Engineered botulinum neurotoxin

Abstract

Disclosed herein are modified BoNT / B4 receptor binding domains (B4-H) having amino acid mutations. C ) a botulinum neurotoxin (BoNT) polypeptide, wherein the amino acid mutation modifies the binding of the BoNT to the human SytII receptor. Specific mutations and combinations of mutations are disclosed. Also disclosed are isolated modified H C , H containing such modifications C Polypeptides, chimeric molecules, pharmaceutical compositions, and methods of making and using the same. Methods of identifying additional such modified receptor binding domains are also disclosed.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 378,967, filed August 24, 2016, the contents of which are incorporated herein by reference in their entirety.

[0003] US government support

[0004] This invention was made with U.S. Government support under NIH 1R21NS082830 awarded by the National Institute of Health. The U.S. Government has certain rights in this invention.

[0005] Sequence Listing

[0006] This specification refers to the sequence listing (submitted electronically as a .txt file named "0342941_0602_SL.TXT" on August 24, 2017). The .txt file was generated on August 22, 2017 and is 106,905 bytes in size. The entire contents of the sequence listing are incorporated herein by reference. Technical Field

[0007] The present invention relates to the field of modified neurotoxins and their use for the treatment of medical and / or aesthetic conditions. Background Art

[0008] Botulinum neurotoxins belong to a family of bacterial toxins that include seven major serotypes (BoNT / AG) (1). These toxins act by blocking the release of neurotransmitters from neurons, thereby causing paralysis in animals and humans. In recent years, BoNTs have been widely used to treat an increasing number of medical conditions: local injection of small amounts of toxin can attenuate neuronal activity in a target area, which can be beneficial in many medical conditions and for cosmetic purposes (7-9).

[0009] BoNT / A and BoNT / B are the only two BoNTs currently approved by the FDA for use in humans (7-9). These are toxins purified from bacteria without any sequence modifications (defined as wild type, WT). As the use of BoNTs has grown, limitations and adverse reactions have been reported. The main limitation is the production of neutralizing antibodies in patients, which renders future treatment ineffective (11). Termination of BoNT use often leaves patients with no other effective options to treat / alleviate their condition. The likelihood of an antibody response is directly related to both the toxin dose and the frequency of injections (11). Therefore, this limitation occurs primarily in the treatment of muscle spasms, which involves relatively high doses of toxin.

[0010] The major adverse reactions are usually associated with treating muscle spasms but not with cosmetic applications. This is because adverse reactions are primarily due to the spread of the toxin to other areas of the body, and the likelihood of toxin spread is directly related to the dose injected. Adverse reactions range from transient, non-serious events such as ptosis and diplopia to life-threatening events and even death. In a petition submitted to the FDA by Dr. Sidney Wolfe in 2008, a total of 180 serious adverse events were documented, including 16 deaths. As a result, the FDA now requires a "black box warning" for all BoNT products, highlighting the risk of toxin spread, following similar warnings issued by the European Union.

[0011] Because both neutralizing antibody production and toxin spread are directly related to the injected dose, there is a strong desire to reduce the toxin dose (while maintaining the same level of toxin activity), which means that the efficacy of individual toxin molecules must be enhanced. Such modified BoNTs with improved specificity for neurons will also reduce any potential off-target effects due to nonspecific entry into other cell types.

[0012] BoNTs are targeted and enter neurons by binding to their specific receptors via their receptor binding domains, which are well defined in the literature (BoNT-H C , Figure 1A , B) 1 Receptor binding determines the efficacy and specificity of BoNT recognition of neurons. Improving the receptor binding ability of BoNTs will enhance their efficacy and specificity in targeting neurons. Receptors have been identified for most BoNTs ( Figure 1C BoNT / B, DC, and G share two homologous synaptic vesicle proteins, synaptotagmin I and II (SytI / II), as their receptors 13-18 , while BoNT / A, E, D, and F use another synaptic vesicle protein, SV2. In addition to the protein receptors, all BoNTs require the lipid co-receptor ganglioside ( Figure 1D ), which are abundant on the surface of neurons 27 Of the two Syt isoforms, Syt II has approximately 10-fold higher binding affinity for BoNT / B than Syt I and is also the predominant isoform expressed in motor nerve terminals, which are the target neurons for BoNTs ( Figure 2A ). Thus, Syt II is considered the primary toxin receptor for BoNT / B, DC, and G, while Syt I is a secondary toxin receptor at motor nerve endings. This is true in rodents and probably in most mammals.

[0013] One might argue that BoNTs are already highly specific for neurons and ask if it is possible to further improve their binding to neurons? The answer is "yes" for humans, at least in part because human Syt II was recently found to have greatly reduced binding and function as a BoNT / B receptor due to a unique amino acid change from rodent (rat / mouse) Syt II within the toxin binding site. This is a change from phenylalanine (F) to leucine (L) at position 54 (mouse Syt II sequence) (e.g., US 9,598,685, Figure 2B Sequence alignment has revealed that the phenylalanine at this position is highly conserved in both Syt I and Syt II of vertebrates including platypus, fish, rodents, and monkeys. 48 Only human and chimpanzee Syt II contain leucine at this position. Due to this residue change, human and chimpanzee Syt II have greatly reduced binding to BoNT / B, DC and G (e.g., US 9,598,685, Figure 2C ), and is significantly less efficient than mouse Syt II in regulating the entry of BoNT / B (compare US 9,598,685, Figure 2D As shown in ). Since human and chimpanzee Syt I still contain phenylalanine at the same position and can bind BoNT / B, DC and G (as shown in US 9,598,685, Figure 2E 1,2-D (shown in Figure 2), so the high affinity receptor for BoNT / B, DC, and G in humans is limited to the minor receptor Syt I. These findings provide an explanation for clinical observations that much higher doses of BoNT / B are required than BoNT / A (which binds to a different receptor) to achieve the same level of therapeutic effect in patients. These observations were previously attributed to other reasons, such as the percentage of active neurotoxin in the formulation used. The recent observation of this difference in binding of BoNT / B to human Syt II relative to Syt II of other species suggests that different residues of BoNT / B may be involved in binding to human Syt II. Therefore, sequence modifications of BoNT / B that are expected to affect binding to rodent Syt II may have unpredictable effects on BoNT / B binding to human Syt II.

[0014] Therefore, there is a need to improve the binding of BoNT / B to the human receptor Syt II as a way to increase its efficacy and specificity in targeting human neurons and to reduce the dose of toxin required for therapeutic applications. Summary of the invention

[0015] As described above, Clostridium botulinum neurotoxins have been found to be useful in medicine for the treatment of pathological conditions and for cosmetic purposes. Wild-type Clostridium botulinum serotype B, strain 4 polypeptides do not bind to the human SytII receptor with high affinity or result in effective inhibition of unwanted neuronal activity. Described herein are site-directed mutagenesis of the serotype B, strain 4 (BoNT / B4) receptor binding domain that significantly increases the expression of the modified receptor binding domain (BoNT / B4 H c ) ability to bind to human SytII receptor. Modified BoNT / B4 H c Increased binding of the domain to the hSytII receptor increases the ability of BoNT polypeptides comprising the domain to inhibit neuronal activity and renders BoNT polypeptides comprising such domains significantly more effective and less likely to provoke an unwanted immune response for therapeutic and cosmetic uses.

[0016] In one aspect, then, described herein are botulinum neurotoxin (BoNT) polypeptides 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 that binds human SytII (B4-H c ).

[0017] In one embodiment, the modified receptor binding domain of a BoNT polypeptide comprises the substitution mutations V1113K, S1117P, S1196A, and I1197P.

[0018] In another embodiment, the modified receptor binding domain of the BoNT polypeptide further comprises 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. c ) comprises two substitution mutations corresponding to substitution mutations in serotype B, strain 4, wherein the substitution mutations are selected from the group consisting of Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C and Y1183P.

[0019] In another embodiment, Clostridium botulinum serotype B, strain 4 (B4-H c) polypeptide further comprises two or more substitution mutations corresponding to substitution mutations in serotype B, strain 4, wherein one of the substitution mutations is selected from Q1191M, Q1191C, Q1191V, Q1191L and Q1191Y.

[0020] In another embodiment, Clostridium botulinum serotype B, strain 4 (B4-H c ) polypeptide further comprises 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.

[0021] In another embodiment, Clostridium botulinum serotype B, strain 4 (B4-H c ) polypeptide further comprises two substitution mutations selected from the group consisting of Q1191M and W1178Q, Q1191C and W1178Q, Q1191V and W1178Q, Q1191L and W1178Q, Q1191Y and W1178Q, Q1191M and Y1183P, Q1191M and Y1183C ... In another embodiment, the Clostridium botulinum serotype B, strain 4 (B4-H c ) polypeptide further comprises two substitution mutations corresponding to Q1191M and W1178Q in serotype B, strain 4. In another embodiment, Clostridium botulinum serotype B, strain 4 (B4-H c ) polypeptide further comprises two substitution mutations corresponding to Q1191M and W1183P in serotype B, strain 4. In another embodiment, Clostridium botulinum serotype B, strain 4 (B4-H c ) The modified receptor binding domain of the polypeptide also comprises two substitution mutations corresponding to Q1191M and W1183C in serotype B, strain 4.

[0022] In another embodiment, Clostridium botulinum serotype B, strain 4 (B4-H c ) polypeptide further comprises three substitution mutations. In another embodiment, the three additional substitution mutations are at positions corresponding to Q1191, W1178, and Y1183 of serotype B, strain 4. In another embodiment, the three additional substitution mutations correspond to Q1191M, W1178Q, and Y1183P of serotype B, strain 4.

[0023] In another embodiment, the modified receptor binding domain further comprises a substitution mutation corresponding to a substitution mutation in serotype B, strain 4 selected from the group consisting of: Q1191C; Q1191V; Q1191L; Q1191Y; Q1191M; Y1199W; Y1199E; and Y1199H.

[0024] In another embodiment, the modified receptor binding domain further comprises a substitution mutation corresponding to the substitution mutation Q1191M in serotype B, strain 4.

[0025] In another embodiment, the modified receptor binding domain further comprises two substitution mutations corresponding to substitution mutations in serotype B, strain 4 selected from: a first mutation selected from Q1191C; Q1191; Q1191L; Q1191Y; and Q1191M; and a second mutation selected from: Y1199W; Y1199E; and Y1199H.

[0026] In another embodiment, the modified receptor binding domain further comprises one or more substitution mutations corresponding to substitution mutations in serotype B, strain 4, selected from the group consisting of: W1178Y; W1178Q; W1178A; W1178S; Y1183C; Y1183P and combinations thereof.

[0027] In another embodiment, the modified receptor binding domain comprises two substitution mutations corresponding to Q1191M and Y1199W, Q1191M and W1178Q, Q1191C and Y1199W, Q1191C and W1178Q, Q1191V and Y1199W, or Q1191V and W1178Q in serotype B, strain 4.

[0028] In another embodiment, the modified (BH c ) comprises three substitution mutations at positions corresponding to Q1191, W1178 and Y1183 of serotype B, strain 4. In another embodiment, the three substitution mutations correspond to Q1191M, W1178Q and Y1183C of serotype B, strain 4.

[0029] Also provided herein is a nucleic acid encoding a BoNT polypeptide as described in any aspect or embodiment herein, or a nucleic acid vector comprising such a nucleic acid operably linked to a sequence allowing expression of the BoNT polypeptide. Also provided herein is a cell comprising such a nucleic acid or nucleic acid vector, and / or a cell expressing such a BoNT polypeptide.

[0030] In another aspect, described herein is 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 ), which comprises substitution mutations V1113K, S1117P, S1196A and I1197P, and further comprises a substitution mutation at the position corresponding to S1201 of serotype B, strain 4.

[0031] In one embodiment of any of the preceding aspects, the protease domain, translocation domain, and protease cleavage site of the BoNT polypeptide are from a serotype selected from the group consisting of A, B, C, D, E, F, G, and combinations thereof.

[0032] In another embodiment of any of the preceding aspects, the protease domain, translocation domain, and protease cleavage site are from serotype B, strain 1.

[0033] In another embodiment of any of the preceding aspects, the protease domain, translocation domain, and protease cleavage site are from serotype A, strain 1.

[0034] In another embodiment, the protease cleavage site is from serotype A, serotype B, or is a chimeric cleavage site from serotypes A and B.

[0035] In another aspect, described herein is a modified receptor binding domain comprising a heavy chain of Clostridium botulinum serotype B, strain 4 that binds to human SytII (B4-H c ) polypeptide.

[0036] In one embodiment, the modified receptor binding domain comprises the substitution mutations V1113K, S1117P, S1196A, and I1197P.

[0037] In another embodiment, the modified receptor binding domain further comprises one or more substitution mutations corresponding to substitution mutations in serotype B, strain 4, wherein the substitution mutations are selected from the group consisting of: Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

[0038] In another embodiment, the modified (B4-H c ) comprises two substitution mutations corresponding to substitution mutations in serotype B, strain 4, wherein the substitution mutations are selected from the group consisting of: Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C and Y1183P.

[0039] In another embodiment, the modified B4-H c Also comprising two or more substitution mutations corresponding to the 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.

[0040] In another embodiment, the modified B4-H c It also comprises 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.

[0041] In another embodiment, the modified B4-H cThe present invention also comprises two substitution mutations selected from the group consisting of Q1191M and W1178Q, Q1191C and W1178Q, Q1191V and W1178Q, Q1191L and W1178Q, Q1191Y and W1178Q, Q1191M and Y1183P, Q1191M and Y1183C, Q1191C and Y1178Q in strain 4 corresponding to serotype B. Substitution mutations of 1183P, 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.

[0042] In another embodiment, the two substitution mutations correspond to Q1191M and W1178Q in serotype B, strain 4.

[0043] In another embodiment, the two substitution mutations correspond to Q1191M and W1183P in serotype B, strain 4.

[0044] In another embodiment, the two substitution mutations correspond to Q1191M and W1183C in serotype B, strain 4.

[0045] In another embodiment, the modified (B4-H c ) further comprises three substitution mutations. In another embodiment, the three additional substitution mutations are at positions corresponding to Q1191, W1178, and Y1183 of serotype B, strain 4. In another embodiment, the three additional substitution mutations correspond to Q1191M, W1178Q, and Y1183P of serotype B, strain 4.

[0046] In another aspect, described herein is a chimeric molecule comprising a first portion that is a modified receptor binding domain of Clostridium botulinum serotype B, strain 4 (B4-H) linked to a second portion. c ), wherein the modified B4-H c Binds to human SytII.

[0047] In one embodiment, the modified receptor binding domain of Clostridium botulinum serotype B, strain 4 (B4-H c ) comprises the substitution mutations V1113K, S1117P, S1196A and I1197P.

[0048] In another embodiment, the modified receptor binding domain of Clostridium botulinum serotype B, strain 4 (B4-Hc ) 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.

[0049] In another embodiment, the modified B4-H c Also included are two substitution mutations selected from the group consisting of: Q1191M, Q1191C, Q1191V, Q1191L, Q1191Y, W1178Y, W1178Q, W1178A, W1178S, Y1183C and Y1183P.

[0050] In another aspect, described herein is a chimeric molecule comprising a first portion that is a modified receptor binding domain of Clostridium botulinum serotype B, strain 4 (B4-H) linked to a second portion. c ), wherein the modified B4-H c The invention comprises two or more substitution mutations corresponding to substitution mutations in serotype B, strain 4 and binds to human SytII, wherein one of the substitution mutations is selected from the group consisting of: Q1191M, Q1191C, Q1191V, Q1191L and Q1191Y.

[0051] In one embodiment, one of the substitution mutations corresponds to W1178Y, W1178Q, W1178A, W1178S, Y1183C or Y1183P in serotype B, strain 4.

[0052] In another embodiment, the two substitution mutations correspond to serotype B, 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, and W1178Q and Y1183C.

[0053] In another embodiment, the modified (BH c) comprises three substitution mutations. In another embodiment, the three substitution mutations are at positions corresponding to Q1191, W1178, and Y1183 of serotype B, strain 4. In another embodiment, the three substitution mutations correspond to Q1191M, W1178Q, and Y1183P of serotype B, strain 4.

[0054] In another embodiment, the first moiety and the second moiety are covalently linked.

[0055] In another embodiment, the first moiety and the second moiety are non-covalently linked.

[0056] In another embodiment, the second moiety is selected from the group consisting of a small molecule, a nucleic acid, a short polypeptide, and a protein.

[0057] In another embodiment, the second moiety is a biologically active molecule.

[0058] In another embodiment, the second moiety is a therapeutic polypeptide or a non-polypeptide drug.

[0059] In another aspect, described herein is a pharmaceutical composition comprising a botulinum neurotoxin (BoNT) polypeptide or chimeric molecule as described herein, or comprising a nucleic acid or nucleic acid vector encoding a BoNT polypeptide as described herein.

[0060] In one embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0061] In another aspect, described herein is a kit comprising a pharmaceutical composition as described herein. The kit may include instructions for therapeutic administration of the pharmaceutical composition.

[0062]

[0013] In another aspect, described herein is a method of producing a botulinum neurotoxin (BoNT) polypeptide, comprising the step of culturing a cell as described herein comprising a nucleic acid or a nucleic acid vector encoding a BoNT polypeptide or a chimeric BoNT polypeptide as described herein under conditions whereby the BoNT polypeptide or chimeric BoNT polypeptide is produced.

[0063] In one embodiment, the method further comprises one or more of the following steps: recovering the BoNT polypeptide from the culture; purifying the BoNT polypeptide; activating the BoNT polypeptide; or formulating the BoNT polypeptide.

[0064] In another aspect, described herein is a method for treating a disorder associated with unwanted neuronal activity comprising administering to a subject a therapeutically effective amount of a BoNT polypeptide or chimeric BoNT polypeptide as described herein, or a pharmaceutical composition comprising such a polypeptide, thereby contacting one or more neurons exhibiting the unwanted neuronal activity, thereby treating the disorder.

[0065] In one embodiment, the disorder is selected from the group consisting of: spasmodic dystonia, spasmodic torticollis, laryngeal dystonia, oromandibular dystonia, tongue dystonia, cervical dystonia, local hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorders, cerebral palsy, local spasms and other speech disorders, spastic colitis, neurogenic bladder, anal spasm, limb spasms, tics, tremors, bruxism, anal fissures, achalasia, dysphagia and other dystonias and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, secretory disorders, pain caused by muscle spasms, headaches, migraines, and dermatological or aesthetic / cosmetic disorders.

[0066] In another aspect, described herein is a botulinum neurotoxin (BoNT) polypeptide as described herein, a chimeric BoNT polypeptide as described herein, or a pharmaceutical composition comprising such a BoNT polypeptide or chimeric polypeptide for use in medicine.

[0067] In another aspect, described herein are botulinum neurotoxin (BoNT) polypeptides as described herein, chimeric BoNT polypeptides as described herein, or pharmaceutical compositions comprising such BoNT polypeptides or chimeric polypeptides for use in treating a disorder associated with unwanted neuronal activity.

[0068] definition

[0069] As used herein, the term "binding affinity" refers to the strength of the binding activity of a molecule to a specific receptor system. Typically, high binding affinity is generated by larger intermolecular forces between a binding domain and its receptor system, while low binding affinity involves smaller intermolecular forces between a ligand and its receptor. High binding affinity involves a longer residence time of the binding domain at its receptor binding site than in the case of low binding affinity. Therefore, a molecule with high binding affinity means that a lower concentration of the molecule is required to maximize the occupation of the binding site of the receptor system and trigger a physiological response. In contrast, low binding affinity means that a relatively high concentration of the molecule is required before the receptor binding site of the receptor system is occupied to the maximum and the maximum physiological response is achieved. Therefore, a botulinum neurotoxin as described herein with increased binding activity due to high binding affinity will allow the administration of a reduced dose of toxin, thereby reducing or preventing unwanted side effects associated with the dispersion of the toxin to non-targeted areas.

[0070] One parameter commonly used to measure binding affinity is defined as the binding K D Because K D The dissociation constant (K 解离 ) and the association constant (K 缔合 ), K D The lower the value, the higher the binding affinity.

[0071] As used herein, the term "significantly enhanced binding" when used to describe the binding affinity of a Clostridium botulinum neurotoxin molecule or a BoNT / B-Hc domain or receptor binding fragment thereof described herein to a specific receptor (e.g., human Syt II or human Syt I) refers to an increase in binding affinity to a specific receptor that is significantly increased (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the binding affinity of the wild-type molecule) compared to the non-substituted version of the molecule. In one embodiment, the enhanced binding of the substituted molecule is achieved by increasing the binding affinity of the substituted molecule to that of the unsubstituted molecule (e.g., a BoNT / B-Hc domain having a naturally occurring BoNT H C In other words, the K D K of the unsubstituted molecule D In one embodiment, the enhanced binding is demonstrated by a binding affinity that is significantly higher (e.g., 1.5X, 2.0X, 2.5X, 3.0X, etc.) than the binding affinity of the unsubstituted molecule. In other words, the K of the substituted molecule is D Compared to the K of the unsubstituted molecule D In one embodiment, the enhanced binding demonstrated is between about 0.59 μM and 5.82 μM K D In one embodiment, the demonstrated enhanced binding is between about 0.59 μM and 4.3 μM K. D In one embodiment, the enhanced binding is achieved by K D ≤5.82 μM. In one embodiment, the enhanced binding is confirmed by K D ≤4.30 μM. In one embodiment, the enhanced binding is confirmed by K D In one embodiment, the enhanced binding is demonstrated by a K of about 0.59 μM. D Confirmed.

[0072] In one embodiment, the substituted molecule has a K of human Syt II. D≤10 μM, preferably ≤9 μM, 8 μM, 7 μM, 6 μM, 5 μM, 4 μM, 3 μM or 2 μM, more preferably ≤1 μM or 0.6 μM. In one embodiment, the substituted molecule has a K of ≤10 μM for human Syt I. D ≤10 μM, preferably ≤9 μM, 8 μM, 7 μM, 6 μM, 5 μM, 4 μM, more preferably ≤3 μM. In one embodiment, the substituted molecule has a K of ≤10 μM for human Syt II. D ≤7 μM, and the substituted molecule has a K of 1.5 for human Syt I D ≤6 μM. In a preferred embodiment, the substituted molecule has a K of 1.17kDa for human Syt II. D ≤1μM, and the substituted molecule has a K of 1.5 for human Syt I D ≤3μM.

[0073] In one embodiment, the enhanced binding results in detectable binding to human Syt I in the absence of co-receptor gangliosides, such as exemplified by the experiments described herein.

[0074] When used to describe BoNT / BH produced by the point mutations described herein C When referring to the binding affinity of a fragment, the terms "significantly enhanced binding" or "significantly reduced binding" refer to an increase or decrease, respectively, in the binding affinity of a modified binding domain (e.g., expressed as an isolated fragment or in the context of a larger neurotoxin polypeptide construct) to a specific receptor (e.g., human Syt II or human Syt I as discussed directly above).

[0075] As used herein, the term "significantly reduced binding" when used to describe the binding affinity of a botulinum neurotoxin molecule or binding fragment thereof described herein (e.g., BoNT / B-Hc) to a specific receptor (e.g., human Syt I or human Syt II) refers to a decrease in binding affinity to a specific receptor that is significantly reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the binding affinity of the wild-type molecule) compared to the non-substituted version of the molecule. In one embodiment, the reduced binding of the substituted molecule results in a decrease in binding affinity to the unsubstituted neurotoxin (e.g., a BoNT / B-Hc having a naturally occurring C The binding affinity of the substituted molecule is an order of magnitude or lower than that of the neurotoxin. In other words, the K D K of unsubstituted neurotoxin DIn one embodiment, the reduced binding of the substituted molecule results in a binding affinity that is significantly lower (e.g., 1.5X, 2.0X, 2.5X, 3.0X, etc.) than the binding affinity of the unsubstituted molecule. In other words, the K of the substituted molecule is D Compared to the K of the unsubstituted molecule D Significantly higher (eg, 1.5X, 2.0X, 2.5X, 3.0X, etc.). In one embodiment, the significantly reduced binding results in a detectable loss of binding to human Syt I in the absence of a co-receptor ganglioside.

[0076] As used herein, the term "botulinum neurotoxin" means any polypeptide that can execute the overall cellular mechanism whereby a botulinum toxin enters neurons and inhibits neurotransmitter release. The mechanism encompasses binding of the botulinum toxin to low or high affinity receptor complexes, internalization of the toxin, translocation of the toxin light chain into the cytoplasm, and enzymatic modification of botulinum toxin substrates. The terms "polypeptide" and "protein" are used interchangeably herein when referring to botulinum neurotoxin molecules and fragments thereof.

[0077] As the term is used herein, a "modified receptor binding domain" or a "modified H C "Promote the binding of the botulinum neurotoxin molecule containing it to the receptor of the botulinum neurotoxin located on the surface of the target cell. Such molecules are usually produced by genetic recombination technology. The modified H C The term "binding activity" as used herein means that one molecule directly or indirectly contacts another molecule through at least one intermolecular or intramolecular force, including but not limited to covalent bonds, ionic bonds, metallic bonds, hydrogen bonds, hydrophobic interactions, van der Waals interactions, etc. or any combination thereof. "Bound" and "binding" are considered as terms of binding.

[0078] As used herein, the term "botulinum toxin protease domain" means a botulinum toxin domain that can perform the enzymatic target modification step of the intoxication process. Thus, the botulinum toxin protease domain specifically targets a botulinum toxin substrate and encompasses proteolytic cleavage of a botulinum toxin substrate, such as a SNARE protein such as a SNAP-25 substrate, a VAMP substrate, and a Syntaxin substrate.

[0079] Non-limiting examples of Clostridium botulinum toxin protease domains are provided in Table 1.

[0080] As used herein, the term "clostridium botulinum toxin translocation domain" or "N " refers to a Clostridium botulinum toxin toxin domain that can perform the translocation step of the intoxication process that mediates the translocation of the Clostridium botulinum toxin light chain. Thus, H N Promotes the movement of Clostridium botulinum toxin across membranes and encompasses the movement of Clostridium botulinum toxin light chain through intracellular vesicle membranes into the cytoplasm of cells. N Non-limiting examples include BoNT / AH N 、BoNT / BH N 、BoNT / C1 H N 、BoNT / DH N 、BoNT / EH N 、BoNT / FH N and BoNT / GH N , whose amino acid sequences are provided in Tables 1 and Figure 8 and Figures 10 to 16 middle.

[0081] As used herein, the term "clostridium botulinum receptor binding domain" and "H C "Botulinum toxin" is synonymous with "domain" and means any naturally occurring Clostridium botulinum receptor binding domain that can perform the cell binding step of the intoxication process, including, for example, binding of a botulinum toxin to a Clostridium botulinum toxin-specific receptor system located on the plasma membrane surface of a target cell. It is contemplated that replacement of binding activity can be accomplished, for example, by replacing a modified (e.g., enhanced) H C Domain replacement of the entire Clostridium botulinum H C Struct domain implementation.

[0082] As used herein, the term "botulinum toxin target cell" means a cell that is a naturally occurring cell in which a naturally occurring botulinum toxin is capable of intoxicating, including, but not limited to, motor neurons; sensory neurons; autonomic neurons; for example, sympathetic neurons and parasympathetic neurons; non-peptidergic neurons, such as cholinergic neurons, adrenergic neurons, noradrenergic neurons, serotonergic neurons, GABAergic neurons; and peptidergic neurons, such as substance P neurons, calcitonin gene-related peptide neurons, vasoactive intestinal peptide neurons, neuropeptide Y neurons, and cholecystokinin neurons.

[0083] "Isolated" means material free to varying degrees from the components that normally accompany it as found in its native state. "Isolated" means the degree of separation from the original source or surrounding environment, such as from flanking amino acids (e.g., in the context of an isolated polypeptide fragment) or from a cell or biological sample.

[0084] The term "purified" is used to refer to a substance, such as a polypeptide, that is "substantially pure" relative to other components of a preparation (e.g., other polypeptides). It can mean that the polypeptide is at least about 50%, 60%, 70% or 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure relative to the other components. Rephrasing, the term "substantially pure" or "substantially purified" with respect to a polypeptide means that the preparation contains less than about 20%, more preferably less than about 15%, 10%, 8%, 7%, and most preferably less than about 5%, 4%, 3%, 2%, 1% or less than 1% of one or more other components (e.g., other polypeptides or cellular components).

[0085] As used herein, the term "conservative" or "conservative substitution mutation" refers to a mutation in which an amino acid is substituted for another amino acid with similar properties such that one skilled in the art of peptide chemistry would expect the secondary structure, chemical properties, and / or hydrophilicity of the polypeptide to be substantially unchanged. The following groups of amino acids have historically been substituted for one another as conservative changes: (1) ala, pro, gly, glu, asp, gln, asn, ser, thr; (2) cys, ser, try, thr; (3) val, ile, leu, met, ala, phe; (4) lys, arg, his; (5) phe, tyr, trp, his. Other generally accepted conservative substitutions are as follows:

[0086] Residue Conservative substitution Residue Conservative substitution Ala Ser Leu Ile; Val Arg Lys Lys Arg; Gln Asn Gln; His Met Leu; Ile Asp Glu Phe Met; Leu; Tyr Gln Asn Ser Thr; Gly Cys Ser Thr Ser; Val Glu Asp Trp Tyr Gly Pro Tyr Trp; Phe His Asn; Gln Val Ile; Leu Ile Leu, Val

[0087] The term "substitution mutation" without reference to a specific amino acid can include any amino acid except the wild-type residue commonly found at that position. Such substitutions can be replaced with non-polar (hydrophobic) amino acids such as glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan and proline. Substitutions can be replaced with polar (hydrophilic) amino acids such as serine, threonine, cysteine, tyrosine, asparagine and glutamine. Substitutions can be replaced with charged amino acids, for example, negatively charged amino acids such as aspartic acid and glutamic acid and positively charged amino acids such as lysine, arginine and histidine.

[0088] Substitution mutations described herein will generally be replaced with different naturally occurring amino acid residues, but in some cases, non-naturally occurring amino acid residues may also be substituted. As a term used herein, non-natural amino acids are non-proteinogenic (i.e., non-protein-encoded) amino acids that are naturally occurring or chemically synthesized. Examples include, but are not limited to, β-amino acids (β3 and β2), homologous amino acids, proline and pyruvic acid derivatives, 3-substituted alanine derivatives, glycine derivatives, ring-substituted phenylalanine and tyrosine derivatives, linear core amino acids, diamino acids, D-amino acids, and N-methyl amino acids. In some embodiments, amino acids may be substituted or unsubstituted. The substituted amino acid or substituent may be a halogenated aromatic or aliphatic amino acid, a halogenated aliphatic or aromatic modification on a hydrophobic side chain, or an aliphatic or aromatic modification.

[0089] The term "therapeutically effective amount" refers to an amount sufficient to achieve a significant reduction in the treatment of one or more symptoms of a disorder when administered to a typical subject suffering from the disorder. The significant reduction in the treatment of symptoms is, for example, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100% or greater compared to a control or untreated subject.

[0090] The term "treat" or "treatment" refers to therapeutic treatment, wherein the purpose is to eliminate or alleviate symptoms. Beneficial or desired clinical results include, but are not limited to, symptom elimination, symptom relief, reduction in symptom severity, stabilization of symptom status (i.e., no worsening), and delay or slowing of symptom progression.

[0091] As used herein, "subject" refers to a person or an animal. Typically, the animal is a vertebrate, such as a primate, a rodent, a domestic animal, or a wild animal. Primates include chimpanzees, crab-eating monkeys, spider monkeys, and macaques such as rhesus monkeys. Rodents include mice, rats, marmots, ferrets, rabbits, and hamsters. Domestic animals and wild animals include cows, horses, pigs, deer, bison, buffaloes, cat species (e.g., domestic cats), canine species (e.g., dogs, foxes, wolves), bird species (e.g., chicks, emus, ostriches), and fish (e.g., trout, catfish, and salmon). Patients or subjects include any subset of the foregoing, e.g., all of the above, but exclude one or more groups or species, such as humans, primates, or rodents. In certain embodiments of aspects described herein, the subject is a mammal, such as a primate, such as humans. The terms "patient" and "subject" are used interchangeably herein. The subject may be male or female. The subject can be a fully developed subject (eg, an adult) or a subject undergoing the development process (eg, a child, infant, or fetus).

[0092] Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects representing animal models of disorders associated with unwanted neuronal activity. In addition, the methods and compositions described herein can be used to treat domesticated animals and / or pets. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0094] Figures 1A to 1D .(Published background data)Schematic model showing how BoNT targets neurons ( Figure 1A ), its total protein structure ( Figure 1B ), a list of identified receptors ( Figure 1C ) and the structural model of BoNT / B binding to its receptor Syt and ganglioside ( Figure 1D ). Figure 1A ) Schematic diagram of BoNT action: BoNT recognizes neurons by binding to its specific receptor (step 1), enters neurons via receptor-mediated endocytosis (step 2), and the light chains of BoNT then translocate across the endosomal membrane to the cytosol (step 3), where these light chains act as proteases to cleave target host proteins (step 4). Figure 1A Adapted from Arnon, S. et al., JAMA, 285:1059, 2001 24 . ( Figure 1B )BoNT consists of a light chain and a heavy chain connected by disulfide bonds. The heavy chain can be further divided into two domains: the translocation domain (H N ) and receptor binding domain (H C ). These functional domains can be switched between different BoNTs. For example, BoNT / BH C Can be used to replace BoNT / AH C To produce chimeric toxins. Figure 1C ) List of identified toxin receptors. ( Figure 1D ) Structural model showing the binding of BoNT / B to its protein receptor Syt(I / II) and its lipid co-receptor ganglioside on the cell surface. Figure 1D Adapted from Chai et al., Nature, 444:1096, 2006 31 .

[0095] FIG. 2A to FIG. 2E Bacterial two-hybrid (BACTH) screening identified point mutations that enhance the binding of BoNT / B to h-Syt II. Figure 2A ) Upper panel: Sequence alignment of the BoNT / B binding site in human Syt II (h-Syt II, SEQ ID NO: 6) and mouse Syt II (m-Syt II, SEQ ID NO: 5). Lower panel: H C B and Syt II: A close-up view of the interface between the targeted residues in BoNT / B for mutagenesis studies are labeled in green (light grey). Syt II residues are labeled in purple (dark grey), with F54, F55, and I58 highlighted (inside the box). Figure 2B )H C B Schematic diagram of the construction of mutant library and BATCH assay. ( Figure 2C ) List of positive mutations identified in the BATCH assay that produced blue colonies. ( Figure 2D ) H listed in Figure C C The B mutant was further analyzed by β-galactosidase activity assay, which reflects the level of reconstructed adenylate cyclase. (n=6, *p<0.05). Figure 2E )H at E1191 C B mutations were examined in pull-down assays using immobilized m-Syt II (residues 1-87) or mouse Syt II (1-87) containing the F54L mutation that mimics the h-Syt II sequence. C B mutants were detected by C B. Detection of HA tag fusion by immunoblot analysis. C B mutations are shown in Fig.18 and Fig.21 middle.

[0096] FIG. 3A to FIG. 3C .H C The combined mutations in B enhance its binding to h-Syt II and Syt I. ( Figure 3A )H C B Double mutations were examined in a pull-down assay for their ability to bind m-Syt II versus their ability to bind h-Syt II. The three double mutations that showed robust binding to h-Syt II are marked in red (bold). Figure 3B )H C B Binding to h-Syt II was quantified by BLI assay. WT H C B and three Hs C Representative association and dissociation curves of B mutants: E1191M / S1199Y, E1191V / S1199W, and E1191C / S1199W. The binding parameters of all 12 double mutations are listed in Table 4, and their representative binding traces are shown in Fig.19 middle.( Figure 3C)WT H C B.H C B(E1191M) and H C Binding of B(E1191M / S1199Y) to immobilized GST-tagged h-Syt I (residues 1-80) was examined in a pull-down assay in the presence (+) and absence (-) of ganglioside (Gang1). C Binding of B to h-Syt I requires the presence of co-receptor gangliosides, whereas H C B(E1191M) and H C B(E1191M / S1199Y) binds to h-Syt I in the absence of gangliosides.

[0097] FIG. 4A to FIG. 4B .H C B MY Shows robust binding to humanized neurons expressing h-Syt II. Figure 4A ) Humanized neurons were generated by KD of endogenous Syt I and expressed full-length h-Syt II in cultured rat cortical neurons. Neurons expressing full-length m-Syt II or m-Syt II (F54L) were used as additional controls. These neurons were exposed to WT H C B, followed by immunostaining analysis. C B through H C B Detection of HA tag fusion. Synaptophysin was labeled as a marker of presynaptic terminals. WT H C B binds to synapses of WT neurons but not Syt I KD neurons. Expression of full-length m-Syt II by lentiviral transduction restored H C B, while expression of full-length m-Syt II(F54L) or full-length h-Syt II failed to rescue binding. Figure 4B )Syt I KD abolish H C B MY Binding to neurons. The binding was rescued by expressing full-length m-Syt II, m-Syt II (F54L) or h-Syt II.

[0098] FIG. 5A to FIG. 5C .BoNT / B MY Demonstrated enhanced efficacy in blocking neurotransmission in humanized neurons. Figure 5A ) Humanized neurons such as Figure 4A These neurons were exposed to full-length WT BoNT / B or BoNT / B MY The concentration gradient was maintained for 24 h. Cell lysates were harvested and analyzed by immunoblotting. β-tubulin was used as an internal loading control.MY Cleaved more VAMP2 than WT BoNT / B at the same concentration, indicating that BoNT / B MY More efficiently enters neurons than WT BoNT / B. FIG. 5B to FIG. 5C ) Humanized neurons were exposed to WT BoNT / B or BoNT / B MY The concentration gradient was maintained for 24 hours, and then the mIPSC activity was recorded by the whole-cell patch clamp method. Figure 5B ) shows a representative mIPSC recording of 30 pM toxin. Figure 5C ) Depicts mIPSC activity versus toxin concentration, normalized to neurons not exposed to any toxin. The number of neurons recorded for each data point is noted in (brackets). MY The same number of neurons were recorded. The half-maximal inhibitory concentration (IC 50 ), 89 pM for WT BoNT / B, and 10 pM for BoNT / B MY , was 7.8 pM, demonstrating that enhanced binding to the human receptor increases the efficacy of the toxin in neurons at a functional level.

[0099] FIG. 6A to FIG. 6D .Sequence variation of BoNT / B subtypes affects their ability to bind h-Syt II. Fig. 6A ) Sequence alignment of BoNT / B subtypes in the region surrounding residues E1191 / S1199 in BoNT / B, including BoNT / B1 (SEQ ID NO:9); BoNT / B2 (SEQ ID NO:10); BoNT / B3 (SEQ ID NO:11); BoNT / B4 (SEQ ID NO:12); BoNT / B5 (SEQ ID NO:13); BoNT / B6 (SEQ ID NO:14); BoNT / B7 (SEQ ID NO:15); BoNT / B8 (SEQ ID NO:16). ( Figure 6B )H containing Q1191 and Y1199 C B4 did not bind to h-Syt II in the pull-down assay. Figure 6C ) In the 19 key residues that form the binding pocket of Syt I / II, H C B4 and H C There are six different residues between B. C The corresponding residues in B are substituted for H C All six residues in B4 generate mutant H that can bind to h-Syt II CB4 (referred to as the “six mutations”: V1113K / S1117P / S1196A / I1197P / A1182T / N1185Y). C B1 and H C The sequence alignment between B4 is shown in Figure 7 middle.( Fig.6D )H c B and H c The binding of B4 to immobilized GST-tagged h-Syt I was examined in a pull-down assay. c The binding of B to h-Syt I requires the presence of gangliosides, while H c B4 was able to bind h-Syt I in the absence of gangliosides in a pull-down assay.

[0100] Figure 7 Alignment of the Hc domains of BoNT / B1 (SEQ ID NO: 17) and BoNT / B4 (SEQ ID NO: 18) amino acid sequences. Alignment of the Hc domain polypeptide sequences of BoNT / B1 and B4 highlights those amino acids that differ between these two strains of serotype B. B4 amino acids V1113, S1117, S1196, and I1197 are identified herein as residues that modulate the ability of the B4 Hc domain to bind to the human SytII molecule. Figure 7 What has already been shown in Table 5 is also confirmed by indicating the 19 key residues that form the Syt II binding pocket with asterisks.

[0101] Figure 8 Is the amino acid sequence of BoNT / B-Hc (strain 1; BoNT / B1 okra strain). BoNT / B, residues 857-1291 of strain 1, GenBank: AB232927.1 (SEQ ID NO: 19).

[0102] Fig. 9 is a nucleic acid sequence encoding BoNT / B-Hc (strain B1, okra strain) (residues 857-1291 of BoNT / B, strain 1, based on GenBank: AB232927.1), which has been optimized for expression in E. coli (SEQ ID NO: 20).

[0103] Fig.10 The amino acid sequence of Clostridium botulinum serotype A (1296 a.a.) is shown (SEQ ID NO: 21).

[0104] Fig.11 The amino acid sequence of Clostridium botulinum serotype B (1291 a.a.) is shown (SEQ ID NO: 22).

[0105] Fig.12 The amino acid sequence of Clostridium botulinum serotype C1 (1291 a.a.) is shown (SEQ ID NO: 23).

[0106] Fig.13 The amino acid sequence of Clostridium botulinum serotype D (1276 a.a.) is shown (SEQ ID NO: 24).

[0107] Fig.14 The amino acid sequence of Clostridium botulinum serotype E (1252 a.a.) is shown (SEQ ID NO: 25).

[0108] Fig.15 The amino acid sequence of Clostridium botulinum serotype F (1274 a.a.) is shown (SEQ ID NO: 26).

[0109] Fig.16 The amino acid sequence of Clostridium botulinum serotype G (1297 a.a.) is shown (SEQ ID NO: 27).

[0110] Fig.17 The amino acid sequence of Clostridium botulinum serotype B, strain Eklund 17B (BoNT / B4) Genbank Ref: EF051570.1 (SEQ ID NO: 28) is shown.

[0111] Fig.18 Thirteen different single substitution mutations were shown to mutate BoNT / B1 H C Effect of the domains on binding to mouse and human SytII.

[0112] Fig.19 Biolayer interferometry traces for binding to human Syt II are shown for all 12 combinations between the single mutation E1191M / C / V / Q and the compatible secondary mutations S1199W / Y and W1178Q and the triple mutation E1191M / S1199W / W1178Q. Binding affinity (K) of all 12 mutants to h-Syt II D ) were measured using biolayer interferometry (BLI) and the parameters are listed in Table 4.

[0113] Fig. 20 The first two BoNT / B1 H C BLI assay measurement results of the binding affinity of domain mutants (E1191M / S1199Y and E1191V / S1199Y) to h-Syt I. C B MY) or E1191V / S1199Y(H C B VY ) C B showed K of 2.9 μM and 5.82 μM, respectively. D .

[0114] Fig.21 H examined in the pull-down assay is shown c The binding of B mutant to m-Syt II and h-Syt II, e.g. Figure 2E As described in.

[0115] Fig. 22 The mutant H c Binding of B4 to m-Syt II and h-Syt II.

[0116] FIG. 23A to FIG. 23B Showing BoNT / B produced in E. coli MY Active and neutralized by BoNT / B antibodies. Fig.23A ) Full-length BoNT / B and BoNT / B MY The toxins were purified from E. coli. These toxins were activated by the endoproteinase Lys-C. SDS-PAGE analysis revealed that the WT BoNT / B toxin was about 93% pure, of which about 80% was in its double-chain form, and the BoNT / B MY The toxin was approximately 86% pure, with approximately 99% being in its double-stranded form. Fig. 23B ) Cultured rat cortical neurons were exposed to BoNT / B MY (1 nM in culture medium, 16 hours), BoNT / B MY Preincubation with rabbit polyclonal anti-BoNT / B antibody (1:200 dilution in culture medium) or trivalent horse antiserum against BoNT / A, B, and E (1:40 dilution in culture medium). Neuronal lysates were harvested 16 hours later. Cleavage of VAMP2 was analyzed by immunoblotting assay. BoNT / B MY It is active because it cleaves VAMP2 in neurons. Both rabbit polyclonal anti-BoNT / B antibody and trivalent horse antiserum readily neutralize BoNT / B MY Actin was used as an internal loading control. These results demonstrate that the E1191M / S1199Y mutations do not alter the immunogenicity of BoNT / B.

[0117] FIG. 24A to FIG. 24B Shown are comparisons between Syt I and Syt II and between human Syt I and mouse Syt I at the BoNT / B-binding interface. Fig.24A) Sequence alignment and structural comparison between mouse Syt I (SEQ ID NO:7) and Syt II (SEQ ID NO:8). Asterisks highlight conserved residues that interact with BoNT / B, which can be seen in the modeled structures of BoNT / B (top) bound to Syt I (bottom, light grey) and Syt II (bottom, dark grey). The binding of Syt I to BoNT / B was modeled based on the available BoNT / B-Syt II crystal structure (PDB: 4KBB). Fig. 24B ) Sequence alignment of human Syt I (SEQ ID NO: 4) versus mouse / rat Syt I (SEQ ID NO: 29) within the BoNT / B binding domain. The only difference, E45 in human Syt I and Q in mouse / rat Syt I, is in bold. The model shows that the side chain of residue 45 (indicated by the arrow) points outward from the binding site and does not interact with BoNT / B. DETAILED DESCRIPTION

[0118] Aspects of the invention relate to the production of polypeptides and nucleic acids encoding such polypeptides comprising a modified receptor binding domain of Clostridium botulinum (e.g., serotype B (BH c )), and more particularly, Clostridium botulinum neurotoxin (BoNT) polypeptides having improved binding to their human receptors by incorporating modified receptor binding domains, and nucleic acids encoding such polypeptides. Based on these findings, a new generation of therapeutic BoNTs can be generated that include the modified receptor binding domains identified herein that have improved efficacy and specificity for targeting human neurons relative to currently used WT BoNTs.

[0119] It has been previously determined that BoNT / BH c The binding of polypeptides to human SytII receptor molecules can be significantly enhanced by mutations at residues corresponding to BoNT / B1 Hc residues E1191 and S1199. See PCT / US2016 / 024211, which is incorporated herein by reference in its entirety. In particular, relative to WT, at BoNT / B1 Hc residues E1191 and S1199, C Mutation of the domain at residue E1191 to Q, M, C, V, L, or Y enhanced hSytII binding. Mutation E1191M provided the most dramatic increase in binding, but E1191Q also significantly promoted binding. See, e.g., Figure 2D and Figure 2E .BoNT / B1H cThe combination of the E1191M mutation and the mutation of residue S1199 to Y further increased the binding of the BoNT / B1 molecule to the hSytII receptor. The double mutant showed strong binding to human neurons expressing the hSytII receptor molecule and exhibited enhanced efficacy in blocking neurotransmission. See Figures 4 and 5.

[0120] Note that BoNT serotype B4 (BoNT / B4) WT H c The domain includes glutamine and tyrosine at positions corresponding to E1191 and S1199 of the serotype B1 molecule, thus corresponding to the E1191Q, S1199Y double mutant of the serotype B1 molecule that would be expected to promote strong binding. Interestingly, despite having glutamine and tyrosine at positions in the serotype B1 protein that promote binding of the serotype B1 neurotoxin, the wild-type B4 molecule cannot effectively bind to the hSytII receptor molecule. Therefore, the amino acids of the B4 molecule other than Q1191 and Y1199 must be important for binding to the hSytII molecule.

[0121] As described in the Examples herein, the H of the B4 molecule was found to differ between serotype B1 and serotype B4. c Mutations in four of the 19 amino acids of the domain conferred strong hSytII binding on serotype B4 molecules. Specifically, the B4 amino acids V1113, S1117, S1196, and I1197 were found to be important for B4 binding to hSytII. Mutations of these residues to the corresponding residues of serotype B1 molecules conferred strong binding on serotype B4 molecules - i.e., the B4 mutations V1113K, S1117P, S1196A, and I1197P conferred strong hSytII binding. Thus, a B4 H containing these mutations in addition to the naturally occurring Q1191 and Y1199 would be expected to be a promising candidate for the future study. c The domain mutants can be used in modified botulinum neurotoxin formulations for therapeutic or cosmetic applications. It is also understood that further modifications of the B4 H comprising V1113K, S1117P, S1196A and I1197P mutations are also contemplated. c Mutants can include, for example, mutations found to promote binding of serotype B1 molecules to hSytII alone or in combination, promoting binding of modified serotype B4 Hc domains. Thus, it is also expected that in a B4 mutant comprising V1113K, S1117P, S1196A and I1197P mutations, for example, further mutations of Q1191 to, for example, C, V, L, Y, M and / or further mutations of Y1199 to, for example, W, E or H positively affect binding. As just one example, it is possible that a B4 Hc domain comprising V1113K, S1117P, S1196A and I1197P mutations plus mutation Q1191M cDomain mutant BoNT neurotoxins will also bind strongly and affect neurotransmission activity.

[0122] Included below is a further description of the studies that led to the discoveries described herein and considerations for one skilled in the art to apply them to the methods and compositions disclosed herein.

[0123] Implementation

[0124] The observation that BoNT / B has lower specificity and potency in humans due to its inability to bind to human Syt II may explain why relatively higher doses are required compared to BoNT / A. Higher BoNT / B doses correspond to an increased chance of triggering an antibody response and developing severe side effects. Therefore, improving the binding of BoNT / B to the human receptor Syt II to increase its efficacy and specificity in targeting human neurons would allow for reduced toxin doses to be used in therapeutic applications.

[0125] Aspects of the present invention arise from modifying BoNT serotype B4-H C The present invention relates to the discovery of protein sequence modifications of a fragment containing a receptor binding domain that binds to the human Syt II receptor. Specific modifications that enhance binding have been identified, resulting in a domain that binds to human Syt II with high affinity. Modified BoNT / B4-H C These binding properties are retained in the context of the full-length BoNT protein. Modified receptor binding domains with enhanced binding are incorporated into molecules containing other BoNT domains, thereby generating full-length BoNT molecules with similar enhanced receptor binding. Thus, novel BoNTs with high affinity binding to human Syt II are generated. BoNTs with significantly enhanced binding can be used in similar treatments, albeit at lower doses than currently available BoNT molecules, thus providing a safer treatment approach.

[0126] BoNT polypeptides, including full-length BoNT polypeptides and BoNT polypeptide fragments or domains (e.g., modified BoNT / H C ) and nucleic acid molecules encoding them are specifically included in the present invention. These polypeptides and nucleic acid molecules can be produced by recombinant DNA procedures known in the art. Such polypeptides are generally referred to as "recombinant polypeptides" or "recombinant nucleic acids."

[0127] Botulinum neurotoxin protein

[0128] One aspect of the invention relates to a botulinum neurotoxin (BoNT) protein comprising a modified receptor binding domain as described herein (e.g., of Clostridium botulinum serotype B4). The BoNT protein also comprises a protease domain, a translocation domain, and a protease cleavage site. Typically, these are arranged in a linear amino-carboxyl single polypeptide order of the protease domain, the protease cleavage site, the translocation domain, and the modified receptor binding domain. However, different arrangements of the various domains are expected to function adequately. In one embodiment, the modified receptor binding domain comprises one or more substitution mutations that result in significantly enhanced binding to the human Syt I receptor and / or the human Syt II receptor.

[0129] BoNT proteins may also include domains that can be used to purify the molecule, such as a hexahistidine tag (His6) (SEQ ID NO: 1) or an epitope tag, such as a hemagglutinin (HA) tag (YPYDVPDYA) (SEQ ID NO: 2). Various such domains are known and used in the art for this purpose.

[0130] BoNT has Figure 1B The overall structure is shown in Figure 1. BoNT consists of three domains, each with a specific and independent function: the protease domain (also called the light chain), the translocation domain (H N ) and receptor binding domain (H C It has been shown that the domains of various strains of Clostridium botulinum neurotoxins are largely interchangeable (as demonstrated by naturally occurring chimeric toxins such as BoNT / CD, which consists of the light chain and H chain of BoNT / C). N and H of BoNT / D(52) C The protein may be in a single-chain form or a double-chain form. The double-chain form is produced by the processing of a naturally occurring protease at a protease cleavage site located between the protease domain and the translocation domain. After the protease processing, the protein is maintained in a double-chain form by the presence of a disulfide bond.

[0131] Strains of Clostridium botulinum produce seven antigenically distinct types of botulinum toxins, which have been identified by studying outbreaks of botulism in humans (BoNT / A, BoNT / B, BoNT / E, and BoNT / F), animals (BoNT / C1 and BoNT / D), or isolated from soil (BoNT / G). Although all seven BoNT serotypes have similar structural and pharmacological properties, each also exhibits heterogeneous bacteriological characteristics. The genetic diversity of Clostridium botulinum strains is described in detail in Hill et al. (Journal of Bacteriology, Vol. 189, No. 3, pp. 818-832 (2007)) (53), the contents of which are incorporated herein by reference. In one embodiment, a BoNT of the invention has domains that are all of the same serotype (A, B, C, D, E, F, or G). In one embodiment, one or more of those domains of the same serotype differ in their strains and / or subtypes.

[0132] Toxins from various serotypes / strains of Clostridium botulinum share the same functional domain organization and overall structural architecture. Each of the botulinum toxins is translated as a single-chain polypeptide of approximately 150 kDa, which is subsequently cleaved by proteolytic cleavage within the disulfide loop by a naturally occurring protease such as an endogenous Clostridium botulinum toxin protease or a naturally occurring protease produced in the environment. This post-translational processing produces a two-chain molecule comprising an approximately 50 kDa light chain (LC) and an approximately 100 kDa heavy chain (HC), which are bound together by a single disulfide bond and non-covalent interactions. Each mature two-chain molecule contains three functionally distinct domains: 1) a proteolytic domain located in the LC, which includes a metalloprotease region containing zinc-dependent endopeptidase activity that specifically targets a core component of the neurotransmitter release apparatus; 2) a metalloprotease region contained in the HC (HC); N ) a translocation domain within the amino-terminal half of the toxin, which facilitates the release of the LC from intracellular vesicles into the cytoplasm of the target cell; and 3) a binding domain found within the carboxyl-terminal half of the HC, which determines the binding activity and binding specificity of the toxin to the receptor complex located on the surface of the target cell. The locations of the specific domains within the toxins of various serotypes / strains are provided in Table 1:

[0133] Table 1

[0134] Clostridium botulinum toxin domains from various serotypes / strains

[0135]

[0136] The complete amino acid sequence of the toxin is provided in Figures 10 to 16 The complete amino acid sequence of the BoNT / B4 neurotoxin polypeptide (also known as the Eklund strain) is provided in Fig.17middle.

[0137] The binding, translocation and protease activity of these three functional domains are all required for toxicity. The overall cellular intoxication mechanism by which Clostridium botulinum toxin enters neurons and inhibits neurotransmitter release is similar regardless of serotype or subtype. Without wishing to be bound by theory, the intoxication mechanism involves at least four steps: 1) receptor binding, 2) complex internalization, 3) light chain translocation, and 4) protease target modification. When the H of Clostridium botulinum toxin CThe process is started when the domain is combined with the toxin-specific receptor on the plasma membrane surface of the target cell. It is believed that the binding specificity of the receptor complex is partially realized by the specific combination of gangliosides and protein receptors. Once combined, the toxin / receptor complex is internalized by endocytosis and the internalized vesicle is sorted to a specific intracellular pathway. The translocation step is triggered by the acidification of the vesicle compartment. After translocation, the light chain endopeptidase of the toxin is released from the intracellular vesicle into the cytosol, wherein it specifically targets one of the three proteins, referred to as the core components of the neurotransmitter release device (vesicle-associated membrane protein (VAMP) / synaptobrevin, 25kDa synaptosome-associated protein (SNAP-25) and syntaxin). These core components are necessary for the synaptic vesicle docking and fusion of nerve endings, and constitute members of the soluble N-ethylmaleimide-sensitive factor-attachment protein-receptor (SNARE) family. BoNT / A and BoNT / E cleave SNAP-25 in the carboxyl-terminal region, releasing a nine- or twenty-six-amino acid segment, respectively, and BoNT / C1 also cleaves SNAP-25 near the carboxyl terminus. The botulinum serotypes BoNT / B, BoNT / D, BoNT / F, and BoNT / G, as well as tetanus toxin, act on the conserved central portion of VAMP and release the amino-terminal portion of VAMP into the cytosol. BoNT / C1 cleaves syntaxin at a single site near the cytoplasmic plasma membrane surface. Selective proteolysis of synaptic SNAREs explains the blockade of neurotransmitter release caused by Clostridium botulinum toxin in vivo. SNARE protein targets of botulinum toxin are common in exocytosis in various non-neuronal types; in these cells, as in neurons, light chain peptidase activity inhibits exocytosis, see, e.g., Yann Humeau et al., How Botulinum and Tetanus Neurotoxins Block Neurotransmitter Release, 82(5) Biochimie. 427-446 (2000); Kathryn Turton et al., Botulinum and Tetanus Neurotoxins: Structure, Function and Therapeutic Utility, 27(11) Trends Biochem. Sci. 552-558. (2002); Giovanna Lalli et al., The Journey of Tetanus and Botulinum Neurotoxins in Neurons, 11(9) Trends Microbiol. 431-437, (2003).

[0138] Domains and chimeric neurotoxins

[0139] The botulinum neurotoxins described herein comprise a modified receptor binding domain (H C ). The modified receptor binding domain exhibits significantly enhanced binding to a human receptor that is normally bound and utilized by one or more strains of Clostridium botulinum toxin (e.g., SytII, SytI). The modified receptor binding domain would be a modified serotype B4 receptor binding domain (as described herein). This can be the same or a different serotype, strain / subtype as one or more other domains within the BoNT. Examples of specifically modified receptor binding domains are provided herein. The invention also encompasses the isolated modified receptor binding domain polypeptides described herein, as well as isolated nucleic acid molecules encoded thereby.

[0140] The botulinum neurotoxins of the present invention also comprise a protease domain, also referred to in the art as a light chain variant. The light chain variant can be a naturally occurring light chain variant, such as a botulinum toxin light chain isotype and a botulinum toxin light chain subtype; or a non-naturally occurring botulinum toxin light chain variant, such as a conservatively substituted botulinum toxin light chain variant. The protease domain can be any serotype (A, B, C, D, E, F, or G), strain, or subtype (as described herein). This can be the same or different serotype, strain / subtype as one or more other domains within the BoNT. In one embodiment, the protease domain is serotype B. In one embodiment, the protease domain is serotype A.

[0141] The botulinum neurotoxin of the present invention further comprises a toxin translocation domain (H N The toxin translocation domain can be any serotype (A, B, C, D, E, F, or G), strain, or subtype (as described herein). This can be the same or a different serotype, strain / subtype as one or more other domains within the BoNT. In one embodiment, H N is serotype B. In one embodiment, H N It is serotype A.

[0142] The various domains described herein (e.g., H N , H C or protease domains) include but are not limited to naturally occurring variants, such as isoforms and subtypes; non-naturally occurring variants, such as conservative substitution mutations. Non-naturally occurring variants refer to domains having at least one amino acid change from the corresponding region of the reference sequence (e.g., from Table 1 or Figure 8 or 10 to 17 and may be described as a percent identity to the corresponding region of the reference sequence.

[0143] Although it is to be understood that the botulinum neurotoxin variants described herein will encompass modified serotype B4 H C Although the present invention relates to a BoNT-1, a naturally occurring C. botulinum toxin domain may be present in the form of a light chain, ... N or H C ) can function in substantially the same manner as the reference botulinum toxin domain on which the naturally occurring Clostridium botulinum domain variant is based, and can be substituted for the reference botulinum toxin domain.

[0144] A non-limiting example of a naturally occurring botulinum toxin domain variant is a botulinum toxin domain isoform, such as a BoNT / A domain isoform, a BoNT / B domain isoform, a BoNT / C1 domain isoform, a BoNT / D domain isoform, a BoNT / E domain isoform, a BoNT / F domain isoform, and a BoNT / G domain isoform. A botulinum toxin domain isoform can function in substantially the same manner as the reference botulinum toxin domain on which the botulinum toxin domain isoform is based, and can be substituted for the reference botulinum toxin domain.

[0145] Another non-limiting example of a naturally occurring Clostridium botulinum toxin domain variant is a Clostridium botulinum toxin domain subtype, such as domains from subtypes BoNT / A1, BoNT / A2, BoNT / A3, BoNT / A4, BoNT / A5; domains from subtypes BoNT / B1, BoNT / B2, BoNT / B3, BoNT / B4, BoNT / B5, BoNT / B6, BoNT / B7; domains from subtypes BoNT / C1-1, BoNT / C1-2, BoNT / DC; domains from subtypes BoNT / E1, BoNT / E2, BoNT / E3, BoNT / E4, BoNT / E5, BoNT / E6, BoNT / E7, BoNT / E8; and domains from subtypes BoNT / F1, BoNT / F2, BoNT / F3, BoNT / F4, BoNT / F5, BoNT / F6, BoNT / F7. A botulinum toxin domain subtype can function in substantially the same manner as, and can be substituted for, a reference botulinum toxin domain on which the botulinum toxin domain subtype is based.

[0146] As used herein, the term "non-naturally occurring variant" (e.g., a Clostridium botulinum toxin light chain variant, H C and H N ) refers to a Clostridium botulinum domain produced by artificial manipulation, including, but not limited to, a domain produced by genetic engineering using random mutagenesis or rational design and a Clostridium botulinum domain produced by chemical synthesis. Non-limiting examples of non-naturally occurring Clostridium botulinum domain variants include, for example, conservative Clostridium botulinum domain variants. As used herein, the term "conservative Clostridium botulinum domain variant" means that the Clostridium botulinum domain has at least one amino acid substituted with another amino acid or amino acid analog that has at least one property similar to the original amino acid from a reference Clostridium botulinum domain sequence (e.g., Tables 1 and 2). Figure 8 and 10 17). The variant may have one, two, three, four, five or more conservative amino acid substitutions compared to the reference domain sequence. Examples of properties include, but are not limited to, similar size, topology, charge, hydrophobicity, hydrophilicity, lipophilicity, covalent bonding ability, hydrogen bonding ability, physicochemical properties, etc. or any combination thereof. The conservative botulinum toxin domain variant may function in substantially the same manner as the reference botulinum toxin domain on which the conservative botulinum toxin domain variant is based, and may be substituted for the reference botulinum toxin domain in any aspect of the invention.

[0147] The non-naturally occurring botulinum toxin domain variants can substitute one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more) amino acids from the reference botulinum toxin domain on which the naturally occurring botulinum toxin domain is based. The non-naturally occurring botulinum toxin domain variants can also have 95% or greater (e.g., 96%, 97%, 98%, or 99%) amino acid identity with the reference botulinum toxin domain on which the naturally occurring botulinum toxin domain variants are based.

[0148] Various non-naturally occurring Clostridium botulinum neurotoxins or specific domains thereof are described in International Patent Publications WO95 / 32738, WO96 / 33273, WO98 / 07864 and WO99 / 17806, each of which is incorporated herein by reference. The Clostridium botulinum neurotoxins or specific domains thereof described herein will generally contain naturally occurring amino acid residues, but in some cases, non-naturally occurring amino acid residues may also be present. Thus, so-called "peptide mimetics" and "peptide analogs" may be used in the context of the present invention, which may include non-amino acid chemical structures that mimic the structure of a particular amino acid or peptide. Such mimetics or analogs are generally characterized by exhibiting similar physical characteristics such as size, charge or hydrophobicity, as well as the appropriate spatial orientation found in their natural peptide counterparts. Specific examples of peptidomimetic compounds are compounds in which the amide bond between one or more amino acids is replaced by, for example, a carbon-carbon bond or other non-amide bond, as is well known in the art (see, e.g., Sawyer, in Sawyer, in Peptide Based Drug Design, pp. 378-422, ACS, Washington DC 1995).

[0149] The modified botulinum neurotoxin described herein comprises a modified receptor binding domain (BoNT / BH) of Clostridium botulinum serotype B4. C ), the modified receptor binding domain comprises H relative to the wild type or Eklund strain C The four substitution mutations in the structural domain are: V1113K, S1117P, S1196A and I1197P. These substitution variants cause a significant increase in binding to the human Syt II receptor. BoNT / B4-H used as a reference template in the present invention C The amino acid sequence of the Eklund strain is shown in Fig.17 (See also NCBI Reference Sequence: YP_001893661.1, Genbank Ref: EF051570.1). It is specifically contemplated herein to generate B4-H incorporating the modified c Such as intact BoNT or containing modified BH C A longer molecule of a polypeptide.

[0150] Toxin spread and production of neutralizing antibodies are issues associated with, but not limited to, BoNT / B, which are also observed with BoNT / A. Improvements in the binding affinity of BoNT / A to its receptor SV2 would alleviate these issues. Because BoNT / B binds to Syt I / II with a much higher affinity than BoNT / A binds to SV2, the use of a modified BoNT / B receptor binding domain (BoNT / B H) with the ability to bind to human Syt II is possible.C )Replace BoNT / AH C This can generate modified chimeric BoNT / A that may have greater efficacy and specificity for human neurons than WT BoNT / A (49-51).

[0151] It is further contemplated that the modified B4 H C Replace H for all other BoNT serotypes / strains C Thus, another aspect of the invention is a BoNT polypeptide comprising a modified serotype B4 receptor binding domain (H4) linked to one or more domains of a different serotype (A, C, D, E, F, or G), strain, or subtype to produce a chimeric neurotoxin. C ). H of each BoNT C The regions are well defined and their respective H C Regions can be exchanged among BoNT molecules (e.g., by genetic engineering). Such manipulations are routinely performed by skilled practitioners by replacing the light chain (LC) and translocation domain (H) of other BoNTs. N ) or H N -LC Coded BoNT / BH C Standard PCR fusion of DNA is performed, which is well established in the art. Alternatively, BoNT / BH C The C-terminal portion (designated as H CC ) is replaced by the C-terminal portion, which is the region of each BoNT that contains the binding sites for the protein receptor and ganglioside. The resulting chimeric toxin will have the ability to target human neurons by binding to human Syt I / II. As a non-limiting example, a modified BoNT / B4-H C (or BoNT / B4-H CC ) can be used to replace H C (or H CC ). The resulting polypeptides are encompassed by the present invention. These chimeric toxins can have greater efficacy and specificity in targeting human neurons than WT BoNT / A. Such chimeric BoNT / A toxins can be used for therapeutic applications in humans and provide significant improvements over WT BoNT / A.

[0152] One aspect relates to an isolated, purified modified receptor binding domain polypeptide as described herein. In one embodiment, the modified receptor binding domain is a modified BoNT / B4-H C In one embodiment, B4-H c Produced from different strains and / or subtypes of BoNT by substituting the amino acids corresponding to the specified positions in B4 described herein. CIt is subtype B1, B2, B3, B5, B6 or B7. In one embodiment, the modified B-Hc does not belong to strain 3, 7 or 8.

[0153] The present invention encompasses modified H genes having amino acid substitutions as described herein for therapeutic use in humans. C (For example, B4-H C ). In one embodiment, the full-length BoNT contains a modified H having amino acid substitutions V1113K, S1117P, S1196A, and I1197P. C (For example, B4-H C ), and further comprising amino acid substitutions at one or more combinations of amino acid residues corresponding to positions Q1191, Y1199, Y1183, and W1178 of B4 (e.g., selected from those listed in Table 2). In one embodiment, the BoNT contains a modified B4 having amino acid substitutions V1113K, S1117P, S1196A, and I1197P and a single additional amino acid substitution as described herein. C In one embodiment, the BoNT contains a modified B4 H having amino acid substitutions V1113K, S1117P, S1196A, and I1197P and two additional amino acid substitutions as described herein. C In one embodiment, the BoNT contains a modified B4 H having amino acid substitutions V1113K, S1117P, S1196A, and I1197P and three additional amino acid substitutions as described herein. C In one embodiment, the modified B-Hc is not from strain 3, 7 or 8.

[0154] The resulting BoNT toxins can have significantly enhanced binding to human Syt II and, therefore, will achieve higher efficacy and specificity for targeting human neurons compared to WT BoNT. The resulting mutants can also maintain similar binding to human SytI, have significantly enhanced binding to human SytI, or have significantly reduced binding to human SytI.

[0155] Polypeptides and chimeric polypeptides

[0156] Another aspect of the invention relates to a polypeptide comprising a modified BoNT / B4 receptor binding domain. In one embodiment, the modified Hc has significantly enhanced binding to human Syt II and / or Syt I in the context of the polypeptide compared to a similar polypeptide having a WT amino acid at the specific position modified in Hc (serotype B4 wild-type counterpart). In one embodiment, the modified serotype B4 Hc has significantly enhanced binding to human Syt II and / or Syt I in the context of the polypeptide. cProduced from different serotypes, strains, and / or subtypes of BoNT by substituting amino acids corresponding to designated positions in B4 described herein. In one embodiment, the modified B-Hc is not of strain 3, 7, or 8. In one embodiment, the resulting polypeptide has the ability to target human neurons by binding to human SytI / II.

[0157] The polypeptide comprising a modified receptor binding domain can be a fusion protein of a receptor binding domain and another functional polypeptide (e.g., a functional polypeptide for a 2-hybrid system (bait or prey, such as T18 or T25, or glutathione S transferase) (GST)). This can also be referred to as a chimeric polypeptide molecule. Alternatively, it can be a fusion of a modified receptor binding domain and a polypeptide tag for identification purposes and / or purification purposes (e.g., a hexahistidine tag (His6) (SEQ ID NO: 1) or an epitope tag such as a hemagglutinin (HA) tag (YPYDVPDYA) (SEQ ID NO: 2) or GST), many of which are known and commonly used in the art. The fusion can have a stretch of amino acids between the respective functional domains that facilitates connection, serves as a cleavage site, or maintains independent conformation and / or function. In one embodiment, the connection retains the function of the modified receptor binding domain. In one embodiment, the connection masks the function of the modified receptor binding domain. Another aspect of the invention relates to a nucleic acid molecule encoding any of these polypeptides.

[0158] Modified BoNT / B4 H C It can be covalently linked (e.g., as a fusion protein) or non-covalently linked to other agents (e.g., proteins, small molecules, short polypeptides, nucleic acids). Thus, another aspect of the invention relates to a chimeric molecule comprising a first portion, which is a modified receptor binding domain of Clostridium botulinum (e.g., serotype B4) as described herein, linked to a second portion. The second portion of the molecule can be a biologically active (or "biologically active") molecule, such as a therapeutic agent (e.g., a polypeptide or non-polypeptide drug, such as a small molecule or nucleic acid). The connection between the first portion and the second portion of the molecule can be covalent (e.g., in the form of a fusion protein) or non-covalent. Methods of such connection are known in the art and can be readily applied by a skilled practitioner. One such use of the chimeric molecules of the invention is as a delivery vehicle targeted to human neurons. For example, a modified BoNT / H C It can be linked to other therapeutic agents to serve as a targeting vehicle to deliver the therapeutic agent to human neurons by binding to human Syt I and / or Syt II.

[0159] Modification of the receptor binding domain (Hc)

[0160] As discussed herein, the present invention relates to a modified Hc and polypeptides (e.g., BoNTs or fusion or chimeric polypeptides) comprising the modified Hc. C Modification of the amino acid sequence can be performed by a variety of methods known to the skilled practitioner. Examples include, but are not limited to, targeted mutagenesis (site-directed mutagenesis) or random mutagenesis of each amino acid residue in the region known to bind Syt I / II. These Syt binding regions have been well defined by previous studies on mouse or rat Syt receptors, but have not been clearly identified for BoNT / BH. c Different subtypes of BoNT / B or other serotypes that bind to Syt I / II (DC or G) can be used as templates to generate the B4-H C The corresponding mutations described above are used to produce the same or similar mutations. By comparing the sequences with the B4 subtype, the corresponding positions of the selected residues to be mutated can be easily identified. The present invention covers the resulting polypeptide products, as well as polypeptides comprising the products and nucleic acid molecules encoding the polypeptides and products.

[0161] Amino acid sequence modification C To produce a modified receptor binding domain, a single residue can be mutated to a different amino acid (single-site substitution), multiple residues can be mutated simultaneously (multi-site substitution), one or more residues can be deleted (deletion), and one or more residues can be inserted (insertion), as well as combinations thereof. Methods for mutating proteins are well known in the art (e.g., encoding a BoNT / H C Targeted single-site and multi-site replacement on DNA sequences).

[0162] In one embodiment, based on previous literature on BoNT / B receptor binding domain (29)_ENREF_29 and the reported BoNT / B-Syt II structure (PDB ID: 2NM1), H is modified. CIn one embodiment, one or more residues in the rodent Syt II that contact rodent Syt II or surrounding regions. These include, but are not limited to, positions corresponding to positions Y1181, I1197, S1196, F1204, F1194, S1117, W1178, Y1183, V1118, S1116, V1113, K1192, Y1199, S1201, Q1191, E1245, Y1256, D1115, and E1203 of BoNT / B4. In one embodiment, one or more of these residues are systematically replaced with other amino acids in addition to the V1113K, S1117P, S1196A, and I1197P substitutions. Combinations of modifications are also contemplated, including, but not limited to, mutations of two or more of the positions to any class of amino acids, such as those described herein.

[0163] By combining mutations in these identified key residues, multiple substitutions (e.g., 2 or 3) can be generated. Such multiple substitution mutants may even have further enhanced binding to h-Syt II, and may further maintain or have enhanced binding to human Syt I. In one embodiment, the modified B4 H C The invention has four amino acid substitutions relative to the B4 wild-type sequence: V1113K, S1117P, S1196A and I1197P, and further comprises one or more substitution mutations corresponding to substitution mutations in serotype B, strain 4, wherein the substitution mutations are selected from the group consisting of Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

[0164] Modified B4 H having substitution mutations V1113K, S1117P, S1196A and I1197P as well as the single amino acid substitution mutations shown in Table 2 below are also contemplated. C , and their incorporation into the various polypeptides described herein.

[0165] Table 2

[0166] Q1191M Y1183C Y1199H S1117Y Q1191E Y1183P Y1199S V1118M Q1191C Y1183M Y1199W S1196Y Q1191V W1178Q Y1199F Y1181M Q1191L W1178Y Y1199L Q1191Y W1178A S1201V Q1191T W1178S S1117P Q1191I Y1199E S1117M

[0167] In one embodiment, the modified B4 H is compared to a similar polypeptide having a WT amino acid at a particular position (also referred to herein as a wild-type counterpart). C Possessing enhanced binding to hSyt II. In one embodiment, the mutants generated also maintain or have significantly enhanced binding to human Syt I compared to the wild-type counterpart.

[0168] In one embodiment, H C With two substitution mutations corresponding to those shown in Table 3 for serotype B, strain 4:

[0169] Table 3

[0170] Q1191M and Y1199W Q1191V and Y1199H Q1191M and Y1199E Q1191L and Y1199W Q1191M and Y1199H Q1191L and Y1199E Q1191C and Y1199W Q1191L and Y1199H Q1191C and Y1199E Q1191Y and Y1199W Q1191C and Y1199H Q1191Y and Y1199E Q1191V and Y1199W Q1191Y and Y1199H Q1191V and Y199E

[0171] Nucleic acid molecules

[0172] Another aspect of the invention relates to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide described herein (e.g., a modified receptor binding domain, or a polypeptide comprising a modified receptor binding domain, or a botulinum neurotoxin comprising a modified receptor binding domain described herein). In one embodiment, the nucleic acid molecule comprises Fig. 9 The nucleic acid sequence shown in . Such nucleic acid molecules can be produced by a skilled practitioner, for example, by recombinant DNA technology. The desired amino acid substitution mutation is performed, for example, by modifying the encoding DNA. For example, the nucleic acid sequence encoding the polypeptide described herein is generated by mutating the nucleic acid codon encoding the specified amino acid to the desired amino acid using the genetic code as shown below:

[0173] TTT Phe TCT Ser TAT Tyr TGT Cys TTC Phe TCC Ser TAC Tyr TGC Cys TTA Leu TCA Ser TGA TTG Leu TCG Ser TGG Trp CTT Leu CCT Pro CAT His CGT Arg CTC Leu CCC Pro CAC His CGC Arg CTA Leu CCA Pro CAA Gln CGA Arg CTG Leu CCG Pro CAG Gln CGG Arg ATT Ile ACT Thr AAT Asn AGT Ser ATC Ile ACC Thr AAC Asn AGC Ser ATA Ile ACA Thr AAA Lys AGA Arg ATG Met* ACG Thr AAG Lys AGG Arg GTT Val GCT Ala GAT Asp GGT Gly GTC Val GCC Ala GAC Asp GGC Gly GTA Val GCA Ala GAA Glu GGA Gly GTG Val GCG Ala GAG Glu GGG Gly

[0174] In one embodiment, the nucleic acid sequence is optimized for expression in E. coli (e.g., based on the nucleic acid sequence of GenBank AB232927.1, the relevant portion of which is shown in Fig. 9 middle).

[0175] Another aspect of the present invention relates to a nucleic acid vector comprising a nucleic acid molecule described herein. In one embodiment, the vector is an expression vector. Such expression vectors are referred to herein as expression constructs and comprise a nucleic acid molecule disclosed herein operably linked to an expression vector that can be used to express the nucleic acid molecule in a cell or a cell-free extract. A variety of expression vectors can be employed to express nucleic acid molecules encoding the botulinum neurotoxin of the present invention, including, but not limited to, viral expression vectors; prokaryotic expression vectors; eukaryotic expression vectors, such as yeast expression vectors, insect expression vectors, and mammalian expression vectors; and cell-free extract expression vectors. It should also be understood that expression vectors that can be used to practice aspects of these methods may include those that express the botulinum neurotoxin under the control of a constitutive, tissue-specific, cell-specific, or inducible promoter element, an enhancer element, or both. Non-limiting examples of expression vectors and well-established reagents and conditions for making and using expression constructs from such expression vectors are readily available from commercial suppliers including, but not limited to, BD Biosciences-Clontech, Palo Alto, Calif.; BD Biosciences Pharmingen, San Diego, Calif.; Invitrogen, Inc, Carlsbad, Calif.; EMD Biosciences-Novagen, Madison, Wis.; QIAGEN, Inc., Valencia, Calif.; and Stratagene, La Jolla, Calif. The selection, preparation, and use of appropriate expression vectors are routine procedures well within the skill of those skilled in the art and learned from the teachings herein.

[0176] cell

[0177] Another aspect of the present invention relates to a cell comprising a nucleic acid molecule or an expression construct as described herein. The cell can be used for the propagation of nucleic acids or for the expression of nucleic acids or both. Such cells include, but are not limited to, prokaryotic cells, including, but not limited to, strains of aerobic, microaerophilic, acidophilic, facultative, anaerobic, Gram-negative, and Gram-positive bacterial cells, such as those derived from, for example, Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacteroides fragilis, Clostridium perfringens, Clostridium difficile, Bacillus crescentus, Lactococcus lactis, Methylobacterium extorquens, Neisseria meningitidis, Neisseria meningitidis, Pseudomonas fluorescens, and Salmonella typhimurium; and eukaryotic cells, including, but not limited to, yeast strains, such as those derived from Pichia pastoris, Pichia methanolica, Pichia pastoris, Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Yarrowia lipolytica; insect cells and cell lines derived from insects, such as those derived from Spodoptera frugiperda, Trichoplusia ni, Drosophila melanogaster, and Manduca hornworm. sexta;And mammalian cells and cell lines derived from mammalian cells, such as those derived from mice, rats, hamsters, pigs, cattle, horses, primates and humans. Cell lines can be obtained from the American Type Culture Collection, the European Collection of Cell Cultures, and the German Collection of Microorganisms and Cell Cultures.Non-limiting examples of specific protocols for selecting, preparing, and using appropriate cell lines are described in, for example, INSECT CELL CULTURE ENGINEERING (Mattheus FA Goosen et al., eds., Marcel Dekker, 1993); INSECT CELL CULTURES: FUNDAMENTAL AND APPLIED ASPECTS (JM Vlak et al., eds., Kluwer Academic Publishers, 1996); Maureen A. Harrison and Ian F. Rae, GENERAL TECHNIQUES OF CELL CULTURE (Cambridge University Press, 1997); CELL AND TISSUE CULTURE: LABORATORY PROCEDURES (Alan Doyle et al., eds., John Wiley and Sons, 1998); R. Ian Freshney, CULTURE OF ANIMAL CELLS: A MANUAL OF BASIC TECHNIQUE (Wiley-Liss, Fourth Supplement, 2000); ANIMALCELL CULTURE: A PRACTICAL APPROACH (John RWMasters, ed., Oxford University Press, 3rd Supplement, 2000); MOLECULAR CLONING A LABORATORY MANUAL, supra, (2001); BASIC CELLCULTURE: A PRACTICAL APPROACH (John M. Davis, Oxford Press, 2nd Supplement, 2002); and CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, supra, (2004). These schemes are within the skill of those skilled in the art and conventional procedures obtained from the teachings herein.

[0178] The cell can be used to express the nucleic acid, thereby producing the encoded polypeptide. Therefore, another aspect of the present invention is a method for producing a botulinum neurotoxin protein, an isolated H C or a H comprising the modifications described herein CThe polypeptide is produced by culturing a host cell having therein a nucleic acid encoding the polypeptide in the context of an expression construct. The culturing is performed under conditions suitable for production of the BoNT polypeptide. The expressed polypeptide can be recovered from the culture, purified and formulated by methods known in the art. The expressed polypeptide can also be activated as desired by methods known in the art. One method of activating the expressed polypeptide is by protease cleavage (or nicking) into an active two-chain form. Such methods can be adapted from methods known in the art, such as those disclosed by Peter F. Bonventre and Lloyd L. Klempe (J. Bacteriol 1960, 79(1):23 and Michael L. Dekleva and Bibhuti R. DasGupta (Biochemical and Biophysical Research Communicaitons 1989, 162:767-772).

[0179] Pharmaceutical composition

[0180] Another aspect of the present invention relates to a pharmaceutical composition comprising a Clostridium botulinum neurotoxin or chimeric molecule described herein. In one embodiment, the polypeptide described herein is an active ingredient in a composition comprising a pharmaceutically acceptable carrier (referred to herein as a pharmaceutical composition). "Pharmaceutically acceptable carrier" means any pharmaceutically acceptable means for mixing and / or delivering a targeted delivery composition to a subject. As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, which helps maintain or establish the form in which the BoNT polypeptide is delivered to a subject. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the composition and compatible with administration to a subject, such as a human. Such compositions may be specifically formulated for administration by one or more of a variety of routes of administration as described herein. Supplementary active ingredients may also be incorporated into the composition. When the medicament, formulation or pharmaceutical composition described herein is administered to a subject, a therapeutically effective amount is preferably administered. As used herein, the term "therapeutically effective amount" refers to an amount that causes improvement or cure of a condition. In one embodiment, the pharmaceutical composition is formulated for administration by injection. In one embodiment, the pharmaceutical composition comprises a botulinum neurotoxin encapsulated in microspheres. In one embodiment, the pharmaceutical composition comprises a botulinum neurotoxin formulated for transepithelial delivery. In one embodiment, the pharmaceutical composition comprises a botulinum neurotoxin formulated for slow release.

[0181] In one embodiment, the botulinum neurotoxin, polypeptide or chimeric molecule of the present invention is in the form of a controlled release formulation. Such compositions and methods of administration are provided in U.S. Patent Publication No. 2007 / 0020295, the contents of which are incorporated herein by reference.

[0182] Botulinum neurotoxins can be obtained by establishing and growing a culture of Clostridium botulinum in a fermenter, then harvesting and purifying the fermented mixture according to known procedures. All botulinum toxin serotypes are initially synthesized as inactive single-chain proteins that must be cleaved or nicked by proteases to become neuroactive. The bacterial strains that produce botulinum toxin serotypes A and G have endogenous proteases, so serotypes A and G can be recovered from bacterial cultures primarily in their active form. In contrast, botulinum toxin serotypes C1, D and E are synthesized by non-proteolytic strains and are therefore generally inactive when recovered from culture. Serotypes B and F are produced by proteolytic strains and non-proteolytic strains and can therefore be recovered in either active or inactive form. Proteolytic strains that produce, for example, botulinum toxin serotype B may cleave only a portion of the toxin produced. The exact ratio of nicked molecules to unnicked molecules depends on the length of incubation and the temperature of the culture. Therefore, a certain proportion of a preparation of, for example, botulinum toxin type B toxin may be inactive. In one embodiment, the neurotoxin of the present invention is in an active state. In one embodiment, the neurotoxin is in an inactive state. In one embodiment, a combination of an active neurotoxin and an inactive neurotoxin is envisaged.

[0183] Pill Box

[0184] Also contemplated herein is a kit comprising a BoNT or polypeptide disclosed herein (e.g., in the form of a pharmaceutical composition). The kit may comprise one or more of the compositions described herein packaged in a vial. The kit may also include a delivery vehicle or device for therapeutic administration of the composition and / or instructions for therapeutic administration. The kit may have therein all components packaged into a shaped container.

[0185] Another aspect of the present invention relates to a delivery tool or device for administering a pharmaceutical composition described herein, which is pre-loaded with a pharmaceutical composition (e.g., for single use). Such a device can be a syringe or a microneedle device for delivering the composition. The syringe can be a disposable syringe pre-loaded with an effective amount of the composition. The microneedle device may include one or more microneedles coated with a composition described herein, such as described in U.S. Patent Publication 2010 / 0196445, the contents of which are incorporated herein in their entirety.

[0186] Treatment

[0187] The present invention also includes methods for treating conditions that are typically treated with neurotoxins (e.g., skeletal muscle conditions, smooth muscle conditions, glandular conditions, neuromuscular conditions, autonomic nerve conditions, pain, or aesthetic / cosmetic conditions). Such conditions are associated with unwanted neuronal activity, as determined by a skilled practitioner. The methods include the step of administering a therapeutically effective amount of a BoNT or polypeptide / chimeric molecule described herein (e.g., as a pharmaceutical composition) to an appropriate location in a mammal to reduce unwanted neuronal activity, thereby treating the condition. Administration is by a route that brings an effective amount of the composition into contact with neurons that exhibit the unwanted activity.

[0188] Specific conditions contemplated for treatment by the methods discussed herein include, without limitation, spasmodic dystonia, spasmodic torticollis, laryngeal dystonia, oromandibular dystonia, tongue dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorders, cerebral palsy, focal spasms and other speech disorders, spastic colitis, neurogenic bladder (i.e., all disorders involving urinary incontinence, such as neurogenic detrusor overactivity or idiopathic overactive bladder), prostate cancer and other forms of cancer, anal spasm, limb spasms, tics, tremors, bruxism, anal fissures, achalasia, dysphagia and other dystonias and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhidrosis, excessive salivation, gastrointestinal hypersecretion and other secretory diseases, pain due to muscle spasms, neuropathic pain, inflammatory pain, headaches such as migraines, itching (pruritus), acne. In addition, the present invention can be used to treat dermatological or aesthetic / cosmetic conditions, such as reducing eyebrow furrows, reducing skin wrinkles. The present invention can also be used to treat sports injuries. Other applications include treating conditions such as sialorrhea and hyperhidrosis. In addition, there is a need for BoNT / B as an alternative toxin for treating patients who have developed neutralizing antibodies to BoNT / A.

[0189] In addition, the modified neurotoxins can be administered to treat other neuromuscular disorders using well-known techniques commonly performed with botulinum toxin type A. For example, the present invention can be used to treat pain, such as headaches, pain caused by muscle spasms, and various forms of inflammatory pain. For example, Aoki U.S. Pat. No. 5,721,215 and Aoki U.S. Pat. No. 6,113,915 disclose methods of treating pain using botulinum toxin type A. The disclosures of these two patents are incorporated herein by reference in their entirety.

[0190] Autonomic nervous system disorders can also be treated with modified neurotoxins. For example, glandular dysfunction is an autonomic nervous system disorder. Glandular dysfunction includes excessive sweating and excessive salivation. Respiratory dysfunction is another example of an autonomic nervous system disorder. Respiratory dysfunction includes chronic obstructive pulmonary disease and asthma. Sanders et al. disclose in U.S. Patent No. 5,766,605 the use of naturally occurring botulinum toxin to treat the autonomic nervous system; for example, methods for treating autonomic nervous system disorders such as excessive sweating, excessive salivation, asthma, etc. The disclosure of Sander et al. is incorporated herein by reference in its entirety. In one embodiment, a method substantially similar to the method of Sanders et al. can be used, but using a modified neurotoxin to treat autonomic nervous system disorders, such as the disorders discussed above. For example, a modified neurotoxin can be topically applied to the nasal cavity of a mammal in an amount sufficient to degenerate the cholinergic neurons of the autonomic nervous system, thereby controlling mucus secretion in the nasal cavity.

[0191] Pain that can be treated by modified neurotoxins includes pain caused by muscle tension or spasm or pain that is not related to muscle spasm. For example, Binder discloses in U.S. Pat. No. 5,714,468 that headaches caused by vascular disorders, muscle tension, neuralgia, and neuropathy can be treated with naturally occurring botulinum toxins such as botulinum toxin type A. Binder's disclosure is incorporated herein by reference in its entirety. In one embodiment, a method substantially similar to Binder's method can be used, but using a modified neurotoxin to treat headaches, particularly headaches caused by vascular disorders, muscle tension, neuralgia, and neuropathy. Pain caused by muscle spasm can also be treated by administering a modified neurotoxin. For example, WO2006001676 (Kang Ahn) discloses a method of using botulinum neurotoxin for treating knee pain caused by saphenous nerve entrapment, WO2008059126 (Christine Favre et al.) discloses a method of using botulinum neurotoxin for treating pain induced by chemotherapy, WO2008090287 (Christine Favre et al.) discloses a method of using botulinum neurotoxin for treating pain induced by anti-HIV treatment, WO2009130600 (Christine Favre et al.) discloses a method of using botulinum neurotoxin for treating pain associated with diabetic neuropathy, and WO2007144493 (Michel Auguet et al.) discloses a method of using botulinum neurotoxin in combination with an opioid derivative for treating pain. The disclosures of WO2006001676, WO2008059126, WO2008090287, WO2009130600, WO2007144493 are incorporated herein by reference in their entirety. In addition, the modified neurotoxins can be administered to mammals to treat pain that is not associated with muscle disorders such as spasms. In a broad embodiment, the method of the present invention for treating non-spasm-related pain comprises central or peripheral administration of the modified neurotoxins.

[0192] Acute or chronic pain not associated with muscle spasms can also be alleviated by local peripheral administration of a modified neurotoxin to the site of actual or perceived pain in a mammal. In one embodiment, the modified neurotoxin is administered subcutaneously at or near the site of pain, such as at or near an incision. In some embodiments, the modified neurotoxin is administered intramuscularly at or near the site of pain, such as at or near the site of a bruise in a mammal. In some embodiments, the modified neurotoxin is injected directly into the joints of a mammal for the treatment or alleviation of pain caused by an arthritic condition. Furthermore, frequent repeated injections or infusions of the modified neurotoxin into peripheral pain sites are also within the scope of the present invention.

[0193] The routes of administration for such methods are known in the art and are readily adapted by a skilled practitioner to the methods described herein (e.g., see, e.g., Harrison's Principles of Internal Medicine (1998), Anthony Fauci et al., ed., 14th edition, published by McGraw Hill). As non-limiting examples, treatment of neuromuscular disorders may include the step of locally administering an effective amount of a molecule to a muscle or muscle group, treatment of autonomic disorders may include the step of locally administering an effective molecule to one or more glands, and treatment of pain may include the step of administering an effective amount of a molecule to the site of pain. Additionally, treatment of pain may include the step of administering an effective amount of a modified neurotoxin to the spinal cord.

[0194] It is also contemplated that the modified B4 H described herein may be used C As a delivery vehicle to target neurons and other cell types expressing human synaptotagmin II in humans. For example, a modified H that is covalently or non-covalently linked to another biologically active molecule (e.g., a therapeutic agent) to form a chimeric molecule described herein C Can be used as a targeting vehicle to deliver biologically active molecules to neurons and other cell types that express human synaptotagmin II by binding to human Syt I and / or Syt II. Therefore, another aspect of the present invention relates to the use of the chimeric polypeptide molecules described herein for delivering biologically active molecules to neurons in human subjects. The second part of the molecule can be a biologically active molecule, such as a therapeutic agent (e.g., a polypeptide or a drug). The connection between the first part and the second part of the molecule can be covalent (e.g., in the form of a fusion protein) or non-covalent. Such methods of connection are known in the art and can be easily applied by skilled practitioners. The chimeric polypeptide molecule will be administered by a route that causes the polypeptide to contact neurons expressing the receptor, as described herein, the modified BH C Binds to the neuron (target neuron).

[0195] Identification of receptor binding activity

[0196] Another aspect of the invention relates to a method for identifying a modified BoNT receptor binding domain for its ability to bind to a receptor (e.g., human Syt I or human Syt II, or human SV2). The method utilizes a 2-hybrid assay system and utilizes a fusion protein made of a receptor and a modified Hc fused (expressed as a fusion protein) to the respective "bait" and "prey" subunits used in a 2-hybrid assay (e.g., a Gal4 transcriptional activation system utilizing an activation domain and a DNA binding domain). 2-hybrid assays are typically performed in yeast (Saccharomyces cerevisiae), but similar assay systems have been developed for use in other unicellular organisms such as E. coli. Those systems are equally comparable. In one embodiment, a bacterial adenylate cyclase 2-hybrid assay is used (Karimova et al., Proc Natl Acad Sci USA. 1998 May 12;95(10):5752-5756, the contents of which are incorporated herein by reference). The system uses T18 and T25 as bait and prey, and can utilize E. coli BTH101 cells.

[0197] The modified Hc is expressed as a fusion protein (called the first fusion protein) with T18 in the Escherichia coli of 2-hybrid assay. In the described method, the receptor (or its binding fragment such as h-Syt II aa1-87) is co-expressed as a fusion protein (called the second fusion protein) with T25 in the Escherichia coli of 2-hybrid assay. The described assay utilizes the positive indication of the interaction between the corresponding molecules by their respective fusions. A possible positive indication is color development. The cloned Escherichia coli colonies expressing the first fusion protein and the second fusion protein are grown on a solid culture medium containing a medium (for example, ampicillin, kanamycin, X-gal and IPTG) containing appropriate selectivity and reporting for a period of time to allow positive indications (for example, color generation) to be produced from positive colonies. The combination of the modified binding domain and the receptor fragment will produce positive indications (for example, the expression of the LacZ gene and causing the production of blue in the colonies grown on X-gal flat plates).

[0198] When compared with appropriate controls (positive and negative), such positive indications (e.g., color development) of the analysis colonies are analyzed. The analysis can be performed visually or by non-human machines. Appropriate negative controls (e.g., clone colonies lacking key components of the assay system, such as bait and prey expressed without fusion) that cannot produce obvious positive indications (e.g., LacZ expression) are used. Appropriate strong positive controls can also be used. An example of a strong positive control for human Syt II receptors in the assay is B1-Hc with a substitution mutation (M / C / V or Q) at E1191. Weak positive controls can also be used to identify the strength of binding. An example of a weak positive control for human Syt II receptors in the assay is B1-Hc with a substitution mutation (Q / Y / A or S) at W1178. Identification of positive indications (e.g., color development) indicates that the modified Hc binds to the receptor. Identification of strong positive indications such as strong color development (e.g., stronger than weak positive controls) indicates that Hc binds to the receptor with high affinity.

[0199] The modified Hc used in the assay may be any modified Hc disclosed herein. As a non-limiting example, the modified Hc may contain one, two or three substitution mutations, such as those disclosed herein. The modified Hc may be any serotype / strain or subtype as disclosed herein.

[0200] The embodiments described here and in the following examples are for illustrative purposes only, and various modifications or changes apparent to those skilled in the art are intended to be included within the scope of the present invention.

[0201] Unless otherwise defined herein, scientific and technical terms used in conjunction with this application shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0202] It is to be understood that the invention is not limited to the particular methodology, protocols, reagents, etc. described herein, and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

[0203] Other than in the operating examples, or unless otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term "about." The term "about" when used in conjunction with a percentage to describe the present invention means ±1%.

[0204] In one aspect, the present invention relates to compositions, methods, and one or more components thereof as described herein, which are essential to the present invention, but still include essential or non-essential unspecified elements ("comprising"). In some embodiments, other elements included in the description of the composition, method, or its respective component are limited to those elements that do not substantially affect one or more basic and novel features of the invention ("consisting essentially of ..."). The same applies to the steps within the method and the compositions and components therein. In other embodiments, the inventions, compositions, methods, and their respective components as described herein are intended to exclude any elements that are not considered to be essential elements of the component, composition, or method ("consisting of ...").

[0205] All identified patents, patent applications and publications are expressly incorporated herein by reference for describing and disclosing methods described in such publications that can be used in conjunction with the present invention. These publications only provide disclosures prior to the filing date of the present application. In this regard, it should never be interpreted as an admission that the inventor has no right to advance this disclosure due to existing inventions or any other reason. All statements about dates or descriptions about the contents of these documents are based on information available to the applicant and should not constitute any permission for amendments to the dates or contents of these documents.

[0206] The present invention is further illustrated by the following examples, which should not be construed as further limiting.

[0207] Example

[0208] Example 1. Identification of single mutations that enhance binding of BoNT / B to h-Syt II

[0209] The co-crystal structure of BoNT / B bound to rat Syt II has been solved (19, 20). This provided a good basis for selecting a total of 19 residues at the BoNT / B-Syt II interface for mutagenesis studies ( Figure 2A , Table 5). Our strategy was to saturate each of these 19 positions with all 20 possible amino acids, aiming to identify all single residue mutations that increase binding to h-Syt II. To this end, we utilized the bacterial adenylate cyclase two-hybrid approach (BACTH) (**33). In brief, the H C (H C B) Subcloned into a vector with a split fragment of bacterial adenylate cyclase (designated T18). C B fusion constructs were amplified by PCR using primers with random trinucleotides (NNN) at the selected positions, generating a pool of constructs encoding all 20 possible amino acids at the selected sites ( Figure 2BThis pool of constructs was then co-transformed into bacteria (E. coli strain BTH101) together with a construct constitutively expressing a h-Syt II fragment containing the toxin binding site (residues 1-87) fused to the other half of the split bacterial adenylate cyclase (designated T25). C B binds to h-Syt II to bring T18 and T25 together and restore the activity of adenylate cyclase, which causes the expression of the lacZ gene and the production of blue colonies on X-Gal plates ( Figure 2B ).

[0210] Four positions showed blue colonies E1191 (22.7% of total colonies), W11178 (7.8% of total colonies), Y1183 (4.0% of total colonies) and S1199 (5.1% of total colonies) (Table 6). Plasmids were extracted from these blue colonies and sequenced, revealing mutations at each site: E1191M / C / V / Q / L / Y, Y1183 / C / P, S1199W / E / Y / H, W1178Y / Q / A / S ( Figure 2C ). These mutations were further validated by measuring the level of β-galactosidase in bacteria, which reflects the reconstructed adenylate cyclase activity. The four mutations at the E1191 site, E1191M / C / V / Q, showed the strongest β-galactosidase activity among all the mutations ( Figure 2D ). We further examined the mutation at E1191 using a pull-down assay. A GST-tagged wild-type (WT) mouse Syt II fragment (m-Syt II, residues 1-87) was used as a positive control. A human Syt II fragment was generated by mutating F54 in mouse Syt II (1-87) to L, thereby mimicking the human sequence (designated h-Syt II). The GST-tagged Syt II fragment was immobilized on beads and used to pull down H C B. Bound H C B Detected by immunoblotting analysis through the fused HA tag. E1191M / C / V / Q produced significant binding levels to h-Syt II ( Figure 2E Other mutations at E1191 and other sites did not induce significant binding to h-Syt II ( Fig.18 ). Confirm that you have previously Fig.18 The content displayed in Fig.21 It was also shown that other mutations did not cause significant binding to h-Syt II. Taken together, these data confirm that H containing the E1191M / C / V / Q mutation C B obtains improved ability to bind h-Syt II.

[0211] Example 2.H C Combined mutations in B further enhance its binding to h-Syt II

[0212] Next, we discuss H C Whether binding of B(E1191M / C / V / Q) to h-Syt II could be further enhanced by including secondary mutations at different positions. Double mutations were generated by combining E1191M at positions 1183, 1199, and 1178 with residue changes identified in the BACTH screen ( Figure 2C The ability of these 10 double mutants to bind h-Syt II was analyzed in a pull-down assay ( Figure 3A ). Three of them: E1191M / S1199W, E1191M / S1199Y and E1191M / W1178Q showed strong binding to h-SytII, while the combination of E1191M and Y1183C / P reduced the binding to h-Syt II ( Figure 3A Considering that E1191 is spatially close to Y1183 ( Figure 2A ), suggesting a potential structural conflict when mutating both E1191 and Y1183.

[0213] We next generated all 12 combinations between the single mutation E1191M / C / V / Q and the compatible secondary mutations S1199W / Y and W1178Q as well as the triple mutation E1191M / S1199W / W1178Q. The binding affinities (K) of all 12 mutants for h-Syt II D ) were measured using biolayer interferometry (BLI), with parameters listed in Table 4 and representative traces shown in Fig.19 Briefly, a GST-tagged Syt fragment was immobilized to the probe, which was then exposed to different concentrations of purified H C B (association stage, Figure 3B ), followed by a wash step (dissociation phase, Figure 3B ). C Binding of B to m-Syt II was used as a positive control, which showed K D The WT H C The binding of B to h-Syt II was too weak to be reliably determined beyond the detection limit, and the estimated K D >20μM( Figure 3B , Table 4). The E1191M mutant produced a binding K of 6.7 μM for h-Syt II. D Most double mutants further improved binding affinity, K D Values ​​ranged from 0.59 μM to 4.3 μM ( Fig.19 , Table 4). The first two double mutants, E1191M / S1199Y (K D=0.72 μM) and E1191V / S1199Y (K D =0.59 μM), providing an affinity that is an order of magnitude higher than that of the E1191M mutant ( Figure 3B , Table 4). The E1191Q / W1178Q double mutant was the only mutant that did not bind to h-Syt II. The triple mutant E1191M / S1199W / W1178Q showed similar binding affinity to the double mutant E1191M / S1199W, indicating that adding the third mutation site did not provide further improvement (Table 4).

[0214] Example 3.H C B mutant shows enhanced binding to h-Syt I

[0215] Next, check H C Does the B mutant still bind to h-Syt I. Figure 24 depicts a comparison between Syt I and Syt II and between human Syt I and mouse Syt I at the BoNT / B-binding interface described herein. WT H C Binding of B to Syt I can only be detected in the presence of co-receptor gangliosides in pull-down assays. This is because Syt I exhibits lower binding affinity for BoNT / B than Syt II (15, 28). We found that both the E1191M mutant and the E1191M / S1199Y mutant bound to h-Syt I in the absence of gangliosides in pull-down assays ( Figure 3C ), suggesting that these mutants may have enhanced binding to h-Syt I. We measured the binding affinity of our first two mutants (E1191M / S1199Y and E1191V / S1199Y) to h-Syt I using the BLI assay. C B MY ) or E1191V / S1199Y(H C B VY ) C B showed K of 2.9 μM and 5.82 μM, respectively. D Considering WT H C The binding of B to h-Syt I was too weak to be reliably determined (Table 4, Fig. 20 ), H C The B mutant not only enhances its binding to h-Syt II, but also enhances its binding to h-Syt I. C B VY In comparison, H C B MYShowing higher binding affinity for Sy-I and lower dissociation constants for both Syt I and h-SytII, we selected this mutant for further characterization.

[0216] Example 4.H C B MY Binds to h-Syt II on the surface of neurons

[0217] Next, check H C B MY Whether the mutant can bind to h-Syt II on the surface of physiologically relevant neurons was investigated. To this end, cultured rat cortical neurons expressing Syt I but not Syt II were used (13). Thus, knockout (KD) of Syt I generates neurons without endogenous receptors. Expression of full-length h-Syt II in these Syt I KD neurons generates "humanized" neurons with only h-Syt II as toxin receptor (18). As expected, WT H C B binds strongly to rat neurons, and binding is abolished after knockdown of endogenous Syt I. Expression of full-length m-Syt II, but not full-length h-SytII nor m-Syt II containing the F54L mutation, restores WT H C The combination of B Figure 4A ). On the contrary, H C B MY The robust binding to neurons expressing m-Syt II, h-Syt II, or m-Syt II(F54L) was demonstrated, confirming that H C B MY Has enhanced ability to bind to h-Syt II on the surface of neurons ( Figure 4B ).

[0218] Example 5. BoNT / B mutants exhibit enhanced efficacy in blocking neurotransmission in humanized neurons

[0219] To address the critical question of whether the enhanced binding to h-Syt II translates into improved efficacy at the functional level in neurons, full-length WT BoNT / B and the E1191M / S1199Y double mutant toxin (BoNT / B MY ) were recombinantly produced in E. coli ( FIG. 23 ). Humanized neurons were exposed to WT BoNT / B or BoNT / B MY Toxin gradient. Cleavage of VAMP2 was examined by immunoblot analysis. Figure 5A As shown in Figure 3, at each toxin concentration tested, more VAMP2 was expressed in neurons exposed to BoNT / B compared to neurons exposed to WT BoNT / B. MYlysed in neurons, indicating that BoNT / B MY Targets and enters neurons more efficiently than WT toxin.

[0220] Neurotransmitter release was next monitored by recording miniature inhibitory postsynaptic currents (mIPSCs) using whole-cell patch clamp recordings. The frequency of mIPSCs reflects the activity of neurotransmitter release in the neuronal population. BoNT / B enters the presynaptic terminal to block neurotransmitter release, thereby reducing the frequency of mIPSCs ( Figure 5B ). Humanized neurons were exposed to WT BoNT / B or BoNT / B MY The gradient of Figure 5C As shown in MY The toxin showed greatly enhanced potency, with a half-maximal inhibitory concentration (IC) approximately 11-fold lower than that of the WT toxin. 50 ). These data demonstrate that enhanced binding to the human receptor results in increased efficacy of the toxin in neurons at a functional level.

[0221] Example 6. BoNT / B4 subtype contains Q1191 / Y1199 but does not bind to h-Syt II

[0222] Finally, we explored whether there were any naturally occurring sequence variations in BoNT / B subtypes that could affect binding to h-Syt II. To date, eight subtypes of BoNT / B are known (BoNT / B1-B8, with BoNT / B1 being referred to as the prototypical BoNT / B), with sequence variation as high as 7% (34). Sequence alignment revealed variations at both E1191 and S1199 ( Fig. 6A Interestingly, this was the case for E1191Q (BoNT / B4, BoNT / B8) and S1199Y (BoNT / B2, BoNT / B3, BoNT / B4, BoNT / B7). Even the combination of E1191Q and S1199Y in B4 - this double mutant in BoNT / B1 - resulted in robust binding to h-Syt II (Table 4). However, H C B4 did not bind to h-Syt II in the pull-down assay ( Figure 6B ). This may be due to H C B4 varies from other residues in BoNT / B1. Among the 19 key residues at the BoNT / B-Syt interface, there are four other residues that are different between BoNT / B1 and BoNT / B4 ( Figure 7 ). In fact, H C Substitution of all four residues in B4 with the corresponding residues in BoNT / B1 increased its binding to h-syt II ( Figure 6C ). Confirm that the Figure 6C , Fig. 22 The content also shows that H is replaced by the corresponding residue in BoNT / B1 C All four residues in B4 generate a mutant H that can bind to h-Syt II c B4. Interestingly, in the absence of gangliosides, H C B4 showed strong binding to h-Syt I, indicating that the subtype has a better binding affinity to Syt I than BoNT / B1 ( Fig.6D ).

[0223] Discussion

[0224] By combining rational design based on the available co-crystal structure of the BoNT / B-Syt II complex with the BACTH approach, which saturates all possible single point mutations on each selected target residue, a series of point mutations in BoNT / B1 that improve binding to h-SytII were identified. These point mutations were further examined in different combinations, revealing that double mutants exhibited high affinity for h-Syt II. Furthermore, full-length BoNT / B1 containing two of these mutations showed approximately 11-fold higher potency than WT BoNT / B1 on humanized neurons, demonstrating that enhanced binding to toxin receptors translates into higher potency at a functional level in neurons.

[0225] Residue E1191 of BoNT / B1 was found to be a key position for binding to both h-Syt I and h-Syt II. The residue is located at the periphery of the BoNT / B·Syt II interface, where by placing M / C / V / Q, it is possible that these residues can form new / additional bonds with Syt II, as well as remove potentially unfavorable negative charges, thereby enhancing the overall binding affinity. In addition to h-Syt II, the E1191M mutation also increased the binding affinity to Syt I. E1191L has been previously shown to increase the binding of BoNT / B to Syt I (35). It was also found herein that replacement of E1191 with L or Y showed increased binding to h-Syt II, but the extent of the increase was much less than that of M / C / V / Q. Therefore, these results suggest that E1191 is located at a key position for regulating the binding affinity between BoNT / B and Syt I / II without disrupting the overall binding interface.

[0226] The BACTH screen identified other mutations in BoNT / B that caused only a slight increase in binding to h-Syt II, including W1178Y / Q / A / S, Y1183C / P, and S1199W / E / Y / H. S1199Y was previously reported to increase BoNT / B binding to Syt II, possibly due to a π-stacking interaction between S1199Y and F47 of Synt II (19). Interestingly, sequence analysis of BoNT / B isoforms revealed residue variation in some of these positions. For example, position 1199 can be S, Y, or H, and position 1191 can be E, K, or Q. Such residue changes would be expected to alter the binding affinity to Syt I or Syt II. For example, BoNT / B4 binds h-Syt I more strongly than BoNT / B1, perhaps because it contains a Q at position 1191 and a Y at position 1199. It appears that nature has sampled a range of residues for the ability of the two key positions identified in our BACTH screen to alter toxin-receptor interactions. C Naturally occurring sequence variation within domains provides a rich resource for mining residues that may modulate toxin-receptor interactions.

[0227] BoNT / DC and BoNT / G recognize the same surface residues on Syt I / II as BoNT / B, and both show reduced binding to h-Syt II (18). The co-crystal structure of BoNT / DC in complex with Syt I / II has been solved (36); therefore, a similar approach targeting key residues in the binding interface could identify specific mutations in BoNT / DC that increase its affinity for h-Syt II.

[0228] Of note, humans are known to have a lower sensitivity to BoNT / D due to a single residue change in the human VAMP1 substrate from M48 in mice (mouse VAMP1 sequence number) to I48 in humans. With residue I in that position, BoNT / D exhibits significantly reduced cleavage efficiency (37-39). Considering that BoNT / DC shares the same protease domain as BoNT / D, humans are less sensitive to BoNT / DC due to a single residue change in its receptor and a single residue change in its substrate. While the possibility that these two changes were random events cannot be ruled out, it is possible that BoNT may have been a selective force in human evolution (37).

[0229] The medical use of BoNTs is a notable example of transforming deadly toxins into effective therapeutic agents. As medical applications rapidly expand, there is increasing interest in engineering BoNTs to improve their therapeutic efficacy and reduce their adverse effects (40-44). C Replace H in BoNT / A C The chimeric BoNT / ABs with increased potency in mouse tissues were generated (45, 46). These findings suggest that H C B generates the possibility of chimeric BoNT / AB that potentially further improves the potency of BoNT / A in humans. Therefore, the BoNT / B mutants identified here allow for the development of a new generation of therapeutic toxins with improved efficacy in humans. In addition, such modified BoNT / B and their H C It will also be a valuable scientific tool and delivery vehicle for targeting human neurons.

[0230] Materials and methods

[0231] Materials and constructs .

[0232] The following antibodies were purchased from the indicated suppliers: Synapsin I (clone 46.1, Synaptic Systems), VAMP2 (clone 69.1, Synaptic Systems), HA (16B12, Covance), and β-tubulin III (ab18207, Abcam). Bovine mixed brain gangliosides were purchased from Matreya LLC (Pleasant Gap, PA) and reconstituted in Tris-buffered saline (TBS: 20 mM Tris, 150 mM NaCl) as previously described (14). C B (residues 857-1291, Genbank: ACA46990.1) and H C The cDNA of B4 (Genbank: EF051570) was codon-optimized for E. coli expression and synthesized by Genscript Inc. (New Brunswick, NJ). The following cDNAs were generously provided by the indicated groups: rat Syt I (TC Sudhof, Palo Alto, CA), mouse Syt II (M. Fukuda, Ibaraki, Japan), human Syt I (RB Sutton, Lubbock, TX). Encoding H C The DNA of B was subcloned into the pET28a vector, in which the His6 tag (SEQ ID NO: 1) and the HA tag (YPYDVPDYA) (SEQ ID NO: 2) were fused to its N-terminus. CThe mutations in B were generated by PCR using a site-directed mutagenesis kit (Agilent Technologies, CA). GST-tagged Syt I / II fragments and the SytII F54L mutant were described previously (15, 18, 23).

[0233] Production and activation of full-length BoNT / B and BoNT / B MY .

[0234] Encodes full-length inactive BoNT / B R370A、Y373F The cDNA of (Accession No. B1INP5) was codon optimized for expression in E. coli. It was cloned into pET32a via NdeI / BamHI to add a hexahistidine affinity tag (SEQ ID NO: 1) to the C-terminus. The construct was restored back to its active WT form by standard site-directed mutagenesis (Agilent). E1191M / S1199Y mutations were introduced to produce BoNT / B MY Double mutant. The construct was transformed into E. coli (strain BL21 DE3), grown in mTB + 100 μg / mL ampicillin, and induced with 1 mM IPTG at 16 ° C for 20 hours. Bacteria were harvested and lysed by sonication in 5 mL 0.5 M NaCl and 0.5 μL Benzonase per gram of pellet. The crude lysate was clarified by centrifugation at 4000 × g for 1 hour, and BoNT / B was captured on a HisTrap HP column (GE) and eluted by FPLC (Ge) with 0.1 M imidazole in 0.5 M NaCl in 50 mM Tris pH 8. The eluate was desalted into 125 mM NaCl in 50 mM Tris pH 8 using a HiPrep 26 / 10 desalting column (GE) and then concentrated to 0.6 mg / mL using a 10 kDa MWCO spin filter. The concentrate was treated with 0.1 μg / mL endoproteinase Lys-C at 37°C for 2 hours and then adjusted to 1 M (NH4)2SO4 before final purification by hydrophobic interaction chromatography using Phenyl Sepharose HP (GE). The protein was eluted with a linear gradient from 1 M to 0 M (NH4)2SO4 in 50 mM Tris pH 8. Fractions containing double-chain BoNT / B were pooled, desalted, concentrated, and stored at -80°C.

[0235] BACTH (Bacterial Adenylate Cyclase Two-Hybrid Assay) .

[0236] The BACTH assay was performed according to the manufacturer's instructions (Euromedex). Two compatible plasmids, pUT18C and pKT25, were selected for screening. The H-Syt II luminal domain (residues 1-80) was cloned into pKT25 to generate pKT25-h-Syt II.C B was cloned into pUT18C to produce T18-H C B.H. C B mutant libraries were generated with primers containing random nucleotide triplets (NNN) at the specified positions. Each library was co-transformed into the E. coli indicator strain BTH101 by electroporation with the pKT25-h-Syt II plasmid and screened on LB agar plates containing 100 μg / ml ampicillin, 50 μg / ml kanamycin, 0.5 mM IPTG and 40 μg / ml X-Gal. The plates were incubated at 30°C for 64 hours. Plasmids were extracted from blue colonies and sequenced. The total number of colonies determined the probability of covering all 20 amino acids at the selected mutation site. This was determined by the Clark-Carbon equation: P = 1-(1-f) N Calculated as , where f reflects the number of possible residues (here f = 1 / 20, since there are 20 different amino acids), and N is the total number of colonies. In our assay, using a minimum number of 380 colonies, the probability of covering all 20 amino acids at each position is >99.8%.

[0237] β-Galactosidase activity assay .

[0238] The assay was performed as described previously (47). Briefly, E. coli BTH101 cells harboring the plasmid of interest were inoculated into LB medium containing antibiotics and IPTG (0.5 mM). The culture was grown overnight at 37°C to reach stationary phase. The OD of the culture was recorded before harvesting. 600 . The culture was centrifuged and the cell pellet was washed twice with PBS and resuspended in Z buffer (60mM Na2HPO4, 40mM Na2HPO4, 10mM KCl, 1mM MgSO4 and 20mM DTT). Then, 1:10 (v / v) chloroform and 1:20 (v / v) 0.1% SDS were added and mixed thoroughly to permeabilize the cells. The mixture was then mixed with o-nitrophenyl-p-galactoside (4mg / ml in Z buffer) at a ratio of 5:1 and incubated at 28°C for 10 minutes. The reaction was stopped by adding 80μl of 1M Na2CO3. β-galactosidase activity was calculated as A 420 / OD 600 .

[0239] GST pull-down assay .

[0240] Two types of pull-down assays were performed. The first series was used to rapidly screen mutant H CB Binding of GST-tagged m-SytII (residues 1-87) and a mutant m-Syt II (F54L) that mimics human Syt II (designated h-Syt II). C The E. coli of B was spun down, resuspended in 800 μl TBS, sonicated, and then incubated with 2% Triton X-100 at 4°C for 1 hour. The samples were then spun down in a microcentrifuge at 4°C for 15 minutes. The supernatant was collected and used for pull-down assays by incubating with 10 μg of GST-Syt protein immobilized on glutathione-agarose beads (GE bioscience, Piscataway, NJ) at 4°C for 1 hour. The samples were washed three times in wash buffer (TBS with 0.5% Triton X-100) and analyzed by immunoblotting analysis using anti-HA antibodies. For mutants that showed enhanced binding to h-Syt II, these H C The B mutant was further characterized as a His6-tagged protein ("His6" is disclosed as SEQ ID NO: 1) in a pull-down assay and then purified H C B (100 nM) and immobilized GST-Syt II in 100 μl TBS buffer plus 0.5% Triton X-100 in the presence or absence of gangliosides (60 μg / ml) for 1 hour at 4°C. The beads were washed three times with TBS buffer plus 0.5% Triton X-100. After immunoblotting analysis, 10% of the bound material was subjected to SDS-PAGE.

[0241] Biolayer interferometry .

[0242] H C The binding affinity between B variants and Syt I / Syt II was measured by BLI assay using the Blitz system (ForteBio). Briefly, GST-tagged Syt I or Syt II (20 μg / ml) was immobilized to the Dip and Read TM The anti-GST biosensor (ForteBio) was equilibrated with PBS buffer. The biosensor was then exposed to a series of concentrations of H C B, then washed with PBS. Binding affinity (K D ) were calculated using the Blitz system software according to the manufacturer's instructions (ForteBio).

[0243] Neuronal cultures, lentivirus, and toxin binding / entry assays .

[0244] Rat cortical neurons were prepared from E18-19 embryos as described previously (14). Constructs for Syt IKD, m-Syt II, and h-Syt II expression in neurons were previously described (18). Lentiviruses were added to neuronal cultures at DIV5 (days in vitro), and toxin binding / entry experiments were performed at DIV12-14. Toxins were expressed in high K + Buffer (87mM NaCl, 56mM Kcl, 1.5mM KH2PO4, 8mM Na2HPO4, 0.5mM MgCl2 and 1mM CaCl) was diluted and preheated to 37°C. Neurons were exposed to the above-mentioned buffer containing toxins at 37°C for 5 minutes and then washed with PBS. These neurons were subjected to immunostaining analysis or incubated in non-toxic medium for another 24 hours and then subjected to immunoblotting analysis.

[0245] mIPSC recordings .

[0246] Whole-cell patch clamp recordings were obtained from cortical neurons cultured at DIV 14-18 (DIV 14-18). The pipette solution contained (in mM): 135 CsCl, 10 HEPES, 1 EGTA, 1 Na-GTP, 4 Mg-ATP and 10 QX-314 (pH 7.4, adjusted with CsOH). The resistance of the pipette filled with intracellular solution varied between 4 MΩ and 5 MΩ. After forming the whole-cell configuration and equilibrating the intracellular pipette solution, the series resistance was adjusted to 10 MΩ. Synaptic currents were monitored at a holding potential of -70 mV using an EPC-10 / 2 amplifier (HEKA). The bath solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl, 1 MgCl2, 10 HEPES, 10 glucose (pH 7.4, adjusted with NaOH). By adding AMPA and NMDA receptor blockers CNQX and APV to the extracellular bath, spontaneous inhibitory postsynaptic current (sIPSC) and evoked inhibitory postsynaptic current (eIPSC) were pharmacologically inhibited. Spontaneous miniature inhibitory postsynaptic current (mIPSC) was monitored in the presence of tetrodotoxin (TTX) to block action potentials. Data were analyzed using Clampfit 10 (Molecular Devices), Origin8 software (Mocrocal Inc.), MiniAnalysis software (Synaptosoft) and Igor (Wavemetrics). Statistical analysis was performed with Student's t-test (*P<0.01). All data shown are mean ± SEM.

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[0301] Table 4: Interaction between GST - Syt II / Syt I and H C and B measured by BLI.

[0302]

[0303]

[0304] Table 5: List of 19 key residues in BoNT / B that form the binding pocket for Syt I / II, selected based on the available co-crystal structure of BoNT / B in complex with Syt II (19, 20).

[0305]

[0306] Table 6: Summary of colony numbers at each mutagenesis site from the BACTH screen.

[0307]

[0308] Sequence Listing <110> PRESIDENT AND FELLOWS OF HARVARD COLLEGE <120> Engineered botulinum neurotoxin <130> 0342941-0602 <140> <141> <150> 62 / 378,967 <151> 2016-08-24 <160> 29 <170> PatentIn Version 3.5 <210> 1 <211> 6 <212> PRT <213> Artificial sequence <220> <221> source <223> / note = "Description of artificial sequence: synthetic 6xHis tag" <400> 1 His His His His His 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial sequence <220> <221> source <223> / note = "Description of artificial sequence: synthetic peptide" <400> 2 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 <210> 3 <211> 22 <212> PRT <213> Mus musculus <400> 3 Gly Glu Gly Lys Glu Asp Ala Phe Ser Lys Leu Lys Gln Lys Phe Met 1 5 10 15 Asn Glu Leu His Lys Ile 20 <210> 4 <211> 22 <212> PRT <213> Homo sapiens <400> 4 Gly Glu Gly Lys Glu Asp Ala Phe Ser Lys Leu Lys Glu Lys Phe Met 1 5 10 15 Asn Glu Leu His Lys Ile 20 <210> 5 <211> 22 <212> PRT <213> Mus musculus <400> 5 Gly Glu Ser Gln Glu Asp Met Phe Ala Lys Leu Lys Glu Lys Phe Phe 1 5 10 15 Asn Glu Ile Asn Lys Ile 20 <210> 6 <211> 22 <212> PRT <213> Homo sapiens <400> 6 Gly Glu Ser Gln Glu Asp Met Phe Ala Lys Leu Lys Glu Lys Leu Phe 1 5 10 15 Asn Glu Ile Asn Lys Ile 20 <210> 7 <211> 14 <212> PRT <213> Rat <400> 7 Asp Ala Phe Ser Lys Leu Lys Gln Lys Phe Met Asn Glu Leu 1 5 10 <210> 8 <211> 14 <212> PRT <213> Rat <400> 8 Asp Met Phe Ala Lys Leu Lys Glu Lys Phe Phe Asn Glu Ile 1 5 10 <210> 9 <211> 26 <212> PRT <213> Clostridium botulinum <400> 9 Lys Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser 1 5 10 15 Asp Ser Asp Glu Phe Tyr Asn Thr Ile Gln 20 25 <210> 10 <211> 26 <212> PRT <213> Clostridium botulinum <400> 10 Lys Asp Phe Lys Glu Glu Glu Lys Lys Leu Phe Leu Ala Asn Ile Tyr 1 5 10 15 Asp Ser Asn Glu Phe Tyr Lys Thr Ile Gln 20 25 <210> 11 <211> 26 <212> PRT <213> Clostridium botulinum <400> 11 Lys Asp Phe Lys Lys Lys Glu Glu Lys Leu Phe Leu Ala Asn Ile Tyr 1 5 10 15 Asp Ser Asn Glu Phe Tyr Asn Thr Ile Gln 20 25 <210> 12 <211> 26 <212> PRT <213> Clostridium botulinum <400> 12 Lys Asn Phe Lys Glu Gln Glu Gln Lys Leu Phe Leu Ser Ile Ile Tyr 1 5 10 15 Asp Ser Asn Glu Phe Tyr Lys Thr Ile Gln 20 25 <210> 13 <211> 26 <212> PRT <213> Clostridium botulinum <400> 13 Lys Tyr Phe Lys Lys Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser 1 5 10 15 Asp Ser Asp Glu Phe Tyr Asn Thr Ile Gln 20 25 <210> 14 <211> 26 <212> PRT <213> Clostridium botulinum <400> 14 Lys Asn Phe Lys Lys Lys Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser 1 5 10 15 Asp Ser Asp Glu Phe Tyr Asn Thr Ile Gln 20 25 <210> 15 <211> 26 <212> PRT <213> Clostridium botulinum <400> 15 Lys Asn Phe Lys Gly Gln Glu Glu Lys Leu Phe Leu Ala Asn Ile Tyr 1 5 10 15 Asp Ser Asn Glu Phe Tyr Lys Thr Ile Gln 20 25 <210> 16 <211> 27 <212> PRT <213> Clostridium botulinum <400> 16 Lys Asp Phe Lys Gly Gln Lys Glu Gln Lys Leu Phe Leu Ala Asn Ile 1 5 10 15 His Asp Ser Asn Glu Phe Tyr Lys Thr Ile Gln 20 25 <210> 17 <211> 434 <212> PRT <213> Clostridium botulinum <400> 17 Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn Leu Arg Tyr Lys Asp Asn 1 5 10 15 Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala Lys Val Glu Val Tyr Asp 20 25 30 Gly Val Glu Leu Asn Asp Lys Asn Gln Phe Lys Leu Thr Ser Ser Ala 35 40 45 Asn Ser Lys Ile Arg Val Thr Gln Asn Gln Asn Ile Ile Phe Asn Ser 50 55 60 Val Phe Leu Asp Phe Ser Val Ser Phe Trp Ile Arg Ile Pro Lys Tyr 65 70 75 80 Lys Asn Asp Gly Ile Gln Asn Tyr Ile His Asn Glu Tyr Thr Ile Ile 85 90 95 Asn Cys Met Lys Asn Asn Ser Gly Trp Lys Ile Ser Ile Arg Gly Asn 100 105 110 Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn Gly Lys Thr Lys Ser Val 115 120 125 Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile Ser Glu Tyr Ile Asn Arg 130 135 140 Trp Phe Phe Val Thr Ile Thr Asn Asn Leu Asn Asn Ala Lys Ile Tyr 145 150 155 160 Ile Asn Gly Lys Leu Glu Ser Asn Thr Asp Ile Lys Asp Ile Arg Glu 165 170 175 Val Ile Ala Asn Gly Glu Ile Ile Phe Lys Leu Asp Gly Asp Ile Asp 180 185 190 Arg Thr Gln Phe Ile Trp Met Lys Tyr Phe Ser Ile Phe Asn Thr Glu 195 200 205 Leu Ser Gln Ser Asn Ile Glu Glu Arg Tyr Lys Ile Gln Ser Tyr Ser 210 215 220 Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr Asn Lys Glu 225 230 235 240 Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile Lys Leu Lys 245 250 255 Lys Asp Ser Pro Val Gly Glu Ile Leu Thr Arg Ser Lys Tyr Asn Gln 260 265 270 Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu Tyr Ile Gly Glu Lys Phe 275 280 285 Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp Asp Ile Val 290 295 300 Arg Lys Glu Asp Tyr Ile Tyr Leu Asp Phe Phe Asn Leu Asn Gln Glu 305 310 315 320 Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lys Lys Glu Glu Glu Lys Leu 325 330 335 Phe Leu Ala Pro Ile Ser Asp Ser Asp Glu Phe Tyr Asn Thr Ile Gln 340 345 350 Ile Lys Glu Tyr Asp Glu Gln Pro Thr Tyr Ser Cys Gln Leu Leu Phe 355 360 365 Lys Lys Asp Glu Glu Ser Thr Asp Glu Ile Gly Leu Ile Gly Ile His 370 375 380 Arg Phe Tyr Glu Ser Gly Ile Val Phe Glu Glu Tyr Lys Asp Tyr Phe 385 390 395 400 Cys Ile Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys Pro Tyr Asn 405 410 415 Leu Lys Leu Gly Cys Asn Trp Gln Phe Ile Pro Lys Asp Glu Gly Trp 420 425 430 Thr Glu <210> 18 <211> 434 <212> PRT <213> Clostridium botulinum <400> 18 Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn Leu Arg Tyr Arg Asp Asn 1 5 10 15 Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala Lys Val Glu Val Tyr Asp 20 25 30 Gly Val Lys Leu Asn Asp Lys Asn Gln Phe Lys Leu Thr Ser Ser Ala 35 40 45 Asp Ser Lys Ile Arg Val Thr Gln Asn Gln Asn Ile Ile Phe Asn Ser 50 55 60 Met Phe Leu Asp Phe Ser Val Ser Phe Trp Ile Arg Ile Pro Lys Tyr 65 70 75 80 Arg Asn Asp Asp Ile Gln Asn Tyr Ile His Asn Glu Tyr Thr Ile Ile 85 90 95 Asn Cys Met Lys Asn Asn Ser Gly Trp Lys Ile Ser Ile Arg Gly Asn 100 105 110 Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn Gly Lys Thr Lys Ser Val 115 120 125 Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile Ser Glu Tyr Ile Asn Arg 130 135 140 Trp Phe Phe Val Thr Ile Thr Asn Asn Leu Asp Asn Ala Lys Ile Tyr 145 150 155 160 Ile Asn Gly Thr Leu Glu Ser Asn Met Asp Ile Lys Asp Ile Gly Glu 165 170 175 Val Ile Val Asn Gly Glu Ile Thr Phe Lys Leu Asp Gly Asp Val Asp 180 185 190 Arg Thr Gln Phe Ile Trp Met Lys Tyr Phe Ser Ile Phe Asn Thr Gln 195 200 205 Leu Asn Gln Ser Asn Ile Lys Glu Ile Tyr Lys Ile Gln Ser Tyr Ser 210 215 220 Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr Asn Lys Glu 225 230 235 240 Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile Lys Leu Val 245 250 255 Lys Asp Ser Ser Val Gly Glu Ile Leu Ile Arg Ser Lys Tyr Asn Gln 260 265 270 Asn Ser Asn Tyr Ile Asn Tyr Arg Asn Leu Tyr Ile Gly Glu Lys Phe 275 280 285 Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp Asp Ile Val 290 295 300 Arg Lys Glu Asp Tyr Ile His Leu Asp Phe Val Asn Ser Asn Glu Glu 305 310 315 320 Trp Arg Val Tyr Ala Tyr Lys Asn Phe Lys Glu Gln Glu Gln Lys Leu 325 330 335 Phe Leu Ser Ile Ile Tyr Asp Ser Asn Glu Phe Tyr Lys Thr Ile Gln 340 345 350 Ile Lys Glu Tyr Asp Glu Gln Pro Thr Tyr Ser Cys Gln Leu Leu Phe 355 360 365 Lys Lys Asp Glu Glu Ser Thr Asp Asp Ile Gly Leu Ile Gly Ile His 370 375 380 Arg Phe Tyr Glu Ser Gly Val Leu Arg Lys Lys Tyr Lys Asp Tyr Phe 385 390 395 400 Cys Ile Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys Pro Tyr Lys 405 410 415 Ser Asn Leu Gly Cys Asn Trp Gln Phe Ile Pro Lys Asp Glu Gly Trp 420 425 430 Thr Glu <210> 19 <211> 435 <212> PRT <213> Clostridium botulinum <400> 19 Asn Ser Glu Ile Leu Asn Asn Ile Ile Leu Asn Leu Arg Tyr Lys Asp 1 5 10 15 Asn Asn Leu Ile Asp Leu Ser Gly Tyr Gly Ala Lys Val Glu Val Tyr 20 25 30 Asp Gly Val Glu Leu Asn Asp Lys Asn Gln Phe Lys Leu Thr Ser Ser 35 40 45 Ala Asn Ser Lys Ile Arg Val Thr Gln Asn Gln Asn Ile Ile Phe Asn 50 55 60 Ser Val Phe Leu Asp Phe Ser Val Ser Phe Trp Ile Arg Ile Pro Lys 65 70 75 80 Tyr Lys Asn Asp Gly Ile Gln Asn Tyr Ile His Asn Glu Tyr Thr Ile 85 90 95 Ile Asn Cys Met Lys Asn Asn Ser Gly Trp Lys Ile Ser Ile Arg Gly 100 105 110 Asn Arg Ile Ile Trp Thr Leu Ile Asp Ile Asn Gly Lys Thr Lys Ser 115 120 125 Val Phe Phe Glu Tyr Asn Ile Arg Glu Asp Ile Ser Glu Tyr Ile Asn 130 135 140 Arg Trp Phe Phe Val Thr Ile Thr Asn Asn Leu Asn Asn Ala Lys Ile 145 150 155 160 Tyr Ile Asn Gly Lys Leu Glu Ser Asn Thr Asp Ile Lys Asp Ile Arg 165 170 175 Glu Val Ile Ala Asn Gly Glu Ile Ile Phe Lys Leu Asp Gly Asp Ile 180 185 190 Asp Arg Thr Gln Phe Ile Trp Met Lys Tyr Phe Ser Ile Phe Asn Thr 195 200 205 Glu Leu Ser Gln Ser Asn Ile Glu Glu Arg Tyr Lys Ile Gln Ser Tyr 210 215 220 Ser Glu Tyr Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr Asn Lys 225 230 235 240 Glu Tyr Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile Lys Leu 245 250 255 Lys Lys Asp Ser Pro Val Gly Glu Ile Leu Thr Arg Ser Lys Tyr Asn 260 265 270 Gln Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu Tyr Ile Gly Glu Lys 275 280 285 Phe Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp Asp Ile 290 295 300 Val Arg Lys Glu Asp Tyr Ile Tyr Leu Asp Phe Phe Asn Leu Asn Gln 305 310 315 320 Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lys Lys Glu Glu Glu Lys 325 330 335 Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp Glu Phe Tyr Asn Thr Ile 340 345 350 Gln Ile Lys Glu Tyr Asp Glu Gln Pro Thr Tyr Ser Cys Gln Leu Leu 355 360 365 Phe Lys Lys Asp Glu Glu Ser Thr Asp Glu Ile Gly Leu Ile Gly Ile 370 375 380 His Arg Phe Tyr Glu Ser Gly Ile Val Phe Glu Glu Tyr Lys Asp Tyr 385 390 395 400 Phe Cys Ile Ser Lys Trp Tyr Leu Lys Glu Val Lys Arg Lys Pro Tyr 405 410 415 Asn Leu Lys Leu Gly Cys Asn Trp Gln Phe Ile Pro Lys Asp Glu Gly 420 425 430 Trp Thr Glu 435 <210> 20 <211> 1308 <212> DNA <213> Synthetic sequence <220> <221> Source <223> / note = “Description of synthetic sequence: synthetic polypeptide” <400> 20 aacagcgaaa tcctgaacaa cattattctg aacctgcgct ataaagataa caacctgatt 60 gatctgagcg gctatggcgc gaaagtggaa gtgtatgatg gcgtggaact gaacgataaa 120 aaccagttca aactgaccag ctctgcgaac agcaaaattc gtgtgaccca gaaccagaac 180 attatcttca acagcgtgtt tctggatttt agcgtgagct tttggattcg catcccgaaa 240 tataaaaacg atggcatcca gaactatatc cacaacgaat acaccattat caactgcatg 300 aaaaaaca gcggctggaa aattagcatt cgtggcaacc gtattatttg gaccctgatc 360 gatattaacg gcaaaaccaa aagcgtgttc ttcgaataca acatccgcga agatatcagc 420 gaatacatta accgctggtt ctttgtgacc attaccaaca acctgaacaa cgcgaaaatt 480 tatatcaacg gtaaactgga aagcaacacc gatatcaaag atatccgcga agtgattgcg 540 aacggcgaaa tcatctttaa actggatggc gatattgatc gtacccagtt catctggatg 600 aatacttca gcatcttcaa caccgaactg agccagagca acattgaaga acgctacaaa 660 atccagagct atagcgaata cctgaaagat ttttggggca atccgctgat gtataacaaa 720 gagtattaca tgttcaacgc gggtaacaaa aacagctata tcaaactgaa aaaagatagc 780 ccggtgggcg aaattctgac ccgtagcaaa tataaccaga acagcaaata catcaactat 840 cgcgatctgt atatcggcga aaaatttatc attcgccgca aaagcaacag ccagagcatt 900 aacgatgata tcgtgcgcaa agaagattat atctacctgg atttttcaa cctgaaccag 960 gaatggcgcg tttataccta taatatttc aaaaaagagg aagagaaact gtttctggcc 1020 ccgattagcg atagcgatga atttacaac accatccaaa ttaaagaata cgatgaacag 1080 ccgacctata gctgccagct gctgtttaaa aaagatgaag aaagcaccga tgaaattggc 1140 ctgattggca tccatcgttt ctatgaaagc ggcatcgtgt tcgaagaata taaagattat 1200 ttctgcatca gcaaatggta tctgaaagaa gtgaaacgca aaccgtataa cctgaaactg 1260 ggctgcaact ggcagttat tccgaaagat gaaggctgga ccgaataa 1308 <210> 21 <211> 1296 <212> PRT <213> Read more <400> 21 Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly 1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro 20 25 30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg 35 40 45 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu 50 55 60 Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr 65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu 85 90 95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val 100 105 110 Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys 115 120 125 Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr 130 135 140 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr 165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe 180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu 195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu 210 215 220 You Have His Gly His Arg You Have Tyr Gly Have You Have Asn Pro Asn 225 230 235 240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu 245 250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys 260 265 270 Phe With Asp Ser To Gln Glu Asn Glue To Phe Arg To Tyr Tyr Tyr Asn 275 280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val 290,295,300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320 Tyr Leu Leu Ser Glu Asp Thr Gly Lys Phe Ser Val Asp Lys Leu 325 330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp 340 345 350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn 355 360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr 370 375 380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg 420 425 430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys 435 440 445 Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe 450 455 460 Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu 465 470 475 480 Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu 485 490 495 Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro 500 505 510 Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu 515 520 525 Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu 530 535 540 Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu 545 550 555 560 His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu 565 570 575 Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys 580 585 590 Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu 595 600 605 Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr 610 615 620 Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala 625 630 635 640 Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu 645 650 655 Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala 660 665 670 Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys 675 680 685 Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu 690 695 700 Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys 705 710 715 720 Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu 725 730 735 Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn 740 745 750 Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp 755 760 765 Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile 770 775 780 Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met 785 790 795 800 Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys 805 810 815 Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly 820 825 830 Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp 835 840 845 Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser 850 855 860 Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn 865 870 875 880 Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser 885 890 895 Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn 900 905 910 Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu 915 920 925 Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser 930 935 940 Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn 945 950 955 960 Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val 965 970 975 Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu 980 985 990 Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser 995 1000 1005 Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg 1010 1015 1020 Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln 1025 1030 1035 Lys Pro Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile 1040 1045 1050 Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp 1055 1060 1065 Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu 1070 1075 1080 Ile Lys Asp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys 1085 1090 1095 Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr Tyr Met 1100 1105 1110 Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val 1115 1120 1125 Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val 1130 1135 1140 Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr 1145 1150 1155 Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn Ile 1160 1165 1170 Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys Asn 1175 1180 1185 Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val Glu 1190 1195 1200 Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser 1205 1210 1215 Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn 1220 1225 1230 Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly 1235 1240 1245 Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala 1250 1255 1260 Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu 1265 1270 1275 Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu 1280 1285 1290 Arg Pro Leu 1295 <210> 22 <211> 1291 <212> PRT <213> Clostridium botulinum <400> 22 Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 Lys Val Leu Val Cys To Be Asp Pro Asn To Asn To Asn To Tyr 305 310 315 320 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340 345 350 Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395 400 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Serve Lys Glu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp 435 440 445 Val Asp Asn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn 465 470 475 480 Tyr Ile Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 Lys Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 Thr Phe Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560 Asp Ala Leu Leu Phe Ser Asn Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 Trp Val Lys Gln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 Asn Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620 Gly Leu Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640 Asn Ala Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655 Glu Leu Leu Ile Pro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile 660 665 670 Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685 Arg Asn Glu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr 705 710 715 720 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730 735 Arg Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp 740 745 750 Phe Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln Ala Ile 755 760 765 Asp Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser Tyr Leu Met 770 775 780 Lys Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu Asp Phe Asp Asn 785 790 795 800 Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 Leu Ile Gly Ser Ala Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu 820 825 830 Lys Thr Ile Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile 835 840 845 Leu Ile Glu Met Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855 860 Ile Leu Asn Leu Arg Tyr Lys Asp Asn Asn Leu Ile Asp Leu Ser Gly 865 870 875 880 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Glu Leu Asn Asp Lys 885 890 895 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asn Ser Lys Ile Arg Val Thr 900 905 910 Gln Asn Gln Asn Ile Ile Phe Asn Ser Val Phe Leu Asp Phe Ser Val 915 920 925 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Lys Asn Asp Gly Ile Gln Asn 930 935 940 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser 945 950 955 960 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965 970 975 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg 980 985 990 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Leu Asn Asn Ala Lys Ile Tyr Ile Asn Gly Lys Leu Glu 1010 1015 1020 Ser Asn Thr Asp Ile Lys Asp Ile Arg Glu Val Ile Ala Asn Gly 1025 1030 1035 Glu Ile Ile Phe Lys Leu Asp Gly Asp Ile Asp Arg Thr Gln Phe 1040 1045 1050 Ile Trp Met Lys Tyr Phe Ser Ile Phe Asn Thr Glu Leu Ser Gln 1055 1060 1065 Ser Asn Ile Glu Glu Arg Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr 1070 1075 1080 Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr 1085 1090 1095 Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile Lys Leu Lys 1100 1105 1110 Lys Asp Ser Pro Val Gly Glu Ile Leu Thr Arg Ser Lys Tyr Asn 1115 1120 1125 Gln Asn Ser Lys Tyr Ile Asn Tyr Arg Asp Leu Tyr Ile Gly Glu 1130 1135 1140 Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp 1145 1150 1155 Asp Ile Val Arg Lys Glu Asp Tyr Ile Tyr Leu Asp Phe Phe Asn 1160 1165 1170 Leu Asn Gln Glu Trp Arg Val Tyr Thr Tyr Lys Tyr Phe Lys Lys 1175 1180 1185 Glu Glu Glu Lys Leu Phe Leu Ala Pro Ile Ser Asp Ser Asp Glu 1190 1195 1200 Phe Tyr Asn Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln Pro Thr 1205 1210 1215 Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp 1220 1225 1230 Glu Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Ile 1235 1240 1245 Val Phe Glu Glu Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr 1250 1255 1260 Leu Lys Glu Val Lys Arg Lys Pro Tyr Asn Leu Lys Leu Gly Cys 1265 1270 1275 Asn Trp Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1280 1285 1290 <210> 23 <211> 1291 <212> PRT <213> Clostridium botulinum <400> 23 Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn 1 5 10 15 Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30 Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45 Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60 Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp 65 70 75 80 Ser Asp Lys Asp Pro Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110 Asp Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125 Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140 Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly 145 150 155 160 Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165 170 175 Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile 180 185 190 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr Asn Asp 195 200 205 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met Asp Pro Ile 210 215 220 Leu Ile Leu Met His Glu Leu Asn His Ala Met His Asn Leu Tyr Gly 225 230 235 240 Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile 245 250 255 Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala 260 265 270 Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr 275 280 285 Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu 290 295 300 Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly 305 310 315 320 Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335 Ser Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350 Glu Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365 Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380 Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn 385 390 395 400 Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405 410 415 Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu 420 425 430 Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg Ser Leu Tyr Asn 435 440 445 Lys Thr Leu Asp Cys Arg Glu Leu Leu Val Lys Asn Thr Asp Leu Pro 450 455 460 Phe Ile Gly Asp Ile Ser Asp Val Lys Thr Asp Ile Phe Leu Arg Lys 465 470 475 480 Asp Ile Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser 485 490 495 Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu 500 505 510 Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly 515 520 525 Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu 530 535 540 Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu 545 550 555 560 Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser Ala 565 570 575 Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val Asn Ala Gly 580 585 590 Val Gln Gly Gly Leu Phe Leu Met Trp Ala Asn Asp Val Val Glu Asp 595 600 605 Phe Thr Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp 610 615 620 Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn 625 630 635 640 Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val 645 650 655 Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly 660 665 670 Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys 675 680 685 Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys Asp Ser 690 695 700 Tyr Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile Ile Thr Gln Phe 705 710 715 720 Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly 725 730 735 Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser 740 745 750 Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu 755 760 765 Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg 770 775 780 Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile 785 790 795 800 Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn 805 810 815 Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly Glu Val Asp Lys Leu 820 825 830 Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile Pro Phe Asn Ile 835 840 845 Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr 850 855 860 Phe Asn Asn Ile Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Arg Lys 865 870 875 880 Asn Thr Leu Val Asp Thr Ser Gly Tyr Asn Ala Glu Val Ser Glu Glu 885 890 895 Gly Asp Val Gln Leu Asn Pro Ile Phe Pro Phe Asp Phe Lys Leu Gly 900 905 910 Ser Ser Gly Glu Asp Arg Gly Lys Val Ile Val Thr Gln Asn Glu Asn 915 920 925 Ile Val Tyr Asn Ser Met Tyr Glu Ser Phe Ser Ile Ser Phe Trp Ile 930 935 940 Arg Ile Asn Lys Trp Val Ser Asn Leu Pro Gly Tyr Thr Ile Ile Asp 945 950 955 960 Ser Val Lys Asn Asn Ser Gly Trp Ser Ile Gly Ile Ile Ser Asn Phe 965 970 975 Leu Val Phe Thr Leu Lys Gln Asn Glu Asp Ser Glu Gln Ser Ile Asn 980 985 990 Phe Ser Tyr Asp Ile Ser Asn Asn Ala Pro Gly Tyr Asn Lys Trp Phe 995 1000 1005 Phe Val Thr Val Thr Asn Asn Met Met Gly Asn Met Lys Ile Tyr 1010 1015 1020 Ile Asn Gly Lys Leu Ile Asp Thr Ile Lys Val Lys Glu Leu Thr 1025 1030 1035 Gly Ile Asn Phe Ser Lys Thr Ile Thr Phe Glu Ile Asn Lys Ile 1040 1045 1050 Pro Asp Thr Gly Leu Ile Thr Ser Asp Ser Asp Asn Ile Asn Met 1055 1060 1065 Trp Ile Arg Asp Phe Tyr Ile Phe Ala Lys Glu Leu Asp Gly Lys 1070 1075 1080 Asp Ile Asn Ile Leu Phe Asn Ser Leu Gln Tyr Thr Asn Val Val 1085 1090 1095 Lys Asp Tyr Trp Gly Asn Asp Leu Arg Tyr Asn Lys Glu Tyr Tyr 1100 1105 1110 Met Val Asn Ile Asp Tyr Leu Asn Arg Tyr Met Tyr Ala Asn Ser 1115 1120 1125 Arg Gln Ile Val Phe Asn Thr Arg Arg Asn Asn Asn Asp Phe Asn 1130 1135 1140 Glu Gly Tyr Lys Ile Ile Ile Lys Arg Ile Arg Gly Asn Thr Asn 1145 1150 1155 Asp Thr Arg Val Arg Gly Gly Asp Ile Leu Tyr Phe Asp Met Thr 1160 1165 1170 Ile Asn Asn Lys Ala Tyr Asn Leu Phe Met Lys Asn Glu Thr Met 1175 1180 1185 Tyr Ala Asp Asn His Ser Thr Glu Asp Ile Tyr Ala Ile Gly Leu 1190 1195 1200 Arg Glu Gln Thr Lys Asp Ile Asn Asp Asn Ile Ile Phe Gln Ile 1205 1210 1215 Gln Pro Met Asn Asn Thr Tyr Tyr Tyr Ala Ser Gln Ile Phe Lys 1220 1225 1230 Ser Asn Phe Asn Gly Glu Asn Ile Ser Gly Ile Cys Ser Ile Gly 1235 1240 1245 Thr Tyr Arg Phe Arg Leu Gly Gly Asp Trp Tyr Arg His Asn Tyr 1250 1255 1260 Leu Val Pro Thr Val Lys Gln Gly Asn Tyr Ala Ser Leu Leu Glu 1265 1270 1275 Ser Thr Ser Thr His Trp Gly Phe Val Pro Val Ser Glu 1280 1285 1290 <210> 24 <211> 1276 <212> PRT <213> Clostridium botulinum <400> 24 Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val Asn Asp 1 5 10 15 Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu Ile Thr Thr 20 25 30 Pro Val Lys Ala Phe Met Ile Thr Gln Asn Ile Trp Val Ile Pro Glu 35 40 45 Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro Pro Arg Pro 50 55 60 Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu Ser Thr Asp 65 70 75 80 Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn Tyr Leu Val Val 100 105 110 Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro Glu Asp Thr Phe Asp 115 120 125 Phe Thr Arg His Thr Thr Asn Ile Ala Val Glu Lys Phe Glu Asn Gly 130 135 140 Ser Trp Lys Val Thr Asn Ile Ile Thr Pro Ser Val Leu Ile Phe Gly 145 150 155 160 Pro Leu Pro Asn Ile Leu Asp Tyr Thr Ala Ser Leu Thr Leu Gln Gly 165 170 175 Gln Gln Ser Asn Pro Ser Phe Glu Gly Phe Gly Thr Leu Ser Ile Leu 180 185 190 Lys Val Ala Pro Glu Phe Leu Leu Thr Phe Ser Asp Val Thr Ser Asn 195 200 205 Gln Ser Ser Ala Val Leu Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 220 Ile Ala Leu Met His Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly 225 230 235 240 Ile Asn Ile Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245 250 255 Phe Phe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270 Phe Gly Gly Leu Asp Val Glu Ile Ile Pro Gln Ile Glu Arg Ser Gln 275 280 285 Leu Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu 290 295 300 Asn Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp 305 310 315 320 Lys Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys Asp Asn 325 330 335 Thr Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser Leu Tyr Ser 340 345 350 Asp Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser Ser Gln Tyr Asn 355 360 365 Val Lys Asn Arg Thr His Tyr Phe Ser Arg His Tyr Leu Pro Val Phe 370 375 380 Ala Asn Ile Leu Asp Asp Asn Ile Tyr Thr Ile Arg Asp Gly Phe Asn 385 390 395 400 Leu Thr Asn Lys Gly Phe Asn Ile Glu Asn Ser Gly Gln Asn Ile Glu 405 410 415 Arg Asn Pro Ala Leu Gln Lys Leu Ser Ser Glu Ser Val Val Asp Leu 420 425 430 Phe Thr Lys Val Cys Leu Arg Leu Thr Lys Asn Ser Arg Asp Asp Ser 435 440 445 Thr Cys Ile Lys Val Lys Asn Asn Arg Leu Pro Tyr Val Ala Asp Lys 450 455 460 Asp Ser Ile Ser Gln Glu Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu 465 470 475 480 Thr Asn Val Gln Asn Tyr Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile 485 490 495 Leu Asp Gly Gln Val Pro Ile Asn Pro Glu Ile Val Asp Pro Leu Leu 500 505 510 Pro Asn Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu Ile Val 515 520 525 Phe Tyr Asp Asp Ile Thr Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr 530 535 540 Tyr Leu Glu Ser Gln Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu 545 550 555 560 Thr Thr Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys Ile Tyr Thr 565 570 575 Phe Leu Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val Gln Ala Gly 580 585 590 Leu Phe Leu Asn Trp Ala Asn Glu Val Val Glu Asp Phe Thr Thr Asn 595 600 605 Ile Met Lys Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Val Ile 610 615 620 Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Gly Asn Ser Ala Leu Arg 625 630 635 640 Gly Asn Phe Asn Gln Ala Phe Ala Thr Ala Gly Val Ala Phe Leu Leu 645 650 655 Glu Gly Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Val Phe Thr Phe 660 665 670 Tyr Ser Ser Ile Gln Glu Arg Glu Lys Ile Ile Lys Thr Ile Glu Asn 675 680 685 Cys Leu Glu Gln Arg Val Lys Arg Trp Lys Asp Ser Tyr Gln Trp Met 690 695 700 Val Ser Asn Trp Leu Ser Arg Ile Thr Thr Gln Phe Asn His Ile Asn 705 710 715 720 Tyr Gln Met Tyr Asp Ser Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala 725 730 735 Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn 740 745 750 Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile 755 760 765 Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val 770 775 780 Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn 785 790 795 800 Lys Phe Asp Leu Arg Thr Lys Thr Glu Leu Ile Asn Leu Ile Asp Ser 805 810 815 His Asn Ile Ile Leu Val Gly Glu Val Asp Arg Leu Lys Ala Lys Val 820 825 830 Asn Glu Ser Phe Glu Asn Thr Met Pro Phe Asn Ile Phe Ser Tyr Thr 835 840 845 Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Ser Ile 850 855 860 Asn Asp Ser Lys Ile Leu Ser Leu Gln Asn Lys Lys Asn Ala Leu Val 865 870 875 880 Asp Thr Ser Gly Tyr Asn Ala Glu Val Arg Val Gly Asp Asn Val Gln 885 890 895 Leu Asn Thr Ile Tyr Thr Asn Asp Phe Lys Leu Ser Ser Ser Gly Asp 900 905 910 Lys Ile Ile Val Asn Leu Asn Asn Asn Ile Leu Tyr Ser Ala Ile Tyr 915 920 925 Glu Asn Ser Ser Val Ser Phe Trp Ile Lys Ile Ser Lys Asp Leu Thr 930 935 940 Asn Ser His Asn Glu Tyr Thr Ile Ile Asn Ser Ile Glu Gln Asn Ser 945 950 955 960 Gly Trp Lys Leu Cys Ile Arg Asn Gly Asn Ile Glu Trp Ile Leu Gln 965 970 975 Asp Val Asn Arg Lys Tyr Lys Ser Leu Ile Phe Asp Tyr Ser Glu Ser 980 985 990 Leu Ser His Thr Gly Tyr Thr Asn Lys Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Ile Met Gly Tyr Met Lys Leu Tyr Ile Asn Gly Glu Leu 1010 1015 1020 Lys Gln Ser Gln Lys Ile Glu Asp Leu Asp Glu Val Lys Leu Asp 1025 1030 1035 Lys Thr Ile Val Phe Gly Ile Asp Glu Asn Ile Asp Glu Asn Gln 1040 1045 1050 Met Leu Trp Ile Arg Asp Phe Asn Ile Phe Ser Lys Glu Leu Ser 1055 1060 1065 Asn Glu Asp Ile Asn Ile Val Tyr Glu Gly Gln Ile Leu Arg Asn 1070 1075 1080 Val Ile Lys Asp Tyr Trp Gly Asn Pro Leu Lys Phe Asp Thr Glu 1085 1090 1095 Tyr Tyr Ile Ile Asn Asp Asn Tyr Ile Asp Arg Tyr Ile Ala Pro 1100 1105 1110 Glu Ser Asn Val Leu Val Leu Val Gln Tyr Pro Asp Arg Ser Lys 1115 1120 1125 Leu Tyr Thr Gly Asn Pro Ile Thr Ile Lys Ser Val Ser Asp Lys 1130 1135 1140 Asn Pro Tyr Ser Arg Ile Leu Asn Gly Asp Asn Ile Ile Leu His 1145 1150 1155 Met Leu Tyr Asn Ser Arg Lys Tyr Met Ile Ile Arg Asp Thr Asp 1160 1165 1170 Thr Ile Tyr Ala Thr Gln Gly Gly Glu Cys Ser Gln Asn Cys Val 1175 1180 1185 Tyr Ala Leu Lys Leu Gln Ser Asn Leu Gly Asn Tyr Gly Ile Gly 1190 1195 1200 Ile Phe Ser Ile Lys Asn Ile Val Ser Lys Asn Lys Tyr Cys Ser 1205 1210 1215 Gln Ile Phe Ser Ser Phe Arg Glu Asn Thr Met Leu Leu Ala Asp 1220 1225 1230 Ile Tyr Lys Pro Trp Arg Phe Ser Phe Lys Asn Ala Tyr Thr Pro 1235 1240 1245 Val Ala Val Thr Asn Tyr Glu Thr Lys Leu Leu Ser Thr Ser Ser 1250 1255 1260 Phe Trp Lys Phe Ile Ser Arg Asp Pro Gly Trp Val Glu 1265 1270 1275 <210> 25 <211> 1252 <212> PRT <213> Clostridium botulinum <400> 25 Met Pro Lys Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg 1 5 10 15 Thr Ile Leu Tyr Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr Thr Pro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys 65 70 75 80 Asp Arg Phe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95 Asn Leu Ser Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110 Tyr Leu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp 115 120 125 Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu 130 135 140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170 175 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180 185 190 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu 195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr Gly Ala 210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln Asn Pro Leu 225 230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu Phe Leu Thr Phe Gly 245 250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala Gln Ser Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285 Val Gln Will Be Asn Pro Leu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290,295,300 Ala Lys Tyr Gly Leu Asp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 305 310 315 320 Ile Asn Lys Phe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 Phe Asp Leu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350 Gly Gln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asp Ser Ile 355 360 365 Tyr Asn Ile Is Glu Gly Tyr Asn Ile Asn Leu Lys Val Asn Phe 370 375 380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg With Thr Pro With Thr 385 390 395 400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410 415 Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile Asn Asn Gly 420 425 430 Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn Asp Asp Asn Ile 435 440 445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr Ser Asn Asn Asn Tyr 450 455 460 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn Phe Asn Ser Glu Ser Ala 465 470 475 480 Pro Gly Leu Ser Asp Glu Lys Leu Asn Leu Thr Ile Gln Asn Asp Ala 485 490 495 Tyr Ile Pro Lys Tyr Asp Ser Asn Gly Thr Ser Asp Ile Glu Gln His 500 505 510 Asp Val Asn Glu Leu Asn Val Phe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 Pro Glu Gly Glu Asn Asn Val Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 Leu Leu Glu Gln Pro Lys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 545 550 555 560 Asn Asn Val Asn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575 Gln Gln Val Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590 Val Asp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605 Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala 610 615 620 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu 625 630 635 640 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu Gly Ser 645 650 655 Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn Ala Leu Lys 660 665 670 Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe Ile Val Ser Asn 675 680 685 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln Met 690 695 700 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala Ile Lys Thr Ile Ile Glu 705 710 715 720 Ser Lys Tyr Asn Ser Tyr Thr Leu Glu Glu Lys Asn Glu Leu Thr Asn 725 730 735 Lys Tyr Asp Ile Lys Gln Ile Glu Asn Glu Leu Asn Gln Lys Val Ser 740 745 750 Ile Ala Met Asn Asn Ile Asp Arg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 Tyr Leu Met Lys Leu Ile Asn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 Tyr Asp Glu Asn Val Lys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His 785 790 795 800 Gly Ser Ile Leu Gly Glu Ser Gln Gln Glu Leu Asn Ser Met Val Thr 805 810 815 Asp Thr Leu Asn Asn Ser Ile Pro Phe Lys Leu Ser Ser Tyr Thr Asp 820 825 830 Asp Lys Ile Leu Ile Ser Tyr Phe Asn Lys Phe Phe Lys Arg Ile Lys 835 840 845 Ser Ser Ser Val Leu Asn Met Arg Tyr Lys Asn Asp Lys Tyr Val Asp 850 855 860 Thr Ser Gly Tyr Asp Ser Asn Ile Asn Ile Asn Gly Asp Val Tyr Lys 865 870 875 880 Tyr Pro Thr Asn Lys Asn Gln Phe Gly Ile Tyr Asn Asp Lys Leu Ser 885 890 895 Glu Val Asn Ile Ser Gln Asn Asp Tyr Ile Ile Tyr Asp Asn Lys Tyr 900 905 910 Lys Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Asn Tyr Asp Asn 915 920 925 Lys Ile Val Asn Val Asn Asn Glu Tyr Thr Ile Ile Asn Cys Met Arg 930 935 940 Asp Asn Asn Ser Gly Trp Lys Val Ser Leu Asn His Asn Glu Ile Ile 945 950 955 960 Trp Thr Leu Gln Asp Asn Ala Gly Ile Asn Gln Lys Leu Ala Phe Asn 965 970 975 Tyr Gly Asn Ala Asn Gly Ile Ser Asp Tyr Ile Asn Lys Trp Ile Phe 980 985 990 Val Thr Ile Thr Asn Asp Arg Leu Gly Asp Ser Lys Leu Tyr Ile Asn 995 1000 1005 Gly Asn Leu Ile Asp Gln Lys Ser Ile Leu Asn Leu Gly Asn Ile 1010 1015 1020 His Val Ser Asp Asn Ile Leu Phe Lys Ile Val Asn Cys Ser Tyr 1025 1030 1035 Thr Arg Tyr Ile Gly Ile Arg Tyr Phe Asn Ile Phe Asp Lys Glu 1040 1045 1050 Leu Asp Glu Thr Glu Ile Gln Thr Leu Tyr Ser Asn Glu Pro Asn 1055 1060 1065 Thr Asn Ile Leu Lys Asp Phe Trp Gly Asn Tyr Leu Leu Tyr Asp 1070 1075 1080 Lys Glu Tyr Tyr Leu Leu Asn Val Leu Lys Pro Asn Asn Phe Ile 1085 1090 1095 Asp Arg Arg Lys Asp Ser Thr Leu Ser Ile Asn Asn Ile Arg Ser 1100 1105 1110 Thr Ile Leu Leu Ala Asn Arg Leu Tyr Ser Gly Ile Lys Val Lys 1115 1120 1125 Ile Gln Arg Val Asn Asn Ser Ser Thr Asn Asp Asn Leu Val Arg 1130 1135 1140 Lys Asn Asp Gln Val Tyr Ile Asn Phe Val Ala Ser Lys Thr His 1145 1150 1155 Leu Phe Pro Leu Tyr Ala Asp Thr Ala Thr Thr Asn Lys Glu Lys 1160 1165 1170 Thr Ile Lys Ile Ser Ser Ser Gly Asn Arg Phe Asn Gln Val Val 1175 1180 1185 Val Met Asn Ser Val Gly Asn Asn Cys Thr Met Asn Phe Lys Asn 1190 1195 1200 Asn Asn Gly Asn Asn Ile Gly Leu Leu Gly Phe Lys Ala Asp Thr 1205 1210 1215 Val Val Ala Ser Thr Trp Tyr Tyr Thr His Met Arg Asp His Thr 1220 1225 1230 Asn Ser Asn Gly Cys Phe Trp Asn Phe Ile Ser Glu Glu His Gly 1235 1240 1245 Trp Gln Glu Lys 1250 <210> 26 <211> 1274 <212> PRT <213> Clostridium botulinum <400> 26 Met Pro Val Ala Ile Asn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp 1 5 10 15 Asp Thr Ile Leu Tyr Met Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30 Tyr Tyr Lys Ala Phe Glu Ile Met Arg Asn Val Trp Ile Ile Pro Glu 35 40 45 Arg Asn Thr Ile Gly Thr Asn Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60 Leu Lys Asn Gly Ser Ser Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr 65 70 75 80 Asp Ala Glu Lys Asp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys 85 90 95 Arg Ile Asn Ser Asn Pro Ala Gly Lys Val Leu Leu Gln Glu Ile Ser 100 105 110 Tyr Ala Lys Pro Tyr Leu Gly Asn Asp His Thr Pro Ile Asp Glu Phe 115 120 125 Ser Pro Val Thr Arg Thr Thr Ser Val Asn Ile Lys Leu Ser Thr Asn 130 135 140 Val Glu Ser Ser Met Leu Leu Asn Leu Leu Val Leu Gly Ala Gly Pro 145 150 155 160 Asp Ile Phe Glu Ser Cys Cys Tyr Pro Val Arg Lys Leu Ile Asp Pro 165 170 175 Asp Val Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile 180 185 190 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly 195 200 205 Gly His Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser 210 215 220 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala Arg 225 230 235 240 Gly Val Thr Tyr Glu Glu Thr Ile Glu Val Lys Gln Ala Pro Leu Met 245 250 255 Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu Thr Phe Gly Gly 260 265 270 Gln Asp Leu Asn With Thr Ser On Met Lys Glu Lys With Tyr Asn 275 280 285 The Thr Arg Asn of Glu Val 290,295,300 Asn Ser Ala Pro Pro Glu Tyr Asp With Asn Glu Tyr Lys Asp Tyr Phe 305 310 315 320 Gln Trp Lys Tyr Gly Leu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325 330 335 Asn Glu Asn Lys Phe Asn Glu Ile Tyr Lys Lys Leu Tyr Ser Phe Thr 340 345 350 Glu Ser Asp Leu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr 355 360 365 Phe Ile Lys Tyr Glu Phe Leu Lys Val Pro Asn Leu Leu Asp Asp 370 375 380 Ile Tyr Thr Val Served Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn 385 390 395 400 Asn Arg Gly Gln Ser Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410 415 Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val 420 425 430 Ile Pro Arg Lys Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile Arg Val 435 440 445 Asn Asn Ser Glu Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu 450 455 460 Asn Asp Ile Asn Thr Pro Lys Glu Ile Asp Asp Thr Thr Asn Leu Asn 465 470 475 480 Asn Asn Tyr Arg Asn Asn Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser 485 490 495 Gln Thr Ile Pro Gln Ile Ser Asn Arg Thr Leu Asn Thr Leu Val Gln 500 505 510 Asp Asn Ser Tyr Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser Glu Ile 515 520 525 Glu Glu Tyr Asp Val Val Asp Phe Asn Val Phe Phe Tyr Leu His Ala 530 535 540 Gln Lys Val Pro Glu Gly Glu Thr Asn Ile Ser Leu Thr Ser Ser Ile 545 550 555 560 Asp Thr Ala Leu Leu Glu Glu Ser Lys Asp Ile Phe Phe Ser Ser Glu 565 570 575 Phe Ile Asp Thr Ile Asn Lys Pro Val Asn Ala Ala Leu Phe Ile Asp 580 585 590 Trp Ile Ser Lys Val Ile Arg Asp Phe Thr Thr Glu Ala Thr Gln Lys 595 600 605 Ser Thr Val Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Val 610 615 620 Gly Leu Ala Leu Asn Ile Ile Ile Glu Ala Glu Lys Gly Asn Phe Glu 625 630 635 640 Glu Ala Phe Glu Leu Leu Gly Val Gly Ile Leu Leu Glu Phe Val Pro 645 650 655 Glu Leu Thr Ile Pro Val Ile Leu Val Phe Thr Ile Lys Ser Tyr Ile 660 665 670 Asp Ser Tyr Glu Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn Asn Ser 675 680 685 Leu Ile Glu Arg Glu Ala Lys Trp Lys Glu Ile Tyr Ser Trp Ile Val 690 695 700 Ser Asn Trp Leu Thr Arg Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu 705 710 715 720 Gln Met Tyr Gln Ala Leu Gln Asn Gln Val Asp Ala Ile Lys Thr Ala 725 730 735 Ile Glu Tyr Lys Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg Leu 740 745 750 Glu Ser Glu Tyr Asn Ile Asn Asn Ile Glu Glu Glu Leu Asn Light Light 755 760 765 Val Ser Leu Ala Met Lys Asn Ile Glu Arg Phe Met Thr Glu Ser Ser 770 775 780 Ile Ser Tyr Leu Met Lys Leu Ile Asn Glu Ala Lys Val Gly Lys Leu 785 790 795 800 Lys Lys Tyr Asp Asn His Val Lys Ser Asp Leu Leu Asn Tyr Ile Leu 805 810 815 Asp His Arg Ser Ile Leu Gly Glu Gln Thr Asn Glu Leu Ser Asp Leu 820 825 830 Val Thr Ser Thr Leu Asn Ser Ser Ile Pro Phe Glu Leu Ser Ser Tyr 835 840 845 Thr Asn Asp Lys Ile Leu Ile Ile Tyr Phe Asn Arg Leu Tyr Lys Lys 850 855 860 Ile Lys Asp Ser Ser Ile Leu Asp Met Arg Tyr Glu Asn Asn Lys Phe 865 870 875 880 Ile Asp Ile Ser Gly Tyr Gly Ser Asn Ile Ser Ile Asn Gly Asn Val 885 890 895 Tyr Ile Tyr Ser Thr Asn Arg Asn Gln Phe Gly Ile Tyr Asn Ser Arg 900 905 910 Leu Ser Glu Val Asn Ile Ala Gln Asn Asn Asp Ile Ile Tyr Asn Ser 915 920 925 Arg Tyr Gln Asn Phe Ser Ile Ser Phe Trp Val Arg Ile Pro Lys His 930 935 940 Tyr Lys Pro Met Asn His Asn Arg Glu Tyr Thr Ile Ile Asn Cys Met 945 950 955 960 Gly Asn Asn Asn Ser Gly Trp Lys Ile Ser Leu Arg Thr Val Arg Asp 965 970 975 Cys Glu Ile Ile Trp Thr Leu Gln Asp Thr Ser Gly Asn Lys Glu Asn 980 985 990 Leu Ile Phe Arg Tyr Glu Glu Leu Asn Arg Ile Ser Asn Tyr Ile Asn 995 1000 1005 Lys Trp Ile Phe Val Thr Ile Thr Asn Asn Arg Leu Gly Asn Ser 1010 1015 1020 Arg Ile Tyr Ile Asn Gly Asn Leu Ile Val Glu Lys Ser Ile Ser 1025 1030 1035 Asn Leu Gly Asp Ile His Val Ser Asp Asn Ile Leu Phe Lys Ile 1040 1045 1050 Val Gly Cys Asp Asp Glu Thr Tyr Val Gly Ile Arg Tyr Phe Lys 1055 1060 1065 Val Phe Asn Thr Glu Leu Asp Lys Thr Glu Ile Glu Thr Leu Tyr 1070 1075 1080 Ser Asn Glu Pro Asp Pro Ser Ile Leu Lys Asn Tyr Trp Gly Asn 1085 1090 1095 Tyr Leu Leu Tyr Asn Lys Lys Tyr Tyr Leu Phe Asn Leu Leu Arg 1100 1105 1110 Lys Asp Lys Tyr Ile Thr Leu Asn Ser Gly Ile Leu Asn Ile Asn 1115 1120 1125 Gln Gln Arg Gly Val Thr Glu Gly Ser Val Phe Leu Asn Tyr Lys 1130 1135 1140 Leu Tyr Glu Gly Val Glu Val Ile Ile Arg Lys Asn Gly Pro Ile 1145 1150 1155 Asp Ile Ser Asn Thr Asp Asn Phe Val Arg Lys Asn Asp Leu Ala 1160 1165 1170 Tyr Ile Asn Val Val Asp Arg Gly Val Glu Tyr Arg Leu Tyr Ala 1175 1180 1185 Asp Thr Lys Ser Glu Lys Glu Lys Ile Ile Arg Thr Ser Asn Leu 1190 1195 1200 Asn Asp Ser Leu Gly Gln Ile Ile Val Met Asp Ser Ile Gly Asn 1205 1210 1215 Asn Cys Thr Met Asn Phe Gln Asn Asn Asn Gly Ser Asn Ile Gly 1220 1225 1230 Leu Leu Gly Phe His Ser Asn Asn Leu Val Ala Ser Ser Trp Tyr 1235 1240 1245 Tyr Asn Asn Ile Arg Arg Asn Thr Ser Ser Asn Gly Cys Phe Trp 1250 1255 1260 Ser Ser Ile Ser Lys Glu Asn Gly Trp Lys Glu 1265 1270 <210> 27 <211> 1297 <212> PRT <213> Clostridium botulinum <400> 27 Met Pro Val Asn Ile Lys Asn Phe Asn Tyr Asn Asp Pro Ile Asn Asn 1 5 10 15 Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr 20 25 30 Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu 35 40 45 Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly 50 55 60 Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys 65 70 75 80 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe 85 90 95 Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile 100 105 110 Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys 115 120 125 Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile Ile Gln 130 135 140 Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu Met Thr Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile 165 170 175 Met Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met 180 185 190 Ile Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 220 Ala Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu Phe 245 250 255 Phe Met Gln His Ser Asp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile 275 280 285 Tyr Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn 290 295 300 Ile Val Ser Ser Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu Tyr Lys 305 310 315 320 Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys 325 330 335 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met 340 345 350 Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile Lys Thr 355 360 365 Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile Lys Thr Glu Lys 370 375 380 Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe Asn Ile Ala 385 390 395 400 Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gln Asn Lys Ala Val Asn 405 410 415 Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg 420 425 430 Ile Ala Met Cys Lys Pro Val Met Tyr Lys Asn Thr Gly Lys Ser Glu 435 440 445 Gln Cys Ile Ile Val Asn Asn Glu Asp Leu Phe Phe Ile Ala Asn Lys 450 455 460 Asp Ser Phe Ser Lys Asp Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn 465 470 475 480 Thr Gln Asn Asn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile 485 490 495 Leu Asp Asn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr 500 505 510 Glu Pro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Lys 515 520 525 Gln Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu 530 535 540 Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile Glu Asn Leu Gln Leu 545 550 555 560 Thr Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr Thr 565 570 575 Phe Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val Gly Ala 580 585 590 Ser Leu Phe Val Asn Trp Val Lys Gly Val Ile Asp Asp Phe Thr Ser 595 600 605 Glu Ser Thr Gln Lys Ser Thr Ile Asp Lys Val Ser Asp Val Ser Ile 610 615 620 Ile Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val Gly Asn Glu Thr Ala 625 630 635 640 Lys Glu Asn Phe Lys Asn Ala Phe Glu Ile Gly Gly Ala Ala Ile Leu 645 650 655 Met Glu Phe Ile Pro Glu Leu Ile Val Pro Ile Val Gly Phe Phe Thr 660 665 670 Leu Glu Ser Tyr Val Gly Asn Lys Gly His Ile Ile Met Thr Ile Ser 675 680 685 Asn Ala Leu Lys Lys Arg Asp Gln Lys Trp Thr Asp Met Tyr Gly Leu 690 695 700 Ile Val Ser Gln Trp Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile 705 710 715 720 Lys Glu Arg Met Tyr Asn Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu 725 730 735 Lys Ile Ile Glu Asp Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met 740 745 750 Asn Ile Asn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln Ser 755 760 765 Ile Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser 770 775 780 Ile Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys Leu 785 790 795 800 Lys Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu Tyr Ile Asp 805 810 815 Thr Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn Ile Leu Lys Ser Lys 820 825 830 Val Asn Arg His Leu Lys Asp Ser Ile Pro Phe Asp Leu Ser Leu Tyr 835 840 845 Thr Lys Asp Thr Ile Leu Ile Gln Val Phe Asn Asn Tyr Ile Ser Asn 850 855 860 Ile Ser Ser Asn Ala Ile Leu Ser Leu Ser Tyr Arg Gly Gly Arg Leu 865 870 875 880 Ile Asp Ser Ser Gly Tyr Gly Ala Thr Met Asn Val Gly Ser Asp Val 885 890 895 Ile Phe Asn Asp Ile Gly Asn Gly Gln Phe Lys Leu Asn Asn Ser Glu 900 905 910 Asn Ser Asn Ile Thr Ala His Gln Ser Lys Phe Val Val Tyr Asp Ser 915 920 925 Met Phe Asp Asn Phe Ser Ile Asn Phe Trp Val Arg Thr Pro Lys Tyr 930 935 940 Asn Asn Asn Asp Ile Gln Thr Tyr Leu Gln Asn Glu Tyr Thr Ile Ile 945 950 955 960 Ser Cys Ile Lys Asn Asp Ser Gly Trp Lys Val Ser Ile Lys Gly Asn 965 970 975 Arg Ile Ile Trp Thr Leu Ile Asp Val Asn Ala Lys Ser Lys Ser Ile 980 985 990 Phe Phe Glu Tyr Ser Ile Lys Asp Asn Ile Ser Asp Tyr Ile Asn Lys 995 1000 1005 Trp Phe Ser Ile Thr Ile Thr Asn Asp Arg Leu Gly Asn Ala Asn 1010 1015 1020 Ile Tyr Ile Asn Gly Ser Leu Lys Lys Ser Glu Lys Ile Leu Asn 1025 1030 1035 Leu Asp Arg Ile Asn Ser Ser Asn Asp Ile Asp Phe Lys Leu Ile 1040 1045 1050 Asn Cys Thr Asp Thr Thr Lys Phe Val Trp Ile Lys Asp Phe Asn 1055 1060 1065 Ile Phe Gly Arg Glu Leu Asn Ala Thr Glu Val Ser Ser Leu Tyr 1070 1075 1080 Trp Ile Gln Ser Ser Thr Asn Thr Leu Lys Asp Phe Trp Gly Asn 1085 1090 1095 Pro Leu Arg Tyr Asp Thr Gln Tyr Tyr Leu Phe Asn Gln Gly Met 1100 1105 1110 Gln Asn Ile Tyr Ile Lys Tyr Phe Ser Lys Ala Ser Met Gly Glu 1115 1120 1125 Thr Ala Pro Arg Thr Asn Phe Asn Asn Ala Ala Ile Asn Tyr Gln 1130 1135 1140 Asn Leu Tyr Leu Gly Leu Arg Phe Ile Ile Lys Lys Ala Ser Asn 1145 1150 1155 Ser Arg Asn Ile Asn Asn Asp Asn Ile Val Arg Glu Gly Asp Tyr 1160 1165 1170 Ile Tyr Leu Asn Ile Asp Asn Ile Ser Asp Glu Ser Tyr Arg Val 1175 1180 1185 Tyr Val Leu Val Asn Ser Lys Glu Ile Gln Thr Gln Leu Phe Leu 1190 1195 1200 Ala Pro Ile Asn Asp Asp Pro Thr Phe Tyr Asp Val Leu Gln Ile 1205 1210 1215 Lys Lys Tyr Tyr Glu Lys Thr Thr Tyr Asn Cys Gln Ile Leu Cys 1220 1225 1230 Glu Lys Asp Thr Lys Thr Phe Gly Leu Phe Gly Ile Gly Lys Phe 1235 1240 1245 Val Lys Asp Tyr Gly Tyr Val Trp Asp Thr Tyr Asp Asn Tyr Phe 1250 1255 1260 Cys Ile Ser Gln Trp Tyr Leu Arg Arg Ile Ser Glu Asn Ile Asn 1265 1270 1275 Lys Leu Arg Leu Gly Cys Asn Trp Gln Phe Ile Pro Val Asp Glu 1280 1285 1290 Gly Trp Thr Glu 1295 <210> 28 <211> 1291 <212> PRT <213> Clostridium botulinum <400> 28 Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15 Asp Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60 Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 75 80 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 90 95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu 115 120 125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile Ser Asn 130 135 140 Pro Gly Glu Val Glu Gln Lys Lys Gly Ile Phe Ala Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 Phe Met Gln Ser Thr Asp Thr Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln Asp Pro Ser Ile Ile Ser Pro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290,295,300 Lys Val Leu Val Cys To Be Asp Pro Asn To Asn To Asn To Tyr 305 310 315 320 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330 335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asn Lys Leu Tyr Lys Ser Leu 340 345 350 Met Phe Gly Phe Thr Glu Ile Asn Ile Ala Glu Asn Tyr Lys Ile Lys 355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys Ile Lys 370 375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly Phe Asn Ile 385 390 395 400 Ser Asp Lys Asn Met Gly Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Serve Lys Glu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys Lys Ser Val Lys Val Pro Gly Ile Cys Ile Asp 435 440 445 Val Asp Asn Glu Asn Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser 450 455 460 Asp Asp Leu Ser Lys Asn Glu Arg Val Glu Tyr Asn Thr Gln Asn Asn 465 470 475 480 Tyr Ile Gly Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp 485 490 495 Leu Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu Thr 500 505 510 Asp Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala Ile Lys 515 520 525 Lys Val Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu Tyr Ser Gln 530 535 540 Thr Phe Pro Leu Asn Ile Arg Asp Ile Ser Leu Thr Ser Ser Phe Asp 545 550 555 560 Asp Ala Leu Leu Val Ser Ser Lys Val Tyr Ser Phe Phe Ser Met Asp 565 570 575 Tyr Ile Lys Thr Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly 580 585 590 Trp Val Lys Gln Ile Val Asp Asp Phe Val Ile Glu Ala Asn Lys Ser 595 600 605 Ser Thr Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile 610 615 620 Gly Leu Ala Leu Asn Val Gly Asp Glu Thr Ala Lys Gly Asn Phe Glu 625 630 635 640 Ser Ala Phe Glu Ile Ala Gly Ser Ser Ile Leu Leu Glu Phe Ile Pro 645 650 655 Glu Leu Leu Ile Pro Val Val Gly Val Phe Leu Leu Glu Ser Tyr Ile 660 665 670 Asp Asn Lys Asn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys 675 680 685 Arg Val Glu Lys Trp Ile Asp Met Tyr Gly Leu Ile Val Ala Gln Trp 690 695 700 Leu Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr 705 710 715 720 Lys Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr 725 730 735 Lys Tyr Asn Ile Tyr Ser Glu Glu Glu Lys Ser Asn Ile Asn Ile Asn 740 745 750 Phe Asn Asp Ile Asn Ser Lys Leu Asn Asp Gly Ile Asn Gln Ala Met 755 760 765 Asp Asn Ile Asn Asp Phe Ile Asn Glu Cys Ser Val Ser Tyr Leu Met 770 775 780 Lys Lys Met Ile Pro Leu Ala Val Lys Lys Leu Leu Asp Phe Asp Asn 785 790 795 800 Thr Leu Lys Lys Asn Leu Leu Asn Tyr Ile Asp Glu Asn Lys Leu Tyr 805 810 815 Leu Ile Gly Ser Val Glu Asp Glu Lys Ser Lys Val Asp Lys Tyr Leu 820 825 830 Lys Thr Ile Ile Pro Phe Asp Leu Ser Thr Tyr Thr Asn Asn Glu Ile 835 840 845 Leu Ile Lys Ile Phe Asn Lys Tyr Asn Ser Glu Ile Leu Asn Asn Ile 850 855 860 Ile Leu Asn Leu Arg Tyr Arg Asp Asn Asn Leu Ile Asp Leu Ser Gly 865 870 875 880 Tyr Gly Ala Lys Val Glu Val Tyr Asp Gly Val Lys Leu Asn Asp Lys 885 890 895 Asn Gln Phe Lys Leu Thr Ser Ser Ala Asp Ser Lys Ile Arg Val Thr 900 905 910 Gln Asn Gln Asn Ile Ile Phe Asn Ser Met Phe Leu Asp Phe Ser Val 915 920 925 Ser Phe Trp Ile Arg Ile Pro Lys Tyr Arg Asn Asp Asp Ile Gln Asn 930 935 940 Tyr Ile His Asn Glu Tyr Thr Ile Ile Asn Cys Met Lys Asn Asn Ser 945 950 955 960 Gly Trp Lys Ile Ser Ile Arg Gly Asn Arg Ile Ile Trp Thr Leu Ile 965 970 975 Asp Ile Asn Gly Lys Thr Lys Ser Val Phe Phe Glu Tyr Asn Ile Arg 980 985 990 Glu Asp Ile Ser Glu Tyr Ile Asn Arg Trp Phe Phe Val Thr Ile Thr 995 1000 1005 Asn Asn Leu Asp Asn Ala Lys Ile Tyr Ile Asn Gly Thr Leu Glu 1010 1015 1020 Ser Asn Met Asp Ile Lys Asp Ile Gly Glu Val Ile Val Asn Gly 1025 1030 1035 Glu Ile Thr Phe Lys Leu Asp Gly Asp Val Asp Arg Thr Gln Phe 1040 1045 1050 Ile Trp Met Lys Tyr Phe Ser Ile Phe Asn Thr Gln Leu Asn Gln 1055 1060 1065 Ser Asn Ile Lys Glu Ile Tyr Lys Ile Gln Ser Tyr Ser Glu Tyr 1070 1075 1080 Leu Lys Asp Phe Trp Gly Asn Pro Leu Met Tyr Asn Lys Glu Tyr 1085 1090 1095 Tyr Met Phe Asn Ala Gly Asn Lys Asn Ser Tyr Ile Lys Leu Val 1100 1105 1110 Lys Asp Ser Ser Val Gly Glu Ile Leu Ile Arg Ser Lys Tyr Asn 1115 1120 1125 Gln Asn Ser Asn Tyr Ile Asn Tyr Arg Asn Leu Tyr Ile Gly Glu 1130 1135 1140 Lys Phe Ile Ile Arg Arg Lys Ser Asn Ser Gln Ser Ile Asn Asp 1145 1150 1155 Asp Ile Val Arg Lys Glu Asp Tyr Ile His Leu Asp Phe Val Asn 1160 1165 1170 Ser Asn Glu Glu Trp Arg Val Tyr Ala Tyr Lys Asn Phe Lys Glu 1175 1180 1185 Gln Glu Gln Lys Leu Phe Leu Ser Ile Ile Tyr Asp Ser Asn Glu 1190 1195 1200 Phe Tyr Lys Thr Ile Gln Ile Lys Glu Tyr Asp Glu Gln Pro Thr 1205 1210 1215 Tyr Ser Cys Gln Leu Leu Phe Lys Lys Asp Glu Glu Ser Thr Asp 1220 1225 1230 Asp Ile Gly Leu Ile Gly Ile His Arg Phe Tyr Glu Ser Gly Val 1235 1240 1245 Leu Arg Lys Lys Tyr Lys Asp Tyr Phe Cys Ile Ser Lys Trp Tyr 1250 1255 1260 Leu Lys Glu Val Lys Arg Lys Pro Tyr Lys Ser Asn Leu Gly Cys 1265 1270 1275 Asn Trp Gln Phe Ile Pro Lys Asp Glu Gly Trp Thr Glu 1280 1285 1290 <210> 29 <211> twenty two <212> PRT <213> unknown <220> <221> source <223> / note = "Unknown description: mouse / rat Syt I sequence" <400> 29 Gly Glu Gly Lys Glu Asp Ala Phe Ser Lys Leu Lys Gln Lys Phe Met 1 5 10 15 Asn Glu Leu His Lys Ile 20

Claims

1. A polypeptide comprising a modified receptor binding domain of the heavy chain of Clostridium botulinum serotype B, strain 4 (B4-H c ), the modified receptor binding domain is based on the same amino acid sequence as the reference B4-Hc consisting of the amino acid sequence shown in SEQ ID NO: 18, wherein, Relative to the reference B4-Hc consisting of the amino acid sequence shown in SEQ ID NO:18, the amino acid sequence of the modified receptor binding domain is modified to replace mutations V1113K, S1117P, S1196A and I1197P, and wherein relative to the reference B4-Hc, the modification enhances the binding of the modified B4-Hc to human SytII.

2. A botulinum neurotoxin (BoNT) polypeptide comprising: a) protease domain; b) a protease cleavage site; c) a translocation domain; and d) Modified receptor binding domain of the heavy chain of Clostridium botulinum serotype B, strain 4 (B4-H c ), the modified receptor binding domain is based on the same amino acid sequence as the reference B4-Hc consisting of the amino acid sequence shown in SEQ ID NO: 18, wherein, Relative to the reference B4-Hc consisting of the amino acid sequence shown in SEQ ID NO:18, the amino acid sequence of the modified receptor binding domain is modified to replace mutations V1113K, S1117P, S1196A and I1197P, and wherein relative to the reference B4-Hc, the modification enhances the binding of the modified B4-Hc to human SytII.

3. A chimeric molecule comprising a first portion, the first portion being a modified receptor binding domain of the heavy chain of Clostridium botulinum serotype B, strain 4 (B4-H c ), the modified receptor binding domain is based on the same amino acid sequence as the reference B4-Hc consisting of the amino acid sequence shown in SEQ ID NO: 18, wherein, Relative to the reference B4-Hc consisting of the amino acid sequence according to SEQ ID NO: 18, the amino acid sequence of the modified receptor binding domain is modified to substitute mutations V1113K, S1117P, S1196A and I1197P, wherein relative to the reference B4-Hc, the modification enhances the binding of the modified B4-Hc to human SytII, Wherein, the second part is selected from the group consisting of small molecules and bioactive molecules.

4. The chimeric molecule of claim 3, wherein the second moiety is selected from the group consisting of a nucleic acid and a protein. The chimeric molecule of claim 3 , wherein the second portion is a polypeptide. The chimeric molecule of claim 3 , wherein the biologically active molecule is a therapeutic polypeptide or a non-polypeptide drug.

7. The polypeptide, BoNT polypeptide, or chimeric molecule of any one of claims 1-6, wherein: The modification further comprises one or more substitution mutations selected from the group consisting of: Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P and combinations thereof.

8. The polypeptide, BoNT polypeptide, or chimeric molecule of any one of claims 1-6, wherein: The modification further comprises two substitution mutations selected from the group consisting of: Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, and Y1183P.

9. The polypeptide, BoNT polypeptide, or chimeric molecule of any one of claims 1-6, wherein the modification further comprises: Two or more substitution mutations, the substitution mutations being selected from the group consisting of Q1191M, Q1191C, Q1191V, Q1191L, Q1191Y, Y1199W, Y1199E, Y1199H, W1178Y, W1178Q, W1178A, W1178S, Y1183C, Y1183P, Y1199F and Y1199L.

10. The polypeptide, BoNT polypeptide, or chimeric molecule of any one of claims 1-6, wherein the modification further comprises: Two substitution mutations, the two 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; W1178Q and Y1183P; and W1178Q and Y1183C.

11. The polypeptide, BoNT polypeptide, or chimeric molecule of claim 10, wherein the two substitution mutations correspond to: i) Q1191M and W1178Q; or ii) Q1191M and Y1183P; or iii) Q1191M and Y1183C.

12. The polypeptide, BoNT polypeptide, or chimeric molecule of any one of claims 1-6, wherein: The modification further comprises three substitution mutations, and the three substitution mutations are selected from the group consisting of Q1191C, Q1191V, Q1191L, Q1191Y, Q1191M, W1178Y, W1178Q, W1178A, W1178S, Y1183C and Y1183P.

13. The polypeptide, BoNT polypeptide, or chimeric molecule of any one of claims 1-6, wherein: The modification also includes three substitution mutations, and the three substitution mutations correspond to Q1191M, W1178Q and Y1183P.

14. The polypeptide, BoNT polypeptide, or chimeric molecule of any one of claims 1-6, further comprising a substitution mutation corresponding to S1201V.

15. The BoNT polypeptide of claim 2, wherein: i) 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; or ii) 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, wherein the protease domain, translocation domain, and protease cleavage site are from serotype B, strain 1; or iii) 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, wherein the protease domain, translocation domain, and protease cleavage site are from serotype A, strain 1; or iv) 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 a combination thereof, wherein the protease cleavage site is from serotype A, serotype B, or a chimeric cleavage site of serotype A and serotype B.

16. The chimeric molecule of any one of claims 3 to 6, wherein The first portion and the second portion are covalently linked.

17. The chimeric molecule of any one of claims 3 to 6, wherein The first portion and the second portion are non-covalently linked.

18. A nucleic acid encoding a polypeptide as claimed in any one of claims 1 or 7-13, a BoNT polypeptide as claimed in any one of claims 2 or 7-13, or a chimeric molecule as claimed in any one of claims 3-10 or 12-13. A nucleic acid vector comprising the nucleic acid according to claim 18 .

20. A cell comprising the nucleic acid of claim 18 or the nucleic acid vector of claim 19.

21. A cell expressing the polypeptide of any one of claims 1 or 7-13, the BoNT polypeptide of any one of claims 2 or 7-13, or the chimeric molecule of any one of claims 3-10 or 12-13.

22. A pharmaceutical composition comprising the polypeptide of any one of claims 1 or 7-13, or the botulinum neurotoxin (BoNT) polypeptide of any one of claims 2 or 7-15, or the chimeric molecule of any one of claims 3-10, 12-13, or 16-17, or the nucleic acid vector of claim 19.

23. The pharmaceutical composition of claim 22, further comprising a pharmaceutically acceptable excipient.

24. A kit comprising the pharmaceutical composition of claim 22 or 23 and instructions for therapeutic administration of the pharmaceutical composition.

25. A method of producing a botulinum neurotoxin (BoNT) polypeptide, the method comprising the step of culturing the cell of claim 21 under conditions where the BoNT polypeptide is produced.

26. The method of claim 25, further comprising one or more of the following steps: i) - recovering the BoNT polypeptide from the culture grown in the culturing step; ii)-purifying the BoNT polypeptide; iii) -activating the BoNT polypeptide; and / or iv) - formulating the BoNT polypeptide.

27. Use of the polypeptide of any one of claims 1 or 7-13, or the botulinum neurotoxin (BoNT) polypeptide of any one of claims 2 or 7-15, the pharmaceutical composition of claim 22 or 23, or the chimeric molecule of any one of claims 3-10, 12-13, or 16-17 for the preparation of a medicament for treating a disorder associated with unwanted neuronal activity, wherein: The disorder is selected from the group consisting of eyelid disorders, focal spasms and other speech disorders, and secretory disorders.

28. Use of a polypeptide as claimed in any one of claims 1 or 7-13, or a botulinum neurotoxin (BoNT) polypeptide as claimed in any one of claims 2 or 7-15, a pharmaceutical composition as claimed in claim 22 or 23, or a chimeric molecule as claimed in any one of claims 3-10, 12-13, or 16-17, in the preparation of a medicament for treating a condition associated with unwanted neuronal activity, wherein the condition is selected from the group consisting of spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dystonia, tongue dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, cerebral palsy, spastic colitis, neurogenic bladder, anal spasm, limb spasm, tics, tremors, bruxismus, anal fissures, achalasia, dysphagia and other dystonias and other conditions characterized by involuntary movements of muscle groups, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, pain caused by muscle spasms, headaches, migraines, and dermatological or aesthetic / cosmetic conditions.

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