Engineered botulinum neurotoxin

Abstract

Disclosed herein are botulinum neurotoxin (BoNT) polypeptides with a modified BoNT/B4 receptor binding domain (B4-H C ) having amino acid mutations that modify the binding of the BoNT to the human SytII receptor. Specific mutations and combinations of mutations are disclosed. Isolated modified H C s, polypeptides comprising such modified H C s, chimeric molecules, pharmaceutical compositions, and methods of making and using the same are also disclosed. Methods of identifying additional such modified receptor binding domains, are further disclosed.

1 . A botulinum neurotoxin (BoNT) polypeptide comprising:
 a) a protease domain;   b) a protease cleavage site;   c) a translocation domain; and   d) a modified receptor binding domain of  Clostridium botulinum  serotype B, strain 4 (B4-H c ) that binds human SytII.   
     
     
         2 . The BoNT polypeptide of  claim 1  wherein the modified receptor binding domain comprises substitution mutations V1113K, S1117P, S1196A, and I1197P. 
     
     
         3 . The polypeptide of  claim 2 , further comprising one or more substitution mutations corresponding to substitution mutations in serotype B, strain 4, selected from the group consisting of:
 Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.   
     
     
         4 . The polypeptide of  claim 3 , wherein the modified (B4-H c ) comprises two substitution mutations corresponding to substitution mutations in serotype B, strain 4, selected from the group consisting of:
 Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, and Y1183P.   
     
     
         5 . A polypeptide of  claim 2  comprising a modified receptor binding domain of  Clostridium botulinum  serotype B, strain 4 (B4-H c ) the polypeptide further comprising two or more substitution mutations corresponding to substitution mutations in serotype B, strain 4, wherein one of the substitution mutations is selected from the group consisting of Q1191M, Q1191C, Q1191V, Q1191L, and Q1191Y. 
     
     
         6 . The polypeptide of  claim 2 , further comprising two or more substitution mutations corresponding to substitution mutations in serotype B, strain 4, wherein one of the substitution mutations corresponds to Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, Y1199F or Y1199L in serotype B, strain 4. 
     
     
         7 . The polypeptide of  claim 2 , further comprising two substitution mutations, selected from the substitution mutations corresponding to Q1191M and WI 178Q, Q1191C and W1178Q, Q1191V and W1178Q, Q1191L and W1178Q, Q1191Y and W1178Q, Q1191M and Y1183P, Q1191M and Y1183C, Q1191C and Y1183P, Q1191C and Y1183C, Q1191V and Y1183P, Q1191V and Y1183C, Q1191L and Y1183P, Q1191L and Y1183C, Q1191Y and Y1183P, Q1191Y and Y1183C, W1178Q and Y1183P, and W1178Q and Y1183C in serotype B, strain 4. 
     
     
         8 . The polypeptide of  claim 5 , wherein the two substitution mutations correspond to Q1191M and W1178Q in serotype B, strain 4. 
     
     
         9 . The polypeptide of  claim 5 , wherein the two substitution mutations correspond to Q1191M and W1183P in serotype B, strain 4. 
     
     
         10 . The polypeptide of  claim 5 , wherein the two substitution mutations correspond to Q1191M and W1183C in serotype B, strain 4. 
     
     
         11 . The polypeptide of  claim 2 , wherein the modified (B4-H c ) further comprises three substitution mutations. 
     
     
         12 . The polypeptide of  claim 11 , wherein the three further substitution mutations are at positions that correspond to Q1191, W1178 and Y1183 of serotype B, strain 4. 
     
     
         13 . The polypeptide of  claim 11 , wherein the three further substitution mutations correspond to Q1191M, W1178Q and Y1183P of serotype B, strain 4. 
     
     
         14 . A nucleic acid encoding a BoNT polypeptide of  claim 1 . 
     
     
         15 . A nucleic acid vector comprising the nucleic acid of  claim 14 . 
     
     
         16 . A cell comprising the nucleic acid of  claim 14 . 
     
     
         17 . A cell expressing the BoNT polypeptide of  claim 1 . 
     
     
         18 . A botulinum neurotoxin (BoNT) polypeptide comprising:
 a) a protease domain;   b) a protease cleavage site;   c) a translocation domain; and   d) a modified receptor binding domain of  Clostridium botulinum  serotype B, strain 4 (B4-H c ), comprising substitution mutations V113K, S1117P, S1196A, and I1197P, and further comprising a substitution mutation at a position corresponding to S1201 of serotype B, strain 4.   
     
     
         19 . The BoNT polypeptide of  claim 1 , wherein the protease domain, translocation domain, and protease cleavage site are from a serotype selected from the group consisting of A, B, C, D, E, F, G, and combinations thereof. 
     
     
         20 . The BoNT polypeptide of  claim 19 , wherein the protease domain, translocation domain, and protease cleavage site are from serotype B, strain 1. 
     
     
         21 . The BoNT polypeptide of  claim 19 , wherein the protease domain and translocation domain are from serotype A, strain 1. 
     
     
         22 . The BoNT polypeptide of  claim 19 , wherein the protease cleavage site is from serotype A, serotype B, or is a chimeric cleavage site of serotypes A and B. 
     
     
         23 . A polypeptide comprising a modified receptor binding domain of  Clostridium botulinum  serotype B, strain 4 heavy chain (B4-H c ) that binds human SytII. 
     
     
         24 . The polypeptide of  claim 23  wherein the modified receptor binding domain comprises substitution mutations V1113K, S1117P, S1196A, and I1197P. 
     
     
         25 . The polypeptide of  claim 24 , further comprising one or more substitution mutations corresponding to substitution mutations in serotype B, strain 4, selected from the group consisting of:
 Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.   
     
     
         26 . The polypeptide of  claim 25 , wherein the modified (B4-H c ) comprises two substitution mutations corresponding to substitution mutations in serotype B, strain 4, selected from the group consisting of:
 Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, and Y1183P.   
     
     
         27 . A polypeptide of  claim 24  comprising a modified receptor binding domain of  Clostridium botulinum  serotype B, strain 4 (B4-H c ) the polypeptide further comprising two or more substitution mutations corresponding to substitution mutations in serotype B, strain 4, wherein one of the substitution mutations is selected from the group consisting of Q1191M, Q1191C, Q1191V, Q1191L, and Q1191Y. 
     
     
         28 . The polypeptide of  claim 24 , further comprising two or more substitution mutations corresponding to substitution mutations in serotype B, strain 4, wherein one of the substitution mutations corresponds to Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, Y1199F or Y1199L in serotype B, strain 4. 
     
     
         29 . The polypeptide of  claim 24 , further comprising two substitution mutations, selected from the substitution mutations corresponding to Q1191M and W1178Q, Q1191C and W1178Q, Q1191V and W1178Q, Q1191L and W1178Q, Q1191Y and W1178Q, Q1191M and Y1183P, Q1191M and Y1183C, Q1191C and Y1183P, Q1191C and Y1183C, Q1191V and Y1183P, Q1191V and Y1183C, Q1191L and Y1183P, Q1191L and Y1183C, Q1191Y and Y1183P, Q1191Y and Y1183C, WI 178Q and Y1183P, and WI 178Q and Y1183C in serotype B, strain 4. 
     
     
         30 . The polypeptide of  claim 27 , wherein the two substitution mutations correspond to Q1191M and W1178Q in serotype B, strain 4. 
     
     
         31 . The polypeptide of  claim 27 , wherein the two substitution mutations correspond to Q1191M and W1183P in serotype B, strain 4. 
     
     
         32 . The polypeptide of  claim 27 , wherein the two substitution mutations correspond to Q1191M and W1183C in serotype B, strain 4. 
     
     
         33 . The polypeptide of  claim 24 , wherein the modified (B4-H c ) further comprises three substitution mutations. 
     
     
         34 . The polypeptide of  claim 33 , wherein the three further substitution mutations are at positions that correspond to Q1191, W1178 and Y1183 of serotype B, strain 4. 
     
     
         35 . The polypeptide of  claim 33 , wherein the three further substitution mutations correspond to Q1191M, W1178Q and Y1183P of serotype B, strain 4. 
     
     
         36 . A nucleic acid encoding a polypeptide of  claim 23 . 
     
     
         37 . A nucleic acid vector comprising the nucleic acid of  claim 36 . 
     
     
         38 . A cell comprising the nucleic acid of  claim 36 . 
     
     
         39 . A cell expressing the polypeptide or chimeric molecule of  claim 23 . 
     
     
         40 . A chimeric molecule comprising a first portion that is a modified receptor binding domain of Clostridial botulinum serotype B, strain 4 (B4-H c ) linked to a second portion, wherein the modified B4-H c  binds to human SytII. 
     
     
         41 . The chimeric molecule of  claim 40  wherein the modified receptor binding domain of Clostridial botulinum serotype B, strain 4 (B4-H c ) comprises substitution mutations V1113K, S1117P, S1196A, and I1197P. 
     
     
         42 . The chimeric molecule of  claim 41  wherein the modified receptor binding domain of Clostridial botulinum serotype B, strain 4 (B4-H c ) further comprises one or more substitution mutations selected from the group consisting of:
 Q1191M; Q1191C; Q1191V; Q1191L; Q1191Y; W1178Y; W1178Q; W1178A; W1178S; Y1183C; Y1183P and combinations thereof. 
 
     
     
         43 . The chimeric molecule of  claim 41 , wherein the modified B4-H c  comprises two substitution mutations selected from the group consisting of: Q1191M, Q1191C, Q1191V, Q1191L, Q1191Y, W1178Y, W1178Q, W1178A, W1178S, Y1183C and Y1183P. 
     
     
         44 . A chimeric molecule comprising a first portion that is a modified receptor binding domain of Clostridial botulinum serotype B, strain 4 (B4-H c ) linked to a second portion, wherein the modified B4-H c  comprises two or more substitution mutations corresponding to substitution mutations in serotype B, strain 4 and binds human SytII, wherein one of the substitution mutations is selected from the group consisting of: Q1191M; Q1191C; Q1191V; Q1191L; and Q1191Y. 
     
     
         45 . The chimeric molecule of  claim 44 , wherein one of the substitution mutations corresponds to W1178Y, W1178Q, W1178A, W1178S, Y1183C, or Y1183P in serotype B, strain 4. 
     
     
         46 . The chimeric molecule of  claim 44 , wherein two substitution mutations correspond to Q1191M and W1178Q, Q1191C and W1178Q, Q1191V and W1178Q, Q1191L and W1178Q, Q1191Y and W1178Q, Q1191M and Y1183P, Q1191M and Y1183C, Q1191C and Y1183P, Q1191C and Y1183C, Q1191V and Y1183P, Q1191V and Y1183C, Q1191L and Y1183P, Q1191L and Y1183C, Q1191Y and Y1183P, Q1191Y and Y1183C, W1178Q and Y1183P, or W1178Q and Y1183C in serotype B, strain 4. 
     
     
         47 . The chimeric molecule of  claim 44 , wherein the modified (B-H c ) comprises three substitution mutations. 
     
     
         48 . The chimeric molecule of  claim 47 , wherein the three substitution mutations are at positions that correspond to Q1191, W1178 and Y1183 of serotype B, strain 4. 
     
     
         49 . The chimeric molecule of  claim 47 , wherein the three substitution mutations correspond to Q1191M, WI 178Q and Y1183P of serotype B, strain 4. 
     
     
         50 . The chimeric molecule of  claim 40 , wherein the first portion and the second portion are linked covalently. 
     
     
         51 . The chimeric molecule of  claim 40 , wherein the first portion and the second portion are linked non-covalently. 
     
     
         52 . The chimeric molecule of  claim 40 , wherein the second portion is selected from the group consisting of a small molecule, a nucleic acid, a short polypeptide and a protein. 
     
     
         53 . The chimeric molecule of  claim 40 , wherein the second portion is a bioactive molecule. 
     
     
         54 . The chimeric molecule of  claim 53 , wherein the second portion is a therapeutic polypeptide or non-polypeptide drug. 
     
     
         55 . A nucleic acid encoding a chimeric polypeptide of  claim 40 . 
     
     
         56 . A nucleic acid vector comprising the nucleic acid of  claim 55 . 
     
     
         57 . A cell comprising the nucleic acid of  claim 55 . 
     
     
         58 . A cell expressing the polypeptide or chimeric molecule of  claim 40 . 
     
     
         59 . A pharmaceutical composition comprising the botulinum neurotoxin (BoNT) polypeptide or polypeptide of  claim 1 . 
     
     
         60 . The pharmaceutical composition of  claim 59 , further comprising a pharmaceutically acceptable excipient. 
     
     
         61 . A kit comprising a pharmaceutical composition of  claim 59  and directions for therapeutic administration of the pharmaceutical composition. 
     
     
         62 . A method to produce a botulinum neurotoxin (BoNT) polypeptide, the method comprising the steps of culturing the cell of  claim 17  under conditions wherein said BoNT polypeptide is produced. 
     
     
         63 . The method of  claim 62  further comprising one or more of the following steps:
 recovering the BoNT polypeptide from the culture, 
 purifying the BoNT polypeptide, 
 activating the BoNT polypeptide, and/or 
 formulating the BoNT polypeptide. 
 
     
     
         64 . A method for treating a condition associated with unwanted neuronal activity comprising administering a therapeutically effective amount of the BoNT polypeptide of  claim 1  to a subject to thereby contact one or more neurons exhibiting unwanted neuronal activity, to thereby treat the condition. 
     
     
         65 . The method of  claim 64 , wherein the condition is selected from the group consisting of, spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity and other voice disorders, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, secretory disorders, pain from muscle spasms, headache pain, migraine, and dermatological or aesthetic/cosmetic conditions. 
     
     
         66 . The botulinum neurotoxin (BoNT) polypeptide of  claim 1  for use in medicine. 
     
     
         67 . The botulinum neurotoxin (BoNT) polypeptide of  claim 1  for use in treating a condition associated with unwanted neuronal activity.