SE2050326A1 - Engineered botulinum neurotoxin serotype E - Google Patents
Engineered botulinum neurotoxin serotype EInfo
- Publication number
- SE2050326A1 SE2050326A1 SE2050326A SE2050326A SE2050326A1 SE 2050326 A1 SE2050326 A1 SE 2050326A1 SE 2050326 A SE2050326 A SE 2050326A SE 2050326 A SE2050326 A SE 2050326A SE 2050326 A1 SE2050326 A1 SE 2050326A1
- Authority
- SE
- Sweden
- Prior art keywords
- bont
- amino acid
- substitution
- acid sequence
- replaces
- Prior art date
Links
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/43—Enzymes; Proenzymes; Derivatives thereof
- A61K38/46—Hydrolases (3)
- A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
- A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
- A61K38/4893—Botulinum neurotoxin (3.4.24.69)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P21/00—Drugs for disorders of the muscular or neuromuscular system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
Abstract
A modified botulinum neurotoxin serotype E (BoNT/E) Heavy Chain Binding domain (He), wherein multiple amino acid sequences are substituted into the BoNT/E He sequence, thereby forming a synaptic associated vesicle 2C (SV2C) receptor binding site in the modified BoNT/E He.
Description
1 ENGINEERED BOTULINUM NEUROTOXIN SEROTYPE E TECHNICAL FIELD
id="p-1"
[001] The present invention relates to an engineered botulinum neurotoxin serotype E(BoNT/E), wherein multiple amino acid sequences have been substituted into the Heavy ChainBinding domain (HC) forming a synaptic associated vesicle 2C (SV2C) receptor binding site in the BoNT/E HC.
BACKGROUND ART
id="p-2"
[002] Botulinum neurotoxins (BoNTs) are a family of proteins produced mainly by clostridiumbotulinum. BoNTs are expressed as a single ”150 kDa polypeptide that is subsequently cleavedinto an active di-chain molecule consisting of a ”50 kDa light-chain (LC) and a ”100 kDa heavy-chain (HC) (Swaminathan, 2011). The LC and HC remain bound by a single disulphide-bond.The LC comprises a single zinc-dependent protease domain and the HC comprises two distinctdomains, the translocation domain (HN) and receptor-binding domain (HC).
id="p-3"
[003] BoNTs target and block synaptic vesicle exocytosis at neuronal terminals which resultsin paralysis of the associated muscle (Dong, I\/|asuyer & Stenmark, 2019). This is achievedthrough a three-step intoxication process; first the HC targets and binds to receptors on theneuronal cell surface. The toxin is then able to enter the cell by endocytosis where it remainswithin the early endosome. Here the HN enables transport of the LC across the endosomalmembrane and into the cytosol. Once released into the cytosol, the LC cleaves a SNARE(soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) protein resulting inthe inhibition of SNARE-mediated exocytosis, halting the release of acetylcholine into theneuromuscularjunction (Dong, I\/|asuyer & Stenmark, 2019).
id="p-4"
[004] Several different BoNT serotypes have been identified on the basis of neutralizationusing specific antisera (Schiavo, I\/|atteoli & Montecucco, 2000). Serotypes A through G(BoNT/A - /G) are well established, while more recently several new BoNT serotypes havebeen identified including BoNT/X, BoNT/En, and BoNT/Wo (Tanizawa et al., 2014; Zornetta etal., 2016; Zhang et al., 2017, 2018).
id="p-5"
[005] I\/lost BoNT serotypes bind both a protein receptor and a polysialoganglioside (Rummel, 2017). Depending on the specific serotype involved, BoNTs bind one of two possible 2 protein receptors: synaptotagmin or synaptic vesicle glycoprotein 2 (SV2). SV2 is the receptorfor BoNT/A, /E, /D, and /F (Dong et al., 2006; I\/|ahrhold et al., 2006; Dong et al., 2008; Rummelet al., 2009; Peng et al., 2011). SV2 proteins are produced as one ofthree isoforms (SV2A,SV2B, SV2C), each ofwhich are glycosylated and contain 12 transmembrane regions alongwith two globular loops, one luminal and the other cytoplasmic (Bartholome et al., 2017). Theexpression levels of each SV2 isoform differs between different neuronal cell types and evensub-populations within a particular cell type. ln particular it has been determined that SV2C isexpressed at greater levels than either SV2A or SV2B in motor neurons (Pellett, Tepp &Johnson, 2019). While BoNT/A subtypes can utilise all three SV2 isoforms to enter neuronalcells, the preferred isoform with the greatest affinity is SV2C. BoNT/E binds predominantlySV2A but also SV2B (Dong et al., 2008; Rummel et al., 2009). SV2A is found in relatively lowlevels within motor neurons however it is upregulated within motor neurons which areassociated with slow-type muscle fibres (Chakkalakal et al., 2010).
id="p-6"
[006] Another major difference between serotypes relates to the observed translocationrate. For example, it has been determined that BoNT/E is capable oftranslocating its LC fasterthan that of BoNT/A (Wang et al., 2008). An increased translocation rate is one factor whichdetermines the onset time. The mechanism oftranslocation is not yet understood but it islikely to involve a conformational change of the HNwhich forms a full or partial pore, allowingan unfolded LC through (Pirazzini et al., 2015). The crystal structure of BoNT/E revealed adomain organisation different from what was previously observed for BoNT/A. The differentdomain organisation of BoNT/E, when compared to BoNT/A, might be responsible for theenhanced translocation rate observed (Lacy et al., 1998; Lacy & Stevens, 1999; Kumaran et al.,2009)[007] I\/|edical applications of botulinum neurotoxins (BoNTs) are remarkably effective andwell tolerated, but some limitations and adverse effects have also been reported. Manymedical and cosmetic applications would benefit from a faster onset of action. The mostwidely used commercial BoNT has an onset time of approximately 2-3 days. An alternativetoxin serotype, BoNT/E, is currently being explored for its fast onset time of approximately 12hours. One drawback of using BoNT/E is that it targets only a subset of the available receptors found on neuronal cells (SV2A and SV2B) which does not include SV2C, compared with BoNT/Awhich can utilise all isoforms (SV2A, SV2B, SV2C). 3[008] A desirable property for a therapeutic BoNT would be a fast onset rate. BoNT/E mayhave a faster onset rate than BoNT/A but it is unable to utilise SV2C which may potentiallyreduce its potency. The design of a toxin which combines the action of BoNT/E with thepotency of BoNT/A would be highly useful for the therapeutic market.[009] Wang et al., 2008, demonstrated that individually folded domains of BoNTs can betransferred between serotypes to generate fully functional, novel BoNT chimeras. Theyswapped the HC domains between BoNT/A and BoNT/E, forming chimeras EA comprising theLC and HN of BoNT/E fused to the HC of BoNT/A and AE comprising LC-HN/A plus HC/E. Eachchimera was activated and found to retain the expected features contributed by therespective domains from BoNT/A and BoNT/E. Chimera EA proved to be faster-acting andmore potent than BoNT/A or AE at the neuromuscular junction. lt was also speculated that asthe HC is sufficient for recognition by BoNT/A of neuronal ectoreceptors, SV2C, andgangliosides, and as EA bound to the intralumenal domain of SV2C in vitro, the binding andendocytotic uptake route of EA would not differ from those of BoNT/A.[0010] The chimeras formed by Wang et al. do not take into account the potential interactionbetween the HC and the HN domains, which may be important for overall toxin stability andduring toxin translocation. ln addition, by replacing the entire HC domain, the ganglioside-binding site of the BoNT/E may also be modified. Hence, it would be desirable with a BoNT chimera that ensures that any potential interface between the HCand HN domains are maintained and that the ganglioside-binding site from BoNT/E is maintained.
SUMMARY OF THE INVENTION
id="p-11"
[0011] lt is an object of the present invention to provide a novel BoNT/E-like toxin based onthe native sequences of BoNT/A and BoNT/E, which BoNT/E-like toxin recognises SV2C andbinds with greater affinity to SV2C than native BoNT/E. The BoNT/E chimera preferably havinga maintained interface between the HC and HN domains and a maintained ganglioside-bindingsite. The novel BoNT/E - BoNT/A chimeras being produced using an advanced three-dimensional structural design process.
id="p-12"
[0012] This object is thus attained by in a first aspect providing a modified/engineeredbotulinum neurotoxin serotype E (BoNT/E) Heavy Chain Binding domain (HC) (SEQ ID NO: 3) comprising multiple amino acid substitutions in the BoNT/E HC sequence (SEQ ID NO: 2) 4 forming a synaptic associated vesicle 2C (SV2C) receptor binding site in the modified BoNT/E HC, wherein the multiple amino acid substitutions in the modified BoNT/E HC sequence comprise the following substitutions with the amino acid residue numbering of the non- modified BoNT/E full length amino acid sequence (SEQ ID NO: 7): substitution 2a or substitution 2b, wherein substitution 2a replaces amino acids atpositions 925-933 and comprises an amino acid sequence: Lys Tyr Phe Asn Ser Ile SerLeu, and substitution 2b replaces amino acids at positions 925-932 and comprises anamino acid sequence: Lys Tyr Phe Asn Ser Ile Ser; substitution 3, wherein substitution 3 replaces amino acids 956-957 and comprises anamino acid sequence: Tyr Gly; substitution 4a or substitution 4b, wherein substitution 4a replaces amino acids 978-982 and comprises an amino acid sequence: Ser Gln I\/Iet Ile Asn, and substitution 4breplaces amino acids 980-981 and comprises an amino acid sequence: I\/Iet Ile;substitution 5a or substitution 5b1 and 5b2, wherein substitution 5a replaces aminoacids 1033-1039 and comprises an amino acid sequence: Leu Asp Gly Cys Arg Asp ThrHis, substitution 5b1 replaces amino acid 1035 and comprises amino acid: Gly, andsubstitution 5b2 replaces amino acids 1037-1039 and comprises an amino acid sequence: Arg Asp Thr His; substitution 7a or substitution 7b1, 7b2, 7b3 and 7b4, wherein substitution 7a replaces amino acids 1109-1123 and comprises an amino acid sequence: Lys Gly Pro Arg Gly SerVal I\/Iet Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg, substitution 7b1 replaces amino acids 1110-1111 and comprises an amino acid sequence: Gly Pro, substitution 7b2 replaces amino acid 1113 and comprises amino acid: Gly, substitution 7b3 replaces amino acids 1115-1120 and comprises an amino acid sequence: I\/Iet Thr Thr Asn Ile Tyr Leu Asn Ser Ser, and substitution 7b4 replaces amino acid 1123 and comprises amino acid: Arg; substitution 8a or substitution 8b1, 8b2, 8b3 and 8b4, wherein substitution 8a replaces amino acids at position 1244-1252 and comprises an amino acid sequence: Pro Val AspAsp Gly Trp Gly Glu Arg Pro Leu, substitution 8b1 replaces amino acid 1245 andcomprises amino acid: Val, substitution 8b2 replaces amino acid 1247 and comprises amino acid: Asp, substitution 8b3 replaces amino acid 1250 and comprises amino acid: 5Gly, and substitution 8b4 replaces amino acid 1252 and comprises an amino acidsequence: Arg Pro Leu.
id="p-13"
[0013] These six substitutions (substitution 2, 3, 4, 5, 7 and 8) were identified at differentlocations within the HC sequence of BoNT/A1 (botulinum serotype A1) and considered to beessential for binding of the SV2C receptor. The identified amino acid sequences were graftedinto the BoNT/E HC. The identified substitutions may be trimmed. For example, substitution 2acould be replaced with its shorter counterpart substitution 2b. lt is believed, however, that thelonger substitution 2a, results in a BoNT/E HC chimera that binds better to the SV2C receptorthan a BoNT/E HC chimera with the trimmed substitution 2b.[0014] The modified BoNT/E HC may further comprise the following substitutions with theamino acid residue numbering of the non-modified BoNT/E full length amino acid sequence: - substitution 1, wherein substitution 1 replaces amino acid 891-896 and comprises an amino acid sequence: Phe Asn Leu Glu Ser Ser, and/or- substitution 6, wherein substitution 6 replaces amino acid 1097 and comprises aminoacid: Tyr.
id="p-15"
[0015] The modified BoNT/E HC comprising also one or both of these substitutions may bindbetter to the SV2C receptor than a modified BoNT/E HC without these additional substitutions.[0016] The BoNT/E HC may be selected from any subtype E1, E2, E3, E4, E5, E6, E7, E8, E9, E10,E11 or E12.[0017] The BoNT/E subtypes share between 88% and 99% amino acid sequence identity. lt ispossible that not yet identified BoNT/E subtypes will be revealed in the future. The identifiedsubstitutions discussed above grafted into such BoNT/E HC would then also most likely resultin BoNT/E HC chimeras with binding affinity for the SV2C receptor.[0018] According to a second aspect there is provided a polypeptide comprising the modifiedBoNT/E HC described above coupled to any one or more of a protein, a polypeptide, an aminoacid sequence, or a fluorescent probe directly or via a linker.[0019] Such polypeptide is preferably produced recombinantly as the BoNT/E HC needs to beproduced recombinantly. The nucleic acids encoding the modified BoNT/E HC or polypeptidecomprising the modified BoNT/E HC may be DNA or RNA, double-stranded or single stranded.[0020] The protein to be included in the polypeptide may be any protein of interest to be transported to a neuronal cell, and/or internalized into a neuronal cell. 6
id="p-21"
[0021] The polypeptide may in addition to the HC comprise a Heavy Chain Translocationdomain (HN), a Light chain (LC) and a protease site positioned between the LC and HN in thepolypeptide sequence, wherein the HN and the LC, respectively and independently originatefrom any ofthe BoNT serotypes A, B, C, D, DC, E, En, F, G, Wo or X or their subtypes.
id="p-22"
[0022] According to a third aspect there is provided a vector comprising a nucleic acidsequence encoding the modified BoNT/E HC or the polypeptide described above.
id="p-23"
[0023] The vector may further comprise a nucleic acid sequence encoding any other proteinor probe that is to be recombinantly produced together with the modified BoNT/E HC, so as toobtain said protein or probe coupled to the modified BoNT/E HC in one polypeptide. Thevector is preferably an expression vector. The vector may comprise a promoter operablylinked to the nucleic acid. A variety of promoters can be used for expression of thepolypeptides described herein, and are known to the person skilled in the technical field.[0024] An expression vector comprising the nucleic acid can be transferred to a host cell byconventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphateprecipitation) and the transfected cells are then cultured by conventional techniques toproduce the polypeptides described herein. ln some embodiments, the expression ofthepolypeptides described herein is regulated by a constitutive, an inducible or a tissue-specificpromoter.
id="p-25"
[0025] The polypeptides may be produced in any cells, eukaryotic or prokaryotic, or in yeast.The polypeptides according to the invention may further be produced in a cell free system.The skilled person will be readily able to apply the expression system of choice to that person.The expression system used for producing the polypeptides ofthe invention are not limiting tothe scope ofthe invention.
id="p-26"
[0026] Purification and modification of recombinant proteins is well known in the art suchthat the design ofthe polyprotein precursor could include a number of embodiments readilyappreciated by a skilled worker.
id="p-27"
[0027] According to a fourth aspect the modified BoNT/E HC and/or the polypeptide may beused in a therapeutic or cosmetic method.
id="p-28"
[0028] The modified BoNT/E HC and/or the polypeptide targets SV2C and has a higher affinitythan native/non-modified BoNT/E for SV2C.
id="p-29"
[0029] The therapeutic treatment or cosmetic treatment may be a treatment to dampen and/or inactivate muscles. 7
id="p-30"
[0030] With regard to cosmetic methods, the modified BoNT/E HC or the polypeptide maypreferably be used to prevent and/or treat wrinkles, brow furrows or unwanted lines, in orderto reduce said wrinkles, furrows and lines.
id="p-31"
[0031] The therapeutic method may be a treatment and/or prevention of a disorder chosenfrom the group comprising neuromuscular disorders and spastic muscle disorders.
id="p-32"
[0032] The disorder may be chosen from the group comprising spasmodic dysphonia,spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervicaldystonia, foca| hand dystonia, blepharospasm, strabismus, hemifacial spasm, eye|id disorder,cerebral palsy, foca| spasticity and other voice disorders, spasmodic colitis, neurogenicbladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia andother muscle tone disorders and other disorders characterized by involuntary movements ofmuscle groups, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinalsecretions, secretory disorders, pain from muscle spasms, headache pain, sports injuries, anddepression.
id="p-33"
[0033] According to a fifth aspect there is provided a pharmaceutical or cosmetic compositioncomprising the modified BoNT/E HC and/or the polypeptide.
id="p-34"
[0034] The modified BoNT/E HC and/or peptide according to the above may be formulated inany suitable pharmaceutical or cosmetic composition. The pharmaceutical composition mayfurther comprise pharmaceutically acceptable excipients, carriers or other additives. Thecosmetic composition comprising the modified BoNT/E HC and/or peptide may furthercomprise cosmetically acceptable excipients, carriers or other additives.
id="p-35"
[0035] The administration ofthe pharmaceutical or cosmetic composition may be viainjection, wherein the injection may be administered at the site ofthe body where unwantedneuronal activity is present. Typically, compositions for administration by injection aresolutions in sterile isotonic aqueous buffer. Where necessary, the composition can alsoinclude a solubilizing agent and a local anesthetic to ease pain at the site of the injection.[0036] According to a sixth aspect there is provided a method of treating a conditionassociated with unwanted neuronal activity, the method comprising administering atherapeutically effective amount ofthe modified BoNT/E HC or the polypeptide or thepharmaceutical composition to a subject to thereby treat the condition, wherein the conditionis chosen from the group comprising of spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, foca| hand dystonia, 8 blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticityand other voice disorders, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics,tremors, bruxism, anal fissure, achalasia, dysphagia and other muscle tone disorders and otherdisorders characterized by involuntary movements of muscle groups, lacrimation,hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, secretory disorders,pain from muscle spasms, headache pain, sports injuries, and depression, and dermatological or aesthetic/cosmetic conditions.
BRIEF DESCRIPTION OF THE DRAWINGS
id="p-37"
[0037] Fig. 1 shows the structure of a botulinum neurotoxin, which consist of a light chain (LC)and heavy chain (HC). The heavy chain can be divided into two functional domains, thetranslocation domain (HN) and binding domain (HC). The crystal structures of both BoNT/A(PDB code: 3BTA) and BoNT/E (PDB code: 3FFZ) are shown in Fig. 1 and have previously beendetermined and reveal two distinct domain organisations (Lacy et al., 1998; Kumaran et al.,2009)
id="p-38"
[0038] Fig. 2 shows the crystal structure of BoNT/A1 HC in complex with a glycosylated fourthextracellular domain (LD4) of SV2C. PDB: 5JLV (Yao et al., 2016).
id="p-39"
[0039] Fig. 3 shows modified locations within the binding domain of native botulinumneurotoxin serotype E used to generate a modified BoNT/E with binding affinity for SV2C. Thenovel toxin has been called BoNT/E2C or E2C.
id="p-40"
[0040] Fig. 4 shows the protein sequence alignment of BoNT/E3 (Uniprot: A2|2S5) and themodified BoNT/E, E2C. Boxed sequences in the BoNT/E sequence have been replaced bysequences taken from BoNT/A1(Uniprot: PODP|1) and grafted into BoNT/E3 (BoNT/E subtypeE3) to create E2C (inserted sequences indicated with boxes in the E2C-sequence). The residuenumbering is based on the full length sequence of E2C and BoNT/E.
id="p-41"
[0041] Fig. 5 shows the modified locations within the binding domain of native botulinumneurotoxin serotype E used to generate E2C, mapped onto the crystal structure of BoNT/E.PDB: 3FFZ (Kumaran et al., 2009). Only the HC part ofthe structure is shown.
id="p-42"
[0042] Fig. 6a shows the protein sequence alignment of BoNT/A1 (Uniprot: PODP|1) and E2C.
Boxed sequences have been taken from BoNT/A1 and grafted into BoNT/E3 to create E2C. The 9 residue numbering is based on the full length sequence of E2C and the full-length sequence ofBoNT/A1.
id="p-43"
[0043] Fig. 6b shows the protein sequence alignment of BoNT/A1 (Uniprot: PODPI1) and E2C.Boxed sequences have been taken from BoNT/A1 and grafted into BoNT/E3 to create E2C. Theresidue numbering is based on the full-length sequence of E2C and the full-length sequence ofBoNT/A1. Compared to Fig. 6a, the inserted sequences (boxed) have been trimmed to aminimum of amino acids needed for E2C to exhibit binding affinity towards the SVZC receptor.[0044] Fig. 7 is a chromatogram (A280 trace) from size-exclusion purification of HC/EZC using aSuperdex200 26/600 column. The fraction containing HC/EZC is indicated.
id="p-45"
[0045] Fig. 8 shows a SDS-PAGE analysis of purified HC/EZC, 'IV|' denotes the lane containingmolecular weight markers.
Fig. 9 shows (A) Design of N-terminal 6x histidine-tagged and Flag-tagged constructs for a SV2binding assay. 1: indicates a histidine-tag; 2: indicates a flag-tag; 3: indicates a Tobacco EtchVirus protease (TEV) site. Arrow indicates the cleaved bond upon incubation with TEVprotease. (B) Schematic diagram of the SV2-binding ELISA. 4: indicates immobilised GST-tagged SV2 (only the fourth extracellular domain of SV2) on plate; 5: indicates binding oftagged Hcto SV2, and 6: indicates detection of bound HC with anti-flag HRP (Horseradishperoxidase)-conjugated antibody.
id="p-46"
[0046] Fig. 10 shows binding of FLAG-tagged HC/Al (I), H@/E1(o), and HC/EZC (A) toimmobilised GST-tagged SVZC (only the fourth extracellular domain of SVZC) measured usingan ELISA. Data were fit using a four-parameter logistic regression.
id="p-47"
[0047] Figs 11a and 11b show protein sequence alignment of BoNT/E subtypes with E2Cmodifications to enhance SVZC binding. E2C modifications are boxed above the sequences andpositions for substitutions are marked with a box in each subtype E1-E12. (Uniprot: BoNT/El -Q00496; BoNT/E2 - A2|2S6; BoNT/E3 - A2|2S5; BoNT/E4 - P30995; BoNT/E5 - Q9K395;BoNT/E6 - 8Y861; BoNT/E7 - G8|2N7; BoNT/E8 - G8|2N8; BoNT/E9 - K7S9Y2; BoNT/E10 -AOA076KOC0; BoNT/E11 - AOAO76KOB0; BoNT/E12 - W8FNB6;). ln Fig. 11a modifications 1 to 4are shown and in Fig.11b modification 5 to 8 are shown.
id="p-48"
[0048] Fig. 12 shows extended use of HC/EZC. (A) The HC/EZC domain can be fused onto the LCand HN domains of any other BoNT serotype or BoNT-like protein. (B) The HC/EZC can be fused to any other functional domain of interest.
DEFINITIONS
id="p-49"
[0049] As used herein, the term Botulinum neurotoxin "BoNT" encompasses any polypeptideor fragment from a Botulinum neurotoxin. The term BoNT may refer to a full-length BoNT. Theterm BoNT may refer to a fragment of the BoNT that can execute the overall cellularmechanism whereby a BoNT enters a neuron and inhibits neurotransmitter release. The termBoNT may simply refer to a fragment of the BoNT, without requiring the fragment to have anyspecific function or activity.
id="p-50"
[0050] As used herein, the term ”translocation domain” or "HN" means a BoNT domain thatcan execute the translocation step ofthe intoxication process that mediates BoNT light chaintranslocation. Thus, an HN facilitates the movement of a BoNT light chain across a membraneinto the cytoplasm of a cell.
id="p-51"
[0051] As used herein, the term ”binding domain” is synonymous with ”HC domain” andmeans any naturally occurring BoNT receptor binding domain that can execute the cell bindingstep ofthe intoxication process, including, e.g., the binding of the BoNT to a BoNT-specificreceptor system located on the plasma membrane surface of a target cell.
id="p-52"
[0052] ln the present disclosure, the terms ”nucleic acid” and ”gene” are usedinterchangeably to describe a nucleotide sequence, or a polynucleotide, encoding for a polypeptide.
DETAILED DESCRIPTION
id="p-53"
[0053] As illustrated in Fig. 1 botulinum neurotoxins (BoNTs) comprise a light chain (LC), linkedby a single disulphide bridge to the heavy chain (HC). The Heavy chain (HC) holds two of thefunctional domains, with the N-terminal translocation domain (HN) and the C-terminal bindingdomain (HC), while LC is responsible for intracellular catalytic activity. The HC thus comprisesthe receptor binding domains, which are able to specifically and irreversibly bind to thespecific receptors expressed on susceptible neurons, whereas the HN forms a channel thatallows the attached LC to translocate from endosomal-like membrane vesicles into thecytosol.
id="p-54"
[0054] Different BoNT serotypes have different sets of receptor binding sites on the HC,typically two receptor binding sites.
id="p-55"
[0055] Even though medical applications of botulinum neurotoxins (BoNTs) are remarkably effective and well tolerated many medical and cosmetic applications would benefit from a 11 faster onset of action. The most widely used commercial BoNT has an onset time ofapproximately 2-3 days. An alternative toxin serotype, BoNT/E, has an onset time ofapproximately 12 hours. BoNT/E only targets a subset of the available receptors found onneuronal cells (SV2A and SV2B) compared with BoNT/A, which can utilise all isoforms (SV2A,SV2B, SV2C).
id="p-56"
[0056] A desirable property for a therapeutic BoNT would be a fast onset rate. BoNT/E mayhave a faster onset rate than BoNT/A but it is unable to utilise all ofthe available SV2 receptorisoforms, which may potentially reduce its potency.
id="p-57"
[0057] The design of a toxin which combines the action of BoNT/E with the potency of BoNT/Aare known for example from Wang et al., 2008, where the HC domains between BoNT/A andBoNT/E were swapped forming chimeras EA comprising the LC and HN of BoNT/E fused to theHC of BoNT/A and AE comprising LC-HN/A plus HC/E. Each chimera was found to retain theexpected features contributed by the respective domains from BoNT/A and BoNT/E. ChimeraEA proved to be faster-acting and more potent than BoNT/A or AE at the neuromuscularjunction. lt was also speculated that as the HC is sufficient for recognition by BoNT/A ofneuronal ectoreceptors, SV2C, and gangliosides, and as EA bound to the intralumenal domainof SV2C in vitro, the binding and endocytotic uptake route of EA would not differ from thoseof BoNT/A.
id="p-58"
[0058] The chimeras formed by Wang et al. do not take into account the potential interactionbetween the HC and the HN domains, which may be important for overall toxin stability andduring toxin translocation. ln addition, by replacing the entire HC domain, the ganglioside-binding site of the BoNT/E may also be modified. Hence, it would be desirable with a BoNTchimera that ensures that any potential interface between the HCand HN domains aremaintained and that the ganglioside-binding site from BoNT/E is maintained.
id="p-59"
[0059] Below follows a description of a novel BoNT/E-like chimera toxin based on the nativesequences of BoNT/A and BoNT/E and its production. The BoNT/E-like chimera toxin beingproduced via an advanced three-dimensional structural design process, which has resulted in aBoNT/E-like toxin that recognises SV2C and binds with greater affinity to SV2C than nativeBoNT/E The novel toxin has been called BoNT/E2C or E2C. 12METHODS AND EXPERIMENTAL RESULTS Constructs
id="p-60"
[0060] The cDNA encoding the HC domain and full-length (inactive) forms of E2C (HC/E2C andBoNT/E2C respectively), H@/A1(Uniprot: PODPI1, residues 876 to 1296), and HC/E3 (Uniprot:A2|2S5, residues 853 to 1255) were codon-optimised for expression in E. coli (seesupplementary information for DNA and amino acid sequences, SEQ ID NOs: 1, 2, 3, 6, 8, 9, 10,13 (including a tag sequence, aa residue 1-26)), synthesised, and cloned into a pET28a(+)vector containing an N-terminal 6x His-tag, Flag-tag, and TEV protease cleavage site(GenScript, NJ, USA). The Hç/E2C construct comprises residues 853 to 1259 of the full-lengthBoNT/E2C sequence. The full-length inactive BoNT/E2C sequence also contains dual active-sitemutations to ensure it is non-toxic for research and development use (BoNT/E2CR348A/Y351F, see supplementary information for protein sequence and DNA sequence, SEQID NOs: 5 and 12 (including a tag sequence, aa residue 1-26)) (Agarwal, Binz & Swaminathan,2005). For ELISA binding assays, the cDNA encoding human SV2C residues 473 to 567 (SV2C)were codon-optimised for expression in E. coli, synthesised, and cloned into a pGEX-5X-1vector (GenScript, NJ, USA). The construct comprises an N-terminal glutathione S-transferase(GST)-tag, FactorXa protease cleavage site, and SV2C 473 to 567 (GST-SV2C, see supplementary information for amino acid and DNA sequence, SEQ ID NOs: 7 and 11).
Protein Expression[0061] Each construct was transformed into BL21(DE3) E. coli (New England BioLabs, USA). An identical expression protocol was used for all proteins. Transformed cells were grown interrific broth medium containing 50 ug/ml kanamycin (for HC/Al, HC/E3, Hç/E2C) or 100 pg/mlampicillin (GST-SV2C) in a LEX bioreactor system (Epyphite3, Canada). Cells were grown at37°C until they reached an OD600 of 0.6. The temperature was reduced to 16°C and proteinexpression induced at an OD600 of 1.0 by the addition of IPTG to a final concentration of 1mM. After induction cells were allowed to continue to grow for a further 18 hours. Cells were harvested by centrifugation at 6000 x g for 15 min.
Production of Hç/E2C and Hc/E3
id="p-62"
[0062] Both Hç/E2C and Hc/E3 were purified using the same method. The cell paste wasresuspended in 50 mM HEPES, 200 mM NaCl, 25 mM lmidazole, pH 7.4. Cells were lysed with a single pass through an Emulsiflex C3 cell disruptor (Avestin) at 20 kPSl. Lysate was cleared by 13 ultracentrifugation at 158,000 x g for 30 min. Clarified lysate was loaded onto a 5 ml HisTrapHP column (GE Healthcare) and Hç/E2C or HC/E3 was eluted using 50 mM HEPES, 200 mMNaCl, 500 mM lmidazole, pH 7.4. The eluted protein was further purified by size-exclusionchromatography (Superdex200 26/600, GE Healthcare) in 50 mM HEPES, 200 mM NaCl, pH7.4.
Production of H@[A1[0063] The cell paste was resuspended in 100 mM HEPES, 500 mM NaCl, 10 mM imidazole, % glycerol, 0.5 mM TCEP, pH 8.0 and lysed by sonication. Lysate was cleared bycentrifugation at 49,000 x g for 20 min before filtration though a 0.45 um filter. Clarified lysatewas loaded onto a 5 ml HisTrap HP column (GE Healthcare) and eluted using 20 mM HEPES,500 mM NaCl, 500 mM imidazole, 10% glycerol, 0.5 mM TCEP, pH 7.5. The eluted protein wasfurther purified by size-exclusion chromatography (Superdex200 16/600, GE Healthcare) in 20mM HEPES, 300 mM NaCl, 10% glycerol, 0.5 mM TCEP, pH 7.5.
Production of GST-SV2C
id="p-64"
[0064] The cell paste was resuspended in phosphate-buffered saline (PBS, Sigma P4417) andlysed by a single pass through an Emulsiflex C3 cell disruptor (Avestin) at 20 kPSl. Lysate wascleared by ultracentrifugation at 158,000 x g for 30 min. Clarified lysate was loaded onto a 5ml GSTrap 4B column (GE Healthcare) and GST-SV2C was eluted using 50 mM Tris-HCl, 150mM NaCl, 10 mM reduced glutathione, pH 8.0. The eluted protein was further purified by size-exclusion chromatography (Superdex200 16/600, GE Healthcare) in 50 mM Tris-HCl, 100 mMNaCl, pH 8.0.
SV2C Binding Assay
id="p-65"
[0065] 96-well plates (Nunc-lmmunom' IV|icroWell“”' 96-well solid plates) were coated withGST-SV2C (10 ug/ml in 100 mM Tris, pH 8.0) by the addition of 100 ul per well and incubationat 4°C for 16 hours. Wells were washed three times with PBS-T/0.1% BSA (200 ul phosphatebuffered saline, 0.05% [v/v] Tween-20, 0.1% [w/v] bovine serum albumin). Non-specificbinding-sites were blocked by incubation with 200 ul PBS-T/2% BSA for 1 hour at 22°C. Washeswere repeated and 100 ul HC domains were added to the wells (serial dilution withconcentration range of 10 uM to 500 pM for each sample in triplicate). HC domains were left to incubate at 22°C for 1 hour before the wells were washed with 100 ul PBS/0.1% BSA three 14 times. The detection antibody (Monoclonal ANT|-FLAG® I\/|2-Peroxidase (HRP) antibody,Sigma) was diluted 1120000 in PBS/0.1% BSA and 100 pl was added to each well and incubatedat 22°C for 1 hour. The wells were washed with 100 pl PBS/0.1% BSA three times and boundHC domain was quantified using 100 pl Ultra TI\/IB (ThermoScientific) as the substrate for HRP.TI\/IB substrate was left to develop for 180 seconds before the reaction was terminated by theaddition of 100 pl 0.2 M H2SO4. The absorbance of each well was measure at 450 nm. Datafrom three independent experiments each completed in triplicate were processed in R (R- Core-Team, 2019) and fit using a 4-parameter logistic regression model.
Design of Full-length E2C
id="p-66"
[0066] BoNT/A subtypes are known to utilise all three isoforms of SV2 and it is likely that eachisoform shares a common binding mode. We aimed to design and produce a chimeric toxinbased on BoNT/E, which shares the ability of BoNT/A to bind SV2C. ln order to create thenovel toxin (termed BoNT/E2C or E2C) which consisted of a BoNT/E sequence which wasmodified to contain parts of BoNT/A, we first analysed available crystal structures. See Fig. 1,which shows crystal structures of BoNT/A and BoNT/E. The crystal structures of BoNT/A1 HC(HC/A1) in complex with both non-glycosylated and glycosylated SV2C, Fig. 2, have beenpreviously published and released into the PDB as 4JRA and 5JLV, respectively (Benoit et al.,2014; Yao et al., 2016). These structures along with their associated publications highlightsome ofthe interactions between BoNT/A1 and SV2C, which we could investigate for use inthe design of E2C. The fourth luminal domain of SV2C (residues 459 to 578, Uniprot: Q496J9)was found to form mostly backbone-backbone interactions with a beta-strand from theBoNT/A1 HC (residues 1141 to 1146). ln addition, R1156 of BoNT/A1 forms a cation-pi-stackinginteraction with F563 of SV2C. This binding interface is then further complemented byinteractions to an N-linked glycan from SV2 (Yao et al., 2016). These studies used mutagenesisto highlight key residues involved in the BoNT/A-SV2C complex that resulted in decreasedcomplex formation if mutated, which were also used towards our design of a modified BoNT/Etoxin. These included residues: 953, 1064, 1145, 1146, 1156, 1249, 1292, and 1294 (Residuenumbers are based on the full-length sequence of BoNT/Al [Uniprot: P10845]).
id="p-67"
[0067] After identification of the components from BoNT/A1 that are essential for binding ofSV2C, we performed a structural analysis and comparison of BoNT/E in order to integrate these components in a manner which would not cause disruption to other regions of the protein. Proteins from the BoNT/E serotypes differ from BoNT/A as they cannot utilise SV2Cbut are known to interact with SV2A and SV2B (Dong et al., 2008). However, there exists nostructural information regarding the BoNT/E-SV2A/B complex and it is likely to differ from thatof BoNT/A. Structures of BoNT/E alone have been determined which were used for analysisinstead. A full-length crystal structure of BoNT/E is available in the PDB as 3FFZ (Kumaran etal., 2009), see Fig. 1. The sequences of HC/Al and HC/E3 were also aligned using ClustalO(Sievers et al., 2011) and their crystal structures aligned using PyI\/IOL (The PyI\/IOL MolecularGraphics System, Version 2.0, Schrödinger, LLC). The HC sequences share only 50% amino acididentity, while their core structural fold is conserved. I\/lajor structural variations wereobserved between the two proteins at key regions, in particular the region of HC/Al which isinvolved in SV2C-binding via backbone-backbone interactions (noted above) is truncated inHC/E3 which would not allow for the same receptor. Structural analysis was also needed toensure that no structural clashes would occur when grafting regions of BoNT/A1 onto BoNT/E.[0068] The final modifications chosen to apply to the native BoNT/E sequence are shown forthe native BoNT/E3 sequence (see supplementary information for protein and DNA sequence,SEQ ID NOs: 7 and 14) and can be divided into 8 distinct groups of changes(I\/Iodifications/substitutions 1 - 8, Figs 3-5, 6a, 6b, 11a and 11b). Thesemodifications/substitutions are found at different locations within the BoNT/A1 HC sequencebut spatially, they can be all found clustered in one region of the protein structure (Fig. 5). AsBoNT/E (BoNT/E1 - /E12) subtypes share between 88% and 99% amino acid sequence identityit is highly likely that the modifications used to modify the native BoNT/E3 sequence could beapplied to all other BoNT/E subtypes (E1-E12) in order to generate the same effect as withBoNT/E3.
id="p-69"
[0069] Substitution 1 is an optional modification, and corresponds to amino acids 917-922 ofthe full-length sequence BoNT/A1: FNLESS (Phe Asn Leu Glu Ser Ser). These amino acids mayreplace amino acids starting at position 891-896 in the full-length native/non-modified BoNT/Esequence (Figs 6a, 6b).
id="p-70"
[0070] Substitution 2a is a non-optional modification and corresponds to amino acid 951-958of the full-length sequence of BoNT/A1: KYFNSISL (Lys Tyr Phe Asn Ser Ile Ser Leu). Theseamino acids may replace amino acids at positions 925-933in the full-length non-modifiedBoNT/E sequence (Fig. 6a). Substitution 2a may be trimmed to substitution 2b and corresponds to amino acid 951-957 ofthe full-length sequence of BoNT/A1: KYFNSIS (Lys Tyr 16 Phe Asn Ser Ile Ser). These amino acids may replace amino acids at positions 925-932 in thefull-length non-modified BoNT/E sequence (Fig. 6b).
id="p-71"
[0071] Substitution 3 is a non-optional modification and corresponds to amino acid 980-981 ofthe full-length sequence of BoNT/A1: YG (Tyr Gly). These amino acids replace amino acids 956-957 in the non-modified BoNT/E full-length sequence (Figs 6a and 6b).
id="p-72"
[0072] Substitution 4a is a non-optional modification and corresponds to amino acids 1002-1006 ofthe full-length sequence of BoNT/A1: SQMIN (Ser Gln Met Ile Asn). These amino acidsreplace amino acids 978-982 in the non-modified BoNT/E full-length sequence (Fig. 6a).Substitution 4a may be trimmed to substitution 4b and corresponds to amino acids 1004-1005of the full-length sequence of BoNT/A1: MI (Met Ile). These amino acids may replace aminoacids 980-981 in the full-length non-modified BoNT/E sequence (Fig. 6b).
id="p-73"
[0073] Substitution 5a is a non-optional modification and corresponds to amino acid 1057-1064 of the full-length sequence of BoNT/A1: LDGCRDTH (Leu Asp Gly Cys Arg Asp Thr His).These amino acids may replace amino acids 1033-1039 in the full-length non-modified BoNT/Esequence (Figs 6a). Substitution 5a can be trimmed to substitutions 5b1 and 5b2 andcorrespond to amino acids 1059 (G (Gly)) and 1061-1064 (RDTH (Arg Asp Thr His)) of the full-length sequence of BoNT/A1, respectively. These amino acids may replace amino acid 1035and 1037-1039, respectively, in the full-length non-modified BoNT/E sequence (Fig. 6b).[0074] Substitution 6 is an optional modification and corresponds to amino acid 1122 of thefull-length sequence of BoNT/A1: Y (Tyr). The amino acid may replace the amino acid atposition 1097 of the full-length non-modified BoNT/E sequence (Figs 6a and 6b).
id="p-75"
[0075] Substitution 7a is a non-optional modification and corresponds to amino acids 1137-1156 of the full-length sequence of BoNT/A1: KGPRGSVMTTNIYLNSSLYR (Lys Gly Pro Arg GlySer Val Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg). The amino acids may replace theamino acids 1109-1123 of the full-length non-modified BoNT/E sequence. Substitution 7a canbe trimmed to substitutions 7b1, 7b2, 7b3 and 7b4 and correspond to amino acids 1138-1139(GP (Gly Pro)), amino acid 1141 (G (Gly)), amino acid 1144-1151 (MTTNIYLNSS (Met Thr ThrAsn Ile Tyr Leu Asn Ser Ser)), and 1156 (R (Arg)) of the full-length BoNT/A1 sequence. Theamino acids replacing the amino acids 1110-1111, 1113, 1115-1120 and 1123, respectively, ofthe full-length non-modified BoNT/E sequence.
id="p-76"
[0076] Substitution 8a is a non-optional modification and corresponds to amino acids 1286- 1296 of the full-length sequence of BoNT/A1: PVDDGWGERPL (Pro Val Asp Asp Gly Trp Gly Glu 17 Arg Pro Leu). The amino acids may replace the amino acids 1244-1252 of the full-length non-modified BoNT/E sequence. Substitution 8a can be trimmed to substitutions 8b1, 8b2, 8b3 and8b4, which correspond to amino acid 1287 (V (Va|)), amino acid 1289 (D (Asp)), amino acid1292 (G (Gly), and amino acid 1294-1296 (RPL (Arg Pro Leu)) of the full-length BoNT/A1sequence. The amino acids replacing amino acids 1245, 1247, 1250 and 1252, respectively, ofthe full-length non-modified BoNT/E sequence.
id="p-77"
[0077] Using all eight substitutions, the sequence of BoNT/E2C alternates on 15 occasionsbetween a native BoNT/A1 or BoNT/E3 sequence (Figs 4,6a, 11a, 11b) and also includes manynovel and non-natural intra-molecular interfaces. None of the substitutions made to thenative BoNT/E3 sequence are in the vicinity ofthe conserved ganglioside-binding site and thuswe are confident that the interaction with the native ganglioside receptor remains intact. Theresulting molecule named BoNT/E2C or E2C should be able to bind SV2C. The active andinactive sequences are given in Figs 4 and 6 (BoNT/E2C and BoNT/E2C_R348A_Y351F, seesupplementary information for protein and DNA sequence, SEQ ID NOs: 5, 6, 12, 13 (shownwith tag sequence, aa residue 1-26)). As discussed above two of the eight substitutions, 1 and6, are optional substitutions. lncluding all eight substitutions may result in an E2C binding withhigher affinity to the SV2C receptor than an E2C comprising only the six non-optional substitutions.
Production of HQEZC
id="p-78"
[0078] To assess the stability of our design we first produced Hc/E2C, which encompasses allof the sequence modifications and includes all of the receptor-binding sites, by recombinantexpression in E. coli. The expressed protein was soluble and purified by affinitychromatography using Ni-sepharose resin followed by size-exclusion chromatography (SEC).The SEC produced a single distinct peak for Hç/E2C at the predicted size, demonstrating theviability of the engineered construct (Fig. 7). Purified Hç/E2C was analysed by SDS-PAGE and determined to be > 95% pure from inspection (Fig. 8).
Hç/E2C has a greater affinity toward non-glycosylated SV2C than Hcfi
id="p-79"
[0079] To determine whether the engineered E2C protein was capable of targeting SV2C, wedeveloped an enzyme-linked immunosorbent assay (ELISA) (Fig. 9). We first assessed whetherHC domains from BoNT/A1, BoNT/E3, and E2C were capable of binding to recombinantly expressed GST-SV2C which lacked post-translational modifications. The GST-SV2C construct 18 was expressed in E. coli and purified. The GST-SV2C protein then was adsorbed onto thesurface of 96-we|| immunoassay plates and the binding of Hc/E2C, HC/Al, and HC/E3 wasmeasured at different protein concentrations. Previously published literature has establishedthat BoNT/E3 is unable to bind with high-affinity to SV2C while BoNT/A1 is capable of bindingboth glycosylated and non-glycosylated forms of SV2C (Rummel et al., 2009; I\/|ahrhold et al.,2013; Yao et al., 2016). Both HC/Al and Hç/E2C displayed strong binding to SV2C (Kd values of85 nM and 54 nM, respectively) while HC/E3 showed very weak affinity in comparison(approximate Kd 2.6 uIVI) (Fig. 10). Due to the weak affinity of HC/E3, the fit could not bedescribed with a high confidence. These ELISA data clearly demonstrate the ability for E2C to bind SV2C with an affinity higher than HC/Al indicating that the design of E2C was successful.
Applying the E2C modifications to other BoNT/E subtypes
id="p-80"
[0080] BoNT/E (BoNT/E1 - /E12) subtypes share between 88% and 99% amino acid sequenceidentity. lt is therefore likely that the modifications used to generate E2C could be applied toall other BoNT/E subtypes (E1-E12) in order to generate the same effect as with BoNT/E3discussed above. A sequence alignment of all BoNT/E subtypes was performed and thelocation of E2C modification is indicated (Fig. 11a and Fig. 11b). We suggest that production ofany of these BoNT/E subtypes (or similar sequences) with the provided changes would result in a similar effect as E2C based on subtype 3.
Using Hç/E2C as a I\/|odular Domain
id="p-81"
[0081] The main innovation described here is BoNT/E2C, an engineered BoNT/E protein whichis capable of binding to the SV2C protein receptor. The engineered E2C HC domain (HC/E2C)could also be used independently to either generate novel chimeric toxins, that contain anative Lcand HN sequence fused to the engineered Hc/E2C, or generate novel proteins thatcontain other functional domains fused to Hç/E2C (Fig. 12). Chimeric BoNTs containing Hç/E2Cmay show an increased affinity to SV2 isoforms, particularly SV2C, and also an increasedpotency compared with the native BoNT. Novel proteins containing Hc/E2C will allow targeting of functional proteins to cells expressing SV2C.
ConclusionsBoNT/E2C is a BoNT/E-like toxin which has been engineered to recognise the SV2C protein.
This novel design is of great potential and could prove more efficacious than current 19 commercial BoNT formulations. Most commercial BoNT formulations are based on theBoNT/A1 subtype with an onset time of approximately 2 - 3 days. Decreasing this time wouldbe highly beneficial for novel therapeutics. There exists a natural toxin serotype, BoNT/E,which has been shown to have a fast onset time. However, it is likely to be less potent thanBoNT/A1 as it can only utilise a subset of the available protein receptors. The main innovationin BoNT/E2C is the HC domain design, which may also be used in combination with other functional domains from proteins such as BoNTs.
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Claims (11)
1. 23 A modified botulinum neurotoxin serotype E (BoNT/E) Heavy Chain Binding domain (HC) (SEQ ID NO: 3) comprising multiple amino acid substitutions in the BoNT/E HC sequence (SEQ ID NO: 2) forming a synaptic associated vesicle 2C (SV2C) receptor binding site in the modified BoNT/E HC, wherein the multiple amino acid substitutions in the modified BoNT/E HC sequence comprise the following substitutions with the amino acid residue numbering of the native BoNT/E full-length amino acid sequence (SEQ ID NO: 7): substitution 2a or substitution 2b, wherein substitution 2a replaces amino acids atpositions 925-933 and comprises an amino acid sequence: Lys Tyr Phe Asn Ser Ile SerLeu, and substitution 2b replaces amino acids at positions 925-932 and comprises anamino acid sequence: Lys Tyr Phe Asn Ser Ile Ser; substitution 3, wherein substitution 3 replaces amino acids 956-957 and comprises anamino acid sequence: Tyr Gly; substitution 4a or substitution 4b, wherein substitution 4a replaces amino acids 978-982 and comprises an amino acid sequence: Ser Gln I\/Iet Ile Asn, and substitution 4breplaces amino acids 980-981 and comprises an amino acid sequence: I\/Iet Ile;substitution 5a or substitution 5b1 and 5b2, wherein substitution 5a replaces aminoacids 1033-1039 and comprises an amino acid sequence: Leu Asp Gly Cys Arg Asp ThrHis, substitution 5b1 replaces amino acid 1035 and comprises amino acid: Gly, andsubstitution 5b2 replaces amino acids 1037-1039 and comprises an amino acidsequence: Arg Asp Thr His; substitution 7a or substitution 7b1, 7b2, 7b3 and 7b4, wherein substitution 7a replacesamino acids 1109-1123 and comprises an amino acid sequence: Lys Gly Pro Arg Gly SerVal I\/Iet Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg, substitution 7b1 replacesamino acids 1110-1111 and comprises an amino acid sequence: Gly Pro, substitution7b2 replaces amino acid 1113 and comprises amino acid: Gly, substitution 7b3 replacesamino acids 1115-1120 and comprises an amino acid sequence: I\/Iet Thr Thr Asn Ile TyrLeu Asn Ser Ser, and substitution 7b4 replaces amino acid 1123 and comprises aminoacid: Arg; substitution 8a or substitution 8b1, 8b2, 8b3 and 8b4, wherein substitution 8a replacesamino acids 1244-1252 and comprises an amino acid sequence: Pro Val Asp Asp Gly Trp Gly Glu Arg Pro Leu, substitution 8b1 replaces amino acid 1245 and comprises amino 24acid: Val, substitution 8b2 replaces amino acid 1247 and comprises amino acid: Asp,substitution 8b3 replaces amino acid 1250 and comprises amino acid: Gly, andsubstitution 8b4 replaces amino acid 1252 and comprises an amino acid sequence: Arg Pro Leu.
2. The modified BoNT/E HC of claim 1, wherein the multiple amino acid substitutionsfurther comprise the following substitutions with the amino acid residue numbering of thenative BoNT/E full-length amino acid sequence: - substitution 1, wherein substitution 1 replaces amino acid 891-896 and comprises an amino acid sequence: Phe Asn Leu Glu Ser Ser, and/or- substitution 6, wherein substitution 6 replaces amino acid 1097 and comprises amino acid: Tyr.
3. The modified BoNT/E HC of claim 1 or 2, wherein the BoNT/E is selected from anysubtype El, E2, E3, E4, E5, E6, E7, E8, E9, E10, E11 or E12.
4. A polypeptide comprising the modified BoNT/E HC according to any of the precedingclaims coupled to any one or more of a protein, a polypeptide, an amino acid sequence, or a fluorescent probe directly or via a linker.
5. The polypeptide according to claim 4, wherein said polypeptide in addition to the HCcomprises a Heavy Chain Translocation domain (HN), a Light chain (LC) and a protease sitepositioned between the LC and HN in the polypeptide sequence, wherein the HN and the LC,respectively and independently originate from any ofthe BoNT serotypes A, B, C, D, DC, E, En, F, G, Wo or X or their subtypes.
6. A vector comprising a nulecic acid sequence encoding the modified BoNT/E HC of any of the claims 1-3 or the polypeptide according to claim 4 or 5.
7. The modified BoNT/E HC according to any of claims 1-3 or the polypeptide according to claim 4 or 5 for use in a therapeutic or cosmetic method.
8. The modified BoNT/E HC or the polypeptide for use according to claim 7, wherein the therapeutic or cosmetic treatment is a treatment to dampen and/or inactivate muscles.
9. The modified BoNT/E HC or polypeptide for use according to any of the claims 7-8,wherein the disorder is chosen from the group comprising spasmodic dysphonia, spasmodictorticollis, laryngeal dystonia, oromandibular dysphonia, |ingua| dystonia, cervical dystonia,foca| hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebralpalsy, foca| spasticity and other voice disorders, spasmodic colitis, neurogenic bladder,anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia and othermuscle tone disorders and other disorders characterized by involuntary movements of musclegroups, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, secretory disorders, pain from muscle spasms, headache pain, sports injuries, and depression.
10. A pharmaceutical or cosmetic composition comprising the modified BoNT/E HC according to any of claims 1-3 or the polypeptide according to claim 4 or 5.
11. A method of treating a condition associated with unwanted neuronal activity, themethod comprising administering a therapeutically effective amount of the modified BoNT/EHC of any of claims 1-3 or the polypeptide of c|am 4 or 5 or the pharmaceutical composition ofclaim 10 to a subject to thereby treat the condition, wherein the condition is chosen from thegroup comprising spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia,oromandibular dysphonia, |ingua| dystonia, cervical dystonia, foca| hand dystonia,blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, foca| spasticityand other voice disorders, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics,tremors, bruxism, anal fissure, achalasia, dysphagia and other muscle tone disorders and otherdisorders characterized by involuntary movements of muscle groups, lacrimation,hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, secretory disorders,pain from muscle spasms, headache pain, sports injuries, and depression, and dermatological or aesthetic/cosmetic conditions.
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| WO2021190987A1 (en) | 2021-09-30 |
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