comprising CDR1 consisting of the amino acid sequence of amino acid numbers 31 to 35 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of amino acid numbers 50 to 66 of SEQ ID NO: 4 and CDR3 consisting of the amino acid sequence of amino acid numbers 99 to 109 of SEQ ID NO: 4, and a light chain variable region of an anti-human TLR2 antibody comprising CDR1 consisting of the amino acid sequence of amino acid numbers 24 to 39 of SEQ ID NO: 6, CDR2 consisting of the amino acid sequence of amino acid numbers 55 to 61 of SEQ ID NO: 6 and CDR3 consisting of the amino acid sequence of amino acid numbers 94 to 102 of SEQ ID NO: 6; and
2) a heavy chain variable region of an anti-human TLR4 antibody comprising CDR1 consisting of the amino acid sequence of amino acid numbers 521 to 525 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of amino acid numbers 540 to 556 of SEQ ID NO: 4 and CDR3 consisting of the amino acid sequence of amino acid numbers 589 to 598 of SEQ ID NO: 4, and a light chain variable region of an anti-human TLR4 antibody comprising CDR1 consisting of the amino acid sequence of amino acid numbers 648 to 658 of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of amino acid numbers 674 to 680 of SEQ ID NO: 4 and CDR3 consisting of the amino acid sequence of amino acid numbers 713 to 721 of SEQ ID NO: 4.