# US 8,540,987 B2 · Composition for neutralizing botulinus toxin type-A, and human anti-botulinus toxin type-A antibody

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- Number: US 8,540,987 B2
- Document ID: US8540987B2
- Status: Granted
- Kind: B2
- Application number: 12/864,018
- Filed: 2009-01-23
- Published: 2011-01-20
- Granted: 2013-09-24
- Earliest claimed: 2008-01-29
- Assignees: Institute for Antibodies Co., Ltd.; Japan AS Represented By Director-General of National Institute of Infectious Diseases; Osaka Prefecture University Public Corporation
- Applicants: Masachika Azuma; Motohide Takahashi; Shunji Kozaki; Masafumi Mukamoto; Tomoko Kohda; Gene Kurosawa; Yoshikazu Kurosawa
- Inventors: Masachika Azuma; Motohide Takahashi; Shunji Kozaki; Masafumi Mukamoto; Tomoko Kohda; Gene Kurosawa; Yoshikazu Kurosawa
- Web page: https://patentgrep.com/patent/US8540987B2

## CPC

- C07K 16/1282: Clostridium (G)
- A61P 31/04: Antibacterial agents
- A61K 2039/505: {comprising antibodies}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- C07K 2317/21: from primates, e.g. man
- C07K 2317/55: Fab or Fab'
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/565: Complementarity determining region [CDR]
- 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: 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
- 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)

## Abstract

Provided herein is a means which is effective for botulism diseases and the prevention of the botulism diseases. Specifically provided is a plurality of human anti-botulinum toxin type-A antibodies having different epitopes from one another. Also specifically provided is a composition for neutralizing botulinum toxin type-A, which comprises a combination of two or more of the antibodies and which has a high neutralizing activity.

## Claims

### Claim 1

A composition for neutralizing botulinum toxin type-A comprising:
a first human anti-botulinum toxin type-A antibody that recognizes an epitope which is recognized by an antibody having a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 8;
a second human anti-botulinum toxin type-A antibody that recognizes an epitope which is recognized by an antibody having a VH comprising the amino acid sequence of SEQ ID NO: 36 and a VL comprising the amino acid sequence of SEQ ID NO: 40; and
a third human anti-botulinum toxin type-A antibody that recognizes an epitope which is recognized by an antibody having a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 48, or that recognizes an epitope which is recognized by an antibody having a VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 56.

### Claim 2 (depends on 1)

The composition for neutralizing botulinum toxin type-A according to claim 1, wherein:
the first human anti-botulinum toxin type-A antibody is any one of antibodies selected from the group consisting of the following (1) to (3);
the second human anti-botulinum toxin type-A antibody is an antibody of the following (4) or (5); and
the third human anti-botulinum toxin type-A antibody is any one of antibodies selected from the group consisting of the following (6) to (8):
(1) an antibody having a heavy chain complementarity-determining region (CDR) 1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 5, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 6, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 7;
(2) an antibody having a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 9, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 10, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 11, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 13, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 14, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 15;
(3) an antibody having a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 21, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23;
(4) an antibody having a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 25, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 26, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 27, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 29, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 30, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 31;
(5) an antibody having a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 33, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 34, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 35, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 37, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 38, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 39;
(6) an antibody having a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 41, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 42, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 43, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 45, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 46, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 47;
(7) an antibody having a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 49, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 50, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 51, a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 53, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 54, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 55; and
(8) an antibody having a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 57, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 58, a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 59; a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 61, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 62, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 63.

### Claim 3 (depends on 2)

The composition for neutralizing botulinum toxin type-A according to claim 2, wherein:
the first human anti-botulinum toxin type-A antibody is any one of antibodies selected from the group consisting of the above-mentioned (1) to (3);
the second human anti-botulinum toxin type-A antibody is the above-mentioned antibody (4) or (5);
the third human anti-botulinum toxin type-A antibody is the above-mentioned antibody (6); and
the above-mentioned antibody (7) or (8) is further comprised as a fourth human anti-botulinum toxin type-A antibody.

### Claim 4 (depends on 2)

The composition for neutralizing botulinum toxin type-A according to claim 2, wherein:
the first human anti-botulinum toxin type-A antibody is the above-mentioned antibody (1);
the second human anti-botulinum toxin type-A antibody is the above-mentioned antibody (5); and
the third human anti-botulinum toxin type-A antibody is the above-mentioned antibody (6).

### Claim 5 (depends on 4)

The composition for neutralizing botulinum toxin type-A according to claim 4, wherein the above-mentioned antibody (7) is further comprised as a fourth human anti-botulinum toxin type-A antibody.

### Claim 6 (depends on 2)

The composition for neutralizing botulinum toxin type-A according to claim 2, wherein:
the first human anti-botulinum toxin type-A antibody is the above-mentioned antibody (1);
the second human anti-botulinum toxin type-A antibody is the above-mentioned antibody (5); and
the third human anti-botulinum toxin type-A antibody is the above-mentioned antibody (7).

### Claim 7 (depends on 6)

The composition for neutralizing botulinum toxin type-A according to claim 6, wherein the above-mentioned antibody (4) is further comprised as a fourth human anti-botulinum toxin type-A antibody.

### Claim 8 (depends on 2)

The composition for neutralizing botulinum toxin type-A according to claim 2, wherein:
the above-mentioned antibody (1) has a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 8;
the above-mentioned antibody (2) has a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16;
the above-mentioned antibody (3) has a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 24;
the above-mentioned antibody (4) has a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 32;
the above-mentioned antibody (5) has a VH comprising the amino acid sequence of SEQ ID NO: 36 and a VL comprising the amino acid sequence of SEQ ID NO: 40;
the above-mentioned antibody (6) has a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 48;
the above-mentioned antibody (7) has a VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 56; and
the above-mentioned antibody (8) has a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 64.

### Claim 9 (depends on 3)

The composition for neutralizing botulinum toxin type-A according to claim 3, wherein:
the above-mentioned antibody (1) has a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 8;
the above-mentioned antibody (2) has a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16;
the above-mentioned antibody (3) has a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 24;
the above-mentioned antibody (4) has a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 32;
the above-mentioned antibody (5) has a VH comprising the amino acid sequence of SEQ ID NO: 36 and a VL comprising the amino acid sequence of SEQ ID NO: 40;
the above-mentioned antibody (6) has a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 48;
the above-mentioned antibody (7) has a VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 56; and
the above-mentioned antibody (8) has a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 64.

### Claim 10 (depends on 4)

The composition for neutralizing botulinum toxin type-A according to claim 4, wherein:
the above-mentioned antibody (1) has a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 8;
the above-mentioned antibody (2) has a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16;
the above-mentioned antibody (3) has a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 24;
the above-mentioned antibody (4) has a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 32;
the above-mentioned antibody (5) has a VH comprising the amino acid sequence of SEQ ID NO: 36 and a VL comprising the amino acid sequence of SEQ ID NO: 40;
the above-mentioned antibody (6) has a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 48;
the above-mentioned antibody (7) has a VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 56; and
the above-mentioned antibody (8) has a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 64.

### Claim 11 (depends on 5)

The composition for neutralizing botulinum toxin type-A according to claim 5, wherein:
the above-mentioned antibody (1) has a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 8;
the above-mentioned antibody (2) has a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16;
the above-mentioned antibody (3) has a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 24;
the above-mentioned antibody (4) has a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 32;
the above-mentioned antibody (5) has a VH comprising the amino acid sequence of SEQ ID NO: 36 and a VL comprising the amino acid sequence of SEQ ID NO: 40;
the above-mentioned antibody (6) has a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 48;
the above-mentioned antibody (7) has a VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 56; and
the above-mentioned antibody (8) has a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 64.

### Claim 12 (depends on 6)

The composition for neutralizing botulinum toxin type-A according to claim 6, wherein:
the above-mentioned antibody (1) has a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 8;
the above-mentioned antibody (2) has a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16;
the above-mentioned antibody (3) has a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 24;
the above-mentioned antibody (4) has a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 32;
the above-mentioned antibody (5) has a VH comprising the amino acid sequence of SEQ ID NO: 36 and a VL comprising the amino acid sequence of SEQ ID NO: 40;
the above-mentioned antibody (6) has a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 48;
the above-mentioned antibody (7) has a VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 56; and
the above-mentioned antibody (8) has a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 64.

### Claim 13 (depends on 7)

The composition for neutralizing botulinum toxin type-A according to claim 7, wherein:
the above-mentioned antibody (1) has a VH comprising the amino acid sequence of SEQ ID NO: 4 and a VL comprising the amino acid sequence of SEQ ID NO: 8;
the above-mentioned antibody (2) has a VH comprising the amino acid sequence of SEQ ID NO: 12 and a VL comprising the amino acid sequence of SEQ ID NO: 16;
the above-mentioned antibody (3) has a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 24;
the above-mentioned antibody (4) has a VH comprising the amino acid sequence of SEQ ID NO: 28 and a VL comprising the amino acid sequence of SEQ ID NO: 32;
the above-mentioned antibody (5) has a VH comprising the amino acid sequence of SEQ ID NO: 36 and a VL comprising the amino acid sequence of SEQ ID NO: 40;
the above-mentioned antibody (6) has a VH comprising the amino acid sequence of SEQ ID NO: 44 and a VL comprising the amino acid sequence of SEQ ID NO: 48;
the above-mentioned antibody (7) has a VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 56; and
the above-mentioned antibody (8) has a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 64.