Patent Application
20210292379
The present invention provides a method for recombinant production of di-chain clostridial neurotoxins, which avoids the requirement of an activation step.
Bacteria in the genus Clostridia produce highly potent and specific protein toxins, which can poison neurons and other cells to which they are delivered. Examples of such clostridial toxins include the neurotoxins produced by C. tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, as well as those produced by C. baratii and C. butyricum.
Among the clostridial neurotoxins are some of the most potent toxins known. By way of example, botulinum neurotoxins have median lethal dose (LD50) values for mice ranging from 0.5 to 5 ng/kg, depending on the serotype. Both tetanus and botulinum toxins act by inhibiting the function of affected neurons, specifically the release of neurotransmitters. While botulinum toxin acts at the neuromuscular junction and inhibits cholinergic transmission in the peripheral nervous system, tetanus toxin acts in the central nervous system.
Clostridial neurotoxins act by proteolytically cleaving intracellular transport proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin)—see Gerald K (2002) “Cell and Molecular Biology” (4th edition) John Wiley & Sons, Inc. The acronym SNARE derives from the term Soluble NSF Attachment Receptor, where NSF means N-ethylmaleimide-Sensitive Factor. SNARE proteins are integral to intracellular vesicle fusion, and thus to secretion of molecules via vesicle transport from a cell. The protease function is a zinc-dependent endopeptidase activity and exhibits a high substrate specificity for SNARE proteins. Accordingly, once delivered to a desired target cell, the non-cytotoxic protease is capable of inhibiting cellular secretion from the target cell.
In nature, clostridial neurotoxins are synthesised as a single-chain polypeptide that is modified post-translationally by a proteolytic cleavage event to form two polypeptide chains joined together by a disulphide bond. Cleavage occurs at a specific cleavage site, often referred to as the activation site, which is located between the cysteine residues that provide the inter-chain disulphide bond. It is only through this activation event that full potency of the clostridial neurotoxin is achieved. The two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. The H-chain comprises an N-terminal translocation component (HN domain) and a C-terminal targeting component (HC domain). The cleavage site is located between the L-chain and the translocation domain components. Following binding of the HC domain to its target neuron and internalisation of the bound toxin into the cell via an endosome, the HN domain translocates the L-chain across the endosomal membrane and into the cytosol, and the L-chain provides a protease function (also known as a non-cytotoxic protease).
Botulinum neurotoxins are well known for their ability to cause a flaccid muscle paralysis and inhibit cholinergic secretions. These properties have led to botulinum neurotoxins being employed in a variety of medical and cosmetic procedures, including treatment of glabellar lines or hyperkinetic facial lines, headache, hemifacial spasm, hyperactivity of the bladder, hyperhidrosis, nasal labial lines, cervical dystonia, blepharospasm, spasticity and hyperhidrosis.
Currently all approved drugs/cosmetic preparations comprising BoNTs contain naturally occurring neurotoxins, purified from clostridial strains (BoNT/A in the case of DYSPORT®, BOTOX® or XEOMIN®, and BoNT/B in the case of MYOBLOC®). The traditional production of BoNT products is carried out by culture of C. botulinum, followed by isolation and purification of the botulinum neurotoxin complex or complex free neurotoxin. C. botulinum are spore-forming bacteria and therefore require special culture equipment and facilities, which are cumbersome. Recombinant production of BoNT in a heterologous host such as E. coli, would therefore be advantageous. However, a limiting step of recombinant manufacture of clostridial neurotoxins is the activation step.
Indeed, current practice for recombinant clostridial neurotoxin manufacture is to express the clostridial neurotoxin as a single polypeptide chain in a suitable heterologous host such as E. coli (upstream process). This initial step is usually followed by a series of purification steps (eg by chromatography) and an activation step requiring the addition of a suitable protease which converts the single chain inactive (or hardly active) clostridial neurotoxin into a di-chain fully active form (downstream process). The activation step requires a specific and controlled cleavage of the clostridial neurotoxin activation loop. This cleavage is achieved by using a suitable protease to produce the desired di-chain clostridial neurotoxin, comprising a light chain and a heavy linked by a disulfide bond. This activation step has proved a very challenging stage of clostridial neurotoxin production. In particular, cleavage events can occur outside the activation loop and lead to the generation of truncated clostridial neurotoxins which must then be separated from the full length di-chain clostridial neurotoxins. In addition, following an incubation period the activating protease has to be removed from the activated toxin in order to avoid contaminating the final pharmaceutical product.
Issues that can be encountered at the activation stage include:
A method for recombinant manufacture of clostridial neurotoxins which would bypass the requirement for an activation step would therefore be of great benefit.
Maisey et al. 1988 (MAISEY, E. Anne, et al. “Involvement of the constituent chains of botulinum neurotoxins A and B in the blockade of neurotransmitter release.” European Journal of Biochemistry 177.3 (1988): 683-691.) attempted to form di-chain BoNT/A and B using previously purified toxin that had been unfolded with the resulting domains refolded separately. When the separate domains where combined they found >70% of toxin did form di-chain toxin however potency was greatly reduced. In their discussion they suggest that this reduced potency is likely to be attributed to the presence of the free domains, non-covalent associations or incorrect disulfide formation.
US2006/0024794 A1 addresses the possibility of co-expressing BoNT domains to produce a di-chain toxin in insect cells using a baclovirus expression system. However, the data presented in particular in FIGS. 10 and 11 of US2006/0024794 A1 show that although a small proportion of di-chain neurotoxin is formed the majority of the clostridial neurotoxin remains as free light chain and heavy chain.
There is therefore a need in the art for improved methods for the recombinant production of di-chain clostridial neurotoxins.
In a first aspect, the present invention provides a method for producing a di-chain clostridial neurotoxin, comprising separately expressing in a heterologous host cell a first gene encoding a clostridial neurotoxin light chain and a second gene encoding a clostridial neurotoxin heavy chain, wherein said first and second genes are expressed in an oxidizing environment of said host cell.
In a second aspect, the present invention provides a cell comprising a first gene encoding a clostridial neurotoxin light chain, and a second gene encoding a clostridial neurotoxin heavy chain, wherein said first and second genes are expressed in an oxidizing environment of said cell.
In a third aspect, the present invention provides a kit comprising
In a fourth aspect, the present invention provides a di-chain clostridial neurotoxin obtained by the method according to the invention.
In a fifth aspect, the present invention provides a pharmaceutical composition comprising a di-chain clostridial neurotoxin according to the invention.
In a sixth aspect, the present invention provides the use of a host cell which has an oxidative cytoplasm for producing a di-chain clostridial neurotoxin, wherein said host cell comprises a first gene encoding a clostridial neurotoxin light chain and a second gene encoding a clostridial neurotoxin heavy chain, wherein said first and second genes are expressed in the oxidative cytoplasm of said host cell.
The present invention is based on the finding by the inventors that co-expressing clostridial neurotoxin light and heavy chains separately within an oxidizing environment of a heterologous host cell, allows the two domains to fold together to form a di-chain clostridial neurotoxin with a drastically increased efficiency.
In a first aspect, the present invention provides a method for producing a di-chain clostridial neurotoxin, comprising separately expressing in a heterologous host cell a first gene encoding a clostridial neurotoxin light chain and a second gene encoding a clostridial neurotoxin heavy chain, wherein the first and second genes are expressed in an oxidizing environment of said host cell.
The term “oxidizing environment” as used herein means a cellular environment that promotes cystine formation (oxidised dimer of cysteine). This is generally achieved through the balance of differing redox proteins such as but not limited to thioredoxin based proteins (e.g DsbA) and glutathione. Non-limiting examples of oxidising environments are the periplasm of Gram negative bacteria or the endoplasmic reticulum of eukaryotic expression systems such as Chinese hamster ovary (CHO), insect or yeast cells.
Numerous prokaryotic and eukaryotic expression systems are known in the state of the art. The host cell can be selected, for example, from prokaryotic cells such as Escherichia coli and Bacillus megaterium, or from eukaryotic cells such as Saccharomyces cerevisiae and Pichia pastoris. Although higher eukaryotic cells, such as insect cells or mammal cells, may be used as well, host cells are nevertheless preferred, which, like C. botulinum, do not possess glycosylation apparatus.
In a preferred embodiment, the host cell is a prokaryote cell. In a more preferred embodiment, the oxidizing environment is the cytoplasm of the prokaryote cell.
Disulfide bonds are formed by the oxidation of sulfhydryl groups between two cysteine side chains resulting in a covalent bond. In nature, cells have enzymes dedicated to reducing disulfide bonds in their cytoplasm (reducing cytoplasm) and the formation of disulphide bonds occurs in extra-cytoplasmic environments such as the periplasm in gram negative bacteria or the endoplasmic reticulum (ER) in eukaryotes. Therefore, production of recombinant proteins requiring disulfide bonds in the cytoplasm of cells such as E. coli is challenging.
The cytoplasm of bacterial cells can be rendered oxidizing through genetic engineering, eg by expressing in the cytoplasm genes involved in disulphide bond formation and/or repressing genes involved in disulphide bond reduction and/or modifying such genes. For example, introducing mutations into genes of the thioredoxin (trxB) and/or glutathione (gor or gshA) pathways and/or cytoplasmically over-expressing DsbC can render the cytoplasmic environment oxidadizing and allow for the formation of disulphide bonds (Bessette, Paul H., et al. “Efficient folding of proteins with multiple disulfide bonds in the Escherichia coli cytoplasm.” Proceedings of the National Academy of Sciences 96.24 (1999): 13703-13708; Lobstein, Julie, et al. “SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm.” Microbial cell factories 11.1 (2012): 1).
Examples of commercially available E. coli strains with oxidizing environment include:
In a preferred embodiment, the cell is a prokaryote cell in which at least one gene involved in disulphide bond formation is overexpressed in the cytoplasm as compared to an otherwise identical wild-type cell and/or at least one gene involved in disulphide bond reduction is repressed as compared to an otherwise identical wild-type cell. In one embodiment, the prokaryote cell is an E. coli cell from a strain selected from AD494, BL21trxB, Origami, Rosetta-gami and SHuffle strains. In a preferred embodiment, the prokaryote cell is an E. coli cell from a Origami or SHuffle strain.
The term “neurotoxin” as used herein means any polypeptide that enters a neuron and inhibits neurotransmitter release. This process encompasses the binding of the neurotoxin to a low or high affinity receptor, the internalisation of the neurotoxin, the translocation of the endopeptidase portion of the neurotoxin into the cytoplasm and the enzymatic modification of the neurotoxin substrate. More specifically, the term “neurotoxin” encompasses any polypeptide produced by Clostridium bacteria (“clostridial neurotoxins”) that enters a neuron and inhibits neurotransmitter release, and such polypeptides produced by recombinant technologies or chemical techniques. It is this di-chain form that is the active form of the toxin. The two chains are termed the heavy chain (H-chain), which has a molecular mass of approximately 100 kDa, and the light chain (L-chain), which has a molecular mass of approximately 50 kDa. The L-chain comprises the endopeptidase activity. The H-chain comprises two functionally distinct domains each having molecular weight of approximately 50 kDa: the “HC domain” that enables the binding of the neurotoxin to a receptor located on the surface of the target cell, and the “HN domain” that enables translocation of the light chain (endopeptidase) into the cytoplasm. The HC domain consists of two structurally distinct subdomains, the “HCN subdomain” (N-terminal part of the HC domain) and the “HCC subdomain” (C-terminal part of the HC domain), each of which has a molecular weight of approximately 25 kDa. The term “di-chain clostridial neurotoxin” as used herein means an active neurotoxin consisting of a clostridial neurotoxin light chain and heavy chain which are linked by a disulphide bond. It is understood that a di-chain clostridial neurotoxin according to the invention is capable of binding to a target cell, of translocating the light chain into the cytoplasm of the target cell and of cleaving a SNARE protein, thereby impairing the target's cell's secretion ability.
Different botulinum neurotoxin (BoNT) serotypes can be distinguished based on inactivation by specific neutralising anti-sera, with such classification by serotype correlating with percentage sequence identity at the amino acid level. BoNT proteins of a given serotype are further divided into different subtypes on the basis of amino acid percentage sequence identity. An example of a BoNT/A amino acid sequence is provided as SEQ ID NO: 1 (UniProt accession number A5HZZ9). An example of a BoNT/B amino acid sequence is provided as SEQ ID NO: 2 (UniProt accession number B1INP5). An example of a BoNT/C amino acid sequence is provided as SEQ ID NO: 3 (UniProt accession number P18640). An example of a BoNT/D amino acid sequence is provided as SEQ ID NO: 4 (UniProt accession number P19321). An example of a BoNT/E amino acid sequence is provided as SEQ ID NO: 5 (accession number WP_003372387). An example of a BoNT/F amino acid sequence is provided as SEQ ID NO: 6 (UniProt accession number Q57236). An example of a BoNT/G amino acid sequence is provided as SEQ ID NO: 7 (accession number WP_039635782). An example of a Tetanus neurotoxin (TeNT) amino acid sequence is provided as SEQ ID NO: 8 (UniProt accession number P04958).
An example of a nucleic acid sequence encoding a BoNT/A is provided as SEQ ID NO: 9. An a nucleic acid sequence encoding a BoNT/B is provided as SEQ ID NO: 10. An a nucleic acid sequence encoding a BoNT/C is provided as SEQ ID NO: 11. An a nucleic acid sequence encoding a BoNT/D is provided as SEQ ID NO: 12. An a nucleic acid sequence encoding a BoNT/E is provided as SEQ ID NO: 13. An a nucleic acid sequence encoding a BoNT/F is provided as SEQ ID NO: 14. An a nucleic acid sequence encoding a BoNT/G sequence is provided as SEQ ID NO: 15. An a nucleic acid sequence encoding a Tetanus neurotoxin (TeNT) sequence is provided as SEQ ID NO: 16.
Exemplary L, HN, HCN and HCC amino acid domains are shown in table 1.
Exemplary nucleic acid sequences encoding L, HN, HCN and HCC domains are shown in table 2.
The above-identified reference sequences should be considered a guide, as slight variations may occur according to sub-serotypes. By way of example, US 2007/0166332 (hereby incorporated by reference in its entirety) cites slightly different clostridial sequences”.
In one embodiment, the clostridial neurotoxin light chain is from a BoNT type A, type B, type C1, type D, type E, type F or type G, or a TeNT.
In one embodiment, the clostridial neurotoxin heavy chain is from a BoNT type A, type B, type C1, type D, type E, type F or type G, or a TeNT.
In one embodiment, the clostridial neurotoxin light chain is from a BoNT type A, type B, type C1, type D, type E, type F or type G, or a TeNT, and the clostridial neurotoxin heavy chain is from a BoNT type A, type B, type C1, type D, type E, type F or type G, or a TeNT.
In one embodiment, the clostridial neurotoxin light and heavy chains are from the same serotype or subtype.
In one embodiment, the clostridial neurotoxin light and heavy chains are from different serotypes or subtypes.
In one embodiment, the clostridial neurotoxin light chain comprises a sequence selected from:
It is understood that a clostridial neurotoxin light chain is capable of cleaving a SNARE protein.
In one embodiment, the clostridial neurotoxin heavy chain comprises a sequence selected from:
It is understood that a clostridial neurotoxin heavy chain is capable of binding to a target cell and of translocating the light chain into the cytoplasm of the target cell.
It is also understood that the HN, HCN and HCC domains of the clostridial neurotoxin heavy chain according to the invention can be from the same or from different clostridial serotypes or subtypes.
In one embodiment, the clostridial neurotoxin heavy chain comprises a HN, a HCN and a HCC domain, wherein
The “percent sequence identity” between two or more nucleic acid or amino acid sequences is a function of the number of identical nucleotides/amino acids at identical positions shared by the aligned sequences. Thus, % identity may be calculated as the number of identical nucleotides/amino acids at each position in an alignment divided by the total number of nucleotides/amino acids in the aligned sequence, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.
The light and/or heavy chains can be from a mosaic neurotoxin. The term “mosaic neurotoxin” as used in this context refers to a naturally occurring clostridial neurotoxin that comprises at least one functional domain from another type of clostridial neurotoxins (e.g. a clostridial neurotoxin of a different serotype), said clostridial neurotoxin not usually comprising said at least one functional domain. Examples of mosaic neurotoxins are naturally occurring BoNT/DC and BoNT/CD. BoNT/DC comprises the L chain and HN domain of serotype D and the HC domain of serotype C, whereas BoNT/CD consists of the L chain and HN domain of serotype C and the HC domain of serotype D.
The light and/or heavy chains can be from a modified neurotoxin and derivatives thereof, including but not limited to those described below. A modified neurotoxin or derivative may contain one or more amino acids that has been modified as compared to the native (unmodified) form of the neurotoxin, or may contain one or more inserted amino acids that are not present in the native (unmodified) form of the toxin. By way of example, a modified clostridial neurotoxin may have modified amino acid sequences in one or more domains relative to the native (unmodified) clostridial neurotoxin sequence. Such modifications may modify functional aspects of the neurotoxin, for example biological activity or persistence. Thus, in one embodiment, the first neurotoxin and/or the second neurotoxin is a modified neurotoxin, or modified neurotoxin derivative.
A modified neurotoxin retains at least one of the functions of a neurotoxin, selected from the ability to bind to a low or high affinity neurotoxin receptor on a target cell, to translocate the endopeptidase portion of the neurotoxin (light chain) into the cell cytoplasm and to cleave a SNARE protein. Preferably, a modified neurotoxin retains at least two of these functions. More preferably a modified neurotoxin retains these three functions.
A modified neurotoxin may have one or more modifications in the amino acid sequence of the heavy chain (such as a modified HC domain), wherein said modified heavy chain binds to target nerve cells with a higher or lower affinity than the native (unmodified) neurotoxin. Such modifications in the HC domain can include modifying residues in the ganglioside binding site of the HC domain or in the protein (SV2 or synaptotagmin) binding site that alter binding to the ganglioside receptor and/or the protein receptor of the target nerve cell. Examples of such modified neurotoxins are described in WO 2006/027207 and WO 2006/114308, both of which are hereby incorporated by reference in their entirety.
A modified neurotoxin may have one or more modifications in the amino acid sequence of the light chain, for example modifications in the substrate binding or catalytic domain which may alter or modify the SNARE protein specificity of the modified LC. Examples of such modified neurotoxins are described in WO 2010/120766 and US 2011/0318385, both of which are hereby incorporated by reference in their entirety.
A modified neurotoxin may comprise one or more modifications that increases or decreases the biological activity and/or the biological persistence of the modified neurotoxin. For example, a modified neurotoxin may comprise a leucine- or tyrosine-based motif, wherein said motif increases or decreases the biological activity and/or the biological persistence of the modified neurotoxin. Suitable leucine-based motifs include xDxxxLL, xExxxLL, xExxxlL, and xExxxLM (wherein x is any amino acid). Suitable tyrosine-based motifs include Y-x-x-Hy (wherein Hy is a hydrophobic amino acid). Examples of modified neurotoxins comprising leucine- and tyrosine-based motifs are described in WO 2002/08268, which is hereby incorporated by reference in its entirety.
In one embodiment, the clostridial neurotoxin is a retargeted neurotoxin. The term “retargeted neurotoxin” (also referred to as “targeted secretion inhibitors”, “TSIs”, “TVEMPs” or “TEMs”) as used herein means a clostridial neurotoxin comprising a Targeting Moiety (TM) which binds to a non clostridial receptor. The TM can replace part or all of the HC or HCC domain of the clostridial neurotoxin heavy chain. Examples of retargeted neurotoxins are disclosed in WO96/33273, WO98/07864, WO00/10598, WO01/21213, WO01/53336; WO02/07759 WO2005/023309, WO2006/026780, WO2006/099590, WO2006/056093, WO2006/059105, WO2006/059113, WO2007/138339, WO2007/106115, WO2007/106799, WO2009/150469, WO2009/150470, WO2010/055358, WO2010/020811, WO2010/138379, WO2010/138395, WO2010/138382, WO2011/020052, WO2011/020056, WO2011/020114, WO2011/020117, WO2011/20119, WO2012/156743, WO2012/134900, WO2012/134897, WO2012/134904, WO2012/134902, WO2012/135343, WO2012/135448, WO2012/135304, WO2012/134902, WO2014/033441, WO2014/128497, WO2014/053651, WO2015/004464, all of which are herein incorporated by reference.
In one embodiment, the gene encoding a clostridial neurotoxin light chain and the gene encoding a clostridial neurotoxin heavy chain are present on the same vector.
In one embodiment, the gene encoding a clostridial neurotoxin light chain and the gene encoding a clostridial neurotoxin heavy chain are present on different vectors.
In principle, any expression vectors can be used to achieve co-expression in E. coli for example pK7, pJ401, pBAD or pET vectors. When using separate vectors to express each domain it is preferable to use different antibiotic resistance markers and origins of replication to help plasmid stability. With the single vector approach it is generally beneficial to have genes under control of separate promoters and ribosome binding sites but this is not essential. Finally, both strategies can control both genes by the same type of promoter or can utilise different ones for each e.g. a T7-lac, T5-lac, rhaBAD and araBAD promoter.
In one embodiment, the gene encoding a clostridial neurotoxin light chain and the gene encoding a clostridial neurotoxin heavy chain are prepared as part of DNA or RNA vector(s), preferably DNA vector(s), comprising a promoter and a terminator. Suitable promoter and terminator sequences are well known in the art.
The choice of promoter depends in this case on the expression systems used for expression. In general, constitutive promoters are preferred, but inducible promoters may likewise be used. The construct produced in this manner includes at least one part of a vector, in particular regulatory elements, the vector, for example, being selected from A-derivates, adenoviruses, baculoviruses, vaccinia viruses, SV40-viruses and retroviruses. The vector is preferably capable of expressing the genes in a given host cell.
In one embodiment, the vector has a promoter selected from:
The genes of the invention may be made using any suitable process known in the art. Thus, the genes may be made using chemical synthesis techniques. Alternatively, the genes of the invention may be made using molecular biology techniques.
The genes of the present invention are preferably designed in silico, and then synthesised by conventional gene synthesis techniques.
The above-mentioned genes are optionally modified for codon-biasing according to the ultimate host cell (e.g. E. coli) expression system that is to be employed.
In one embodiment, the method according to the invention further comprises a step of recovering the di-chain clostridial neurotoxin from the host cell. In particular, the method may include a step of lysing the host cell to provide a host cell homogenate, and a step of isolating the di-chain clostridial toxin protein. In one embodiment, the method according to the invention may further comprise a step of introducing the gene encoding a clostridial neurotoxin light chain and a gene encoding a clostridial neurotoxin heavy chain into the host cell. For example, the genes of the invention may be introduced into the cell in the form of expression vector(s) as described herein.
Typically the di-chain clostridial neurotoxin is purified and/or concentrated after recovery from the host cell. Any suitable method(s) may be used for the recovery, purification and/or concentration of the di-chain clostridial neurotoxin. Standard techniques for recovery, purification and/or concentration are known in the art, for example chromatography methods and/or electrophoresis.
The di-chain clostridial neurotoxin may comprise one or more N-terminal and/or C-terminal located purification tags to assist in the purification of the polypeptide. Whilst any purification tag may be employed, the following are preferred: His-tag (e.g. 6×histidine), preferably as a C-terminal and/or N-terminal tag; MBP-tag (maltose binding protein), preferably as an N-terminal tag; GST-tag (glutathione-S-transferase), preferably as an N-terminal tag; His-MBP-tag, preferably as an N-terminal tag; GST-MBP-tag, preferably as an N-terminal tag; Thioredoxin-tag, preferably as an N-terminal tag; and/or CBD-tag (Chitin Binding Domain), preferably as an N-terminal tag.
One or more peptide spacer/linker molecules may be included in the di-chain clostridial neurotoxin. For example, a peptide spacer may be employed between a purification tag and the rest of the polypeptide molecule.
In a further aspect, the present invention provides a cell comprising a first genes encoding a clostridial neurotoxin light chain and a second gene encoding a clostridial neurotoxin heavy chain, wherein the first and second genes are expressed in an oxidizing environment of the cell.
In a preferred embodiment, said cell is a prokaryote cell. In a more preferred embodiment, the oxidizing environment is the cytoplasm of the prokaryote cell.
In a preferred embodiment, the cell is a prokaryote cell in which at least one gene involved in disulphide bond formation is overexpressed by in the cytoplasm as compared to an otherwise identical wild-type cell and/or at least one gene involved in disulphide bond reduction is repressed as compared to an otherwise identical wild-type cell.
In one embodiment, the prokaryote cell is an E. coli cell from strain selected from AD494, BL21trxB, Origami, Rosetta-gami and SHuffle strains. In a preferred embodiment, the prokaryote cell is an E. coli cell from an Origami or Shuffle strain.
In one embodiment, the first gene encoding a clostridial neurotoxin light chain and the second gene encoding a clostridial neurotoxin heavy chain are present on the same vector.
In one embodiment, the first gene encoding a clostridial neurotoxin light chain and the second gene encoding a clostridial neurotoxin heavy chain are present on different vectors.
In a further aspect, the present invention provides a kit comprising
In a further aspect, the present invention provides a di-chain clostridial neurotoxin obtained by the method according to the invention.
In a further aspect, the present invention provides a pharmaceutical composition comprising a di-chain clostridial neurotoxin according to the invention. Preferably, the pharmaceutical composition comprises a di-chain clostridial neurotoxin according to the invention together with at least one component selected from a pharmaceutically acceptable carrier, excipient, adjuvant, propellant and/or salt.
In another aspect, the invention provides a di-chain clostridial neurotoxin according to the invention or pharmaceutical composition according to the invention for use in therapy.
In another aspect, the invention provides a method of treatment comprising the administration of a suitable dose of a di-chain clostridial neurotoxin according to the invention or pharmaceutical composition according to the invention to a patient in need thereof.
A di-chain clostridial neurotoxin according to the invention is suitable for use in treating a condition associated with unwanted neuronal activity, for example a condition selected from the group consisting of spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, lingual dystonia, cervical dystonia, focal hand dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasticity and other voice disorders, spasmodic colitis, neurogenic bladder, anismus, limb spasticity, tics, tremors, bruxism, anal fissure, achalasia, dysphagia and other muscle tone disorders and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, secretory disorders, pain from muscle spasms, headache pain, migraine and dermatological conditions.
In another aspect, the invention provides a non-therapeutic use of a di-chain clostridial neurotoxin according to the invention for treating an aesthetic or cosmetic condition.
The di-chain clostridial neurotoxin according to the invention may be formulated for oral, parenteral, continuous infusion, inhalation or topical application. Compositions suitable for injection may be in the form of solutions, suspensions or emulsions, or dry powders which are dissolved or suspended in a suitable vehicle prior to use.
In the case of a di-chain clostridial neurotoxin according to the invention that is to be delivered locally, the chimeric neurotoxin may be formulated as a cream (e.g. for topical application), or for sub-dermal injection.
Local delivery means may include an aerosol, or other spray (e.g. a nebuliser). In this regard, an aerosol formulation of a chimeric neurotoxin enables delivery to the lungs and/or other nasal and/or bronchial or airway passages.
Di-chain clostridial neurotoxins according to the invention may be administered to a patient by intrathecal or epidural injection in the spinal column at the level of the spinal segment involved in the innervation of an affected organ.
A preferred route of administration is via laparoscopic and/or localised, particularly intramuscular, injection.
The dosage ranges for administration of the di-chain clostridial neurotoxins according to the invention are those to produce the desired therapeutic effect. It will be appreciated that the dosage range required depends on the precise nature of the di-chain clostridial neurotoxin or composition, the route of administration, the nature of the formulation, the age of the patient, the nature, extent or severity of the patient's condition, contraindications, if any, and the judgement of the attending physician. Variations in these dosage levels can be adjusted using standard empirical routines for optimisation.
Fluid dosage forms are typically prepared utilising the di-chain clostridial neurotoxin according to the invention and a pyrogen-free sterile vehicle. The di-chain clostridial neurotoxin, depending on the vehicle and concentration used, can be either dissolved or suspended in the vehicle. In preparing solutions the di-chain clostridial neurotoxin can be dissolved in the vehicle, the solution being made isotonic if necessary by addition of sodium chloride and sterilised by filtration through a sterile filter using aseptic techniques before filling into suitable sterile vials or ampoules and sealing. Alternatively, if solution stability is adequate, the solution in its sealed containers may be sterilised by autoclaving. Advantageously additives such as buffering, solubilising, stabilising, preservative or bactericidal, suspending or emulsifying agents and or local anaesthetic agents may be dissolved in the vehicle.
Dry powders, which are dissolved or suspended in a suitable vehicle prior to use, may be prepared by filling pre-sterilised ingredients into a sterile container using aseptic technique in a sterile area. Alternatively the ingredients may be dissolved into suitable containers using aseptic technique in a sterile area. The product is then freeze dried and the containers are sealed aseptically.
Parenteral suspensions, suitable for intramuscular, subcutaneous or intradermal injection, are prepared in substantially the same manner, except that the sterile components are suspended in the sterile vehicle, instead of being dissolved and sterilisation cannot be accomplished by filtration. The components may be isolated in a sterile state or alternatively it may be sterilised after isolation, e.g. by gamma irradiation.
Administration in accordance with the present invention may take advantage of a variety of delivery technologies including microparticle encapsulation, viral delivery systems or high-pressure aerosol impingement.
In a further aspect, the invention provides the use of a host cell which has an oxidative cytoplasm for producing a di-chain clostridial neurotoxin, wherein the host cell comprises a first gene encoding a clostridial neurotoxin light chain and a second gene encoding a clostridial neurotoxin heavy chain, wherein the first and second genes are expressed in the cytoplasm of the host cell.
This disclosure is not limited by the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a clostridial neurotoxin” includes a plurality of such candidate agents and reference to “the clostridial neurotoxin” includes reference to one or more clostridial neurotoxins and equivalents thereof known to those skilled in the art, and so forth.
The invention will now be described, by way of example only, with reference to the following Figures and Examples.
Primers were designed to amplify separately the light chain (Table 3—Primers 1 and 2) and the heavy chain (Table 3—Primers 3 and 4) of endonegative BoNT/A1(0) ensuring that a stop codon would be incorporated at the end of the Light chain (LC) and a start codon at the beginning of the Heavy chain (HC). Also included were the restriction sites NcoI (fwrd) and BamHI (rev) to allow the LC to be ligated into MSC 1 of the pETDuet vector (Millipore #71146) while NdeI (fwrd) and XhoI (rev) were used to be able to ligate the HC into MSC 2. Genes were amplified with Q5 Hot start HF master mix (NEB #M0494S) using BoNT/A1(0) template DNA shown in Table 4. The amplified LC and pETDuet vector were then digested with NcoI (NEB #R3193) and BamHI (NEB #R3136) and ligated using NEB T4 DNA Ligase (#M02025).
Next, the resulting pETDuet/LC vector and the amplified HC gene were digested with Xho1 (NEB #R0146S) and Nde (NEB #R0111S) and ligated together resulting in the desired final construct.
To test co-expression of BoNT/A1(0) the vector was transformed into Shuffle T7 ((NEB #C3026H), Shuffle T7 Express cells (NEB #C3029), BL21(DE3) (C25271) and Origami 2 cells (Merks #714083) as instructed and the resulting colonies were stored as microbank beads at −80° C. Note all cloning and transformation steps followed manufacturer's instructions.
For the expression, 100 ml of modified TB (mTB) (Melford #T1703) containing 50 μg/ml Ampicillin in 250 ml baffled flasks were set up for each of the overnight cultures. These were inoculated with one microbank bead for each of the cell lines and grown overnight at 30° C. for 20 hours while shaking at 225 rpm. The next day the main cultures were set up using 900 ml of mTB+50 μg/ml Ampicillin in 2.5 L baffled flasks which were inoculated with 10 ml of the overnight culture. Cell density was allowed to reach an OD600 of 1 by growing at 30° C. while shaking at 225 rpm. Once the desired OD was reached the temperature was allowed to drop to 16° C. (1 hour) before inducing with 1 mM IPTG (Sigma #I6758). Expression cultures were incubated at 16° C. for a further 20 hours prior to recovering cells at 6000 rpm for 30 minutes.
Recovered cells from the expressions were re-suspended with 6 ml/g using 25 mM Tris, 150 mM NaCl pH 8 and then soluble protein was extracted by one pass through a constant systems homogenisior at 20 Kpsi. Cell debris was removed by centrifugation at 12 000 rpm for 30 minutes and then the clarified lysate was assessed by Western blot (
Briefly, clarified lysates were diluted 1:10 with either ThermoFishers NuPAGE® LDS Sample Buffer (4×) #NP0007+0.1 M DTT (Sigma) for the reduced samples or Novex® Tris-Glycine SDS Sample Buffer (2×) #LC2676 for the non-reduced samples. Following heating at 95° C. for 10 minutes, SDS PAGE electrophoresis was performed on these samples using 4-12% Bis Tris acrylamide gels. Proteins were transferred to 0.2 μM nitrocellulose membranes prior to blotting with polyclonal in-house antibodies raised against either the LC of BoNT/A1 or full length BoNT/A1—preference towards HC. Antibody binding was detected using an Anti-Rabbit IgG—Peroxidase antibody (Sigma #A0545-1ML) and visualized using Super Signal West Dura extended duration substrate.
The results presented in
These results confirm that intracellular formation of the BoNT/A1(0) disulphide bridge following co-expression of the light and heavy chains is feasible in all the strains and that minimal amounts of free LC are present when using expression strains containing an oxidative cytoplasm as compared when a strain with a reducing cytoplasm is used (BL21 (DE3)).
3 Litres of BoNT/A1(0) culture were again co-expressed in Shuffle T7 cells and lysed as detailed in example 1. The resultant full length BoNT/A1(0) was purified from clarified lysate using 3 chromatography steps as follows:
The clarified lysate was diluted in half by the addition of 25 mM Tris, 2 M (NH4)2SO4 pH 8 to bring the (NH4)2SO4 concentration up to 1 M. The sample was then loaded onto a pre equilibrated 10 ml Butyl HP column (2×5 ml HiTrap Butyl HP, GE Healthcare #28-4110-05) at 150 cm/hr. Following a 10 column volume (CV) wash using 25 mM Tris, 1 M (NH4)2SO4 pH 8, any bound proteins were eluted over a 25 CV linear gradient down to 25 mM Tris, 35 mM NaCl pH 8 collecting 5 ml fractions. Fractions were then analysed by SDS PAGE and those that contained the target toxin were pooled.
The Butyl HP pool was buffer exchanged into a low salt buffer so that it could be loaded onto a Q HP column. This was achieved by performing several runs of buffer exchange into 25 mM Tris, 20 mM NaCl pH 8 using a HiPrep26/10 desalting column (GE healthcare, #17-5087-01) and following manufacturer's instructions.
The sample was then loaded onto a pre equilibrated 4.7 ml HiScreen Q HP column (GE healthcare, #28-9505-11) at 75 cm/hr. Following a 5 CV wash with 25 mM Tris, 20 mM NaCl pH 8, bound proteins were eluted over a 25 CV linear gradient up to 25 mM Tris, 300 mM NaCl pH 8 collecting 2.5 ml fractions. Following analysis by SDS PAGE, the fractions containing target protein were pooled.
The Q HP pool was conditioned for the Phenyl HP column by diluting the sample in half with 25 mM Tris, 2 M (NH4)2SO4 pH 8 to bring the (NH4)2SO4 up to 1 M. The sample was loaded onto a pre equilibrated 1 ml Phenyl HP (GE Healthcare #17-1351-01) column at 150 cm/hr and then the column was washed with 3 CV of 25 mM Tris, 1 M (NH4)2SO4 pH 8. Elution of bound proteins used a 25 CV linear gradient down to 25 mM Tris, 35 mM NaClpH 8 collecting 0.5 ml fractions. Following analysis by SDS PAGE, fractions containing the target protein were pooled resulting in the final product as shown in
To be used as a control, single chain recombinant BoNT/A1(0) was also expressed and purified. To achieve this, BoNT/A1(0) (Table 4—LC+Activation loop+HC) was inserted into pJ401 so that it could be expressed as a single chain product using the BLR (DE3) E. coli expression strain (Novagen #69053).
For the expression, 100 ml of modified TB (mTB) (Melford #T1703) containing 30 μg/ml Kanamycin in 250 ml baffled flasks was set up for the overnight culture. This was inoculated with one microbank bead grown overnight at 37° C. for 20 hours shaking at 225 rpm. The next day the main cultures were set up using 15×1 L of mTB+30 μg/ml Kanamycin in 2.5 L baffled flasks which were each inoculated with 10 ml of the overnight culture. Cell density was allowed to reach an OD600 of 0.5 by growing at 37° C. while shaking at 225 rpm. Once the desired OD was reached the temperature was allowed to drop to 16° C. (1 hour) before inducing with 1 mM IPTG (Sigma #I6758). Expression cultures were incubated at 16° C. for a further 20 hours prior to recovering cells at 5000 rpm for 20 minutes.
Recovered cells were lysed and toxin purified as with the Co-expressed BoNT/A1(0). The only 2 differences were that the purification was performed at a larger scale and also required an activation step between the 2nd and 3rd columns:
200 ml Butyl HP->53 ml Q HP->Activation (See below)->10 ml Phenyl HP
Activation stage—The Q HP pool (0.46 mg/ml by A280) was incubated with 92 μg (0.8 μg Lys-C/ml of sample) of Lys-C(Sigma #P2289) at 4° C. for 20 hours. Following activation the sample was immediately diluted in half with 25 mM Tris pH 8, 2 M (NH4)2SO4 so that it could be loaded onto the Phenyl HP, purification was then continued as with Example 1.
The two final products resulting from the Phenyl HP column were assessed by SDS-PAGE (
The results shown in
The purified samples were also compared on the optim which is a device that measures Intrinsic florescence and Light scattering giving an indication on folding and stability (
The Optim results show that Co-expressed BoNT A1(0) and single-chain expressed BoNT/A1(0) have very similar transition points in BCM, SLS at 266 and 473 nm which are readouts for melting temperature and small and large particle aggregation respectively.
The SEC results shows that Co-expressed BoNT A1(0) and single-chain expressed BoNT/A1(0) have identical monomer peaks with minimal aggregation in both.
Primers were designed to mutate two residues (Q224E/Y227H) within the LC domain (SEQ ID NO 17) of the BoNT/A1(0) pETDUET co-expression vector, in order to restore zinc-binding essential to the proteolytic activity of this domain. The resulting pETDUET vector will co-express active BoNT/A1 LC and HC, therefore allowing confirmation of potency in a cell-based system. The mutations were introduced using quick change lightning mutagenesis (#210514—Agilent technologies) following manufacturer's instructions.
The resulting vector was transformed into Shuffle T7 cells and expression/purification were performed as described in Example 1—co-expression of BoNT/A1(0) and Example 2—purification of co-expressed BoNT/A1(0). Note, that this molecule only required the first two chromatography columns, Butyl HP and Q HP as it does not require an activation step.
Co-expressed full length BoNT/A1 was then tested on the Rat Ctx Glutamate Release assay which will confirm translocation and snare cleavage by inhibition of glutamate release as a result of BoNT activity. Commercial native BoNT/A1 (LIST biological laboratories) was used as a control against the co-expressed BoNT/A1.
The glutamate release assay showed that co-expressed BoNT/A1 inhibits glutamate release with potency comparable to that of the native BoNT/A. This therefore demonstrates that co-expression is a viable method for production of fully active di-chain clostridial neurotoxin capable of performing all required steps for intoxication (binding and internalisation at the neuronal endplate, translocation of the light chain from the endosome into the cytoplasm and proteolytic cleavage of the target SNARE protein).
| Number | Date | Country | Kind |
|---|---|---|---|
| 16189221.1 | Sep 2016 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2017/073030 | 9/13/2017 | WO | 00 |
