# US 10,766,950 B2 · Methods, compositions and kits for treating a subject using a recombinant neutralizing binding protein

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- Number: US 10,766,950 B2
- Document ID: US10766950B2
- Status: Granted
- Kind: B2
- Application number: 16/237,877
- Filed: 2019-01-02
- Published: 2019-07-25
- Granted: 2020-09-08
- Earliest claimed: 2013-12-26
- Assignees: Tufts University
- Applicants: Tufts University
- Inventors: Charles B. Shoemaker
- Web page: https://patentgrep.com/patent/US10766950B2

## CPC

- C07K 16/1282: Clostridium (G)
- C07K 16/1228: Enterobacterales (O), e.g. Citrobacter (G), Serratia (G), Proteus (G), Providencia (G), Morganella (G) or Yersinia (G)
- C07K 16/1278: Bacillus (G)
- C07K 16/16: against material from plants
- C07K 16/18: against material from animals or humans
- A61K 2039/505: {comprising antibodies}
- A61K 2039/507: {Comprising a combination of two or more separate antibodies}
- C07K 2317/22: from camelids, e.g. camel, llama or dromedary
- C07K 2317/31: multispecific
- C07K 2317/569: Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
- C07K 2317/622: Single chain antibody (scFv)
- 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 2319/40: containing a tag for immunodetection, or an epitope for immunisation
- 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)

## Abstract

Methods, compositions and kits are provided for treating a subject exposed to or at risk for exposure to a disease agent using a pharmaceutical composition including at least one recombinant binding protein or a source of expression of the binding protein, wherein the binding protein neutralizes a disease agent that is a toxin, for example a Clostridium difficile toxin, a Shiga toxin, a ricin toxin, or an anthrax toxin.

## Claims

### Claim 1

An isolated recombinant binding protein comprising an amino acid sequence selected from SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, or SEQ ID NO: 122, which specifically binds to Anthrax protective antigen (PA) toxin.

### Claim 2

A composition comprising at least one recombinant binding protein that specifically binds to Anthrax protective antigen (PA) toxin, wherein the at least one binding protein comprises an amino acid sequence selected from SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, or SEQ ID NO: 122.

### Claim 3

A composition comprising at least one recombinant binding protein that specifically binds to Anthrax protective antigen (PA) toxin, wherein the at least one binding protein is encoded by a polynucleotide sequence comprising a nucleotide sequence selected from SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, or SEQ ID NO: 123.

### Claim 4 (depends on 2)

The composition of claim 2, further comprising a pharmaceutically acceptable carrier, excipient, or vehicle.

### Claim 5

A multimeric binding protein comprising at least two recombinant camelid heavy-chain-only antibody monomers (VHHs) which specifically bind Anthrax protective antigen (PA) toxin and which comprise an amino acid sequence selected from SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, or SEQ ID NO: 122; wherein the at least two VHHs are separated by spacers.

### Claim 6 (depends on 5)

The multimeric binding protein of claim 5, comprising two VHHs which bind Anthrax PA toxin.

### Claim 7 (depends on 5)

The multimeric binding protein of claim 5, further comprising one or more epitope tags to which an anti-epitope tag antibody specifically binds.

### Claim 8 (depends on 5)

The multimeric binding protein of claim 5, wherein the spacer comprises a peptide having an amino acid sequence selected from GGGGS (SEQ ID NO: 54), GGGGSGGGGSGGGGS (SEQ ID NO: 55), or a combination thereof.

### Claim 9 (depends on 7)

The multimeric binding protein of claim 7, wherein the one or more epitope tags comprises the amino acid sequence Gly Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg of SEQ ID NO: 15.

### Claim 10 (depends on 5)

A pharmaceutical composition comprising the multimeric binding protein of claim 5 and a pharmaceutically acceptable carrier, excipient, or vehicle.

### Claim 11 (depends on 4)

A method of targeting and binding to Anthrax protective antigen (PA) toxin, said method comprising contacting said PA toxin with the composition of claim 4 in an amount effective to target and bind to said Anthrax PA toxin.

### Claim 12 (depends on 11)

The method according to claim 11, wherein the Anthrax PA toxin is targeted and bound in vitro or in vivo.

### Claim 13 (depends on 4)

A method of treating or preventing intoxication of a subject by Anthrax protective antigen (PA) toxin, said method comprising: administering to a subject in need thereof the composition of claim 4, in an amount effective for the binding protein to bind to and neutralize Anthrax PA toxin in the subject after intoxication or prior to the subject's having symptoms of intoxication.