# US 10,441,640 B2 · Botulinum neurotoxins with modified light chain specificity and methods for producing same > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,441,640 B2 - Document ID: US10441640B2 - Status: Granted - Kind: B2 - Application number: 14/824,986 - Filed: 2015-08-12 - Published: 2016-06-02 - Granted: 2019-10-15 - Earliest claimed: 2014-08-12 - Assignees: BioMadison, Inc. - Applicants: Biomadison, Inc - Inventors: Francis Mark Dunning; Ward Tucker - Web page: https://patentgrep.com/patent/US10441640B2 ## CPC - A61K 38/4893: {Botulinum neurotoxin (3.4.24.69)} - A61P 5/00: Drugs for disorders of the endocrine system - C07K 14/43595: {from coelenteratae, e.g. medusae} - C07K 14/47: from mammals - C07K 14/705: Receptors; Cell surface antigens; Cell surface determinants {(tumour specific antigens C07K14/4748)} - C12N 9/52: derived from bacteria {or Archaea} - C12Q 1/37: involving peptidase or proteinase - C07K 2319/33: fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies - C07K 2319/50: containing protease site - C07K 2319/55: containing a fusion with a toxin, e.g. diphteria toxin - C07K 2319/60: containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP] - C07K 2319/70: containing domain for protein-protein interaction - C12Y 304/24069: Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin - 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) - A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS - 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) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES - C12Y: ENZYMES ## Abstract A protease directed to a non-neuronal SNARE protein is described. The protease is produced by selective mutation of a botulinum neurotoxin light chain, and is characterized utilizing a reporting construct that includes all or part of the non-neuronal SNARE protein. Such a protease has utility in the treatment of diseases associated with hypersecretion, where the hypersecretion is mediated by a non-neuronal SNARE protein. ## Claims ### Claim 1 A method of identifying a first protease with substrate specificity for a non-neuronal SNARE protein, comprising: providing a first reporting construct comprising a first non-neuronal SNARE protein or a fragment thereof that comprises a protease cleavage site of the first non-neuronal SNARE protein, interposed between a first reporter and a second reporter, wherein the first non-neuronal SNARE protein or fragment thereof is dimensioned to provide an energy transfer between the first reporter and the second reporter to generate a first signal; providing a library comprising a plurality of proteases each comprising a mutated botulinum toxin light chain peptide, wherein each of the plurality of proteases has a different non-native exosite recognition sequence; contacting individual proteases of the library with the first reporting construct; and characterizing a first signal obtained from the first reporting construct, wherein change of the first signal indicates identification of a first protease having substrate specificity for the first non-neuronal SNARE protein. ### Claim 2 (depends on 1) The method of claim 1, comprising the steps of: providing a second reporting construct comprising a second non-neuronal SNARE protein or a fragment thereof that comprises a protease cleavage site of the second non-neuronal SNARE protein, interposed between a third reporter and a fourth reporter, wherein the second non-neuronal SNARE protein or fragment thereof is dimensioned to provide an energy transfer between the third reporter and the fourth reporter to generate a second signal that is distinguishable from the first signal; providing the library of proteases; contacting individual proteases of the library with the second reporting construct; and characterizing a second signal obtained from the second reporting construct, wherein change of the second signal indicates identification of a second protease having substrate specificity for the second non-neuronal SNARE protein. ### Claim 3 (depends on 2) The method of claim 2, wherein the first signal is characterized during a first time interval and the second signal is characterized during a second time interval, and wherein the first time interval and the second time interval at least partially overlap. ### Claim 4 (depends on 1) The method of claim 1, comprising contacting each of the proteases with a control reporting construct, wherein the control reporting construct comprises a fifth reporter and a sixth reporter and a control enzyme substrate sequence interposed between the fifth reporter and the sixth reporter, wherein the control enzyme substrate sequence is dimensioned to provide an energy transfer between the fifth reporter and the sixth reporter to generate a control signal, and wherein the control enzyme substrate sequence is not derived from a botulinum toxin substrate or from a non-neuronal SNARE protein. ### Claim 5 (depends on 4) The method of claim 4, wherein the control signal is distinguishable from the first signal. ### Claim 6 (depends on 1) The method of claim 1, wherein at least one of the mutated light chain peptides of the library is supplied as a first lysate, wherein the first lysate is produced from a first cell that has been induced, wherein the first cell comprises an inducible expression vector encoding for the given mutated light chain peptide. ### Claim 7 (depends on 6) The method of claim 6, comprising a step of exposing the first reporting construct to a second lysate, wherein the second lysate is produced from a second cell, and wherein the second cell comprises the inducible expression vector and has not been induced.