# US 11,897,921 B2 · Propeptide fusion comprising a mutated clostridium botulinum neurotoxin and a VHH domain

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- Number: US 11,897,921 B2
- Document ID: US11897921B2
- Status: Granted
- Kind: B2
- Application number: 14/964,146
- Filed: 2015-12-09
- Published: 2016-06-09
- Granted: 2024-02-13
- Earliest claimed: 2014-12-09
- Assignees: New York University
- Applicants: New York University
- Inventors: Konstantin Ichtchenko; Edwin Vazquez-Cintron; Philip A. Band; Timothy J. Cardozo
- Web page: https://patentgrep.com/patent/US11897921B2

## CPC

- C07K 14/33: from Clostridium (G)
- A61P 25/00: Drugs for disorders of the nervous system
- A61P 39/06: Free radical scavengers or antioxidants
- C07K 16/1282: Clostridium (G)
- A61K 2039/505: {comprising antibodies}
- C07K 2317/622: Single chain antibody (scFv)
- C07K 2319/00: Fusion polypeptide
- C07K 2319/21: containing a His-tag
- C07K 2319/22: containing a Strep-tag
- C07K 2319/41: containing a Myc-tag
- C07K 2319/50: containing protease site
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The present invention is directed to a fusion protein comprising a light chain region of a Clostridial neurotoxin and a heavy chain region of a Clostridial neurotoxin, where the light and heavy chain regions are linked by a disulfide bond. The fusion protein also has a single chain antibody positioned upstream of the light chain region, where the single chain antibody possesses antigen-binding activity. Also disclosed are therapeutic agents, treatment methods, propeptide fusions, isolated nucleic acid molecules, expression systems, host cells, and methods of expressing fusion proteins.

## Claims

### Claim 1

A propeptide fusion comprising:
a light chain of a Clostridium botulinum neurotoxin, wherein the light chain is selected from any one of the following: (i) serotype A of SEQ ID NO:52 comprising E224>A and Y366>A mutations, (ii) serotype A of SEQ ID NO:52 comprising Q162>Y, L256>Y, R257>E, and L322>E mutations, (iii) serotype A of SEQ ID NO:52 comprising Q163>E, E263>L, and L323>I mutations, or (iv) serotype C of SEQ ID NO:54 comprising E230>A, H233>G, and Y375>A mutations;
a heavy chain of a Closlridium botulinum neurotoxin, wherein the light and heavy chains are linked by a disulfide bond;
an intermediate region connecting the light and heavy chain and comprising a highly specific protease cleavage site, wherein the highly specific protease cleavage site has three or more specific adjacent amino acid residues that are recognized by the highly specific protease to enable cleavage; and
a VHH domain comprising FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 structures operably linked to the light chain, wherein the VHH domain possesses antigen binding activity against intra-neuronal targets after translocation into the cytoplasm;
wherein the VHH domain is positioned upstream of the light chain and the light chain is positioned upstream of the heavy chain; and
wherein the propeptide fusion comprises a sequence selected from the group consisting of:
(i) the amino acid sequence comprising amino acids 63-193 of SEQ ID NO: 10 fused with amino acids 218-1517 of SEQ ID NO:10;
(ii) the amino acid sequence comprising amino acids 60-194 of SEQ ID NO: 12 fused with amino acids 219-1518 of SEQ ID NO:12;
(iii) the amino acid sequence comprising amino acids 60-194 of SEQ ID NO: 14 fused with amino acids 219-1518 of SEQ ID NO:14;
(iv) the amino acid sequence comprising amino acids 65-195 of SEQ ID NO:36 fused with amino acids 205-1508 of SEQ ID NO:36;
(v) the amino acid sequence comprising amino acids 267-397 of SEQ ID NO:38 fused with amino acids 407-1710 of SEQ ID NO:38;
(vi) the amino acid sequence comprising amino acids 58-183 of SEQ ID NO:40 fused with amino acids 198-323 of SEQ ID NO:40 fused with amino acids 333-1636 of SEQ ID NO:40;
(vii) the amino acid sequence comprising amino acids 260-385 of SEQ ID NO:42 fused with amino acids 400-525 of SEQ ID NO:42 fused with amino acids 535-1838 of SEQ ID NO:42; and
(viii) the amino acid sequence comprising amino acids 58-183 of SEQ ID NO:44 fused with amino acids 198-323 of SEQ ID NO:44 fused with amino acids 333-1636 of SEQ ID NO:44.