Patent Application
20250034540
The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Apr. 18, 2024, is named “136704-00103.xml” and is 42,685 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.
The present invention relates to Botulinum neurotoxin polypeptides and in particular to a chimeric Botulinum neurotoxin Heavy Chain.
The botulinum neurotoxins (BoNTs) are the most potent protein toxins known to man, and the causative agent of the rare paralytic disease, botulism. This family of bacterial toxins consists of eight serotypes, BoNT/A-G, and the recently described BoNT/X (Montal, 2010; Zhang et al., 2017). They all share a common architecture and are expressed as a protein of 150 kDa that is post-translationally cleaved into a di-chain molecule composed of a light chain (LC, 50 kDa), linked by a single disulphide bridge to the heavy chain (HC, 100 kDa). The HC holds two of the functional domain, with the N-terminal translocation domain (HN) and the C-terminal binding domain (HC), while LC is responsible for intracellular catalytic activity. BoNTs first recognise the cholinergic nerve terminals via specific cell surface receptors, and are then endocytosed within a vesicle. The acidic endosomal environment causes a conformational change that allows translocation of LC within the cytosol, also named toxin translocation. The freed catalytic domain, a zinc-protease, can then specifically target one of three neuronal SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors): BoNT/A, /C and /E cleave SNAP-25; BoNT/B, /D, /F, /G and /X target VAMP (synaptobrevin); syntaxin is cleaved by BoNT/C (Schiavo et al., 2000; Zhang et al., 2017). These three proteins form a complex that mediates the fusion of synaptic vesicle to the plasma membrane (Sudhof and Rothman, 2009). Proteolysis of any of the SNAREs inhibits exocytosis and thus the release of neurotransmitters, effectively causing the flaccid paralysis symptomatic of botulism (Rossetto et al., 2014). The sequence of the three functional domains has previously been described (Lacy D B, et al. 1999.). The catalytic domain is composed of the amino acids 1-437, the translocation domain of amino acids 448-872, and the binding domain of amino acids 873-1295, referring to the BoNT/A sequence in Lacy D B, et al. As all BoNT serotypes and their subtypes are homologous to a large degree, the position of the corresponding domains in any other serotype or subtype will be very similar.
The high potency of these toxins makes them an extremely useful therapeutic agent in the treatment of an increasing range of neuromuscular disorders such as strabismus, cervical dystonia and blepharospasm, as well as other conditions involving the release of acetylcholine such as hyperhydrosis (Chen, 2012). BoNT/A and /B are the only serotypes approved and commercially available as therapeutics. BoNT/A is generally considered to have a higher efficacy in humans and is therefore the serotype of choice in most cases (Bentivoglio et al., 2015). However, treatment with BoNT usually requires repeated injections, as the therapeutic effects of the toxins are only transient. This reportedly led to the emergence of resistance in a small subset of patients developing an immune response to BoNT/A (Lange et al., 2009; Naumann et al., 2013). While BoNT/B represents an alternative, its lower efficacy means that higher doses are required and thus represents a greater risk of immunogenicity (Dressler and Bigalke, 2005). In addition, BoNT/B is also associated with several adverse outcomes such as painful injections, shorter duration of action and more frequent side effects (Bentivoglio et al., 2015). The major adverse effects are also often associated with treating muscle spasms, but not cosmetic applications. This is because the adverse effects are largely due to diffusion of toxins to other regions of the body and the possibility of toxin diffusion is directly related to injected doses. The adverse effects ranges from transient non-serious events such as ptosis and diplopia to life-threatening events, even death.
The binding of BoNT/A and /B to neurons has been characterised in details, and is based on a dual-receptor mechanism, involving a synaptic vesicle protein and a ganglioside anchored on the neuronal membrane. The protein receptor for BoNT/A was identified as SV2 (Dong et al., 2006, Mahrhold et al., 2006). More precisely, BoNT/A can bind to several human SV2 isoforms A, B and C, although the toxin only recognise the N-glycosylated forms of SV2A and SV2B (Yao et al., 2016). The protein receptor for BoNT/B is synaptotagmin (Syt) (Nishiki et al., 1994, 1996; Dong et al., 2003), with a preference for Syt1 over Syt2 in humans (Strotmeier et al., 2012). Ganglioside recognition is the first step of the intoxication process for all BoNTs (Binz and Rummel, 2009), and is mediated by a shared binding mechanism centred on the conserved motif H . . . SxWY . . . G in their sequence. BoNT/A prefers binding to the terminal N-acetylgalactosamine—galactose moiety of GT1b and GD1a (Takamizawa et al. 1986; Schengrund et al. 1991), while data on BoNT/B suggest a preference for the disialyl motif of GD1b and GT1b. The different serotypes vary in their carbohydrate specificity and affinity (Rummel, 2013).
The modular arrangement and distinctive properties of the various BoNT serotypes have made the toxins a target of choice for protein engineering. In particular, several studies have showed that it was possible to swap whole domains between serotypes (Masuyer et al., 2014) and thus obtaining new toxins with unique pharmaceutical potential. For example several molecules consisting of the binding domain of BoNT/B associated with the translocation and catalytic domains of BoNT/A have been produced (Rummel et al., 2011; Wang et al., 2012; Kutschenko et al., 2017).
These so-called chimeric toxins presented attractive pharmacological properties in terms of efficacy and duration of activity, which were associated with the high affinity of BoNT/B for synaptotagmin and the higher expression of this receptor on neurons compared to SV2 (Takamori et al., 2006; Wilhelm et al., 2014).
Because both the generation of neutralizing antibodies and toxin diffusion are directly related to injected doses, lowering toxin doses, while maintaining the same levels of toxin activity, is highly desired, which means the efficacy of individual toxin molecules has to be enhanced. It is therefore an object of the present invention to provide BoNT polypeptides with improved duration and potency, and with less risk of spreading from the site of injection. The inventors have identified a key problem with the previous attempts mentioned above in engineering chimeric BoNT polypeptides. None of the previous attempts took the structural aspect of the polypeptide into account.
Using a structure-based approach and the current knowledge on the receptor binding mechanisms of BoNT/A and /B, the inventors have engineered a new molecule, TriRecABTox (BoNT/TAB) comprising a specifically engineered HC domain (HC/TAB) that is able to recognise a SV2C receptor, a synaptotagmin receptor and a ganglioside receptor. The inventors show that BoNT/TAB can be recombinantly expressed and purified. Using X-ray crystallography, the inventors further demonstrate that BoNT/TAB can bind to its three receptors simultaneously. Thus, BoNT/TAB should recognise neuronal cells with enhanced affinity and has the potential to be a high-efficacy alternative to BoNT/A treatment.
The object above is thus attained by in a first aspect providing a botulinum neurotoxin (BoNT) Heavy Chain Binding domain (HC/TAB), wherein the HC/TAB comprises a) a synaptotagmin (Syt) receptor binding site, and b) a synaptic associated vesicle 2 (SV2) receptor binding site, and c) a ganglioside (Gang) receptor binding site, and wherein said HC/TAB is adapted to synergistically bind to a synaptotagmin (Syt) receptor, a synaptic associated vesicle 2 (SV2) receptor and a ganglioside (Gang) receptor.
The HC/TAB has a N-terminal end (HCN) and a C-terminal end (HCC). According to one embodiment the HCC domain is composed interchangeably of sequences from BoNT serotype A (BoNT/A) and BoNT serotype B (BoNT/B).
According to a further embodiment said HCC end is composed according to a sequence A1B1A2B2A3, where A indicates a sequence from BoNT/A, and B indicates a sequence from BoNT/B.
According to yet a further embodiment the sequences of B1, A2 and B2 comprise mutations and/or deletions to create stable intramolecular interfaces for the entire HC/TAB.
According to yet a further embodiment the sequences forming the Gang receptor binding site originate from any Gang-receptor binding BoNT serotype and their subtypes.
According to yet a further embodiment the sequences forming the Gang receptor binding site originate from BoNT/B.
According to yet a further embodiment the sequences forming the Gang receptor binding site are located in B2.
According to yet a further embodiment the sequences forming the Syt receptor binding site originate from any Syt receptor-binding BoNT serotype and their subtypes.
According to yet a further embodiment the sequences forming the Syt receptor binding site originate from BoNT B, DC or G.
According to yet a further embodiment the sequences forming the Syt receptor binding site are located in B1 and B2.
According to yet a further embodiment the HCN sequence originates from any SV2-receptor binding BoNT serotype and their subtypes
According to yet a further embodiment the HCN sequence originates from BoNT/A.
According to yet a further embodiment the sequences forming the SV2 receptor binding site are located in HCN and in A1 and A3 in the HCC.
According to yet a further embodiment the HC/TAB has an amino acid sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of any of the SEQ. ID.No's 1, 3, 5, 6, 8, 10 or 12.
According to a second aspect, there is provided a polypeptide comprising the HC/TAB according to the first aspect and any embodiment of the first aspect, coupled to any other protein, polypeptide, amino acid sequence or fluorescent probe, directly or via a linker.
According to an embodiment of the second aspect, said polypeptide is a BoNT polypeptide (BoNT/TAB), characterized in that said BoNT/TAB in addition to the HC/TAB comprises a Heavy Chain Translocation domain (HN), a Light chain (LC) and an protease site positioned between the LC and HN in the polypeptide sequence, wherein the HN and the LC, respectively and independently of each other, originate from any of the BoNT serotypes A, B, C, D, DC, E, En, F, G or X and their subtypes, as well as BoNT-like polypeptides.
According to a further embodiment, the polypeptide may comprise any other protein, polypeptide, amino acid sequence or fluorescent probe, linked thereto directly or via a linker.
According to yet a further embodiment the protease site is an exoprotease site. According to yet a further embodiment the exprotease site is a Factor Xa site.
According to yet a further embodiment the polypeptide according the second aspect has an amino acid sequence which is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identical to the sequence of any of the SEQ. ID. No's 1, 3, 5, 6, 8, 10 or 12.
According to a third aspect is provided a vector comprising a nucleic acid sequence encoding a HC/TAB according to the first aspect and any embodiment of the first aspect, or the polypeptide according to the second aspect and any embodiment of the second aspect.
According to a fourth aspect is provided for the use of the HC/TAB according to the first aspect and any embodiment of the first aspect, or the polypeptide according to the second aspect and any embodiment of the second aspect, in a therapeutic method or in a cosmetic method.
According to one embodiment of the fourth aspect, the therapeutic method or cosmetic method is a treatment to dampen and/or inactivate muscles.
According to a further embodiment of the fourth aspect, the therapeutic method is treatment and/or prevention of a disorder chosen from the group comprising neuromuscular disorders, conditions involving the release of acetylcholine, and spastic muscle disorders.
According to yet a further embodiment the disorder is chosen from the group comprising of spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity and other voice disorders, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, ties, tremors, bruxism, anal fissure, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, secretary disorders, pain from muscle spasms, headache pain, sports injuries, and depression.
According to yet a further embodiment the HC/TAB according to the first aspect and any embodiment of the first aspect, or the polypeptide according to the second aspect and any embodiment of the second aspect, may be used in a pharmacological test, to investigate the role of said protein, polypeptide, amino acid sequence or fluorescent probe in a synaptic process.
According to yet a further embodiment the HC/TAB according to the first aspect and any embodiment of the first aspect, or the polypeptide according to the second aspect and any embodiment of the second aspect, may be used as a vehicle for effectively transporting any protein, polypeptide amino acid sequence or fluorescent probe coupled thereto to a neuronal surface.
According to yet a further embodiment the HC/TAB according to the first aspect and any embodiment of the first aspect, or the polypeptide according to the second aspect and any embodiment of the second aspect, may be used as a vehicle for effectively transporting any protein, polypeptide amino acid sequence or fluorescent probe into a neuronal cytosol using a toxin translocation system.
According to a fifth aspect is provided a pharmaceutical or cosmetic composition comprising the HC/TAB according to the first aspect and any embodiment of the first aspect, or the polypeptide according to the second aspect and any embodiment of the second aspect.
According to one embodiment of the fifth aspect, the composition may further comprise pharmaceutically and/or cosmetically acceptable excipients, carriers or other additives.
According to a sixth aspect is provided a kit of parts comprising the composition of the fifth aspect and directions for therapeutic administration of the composition.
According to a seventh aspect is provided a method of treating a condition associated with unwanted neuronal activity, the method comprising administering a therapeutically effective amount of the HC/TAB according to the first aspect and any embodiment of the first aspect, or the polypeptide according to the second aspect and any embodiment of the second aspect, or composition of the fifth aspect, to a subject to thereby treat the condition, wherein the condition is chosen from the group comprising of spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity and other voice disorders, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, ties, tremors, bruxism, anal fissure, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, secretary disorders, pain from muscle spasms, headache pain, sports injuries, and depression, and dermatological or aesthetic/cosmetic conditions.
Protein sequences were aligned with ClustalO (Sievers et al., 2011). The segments of HC/A and HC/B used in the design of HC/TAB are highlighted in black (white writing) and light grey (black writing), respectively. The positions where deletions were included are shown in darker grey (dash).
As used herein, the term Botulinum neurotoxin “BoNT” encompasses any polypeptide or fragment from a Botulinum neurotoxin. The term BoNT may refer to a full-length BoNT. The term BoNT may refer to a fragment of the BoNT that can execute the overall cellular mechanism whereby a BoNT enters a neuron and inhibits neurotransmitter release. The term BoNT may simply refer to a fragment of the BoNT, without requiring the fragment to have any specific function or activity.
As used herein, the term “translocation domain” or “HN” means a BoNT domain that can execute the translocation step of the intoxication process that mediates BoNT light chain translocation. Thus, an HN facilitates the movement of a BoNT light chain across a membrane into the cytoplasm of a cell.
As used herein, the term “binding domain” is synonymous with “HC domain” and means any naturally occurring BoNT receptor binding domain that can execute the cell binding step of the intoxication process, including, e.g., the binding of the BoNT to a BoNT-specific receptor system located on the plasma membrane surface of a target cell.
In the present disclosure, the terms “nucleic acid” and “gene” are used interchangeably to describe a nucleotide sequence, or a polynucleotide, encoding for a polypeptide.
As specified above in the background section, a BoNT comprises a light chain (LC), linked by a single disulphide bridge to the heavy chain (HC). The Heavy chain (HC) holds two of the functional domains, with the N-terminal translocation domain (HN) and the C-terminal binding domain (HC), while LC is responsible for intracellular catalytic activity. The HC thus comprises the receptor binding domains which are able to specifically and irreversibly bind to the specific receptors expressed on susceptible neurons, whereas the HN forms a channel that allows the attached LC to translocate from endosomal-like membrane vesicles into the cytosol. Different BoNT serotypes have different sets of receptor binding sites on the HC, typically two receptor binding sites. The inventors have made use of this knowledge in engineering a novel BoNT HC binding domain (HC/TAB) comprising binding sites for three different receptors.
The inventors have accomplished this by engineering a HC/TAB domain comprising:
The structure of the engineered HC/TAB domain allows the HC/TAB to synergistically bind to a synaptotagmin (Syt) receptor, a synaptic associated vesicle 2 (SV2) receptor and a ganglioside (Gang) receptor. Thus a synergistic binding to three receptors on the neuron cell is accomplished, causing the novel HC/TAB domain to have enhanced affinity as compared to other BoNT HC domains. Thus an overall binding to neurons is improved and consequently the efficacy of the toxin is improved.
The HC further comprises an N-terminal end (HCN) and a C-terminal end (HCC). A key feature of the present invention is the structure of the HCC end of the HC/TAB, which is where the receptor binding domains are located in BoNT.
In one embodiment of the HC/TAB, the HCC end is composed interchangeably of sequences from the BoNT serotype A (BoNT/A) and BoNT serotype B (BoNT/B). By engineering this interchangeable structure, the inventors have been able to optimize a synergistic binding to all three receptors.
In a further embodiment of the invention, the HCC end is composed according to a sequence A1B1A2B2A3, where A indicate a sequence from BoNT/A, and B indicate a sequence from BoNT/B, see
In order to further optimize the HC/TAB according to the above, mutations and deletions have been introduced to create stable intramolecular interfaces, see
According to specific embodiments above, and all of the examples below, the ganglioside receptor binding site originates from BoNT/B, but it is conceivable that it may originate from any Gang receptor-binding BoNT serotype and their subtypes, such as the BoNT serotypes A, B, C, D, DC, E, En, F, G or X, or subtypes thereof, since all of the serotypes have a ganglioside receptor binding site.
According to a preferred embodiment of the present invention, the sequences forming the Gang receptor binding site are located in B2.
The SV2 receptor binding domain normally may originate from any SV2 receptor binding BoNT serotype and their subtypes, and in particular from BoNT serotypes A, D, E and F. In the specific embodiments above and all of the examples below, the SV2 receptor binding domain originates from BoNT/A, but as the skilled person will appreciate, any serotype comprising a SV2 receptor binding domain may be used as the origin for said domain, in accordance with the purpose and intended use of the HC/TAB according to the appended claims.
Part of the SV2 receptor binding domain is present in the HCN end. Thus, as a consequence the HCN sequence may originate from any of the SV2-receptor binding BoNT serotypes and their subtypes. In the specific embodiments above and all of the examples below, the HCN end originates from BoNT/A. However, as the skilled person will appreciate, as long as the SV2 receptor binding domain is functional, the HCN sequence may also originate from any of BoNT serotypes C, D, E, F or G.
Furthermore, according to a preferred embodiment of the present invention, the sequences forming the SV2 receptor binding site are located in HCN and in A1 and A3 in the HCC.
The Syt receptor binding site may originate from any Syt receptor binding BoNT serotype and their subtypes. In particular, the Syt receptor binding site may originate from BoNT serotypes B, chimera DC or G. According to a preferred embodiment of the present invention, the sequences forming the Syt receptor binding site are located in B1 and B2.
The present invention also provides for a polypeptide comprising the HC/TAB according to the above. The polypeptide may thus comprise any other protein, polypeptide, amino acid sequence or fluorescence probe, being coupled to the HC/TAB either directly or via a linker. Hereinafter, a protein, polypeptide or amino acid sequence to be coupled to the HC/TAB is referred to as “protein”.
According to one preferred embodiment, the polypeptide is a recombinant BoNT polypeptide (BoNT/TAB) further comprising a HN and a LC, as well as an exoprotease site positioned between the LC and HN in the polypeptide sequence.
The exoprotease site enables the single-chain polypeptide to be cleaved into a di chain molecule, causing the molecule to become an active toxin. According to an embodiment of the invention, the exoprotease site is a Factor Xa site, although this is not a limiting feature of the polypeptide according to the invention.
According to one embodiment, the BoNT/TAB in its active form is according to the SEQ. ID. No. 5. According to another embodiment, the BoNT/TAB in its active form is according to any of the sequences of SEQ. ID. No's 6, 8, 10 or 12. Preferably, the BoNT/TAB in its active form is according to SEQ. ID. No. 12.
Both the HN and the LC may, respectively and independently, originate from any of the BoNT serotypes A, B, C, D, DC, E, En, F, G or X and their subtypes, as well as BoNT-like polypeptides. New proteins resembling BoNT, i.e. with a similar domain architecture and varying degree of sequence identity, but produced by other organisms than C.-botulinum, are emerging. Thus, the skilled person will be able to choose a HN and/or a LC from any of the BoNT serotypes, their subtypes, or BoNT-like polypeptides.
The mutations and deletions that are introduced in the HC/TAB as specified above, further ensure that an engineered BoNT/TAB may be produced as a soluble protein with the correct structure and required activity.
A polypeptide according to the above is preferably produced recombinantly as the HC/TAB needs to be produced recombinantly.
Thus, the present disclosure also provides for isolated and/or recombinant nucleic acids encoding any of the HC/TAB or polypeptides according to the above. The nucleic acids encoding the HC/TAB or polypeptides of the present disclosure may be DNA or RNA, double-stranded or single stranded. In certain aspects, the subject nucleic acids encoding the isolated polypeptide fragments are further understood to include nucleic acids encoding polypeptides that are variants of any of the HC/TAB or polypeptides described herein. Variant nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions, such as allelic variants.
The present invention also provides for a vector comprising a nucleic acid sequence encoding the HC/TAB according to the above. The vector may further comprise a nucleic acid sequence encoding any other protein or probe that is to be recombinantly produced together with the HC/TAB, so as to obtain said protein or probe coupled to the HC/TAB in one polypeptide. The vector is preferably an expression vector. The vector may comprise a promoter operably linked to the nucleic acid. A variety of promoters can be used for expression of the polypeptides described herein, and are known to the person skilled in the technical field.
An expression vector comprising the nucleic acid can be transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation) and the transfected cells are then cultured by conventional techniques to produce the polypeptides described herein. In some embodiments, the expression of the polypeptides described herein is regulated by a constitutive, an inducible or a tissue-specific promoter.
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 of the invention are not limiting to the scope of the invention.
Purification and modification of recombinant proteins is well known in the art such that the design of the polyprotein precursor could include a number of embodiments readily appreciated by a skilled worker.
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.
It may be advantageous to comprise a HN according to the above in the polypeptide together with the HC/TAB, and replace the LC with the protein of interest, if an internalization of the protein is desired, as the HN then will provide a channel allowing the protein to translocate into the neuronal cell. It may be advantageous to couple the protein of interest directly to the HC/TAB if the neuronal cell surface is the target for the protein. Thus, the following combinations may be obtained, depending on the aimed delivery:
By coupling a cargo protein to the HC/TAB, according to i) above, the cargo protein may be targeted to the neuronal surface. Some internalisation via regular cell surface recycling processes would probably occur, but the neuronal surface would be the main target of such an approach.
By coupling a cargo protein to a HN coupled to the HC/TAB according to ii) above, or to the BoNT/TAB according to iii) above, said cargo proteins may be more effectively transported inside neurons using the toxin translocation system. Once the BoNT toxin has been internalized in the neuron cell in the vesicles, as described in the background, the acidic endosomal environment in the vesicle causes a conformational change that allows translocation of LC from the vesicle into the cytosol of the cell. Thus, said toxin translocation system which is the mechanism for translocating the LC of BoNT from the internalized vesicle into the cytosol, may be used to translocate the above mentioned cargo protein into the cytosol of the neuron cell, by use of the BoNT/TAB. A cargo protein may be coupled to the HN instead of the LC, with an exoprotease site positioned between the cargo protein and HN as disclosed above, or a cargo protein may be coupled to the LC. Both variants will enable a transportation of the cargo protein into the cytosol of the neuronal cell. Thus, both the HC/TAB and the BoNT/TAB may be used as vehicles for transporting any protein to and/or into a neuron. This also provides for the possibility of using the HC/TAB and/or the BoNT/TAB in a pharmacological test to investigate the role of a protein in for instance a synaptic process.
The cargo protein may for instance be any protein tag, such as affinity or fluorescent tags or probes. Thus, any corresponding nucleic acid to such a protein tag may be included in the vector disclosed above. The skilled person will be able to use standard cloning methods in order to comprise any gene of interest in the vector, as well as standard protocols for the protein expression.
The binding domain of BoNT and the cargo protein could be expressed separately with a sortase system that allow their recombination post-translationally. The transpeptidase activity of sortase may thus be used as a tool to produce fusion proteins in vitro and is well within the knowledge of a skilled person within this technical field. In short, a recognition motif (LPXTG) (SEQ ID NO: 15) is added to the C-terminus of a protein of interest while an oligo-glycine motif is added to the N-terminus of the second protein to be ligated. Upon addition of sortase to the protein mixture, the two peptides are covalently linked through a native peptide bond. This method may be used to produce a polypeptide according to the present invention. In the present case, this would mean that the recognition motif is added to the C-terminus of the protein of interest, and the oligo-glycine moif is added to the N-terminus of the HC/TAB or BoNT/TAB.
Additionally, the HC/TAB and/or the BoNT/TAB may be used in a therapeutic method or cosmetic method. Typically, the use of HC/TAB and/or the BoNT/TAB may be very similar to the uses that are already in place for BoNT/A and/or BoNT/B products. These include methods and treatments wherein the purpose of the method and treatment is to dampen and/or inactivate muscles.
The HC/TAB according to the invention enables injections of a BoNT/TAB having a higher affinity to the cell and consequently a higher efficiency. Thus, lower doses are required and a longer duration of action is possible. Therefore, a smaller amount of BoNT/TAB as compared to BoNT/A or BoNT/B, may be injected for the same effect, which decreases adverse effects as less BoNT/TAB will spread from the site of injection. With a higher efficiency, stronger and more efficient binding, and lower dose required, there are less redundant BoNT/TAB available to spread to beyond the injection site. Furthermore, the BoNT could be administered less often with sustained effect, which would also minimize the risk of an immune response and adverse reactions as a consequence thereof.
Typical medical conditions that may be treated and/or prevented with the HC/TAB and/or the BoNT/TAB according to the above are disorders chosen from the group comprising neuromuscular disorders, conditions involving the release of acetylcholine, and spastic muscle disorders. More specifically is may relate to disorders chosen from the group comprising of spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity and other voice disorders, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, ties, tremors, bruxism, anal fissure, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhydrosis, excessive salivation, excessive gastrointestinal secretions, secretary disorders, pain from muscle spasms, headache pain, sports injuries, and depression.
With regards to cosmetic methods, the HC/TAB and/or the BoNT/TAB may preferably be used to prevent and/or treat wrinkles, brow furrows or unwanted lines, in order to reduce said wrinkles, furrows and lines.
The HC/TAB and/or the BoNT/TAB according to the above may be formulated in any suitable pharmaceutical or cosmetic composition. The pharmaceutical composition comprising the HC/TAB and/or the BoNT/TAB may further comprise pharmaceutically acceptable excipients, carriers or other additives. The cosmetic composition comprising the HC/TAB and/or the BoNT/TAB may further comprise cosmetically acceptable excipients, carriers or other additives.
The administration of the pharmaceutical or cosmetic composition may be via injection, wherein the injection is administered at the site of the body where unwanted neuronal activity is present. Typically, compositions for administration by injection are solutions in sterile isotonic aqueous buffer. Where necessary, the composition can also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection.
Furthermore, the pharmaceutical or cosmetic composition may be comprised in a kit with directions for therapeutic administration of the composition. In such a kit, the ingredients of the composition may be supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. The composition may be administered by infusion, and can in that case be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration. A composition for systemic administration may be a liquid, e.g., sterile saline, lactated Ringer's or Hank's solution. In addition, the composition can be in solid forms and re-dissolved or suspended immediately prior to use. Lyophilized forms are also contemplated. The composition can be contained within a lipid particle or vesicle, such as a liposome or microcrystal, which is also suitable for parenteral administration.
Thus, the inventors have developed an engineered BoNT biohybrid adapted to simultaneously bind to all three of the SV2C receptor, the synaptotagmin receptor and the ganglioside receptor. Thereby, a BoNT biohybrid is provided that has a higher potency, efficacy and duration than the BoNT polypeptides of the prior art. Use of the present biohybrid thereby enables administration of lower doses of the toxin than according to the prior art, while maintaining the same effect. Furthermore, use of the present biohybrid enables less frequent administrations than for the BoNT's previously used. Thus, a treatment of a patient with the BoNT biohybrid of the present invention will be more comfortable in that administration does not have to occur as often as in the prior art.
Constructs. The cDNA encoding HC and full-length (inactive) TriRecABTox (HC/TAB and BoNT/TAB, respectively) were codon-optimised for E. coli expression (see supplementary information for DNA sequence), synthesised and cloned into a pET-28a(+) vector with a N-terminal 6×His-tag (GenScript, NJ, USA). The TriRecABTox construct used in our study has three mutations at the catalytic site to avert any safety concerns (E224Q/R363A/Y366F) (Rossetto et al, 2001; Binz et al, 2002). The BoNT/TAB gene encodes for 1311 amino acids, and the HC/TAB gene corresponds to residues [875-1311].
Protein expression and purification. Plasmids carrying the gene of interest were transformed into E. coli BL21 (DE3) cells (New England Biolabs, USA). A similar protocol was used for both proteins. Expressions were carried out by growing cells in terrific broth medium with 50 μg/ml kanamycin at 37° C. for approximately 3 hours and then induced with a 1 mM final concentration of IPTG, and left overnight at 18° C., in a LEX system (Epyphite3, Canada). Cells were harvested and stored at −80° C. Cell lysis for protein extraction was performed with an Emulsiflex-C3 (Avestin, Germany) at 20 kPsi in 25 mM HEPES pH 7.2 with 200 mM NaCl, 25 mM imidazole and 5% (v/v) glycerol. Cell debris were spun down via ultra-centrifugation at 4° C., 267,000 g for 45 min. The protein was first purified by affinity chromatography: the supernatant was loaded onto a 5 ml HisTrap FF column (GE Healthcare, Sweden), washed with 25 mM HEPES pH 7.2, 200 mM NaCl, 25 mM imidazole and 5% (v/v) glycerol, and the protein eluted with 25 mM HEPES PH 7.2, 200 mM NaCl, 250 mM imidazole and 5% (v/v) glycerol. The sample was then dialysed against 25 mM HEPES pH 7.2, 200 mM NaCl, and 5% (v/v) glycerol overnight, before a final size exclusion purification step using a Superdex200 column in a similar buffer (GE Healthcare, Sweden). HC/TAB was kept at 4.5 mg/ml, and BoNT/TAB at 7.3 mg/ml, in 25 mM HEPES pH 7.2 with 200 mM NaCl, 0.025 mM TCEP and 5% glycerol.
Protein characterisation. Protein samples were analysed by gel electrophoresis using NuPAGE 4-12% Bis-Tris gels, and Western blots performed on PVDF membranes (ThermoFisher, Sweden). Primary antibodies against HC/A and HC/B were prepared in-house (raised in rabbit) and probed with an anti-rabbit IgG-Peroxidase antibody (catalogue #SAB3700852, Sigma, Sweden). The poly-histidine tag was probed using an HRP-conjugated monoclonal antibody (ADI.1.10, catalogue #MAI-80218, ThermoFisher, Sweden). TMB substrate (Promega, Sweden) was used for detection. In-house controls purified similarly to HC/TAB and consisting of His-tagged HC/A and HC/B were included for comparison.
Activation of BoNT/TAB. The full-length (inactive) TriRecABTox was designed with a Factor Xa cleavage site (IEGR) between the light and heavy chains for activation into a di-chain form. Activation was performed by incubating 100 μg of BoNT/TAB with 2 μg. of Factor Xa (New England BioLabs, USA) overnight at 4° C. Results of the activation was analysed by gel electrophoresis (as above).
Cloning, expression and purification of SV2C-L4. The interacting part of the fourth luminal domain of synaptic vesicle glycoprotein 2C (SV2C-L4, residues 474-567 Uniprot ID Q496J9) was amplified from cDNA and cloned into a pNIC28-Bsa4 (N-terminal His6 tag with TEV site) vector using LIC cloning. SV2CL4 was expressed in E. coli BL21 (DE3) (New England Biolabs, USA) using a protocol similar to the one described above. His-tagged SV2C-L4 was purified by affinity chromatography on a 2 ml HisTrap HP column (GE Healthcare, Sweden), washed with 20 mM HEPES, pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 50 mM Imidazole, and 0.5 mM TCEP. The protein eluted with 20 mM HEPES, pH 7.5, 500 mM NaCl, 10% (v/v) glycerol, 500 mM Imidazole, and 0.5 mM TCEP. SV2CL4 was then purified further by size exclusion using a Superdex 75 Hiload 16/60 column (GE Healthcare, Sweden) in 20 mM HEPES, pH 7.5, 300 mM NaCl, 10% (v/v) glycerol, and 0.5 mM TCEP.
X-ray crystallography. Samples for crystallisation were prepared by pre-incubation for 15 min at room temperature of HC/TAB at 3.6 mg/ml, with SV2C-L4 at 1 mg/ml (recombinant human SV2C extracellular loop-4 [residues 475-565], 1 mM hSytI peptide (GEGKEDAFSKLKEKFMNELHK (SEQ ID NO: 14), synthesised by GenScript, USA) and 4 mM GD1a oligosaccharide (Elicityl, France).
Crystals were grown with 200 nl of sample mixed with 100 nl of reservoir solution consisting of 20% v/v polyethylene glycol 6000, 0.1 M Citrate pH 5.0 (JCSG-plus screen B9, Molecular Dimensions, United Kingdom) using a sitting drop set-up and incubated at 21° C. Crystals appeared within 2 weeks and were transferred to a cryo-loop and frozen in liquid nitrogen.
Molecular replacement was performed with a model prepared from the coordinates of HC/A in complex with SV2C-L4 (PDB code 4JRA) and of HCB in complex with rat SytII and GD1a (PDB code 4KBB) to determine initial phases for structure solution in PHASER (McCoy et al., 2007). The working models were refined using REFMAC5 (Murshudov et al, 2011) and manually adjusted with COOT (Emsley et al., 2010). Water molecules were added at positions where Fo-Fc electron density peaks exceeded 3σ, and potential hydrogen bonds could be made. Validation was performed with MOLPROBITY (Chen et al., 2010). Ramachandran statistics show that 97.0% of all residues are in the most favoured region, with a single outlier in the disallowed region. Crystallographic data statistics are summarized in Table 1. Figures were drawn with PyMOL (Schrodinger, LLC, USA).
In order to materialise the concept of a three-receptor toxin, the inventors first analysed the structural information available on the BoNT/A and /B molecular interactions with their receptors. Recent work by Yao et al. (2016) and Benoit et al. (2014) provided the X-ray crystal structures of the receptor-binding domain of BoNT/A in complex with SV2C with (PDB 5JLV) and without post-translation modification (PDB 4JRA), respectively. The luminal domain of SV2C (loop4) forms a quadrilateral β-helix that associates with HC/A mainly through backbone-to-backbone interactions with an opened β-strand at the interface of the two subdomains, while the N-glycan of SV2C extends towards HCN (
Several crystal structures of BoNT/B in complex with synaptotagmin have also been described and helped define the toxin's interaction with its receptor (Chai et al., 2006; Jin et al., 2006; Berntsson et al., 2013) (
Additionally, the crystal structures BoNT/A and /Bin complex with their ganglioside receptor (Stenmark et al., 2008; Hamark et al., 2017; Berntsson et al., 2013) provided a detailed description of the carbohydrate binding site for each serotype. The site is highly conserved across the botulinum neurotoxin family and consists of a shallow pocket on the HCC subdomain (
After identification of the components from the two serotypes that are essential for binding to the three different receptors, further structural analysis was performed to integrate them into a single molecule. To this extent, the primary sequences of BoNT/A (Uniprot P10845) and BoNT/B (Uniprot P10844) were aligned with ClustalO (Sievers et al., 2011), and the three-dimensional structures of their binding domain superposed (
The resulting molecule, named TriRecABTox, should be able to bind to the three receptors: SV2, synaptotagmin and gangliosides. Its protein sequence is provided in SEQ. ID. No. 3 (inactive form) and SEQ. ID. No 5 (active form).
The first step towards the characterisation of TriRecABTox was to recombinantly produce the binding domain (HC/TAB) in order to analyse its biochemical properties. For this purpose, the protein sequence was codon-optimised for expression in E. coli. The resulting gene was cloned into a pET-28a(+) vector so as to include a N-terminal poly-histidine tag and facilitate the protein purification process, details are provided in the methods section. The inventors showed that HC/TAB could be expressed and partially purified (
In an effort to evaluate the capacity of HC/TAB to bind to its three receptors, co-crystallisation trials were set up that included HC/TAB with the human SV2C luminal domain [residues 475-565], the human Syt1 peptide [residues 34-53] and the GD1a carbohydrate. Crystals were obtained that diffracted to high resolution (1.5. Å) (
Firstly, the binding domain of the newly designed BoNT/TAB presents the expected fold with its two subdomains: the lectin-like HCN and the β-trefoil fold of HCC (
The HC/TAB-3R structure was compared to that of HC/Å in complex with SV2C. The structure of the SV2C luminal domain is identical in both complexes, with an rmsd of 0.483 Å (over 88 Cα). The two structures were aligned in three-dimension based on the HC domains and showed that SV2C is in the same location, as expected from the inventor's design (
The inventors then compared the HC/TAB-3R structure with that of HC/B in complex with rSyt2. BoNT/B is expected to bind to human synaptotagmin in a similar fashion to its rodent homologues, albeit with varying affinities (Tao et al., 2017). In the crystal structure presented here, hSyt1 also takes on an α-helical arrangement that sits within the same binding groove as rSyt2 in HC/B (
The third receptor contained in the HC/TAB-3R structure corresponds to the GD1a carbohydrate, for which clear electron density was observed from Gal2 to Sia5 (
Overall the results obtained from the HC/TAB-3R crystal structure confirms that a single TriRecABTox molecule is able to simultaneously bind to SV2 receptor, synaptotagmin receptor and its ganglioside receptors in a manner that replicates the binding mechanisms of the parent BoNT/A and /B.
Having established the binding capability of HC/TAB the inventors went on to express and purify the full-length, catalytically inactive, TriRecABTox (BoNT/TAB; SEQ. ID. No. 3). For this purpose, the inventors designed a synthetic gene encoding for 1311 amino acids and containing the three BoNT functional domains, with LC and HN corresponding to the BoNT/A domains, associated with HC/TAB. Three mutations at the catalytic site were included for safety considerations (E2240/R363A/Y366F) (Rossetto et al, 2001; Binz et al, 2002). As per the HC/TAB construct described above, the protein sequence was codon-optimised for expression in E. coli, and cloned into pET-28a(+) with a N-terminal poly-histidine tag. Details are provided in the methods section. The inventors showed that BoNT/TAB could be expressed as a soluble protein of approximately 152 kDa. The initial method used for purification yielded limited amount of non-homogenous material (
Additional characterisation was carried out and confirmed the presence of the histidine-tag, and although the reaction with the probing antibody was very weak compared to the controls (
BoNT/TAB was designed with a Factor Xa cleavage site, IEGR [442-445], between the light and heavy chains (
Altogether the activation assay first provided evidence that the protein produced corresponds to the engineered BoNT/TAB, and secondly that the activation step into a di-chain molecule could be successfully managed. Therefore such step may be included in the production of active full-length TriRecABTox.
Constructs. The cDNA encoding HC/TAB and variants were cloned by GenScript (NJ, USA), in a pET28(a) vector as described previously. BoNT/TAB2.1.3 was cloned in a pET29(a) vector by Toxogen GmbH (Hannover, Germany).
Protein expression and purification. As described previously for HC/TAB variants. BoNT/TAB2.1.3 was produced by Toxogen GmbH (Hannover, Germany), with a protocol similar to the one used for HC/TAB (affinity chromatography and gel filtration). In addition, activation and tag removal of BoNT/TAB2.1.3 was performed with Thrombin at a concentration of 0.05 U/μg, and BoNT/TAB2.1.3 was further purified by gel filtration. Samples were stored in 25 mM HEPES pH 7.2 with 200 mM NaCl, and 5% glycerol.
Protein characterisation. As described previously (gel electrophoresis using NuPAGE 4-12% Bis-Tris gels).
X-ray crystallography. Samples for crystallisation were prepared by pre-incubation for 15 min at room temperature of HC/TAB2.1 at 6.5 mg/ml, with SV2C-L4 at 1 mg/ml (recombinant human SV2C extracellular loop-4 [residues 475-565], 1 mM hSytI peptide (GEGKEDAFSKLKEKFMNELHK (SEQ ID NO: 14), synthesised by GenScript, USA) and 4 mM GD1a oligosaccharide (Elicityl, France). Crystals were grown with 200 nl of sample mixed with 100 nl of reservoir solution consisting of 20% v/v polyethylene glycol 3350, 0.2 M Potassium citrate (JCSG-plus screen B12, Molecular Dimensions, United Kingdom) using a sitting drop set-up and incubated at 21° C. Crystals appeared within 1 week and were transferred to a cryo-loop and frozen in liquid nitrogen. Diffraction data were collected at station 104 of the Diamond Light Source (Didcot, UK), equipped with a PILATUS-6M detector (Dectris, Switzerland). A complete dataset to 1.4 Å was collected from a single crystal at 100° K. Raw data images were processed and scaled with DIALS (Gildea et al, 2014), and AIMLESS (Evans, 2006) using the CCP4 suite 7.0 (CCP4, 1994).
Molecular replacement was performed with the structure of HC/TAB determined previously in PHASER (McCoy et al., 2007). The working models were refined using REFMAC5 (Murshudov et al, 2011) and manually adjusted with COOT (Emsley et al., 2010). Water molecules were added at positions where Fo-Fc electron density peaks exceeded 3σ, and potential hydrogen bonds could be made. Validation was performed with MOLPROBITY (Chen et al., 2010). Ramachandran statistics show that 97.0% of all residues are in the most favoured region, with a single outlier in the disallowed region. Crystallographic data statistics are summarized in Table X1.
Production of an Optimised HC/TAB, HC/TAB2.1
The crystal structure of HC/TAB bound to its three receptors was analysed in order to identify potential sites that could be modified to improve the molecule's stability and function.
In particular, analysis of the local temperature factors (B-factor) within a crystal structure may be interpreted as an indication of the local stability of a protein, with high B-factor suggestive of a disorderly region. From this analysis, a loop at the interface between the two subdomains of HC/TAB, labelled ‘loop 360’, consisting of residues D357 to N362 (SEQ ID: No. 6), was considered for optimisation (See
The plasmid for this new construct was prepared by site-directed mutagenesis (GenScript, USA) and used for recombinant expression of HC/TAB2.1 in E. coli. The protocol used was the same as for the production of HC/TAB (see original method section for expression and purification). We showed that HC/TAB2.1 could be expressed and partially purified using affinity chromatography and size exclusion techniques (
The purified HC/TAB2.1 (SEQ. ID. No. 6) was used in co-crystallisation trials with the human SV2C luminal domain [residues 475-565], the human Syt1 peptide [residues 34-53] and the GD1a carbohydrate. Crystals were obtained that diffracted to high resolution (1.4 Å) and a complete dataset could be collected (Table 2). The structure was solved by molecular replacement using the crystal structure of HC/TAB bound to it three receptors (HC/TAB-3R). The new structure presented all the elements already visible in HC/TAB-3R and provided experimental evidence that HC/TAB2.1 can bind to the three receptors simultaneously, as per HC/TAB. Analysis of the B-factor, showed an improved stability for loop ‘360’ (D357 to Y362;
In order to prepare for future functional analysis of the HC/TAB variants, HC/TAB2.1 was adapted to be compatible with a sortase ligation experiment described recently (Zhang et al, 2017). This experiment allows for a safe and controlled reconstruction of a full-length active BoNT that can be used to test activity. This construct corresponds to a N-terminal truncated HC/TAB2.1 with a cleavable N-terminal His-tagged, and was labelled HC/TAB2.1.1 (SEQ. ID. No. 8). The clone for HC/TAB2.1.1 was prepared (GenScript), used for expression and purification as described previously (
Further analysis of the structural features of HC/TAB2.1 highlighted the presence of a surface-exposed hydrophobic loop which protrudes from the rest of the protein (residues 389-393, SEQ ID: No. 6;
In order to prepare for future functional analysis of BoNT/TAB, a full-length active variant based on the HC/TAB2.1.3 construct was produced and labelled BoNT/TAB2.1.3 (SEQ. ID. No. 12). All steps of the production were carried out in a licensed facility, under contract agreement, at Toxogen GmbH (Hannover, Germany). BoNT/TAB2.1.3 was cloned in a pET29(a) vector and included cleavable C-terminal Strep-and poly-histidine tags, as well as an engineered thrombin cleavage site between the HC and LC domains (SEQ. ID. No. 13), for activation of the product, as described previously. BoNT/TAB2.1.3 could be expressed as a soluble protein, purified and activated with thrombin (
Assays will be performed where the receptor-binding properties of BoNT/TAB will be compared to BoNT/A and/or BoNT/B.
For example, ganglioside receptor-binding assays will be carried out that are adapted from previously described methods. Briefly, in this ELISA the ganglioside receptor of interest (GT1b, GD1b, GD1a, or GM1a) is immobilised on a 96-well microplate (Chen et al., 2008; Willjes et al., 2013), the toxins (or their binding domain) are then applied, and the bound material probed with a monoclonal anti poly-Histidine antibody conjugated to horse radish peroxidase (HRP). This qualitative approach should provide enough information to confirm that the ganglioside receptor-binding characteristics of BoNT/TAB are similar to that of BoNT/B.
Ganglioside receptor binding ELISA. Gangliosides GT1b, GD1b, GD1a, and GM1a are purchased from Carbosynth (Compton, UK). Gangliosides are diluted in methanol to reach a final concentration of 2.5 μg/ml; 100 μL (0.25 μg) is applied to each well of a 96-well PVC assay plates. After evaporation of the solvent at 21° C. (overnight), the wells are washed (3×) with 200 μL of PBS/0.1% (w/v) BSA. Nonspecific binding sites are blocked by incubation for 2 h at 21° C. in 200 μL of PBS/2% (w/v) BSA. Binding assays are performed in 100 μL of PBS/0.1% (w/v) BSA per well for 2 h at 4° C. containing the samples (serial 3-fold dilution ranging from 6 μM to 0.003 μM). Following incubation, wells are washed 3× with PBS/0.1% (w/v) BSA and then incubated with an HRP-anti-His antibody (ThermoFisher #MAI-80218) at a 1:2000 dilution (100μ1/well) for 1 hat 4° C. Finally, after three washing steps with PBS/0.1% (w/v) BSA, bound samples are detected using Ultra TMB (100 μL/well). The reaction is terminated after incubation for 5 min at 21° C. by addition of 100 μL of 1M sulphuric acid. Absorbance at 450 nm is measured with a Tecan Infinite 200 (Mannedorf, Switzerland). Results are analysed with Prism (GraphPad, La Jolla, CA, USA), using a non-linear binding fit.
In order to assess the binding properties to the synaptotagmin receptor, isothermal titration calorimetry (ITC) will be performed, similarly to the assay described by Berntsson et al. (2013). Binding of the hSyt peptides to the toxins will be measured and should provide affinity values (Kd) confirming that BoNT/TAB can bind to the receptor, analogously to BoNT/B.
Isothermal titration calorimetry. Samples are prepared by an additional size exclusion chromatography step (Superdex200, GE Healthcare, Sweden) in 20 mM potassium phosphate pH 7.0, 0.15 M NaCl. Association of Syt peptides to BoNT or their binding domains is measured on an ITC200 (GE Healthcare, Sweden) at 25° C. and 750 rpm. A 200 μL solution of protein (at 20 μM) is added to the cell. Binding is measured upon the addition of peptide (GenScript, USA) with 16 stepwise injections of 2.5 μL each, at a concentration of 200 μM. The first titration is set to 0.5 μL and is subsequently deleted in the data analysis. Data is analysed with the Origin software provided by the manufacturer
The binding to SV2C will be assessed using a pull-down assay such as the one described by Benoit et al. (2014). Briefly, the tagged toxin and non-tagged receptor (or inversely) will be incubated together and loaded onto a Ni-sepharose, then washed and eluted. Results will be visualised by SDS-PAGE.
The potency of BoNT preparation can be evaluated using a mouse Digit Abduction Score (DAS) assay (Broide et al., 2013). This assay measures in vivo the local muscle-weakening efficacy of the toxin after intramuscular injection into mouse or rat hind limb skeletal muscle. The toxin elicits a measurable dose-dependent decrease in the animal's ability to produce a characteristic hind limb startle response. This non-lethal method has been used regularly to estimate the pharmacological properties of different BoNT serotypes or derivatives, such as the recently described recombinant BoNT/B molecules (Elliot et al., 2017). A similar methodology will be used to assess the potency and duration of effect of BoNT/TAB, compared to BoNT/A or /B.
In this study the inventors described how the structural and molecular details of the binding mechanism of BoNT/A and /B were used to engineer a new molecule, TriRecABTox, that possesses enhanced cell recognition capability. A rigorous multi-dimension comparison of BoNT/A and /B structures allowed the inventors to identify the key elements necessary to keep an intact toxin scaffold on which to integrate the receptor binding sites for SV2, synaptotagmin and a ganglioside, in a single molecule. The newly created design, consisting of an alternation of BoNT/A and /B elements, was optimised by including adaptive mutations or deletions to compensate for the newly created non-natural intramolecular interfaces. Such modifications were deemed necessary to ensure that the engineered toxin, BoNT/TAB, could be produced as a soluble protein with the correct structure and required activity.
The inventors first assessed the stability of the design by producing the binding domain on its own, HC/TAB, which holds the modified receptor recognition function, via recombinant expression in E. coli. HC/TAB was expressed with a N-terminal poly-histidine tag as a soluble protein that could be partially purified, thus demonstrating the viability of the engineered construct. In a second step, the inventors proceeded with the production of the full-length BoNT/TAB construct, in a catalytically inactive form. Again, the inventors showed that it could be expressed as a soluble protein of 153 kDa and partially purified with standard liquid chromatography techniques. Presence of the poly-histidine tag on both HC/TAB and BoNT/TAB allowed their purification by affinity chromatography with a Ni-sepharose matrix. Other affinity methods may be used and include an affinity tag that should be preferentially positioned on the N-terminal end of the protein in order to prevent interference with receptor binding. Although the initial preparation showed heterogeneous sample purity, optimisation of the purification process should lead to a product of pharmaceutical standards. It should be added that the active form of BoNT/TAB would have a similar overall structure and binding properties to the inactive molecule used in the present study. The inventors contracted Toxogen GmbH (Hannover, Germany) to produce an active version of BoNT/TAB (BoNT/TAB2.1.3) that was purified successfully with a removable C-terminal tag, so as to not interfere with receptor binding.
In addition, post-translational cleavage of the single-chain BoNT into a di-chain molecule is an essential step for the toxin's activity (DasGupta and Sathyamoorthy, 1985; Shone et al, 1985). While the native toxin is usually activated by a host protease, any recombinant BoNT product needs to be processed with an exopeptidase. Early work on the toxin showed that trypsin could non-specifically cleave BoNT/A to an active di-chain form (Shone et al., 1985), however this may result in unwanted additional degradation of the toxin. More recently, recombinant technologies have allowed the engineering of specific protease recognition motifs within a protein of interest, thus providing better control on the activation strategy of BoNT (Sutton et al, 2005). Here the inventors included a Factor Xa site between LC and HC and observed complete activation of the toxin, thus demonstrating the effectiveness of this enzyme. Future production of BoNT/TAB should incorporate a purification stage that allows for activation of the toxin, followed by removal of the exoprotease from the final product. While Factor Xa appears adequate, other enzymes may be tested and prove successful in achieving acceptable yield of activation. The inventors contracted Toxogen GmbH (Hannover, Germany) to produce an active version of BoNT/TAB (BoNT/TAB2.1.3) that was successfully activated with the thrombin exoprotease and purified to homogeneity.
As a mean to verify the structural integrity of HC/TAB and confirm its enhanced functionality, the inventors co-crystallised the purified sample in complex with human SV2C, human Syt1 and the GD1a carbohydrate. The X-ray crystal structure of the complex was solved to high resolution (1.5 Å), and provided conclusive experimental evidence that a single molecule of HC/TAB could bind to all three receptors simultaneously. Furthermore, comparison to the known structures of HC/A and HC/B with their respective receptors showed that HC/TAB follows an almost identical mechanism of binding.
While the crystal structure demonstrated that HC/TAB could fulfil its purpose, at least in vitro, additional biochemical experiments need to be performed to fully characterise its receptor binding properties. These will include pull-down and ITC assays with the protein receptors, and ganglioside receptor binding ELISA. BoNT/TAB is expected to perform similarly to BoNT/A for SV2 receptor binding, and similarly to BoNT/B with regards to ganglioside receptor and synaptotagmin receptor binding. Additionally, in vivo experiments will provide the main indications on the true potential of BoNT/TAB as a therapeutic. The mouse DAS assay has classically been used to assess BoNT preparations (Broide et al., 2013) and should allow the inventors to determine the efficacy and duration of action of our molecule compared to the currently available products.
Additionally, the design of BoNT/TAB may be further optimised by modifying some sequence elements to improve its biochemical properties and stability. Such alterations may include deletions or mutations that lead to a soluble BoNT still able to simultaneously bind to three receptors. The inventors successfully produced a more stable variant (HC/TAB2.1) and a more soluble variant with higher production yield (HC/TAB2.1.3).
It should be added that from a safety perspective, BoNT/TAB do not represent a novel threat since it is derived from two existing serotypes. It is expected to be recognised by currently available anti-toxins, such as the Botulism Antitoxin Heptavalent BAT or other approved antidotes for BoNT/A and /B.
Serotypes A and B are the only approved BoNTs available on the market. While BoNT/A is the main toxin used therapeutically, molecules with lower immunogenicity and high efficacy would provide safer alternatives (Naumann et al., 2013). Multiple attempts have been made at improving the properties of BoNTs in order to increase their pharmacological potential (Masuyer et al., 2014). A recent successful example include the study by Tao et al. (2017) where mutations engineered in key positions of BoNT/B (E1191M/S1199Y) gave the toxin higher affinity for the human synaptotagmin2 receptor, and showed approximately 11-fold higher efficacy in blocking neurotransmission compared to the wild type. Another approach to improve BoNT efficacy was taken by Elliott et al. (2017) where they analysed the effect of a single mutation (S201P) known to increase the catalytic activity of BoNT/B on its substrate. In this case, the BoNT/B mutant did not present any advantage over the wild type in multiple cell-based assays and in vivo. Altogether these two studies on BoNT/B suggest that the limiting step in the toxin's efficacy resides in the initial neuronal recognition rather than the later intracellular activity.
Earlier studies intending to combine the binding properties of one serotype with the catalytic activity of another led to the design of chimeric molecules where whole domains were swapped (Wang et al., 2008, 2012; Rummel et al., 2011). More particularly, Rummel et al. (2011) and Wang et al. (2012) designed and tested analogous molecules consisting of the HC/B domain associated with the HN+LC domains of BoNT/A. These recombinant toxins were reported to display enhanced potency and induced a lengthier effect in mice compared to the wild type BoNT/A (Kutschenko et al., 2017). Similar observations were obtained when assessing a construct consisting of the C-terminal subdomain (HCc) of BoNT/B coupled with the complementary domains of serotype A (i.e. LC+HN+HCn), and which showed a four-fold higher potency compared to the wild-type (Rummel et al., 2011). All the molecules described above had in common the fact that they would only recognise the two receptors of BoNT/B, synaptotagmin and ganglioside. These results suggest that prolonged effect and higher efficacy may be obtained thanks to a greater intake of LC/A permitted by the higher prevalence of the BoNT/B receptors on neurons. In addition, these chimeric molecules did not take into account the possible inter-domain intra-molecular clashes that may arise from combining domains from different serotypes, and which may affect the potential of these products.
Taking into considerations the results from the latest studies on BoNT engineering, it appears clear that modifying initial cellular recognition is one of the most efficient ways to enhance the pharmacological properties of the therapeutic product. Therefore BoNT/TAB, a single product successfully engineered to recognise SV2 receptor together with the BoNT/B receptors, synaptotagmin and ganglioside, represents a great potential and could yet be more efficacious than the wild type BoNT/A and /B.
The main innovation in BoNT/TAB is the design of the binding domain allowing multiple receptor interactions. Current evidence hints that association of HC/TAB with the translocation and catalytic domains of BoNT/A should provide the molecule with the strongest potency (as designed in BoNT/TAB). However, HC/TAB may still be of interest when combined with the functional domains of other serotypes (
| Number | Date | Country | Kind |
|---|---|---|---|
| 1850213-8 | Feb 2018 | SE | national |
This application is a Continuation Application of U.S. patent application Ser. No.: 16/975,308, filed Aug. 24, 2020, which is a U.S. National Phase of International Application No. PCT/EP2019/054310, filed Feb. 21, 2019, which claims priority to Swedish Application No. 1850213-8 filed Feb. 26, 2018, the contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16975308 | Aug 2020 | US |
| Child | 18615341 | US |
