# US 10,479,826 B2 · Single chain antibody domains for detection of anti-botulinum neurotoxin domains and methods of their use

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- Number: US 10,479,826 B2
- Document ID: US10479826B2
- Status: Granted
- Kind: B2
- Application number: 15/553,532
- Filed: 2016-02-29
- Published: 2018-08-23
- Granted: 2019-11-19
- Earliest claimed: 2015-02-27
- Assignees: City of Hope
- Applicants: City of Hope
- Inventors: Markus Kalkum; Karine Bagramyan; Paul Yazaki
- Web page: https://patentgrep.com/patent/US10479826B2

## CPC

- C07K 16/1282: Clostridium (G)
- G01N 33/5308: {for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites}
- G01N 33/573: for enzymes or isoenzymes
- C07K 2317/22: from camelids, e.g. camel, llama or dromedary
- C07K 2317/31: multispecific
- C07K 2317/56: variable (Fv) region, i.e. VH and/or VL
- C07K 2317/565: Complementarity determining region [CDR]
- C07K 2317/567: Framework region [FR]
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2317/62: comprising only variable region components
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- G01N 2333/952: derived from bacteria
- 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)

## Abstract

Provided herein, inter alia, are single chain antibody binding domains (VHHs) for use in conjunction with the Assay with a Large Immuno-sorbent Surface Area (ALISSA). Engineered and affinity matured VHHs are used as affinity reagents in the ALISSA resulting in an exceptionally sensitive and precise method for the detection of toxins at miniscule concentrations. Thus, provided herein are methods as well as reagents that can be used to detect the presence of botulinum neurotoxins in quantities well below 1 pg/mL which corresponds to a lethal concentration under presumed equal distribution throughout the human body.

## Claims

### Claim 1

A single chain antibody binding domain (VHH) comprising (a) a first complementary determining region amino acid sequence (CDR1) comprising GSGISGIA (SEQ ID NO:5), (b) a second complementary determining region amino acid sequence (CDR2) comprising IFSGGST (SEQ ID NO:6) and (c) a third complementary determining region amino acid sequence (CDR3) comprising RLYGSGDYWVQ (SEQ ID NO:7), wherein the VHH binds to botulinum neurotoxin with a dissociation constant (Kd) of less than 40 pM.

### Claim 2 (depends on 1)

The VHH of claim 1, further comprising a framework (FW) region amino acid sequence comprising (a) QVQLVESGGGLVQVGGSLRLSCVVS (SEQ ID NO:8); (b) MGWYRQAPGKRREMVAD (SEQ ID NO:9); (c) GSVKGRFTISRDNAKKTSYLQMNN AKPEDTGAYYC (SEQ ID NO:10); or (d) GTQVTVSSAHHSED (SEQ ID NO:11).

### Claim 3 (depends on 1)

The VHH of claim 1, further comprising a framework (FW) region amino acid sequence comprising (a) QVQLVESGGGLVQVGGSLRLSCVVS (SEQ ID NO:8); (b) MGWYRQAPGKRREMVAD (SEQ ID NO:9); (c) GSVKGRFTISRDNAKKTSYLQMNN AKPEDTGAYYC (SEQ ID NO:10); and (d) GTQVTVSSAHHSED (SEQ ID NO:11).

### Claim 4 (depends on 1)

The VHH of claim 1, comprising the amino acid sequence QVQLVESGGGLVQVGGSLRLSCVVSGSGISGIAMGWYRQAPGKRREMVADIFSGGSTDYA GSVKGRFTISRDNAKKTSYLQMNNAKPEDTGAYYCRLYGSGDYWVQGTQVTVSSAHHSE D (SEQ ID NO:3).

### Claim 5 (depends on 1)

The VHH of claim 1, wherein the VHH binds to botulinum neurotoxin with a Kd of at least 12 pM.

### Claim 6 (depends on 5)

The VHH of claim 5, wherein said toxin is botulinum neurotoxin serotype A (BoNT/A).

### Claim 7 (depends on 1)

A nucleic acid encoding the VHH of claim 1.

### Claim 8 (depends on 7)

A vector comprising the nucleic acid of claim 7.

### Claim 9 (depends on 8)

A cell comprising the vector of claim 8.

### Claim 10 (depends on 1)

A multimeric single chain antibody binding domain (VHH) construct comprising an N-terminal first VHH and a C-terminal second VHH, wherein the first VHH and the second VHH are independently selected from the VHH of claim 1 or a VHH comprising a CDR1 GFGTWFRFDENT (SEQ ID NO:14), CDR2 VARYPKSGIV (SEQ ID NO:15) and CDR3 NVGEF (SEQ ID NO:16), wherein at least one of the first VHH or the second VHH are the VHH of claim 1.

### Claim 11 (depends on 10)

A nucleic acid encoding the multimeric VHH construct of claim 10.

### Claim 12 (depends on 11)

A vector comprising the nucleic acid of claim 11.

### Claim 13 (depends on 12)

A cell comprising the vector of claim 12.

### Claim 14 (depends on 1)

A method for detecting the presence of a botulinum neurotoxin in a sample comprising:
a) contacting a botulinum neurotoxin with (i) the single chain antibody binding domain (VHH) of claim 1 or (ii) a multimeric VHH construct comprising an N-terminal first VHH and a C-terminal second VHH, wherein the first VHH and the second VHH are independently selected from the VHH of claim 1 or a VHH comprising a CDR1 GFGTWFRFDENT (SEQ ID NO:14), CDR2 VARYPKSGIV (SEQ ID NO:15) and CDR3 NVGEF (SEQ ID NO:16) and wherein at least one of the first VHH or the second VHH are the VHH of claim 1, thereby forming a VHH-botulinum neurotoxin complex; and
b) detecting the VHH-botulinum neurotoxin complex.

### Claim 15 (depends on 1)

A kit comprising:
a) (i) the single chain antibody binding domain (VHH) of claim 1 or (ii) a multimeric VHH construct comprising an N-terminal first VHH and a C-terminal second VHH, wherein the first VHH and the second VHH are independently selected from the VHH of claim 1 or a VHH comprising a CDR1 GFGTWFRFDENT (SEQ ID NO:14), CDR2 VARYPKSGIV (SEQ ID NO:15) and CDR3 NVGEF (SEQ ID NO:16) and wherein at least one of the first VHH or the second VHH are the VHH of claim 1; and
b) a detectable toxin substrate.

### Claim 16 (depends on 1)

A pharmaceutical composition comprising (a)(i) the single chain antibody binding domain (VHH) of claim 1 or (ii) a multimeric VHH construct comprising an N-terminal first VHH and a C-terminal second VHH, wherein the first VHH and the second VHH are independently selected from the VHH of claim 1 or a VHH comprising a CDR1 GFGTWFRFDENT (SEQ ID NO:14), CDR2 VARYPKSGIV (SEQ ID NO:15) and CDR3 NVGEF (SEQ ID NO:16) and wherein at least one of the first VHH or the second VHH are the VHH of claim 1; and (b) one or more pharmaceutically acceptable carriers.

### Claim 17 (depends on 1)

A method for treating botulism in an individual in need thereof, the method comprising administering, to the individual, a therapeutically effective amount of (a) the single chain antibody binding domain (VHH) of claim 1, or (b) a multimeric VHH construct comprising an N-terminal first VHH and a C-terminal second VHH, wherein the first VHH and the second VHH are independently selected from the VHH of claim 1 or a VHH comprising a CDR1 GFGTWFRFDENT (SEQ ID NO:14), CDR2 VARYPKSGIV (SEQ ID NO:15) and CDR3 NVGEF (SEQ ID NO:16) and wherein at least one of the first VHH or the second VHH are the VHH of claim 1.

### Claim 18 (depends on 1)

A complex comprising a botulinum neurotoxin bound to (i) the single chain antibody binding domain (VHH) of claim 1 or (ii) a multimeric VHH construct comprising an N-terminal first VHH and a C-terminal second VHH, wherein the first VHH and the second VHH are independently selected from the VHH of claim 1 or a VHH comprising a CDR1 GFGTWFRFDENT (SEQ ID NO:14), CDR2 VARYPKSGIV (SEQ ID NO:15) and CDR3 NVGEF (SEQ ID NO:16) and wherein at least one of the first VHH or the second VHH are the VHH of claim 1.