JP2019141096A - Methods for manufacture of proteolytically processed polypeptides - Google Patents

Abstract

To provide means and methods for improving neurotoxin polypeptides useful as a means for improving the quality of neurotoxin formulations by reducing the amount of unprocessed and/or partially processed neurotoxin polypeptides.SOLUTION: Provided is a proteolytic active polypeptide comprising the polypeptide having a particular amino acid sequence, and a method using the proteolytic active polypeptide for evaluating the quality of pharmaceutical formulations or at the time of producing pharmaceutical formulations.SELECTED DRAWING: Figure 1

Description

  The present invention relates to novel proteolytically active polypeptides and various uses of such polypeptides in screening and manufacturing methods.

Clostridium botulinum and tetanus bacteria produce extremely potent neurotoxins, ie botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeNT), respectively. These clostridial neurotoxins (CNTs) specifically bind to nerve cells and disrupt neurotransmitter release. Clostridium botulinum secretes seven antigenically distinct serotypes, designated A to G, of Botulinum neurotoxin (BoNT). All serotypes, along with a similar tetanus neurotoxin (TeNT) secreted by tetanus, block synaptic exocytosis by cleaving proteins involved in the formation of SNARE complexes that control cell membrane fusion Zn ²⁺ endoprotease. CNT causes flaccid muscle paralysis observed in botulism and tetanus. In addition, CNT activity has been shown to affect gland secretion. These physiological effects of CNTs on muscle and gland activity are increasingly used in various therapeutic and cosmetic applications. Serotype A botulinum neurotoxin (BoNT / A) was approved for use in humans in the United States in 1989 for the treatment of strabismus, blepharospasm, and other disorders. This is marketed as a botulinum neurotoxin A protein preparation under the trade name BOTOX (Allergan Inc.) and the trade name DYSPORT (Ipsen Ltd), for example. In therapeutic applications, a complex comprising a neurotoxin and additional bacterial proteins is injected directly into the muscle undergoing treatment. At physiological pH, the toxin is released from this protein complex (Eisele et al. 2011, Toxicon 57 (4): 555-65) resulting in the desired pharmacological effect. Improved BoNT / A formulations free of complexed proteins are available under the trade names XEOMIN or Bocouture (Merz Pharmaceuticals GmbH, Frankfurt / Germany). The effect of BoNT is only temporary, which is why repeated administration of BoNT is usually required to maintain the therapeutic effect.

  Each CNT is initially synthesized as an inactive single chain polypeptide. In the case of BoNT, this neurotoxin polypeptide has a molecular weight of about 150 kDa. This post-translational processing of single chain polypeptides requires limited proteolysis in exposed regions called loops (see Table 1) and the formation of adjacent disulfide bridges. An active double-chain neurotoxin is composed of two cleavage products resulting from proteolytic hydrolysis of a single-chain precursor polypeptide: an approximately 50 kDa N-terminal light chain and an approximately 100 kDa heavy chain linked by disulfide bonds. Consists of chains. CNTs are structurally composed of three domains: a catalytic light chain, a translocation domain and a receptor binding domain (C-terminal half) (N-terminal half) (Krieglstein 1990, Eur J See Biochem 188: 39; Krieglstein 1991, Eur J Biochem 202: 41; Krieglstein 1994, J Protein Chem 13: 49; Lacy et al., 1998, Nat.Struct. Biol. 5 (10): 898-902 ). Depending on the number of cleavage sites present in the single chain between the amino acid residues that form the catalytic domain and the amino acid residues that form the translocation domain, the endopeptidase activity is divided into two large cleavage products, That is, in addition to the light and heavy chains, it can result in characteristic short peptides that are the former loop regions that cross-linked what should become light and heavy chains in a single chain of neurotoxins (see the table below). (See 1).

  Neurotoxins are contained in fermentation solutions as a mixture of unprocessed, partially processed, and fully processed polypeptides, all of which are very similar to each other. Because of its chemical and physical properties, the purification of CNTs from fermentation solutions is a major challenge. If endo-proteolytic activity has hydrolyzed the peptide bond between the light chain and the loop while the peptide bond between the loop and the heavy chain N-terminus is still intact, partially Processed neurotoxins typically occur. Furthermore, if endo loop proteolytic activity releases the loop peptide from the heavy chain while the peptide bond between the loop peptide and the C terminus of the light chain has not yet been hydrolyzed, a partial Processed neurotoxins can also be made. Depending on the fermentation conditions and the type of neurotoxin, the fully processed polypeptide without loop peptide is significantly contaminated with 5% to 90% partially processed or unprocessed polypeptide. Can be done. Also, in some cases, this neurotoxin is largely unprocessed and needs to be treated with endopeptidase to become biologically active prior to use in therapy.

  The prior art describes various attempts to treat clostridial neurotoxins with heterologous proteases to reduce the amount of unprocessed or partially processed precursor protein. Trypsin, the most widely used protease to activate clostridial neurotoxins, is useful for activating serotype B (BoNT / B) and E (BoNT / E) clostridial neurotoxins (DasGupta & Sugiyama 1972, Biochem. Biophys. Res. Commun. 48: 108-112; Kozaki et al., 1974, Infect. Immun. 10: 750-756), possibly the C of the heavy chain subunit of BoNT / A Proteolytic action near the ends appears to produce secondary products, and thus appears to break toxin binding to its cellular receptors ((Shone et al., 1985, Eur. J. Bioch. 151: 75-82) More specific cleavage products are theoretically expected from endogenous proteases isolated from natural hosts such as Clostridium botulinum that produce BoNT / A. To the proteolytic activation of Clostridium neurotoxin Various attempts have been made to isolate endogenous proteases: Dekleva and DasGupta (Dekleva & DasGupta, 1989, Biochem. Biophys. Res. Commun. 162: 767-772) have been used to produce BoNT / A botulinum. From the fungal culture, a fraction capable of proteolytically cleaving BoNT / A into heavy and light chain subunits was purified and was further isolated from Clostridium botulinum in later studies by the same authors. An endogenous protease was characterized (Dekleva & DasGupta, 1990, J. Bact. 172: 2498-2503) and a 62 kDa protein composed of a 15.5 kDa polypeptide and a 48 kDa polypeptide was revealed. However, after limited exposure to the 62 kDa protein of Dekleva and DasGupta, significant fragmentation of CNT is observed, indicating that the isolated protease is implicated in Clostridial cell culture and during infection. This suggests that it may not be an unidentified proteolytic enzyme responsible for CNT activation.In fact, other researchers have recently demonstrated clostripain, also known as clostridiopeptidase B (Mitchel & Harrington, 1968, JBC 243: 4683-4692) suggests that it may be involved in the specific activation of CNTs ((Sebaihia et al., 2007, Genome Res. 17 (7) : 1082-1092; WO2009 / 014854). Interestingly, the structure and substrate specificity of this enzyme is reminiscent of the structure and substrate specificity of α-clostripain secreted from Clostridium histolyticum (Dargatz et al. 1993) and its homolog (74% Amino acid identity) exists in Clostridium botulinum (CBO1920). Histolyticus alpha clostripain is a cysteine endopeptidase with strict specificity for arginyl bonds. It is synthesized as an inactive preproenzyme and undergoes autocatalytic cleavage to yield 15.4 kDa and 43 kDa polypeptides that combine to form heterodimeric active enzymes (Dargatz et al 1993). Both the Histolyticus alpha clostripain and Clostridium botulinum 62 kDa proteases require a reducing agent and calcium for full activity and can be affected by the same protease inhibitors. These data strongly suggest that the α-clostripain botulinum ortholog (CBO1920) is an endogenous protease responsible for protein cleavage of the botulinum neurotoxin. The gene coding for clostripain (CPE0846) is also present in C. perfringens and has been found to be positively regulated by the binary system VirR / VirS Shimizu et al. 2002b).

International Publication No. 2009/014854 Pamphlet

Eisele et al., 2011, Toxicon 57 (4): 555-65. Krieglstein 1990, Eur J Biochem 188: 39 Krieglstein 1991, Eur J Biochem 202: 41 Krieglstein 1994, J Protein Chem 13: 49 Lacy et al., 1998, Nat. Struct. Biol. 5 (10): 898-902 DasGupta & Sugiyama 1972, Biochem. Biophys. Res. Commun. 48: 108-112 Kozaki et al., 1974, Infect. Immun. 10: 750-756 Shone et al., 1985, Eur. J. Bioch. 151: 75-82 Dekleva & DasGupta, 1989, Biochem. Biophys. Res. Commun. 162: 767-772 Dekleva & DasGupta, 1990, J. Bact. 172: 2498-2503 Mitchel & Harrington, 1968, JBC 243: 4683-4692 Sebaihia et al., 2007, Genome Res. 17 (7): 1082-1109 Dargatz et al., 1993 Shimizu et al., 2002b

  To date, however, no further definitive experimental evidence has been found, and no protease capable of efficiently converting single-chain precursor CNTs into authentic mature cleavage products, i.e. double-stranded neurotoxins Not yet available in the art. The present invention solves one or more of the above problems.

  Means and methods for reducing the amount of unprocessed and / or partially processed neurotoxin polypeptides and thereby improving the quality of neurotoxin preparations are highly desirable, but are still Not available. Thus, the technical problem underlying the present invention can be viewed as providing means and methods for improving the production of neurotoxin polypeptides by meeting the needs described above. This technical problem is solved by the embodiments characterized in the claims and herein below.

  Accordingly, the present invention relates in one aspect to a proteolytically active polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. In another aspect, the invention relates to a proteolytically active polypeptide consisting of a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. In another aspect, the invention relates to a proteolytically active polypeptide consisting of a polypeptide sequence as set forth in SEQ ID NO: 1.

  The term “proteolytically active polypeptide”, as used herein, relates to the catalytic function of a polypeptide of the invention, and the polypeptide of the invention hydrolyzes peptide bonds. Means you can. In one aspect, “proteolytically active polypeptide” refers to a polypeptide capable of hydrolyzing a polypeptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 4-25. The term “proteolytically inactive polypeptide”, as used herein, relates to the catalytic function of a polypeptide of the invention, and the polypeptide of the invention hydrolyzes peptide bonds. It means that it cannot be disassembled.

One skilled in the art can determine whether a polypeptide according to the sequence definitions described herein is a polypeptide according to the present invention by testing the proteolytic activity of the polypeptide. An assay or test system for determining proteolytic activity comprises contacting a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1 with a test substrate. The test substrate is typically a polypeptide that has been found to be cleavable by a polypeptide of the invention. Preferably, the test substrate is a CNT such as BoNT or a fragment thereof. The test substrate may be, for example, an uncleaved / unprocessed BoNT, referred to herein as “scBoNT”, and for example, serotype A, B, C1, D, E, F, or G (eg, “scBoNT / A”, “scBoNT / B”, etc.) or the test substrate may be a tetanus neurotoxin. Alternatively, the test substrate may be a fragment of a clostridial neurotoxin comprising an amino acid sequence selected from any one of SEQ ID NOs: 4-25. This fragment may be a polypeptide consisting of 50 or more amino acid residues or a peptide consisting of up to 49 amino acid residues. As used throughout this specification, the term “polypeptide” refers to a molecule having 50 or more amino acid residues, while the term “peptide” refers to a molecule having 2-49 amino acid residues. In one aspect, the test substrate is a soluble neurotoxin fragment called LH _N that includes a light chain polypeptide, an exposed loop peptide region, and a translocation domain H _N that is the N-terminal half of the heavy chain polypeptide. In another embodiment, the test substrate is or comprises a peptide selected from any one of SEQ ID NOs: 4-25 (see Table 1). In another embodiment, the test substrate is a chimeric neurotoxin comprising amino acid residues derived from two or more serotypes.

An assay for determining proteolytic activity will typically include determining the extent to which the test substrate is converted to its cleavage product (s). It is observed that one or more cleavage product (s) are observed after contacting the polypeptide with the test substrate, or that an increase in the amount of cleavage product (s) is observed. Indicates that the polypeptide is proteolytically active. The above determination steps may require comparing the substrate and cleavage product (s). The above comparison may require determining the amount of substrate and / or the amount of one or more cleavage product (s) and calculate the ratio of substrate and cleavage product (s) Sometimes it is necessary. In addition, the assay for determining proteolytic activity may comprise a step of comparing the test sample with a reference sample, wherein the reference sample typically comprises (a) at least 50 sequences with the sequence of SEQ ID NO: 1. A polypeptide that contains a polypeptide sequence with% sequence identity and is known to be proteolytically active and a test known to be cleavable by the polypeptide of (b) (a) A substrate. In one aspect, this assay for determining proteolytic activity comprises separating the substrate and cleavage product (s) by electrophoresis or column chromatography and optionally spectroscopically analyzing. It may be advantageous to label the test substrate with one or more labels in order to more easily detect a decrease in test substrate and / or an increase in product (s). The term “label” as used herein means a detectable marker and includes, for example, radioactive labels, antibodies, fluorescent labels. For example, the amount of test substrate and / or cleavage product can be determined by autoradiographic or spectroscopic methods, including methods based on energy resonance transfer between at least two labels. Alternatively, immunological methods such as Western blot or ELISA can be used for detection. Preferred assays for determining the proteolytic activity of the polypeptides of the invention are described herein below in the examples that illustrate the invention. In a particularly preferred embodiment of the invention in 120 minutes at 37 ° C. using a buffer selected from 100 mM Tris-HCl, pH 8.0 or PBS (50 mM Na ₂ HPO ₄ , 150 mM NaCl, pH 7.4). A polypeptide is proteolytically active if more than 20%, preferably more than 95% of the test substrate is converted into cleavage products such as light and heavy chains. The same conditions apply even if the test substrate is not a full length neurotoxin but instead is a fragment of a full length neurotoxin or a derivative of a neurotoxin, for example. In this case it is clear that the cleavage products will be different. However, one skilled in the art can quantify the corresponding cleavage products. In another embodiment, typically 100 ng of proteolytically active polypeptide and a molar ratio of 1: 100 to substrate is used in the assay. In another aspect, samples can be taken intermittently to track catalyst activity over time. This assay can be altered, for example, by using multiple amounts of proteolytically active polypeptides.

  SEQ ID NO: 2 shows the polypeptide sequence of a proteolytically inactive polypeptide (GenBank accession number “CAL82988.1”) derived from botulinum strain ATCC3502 and having an amino acid length of 581 residues. SEQ ID NO: 1 shows a proteolytically active derivative of SEQ ID NO: 2 from which amino acid residues 1-248 of SEQ ID NO: 2 have been deleted.

  The term “polypeptide comprising a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 1” refers to a polypeptide having at least 50% sequence identity to the sequence of SEQ ID NO: 1. In addition, the term refers to a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. The polypeptide is, for example, an internal position of the sequence shown in SEQ ID NO: 1, or its N-terminal side or C-terminal side, or an internal position of an amino acid sequence at least 50% identical to the sequence of SEQ ID NO: 1, or An additional amino acid may be present on the N-terminal side or C-terminal side, and methionine may be present at the N-terminal of the polypeptide. In addition, the term includes, for example, an internal position of the sequence shown in SEQ ID NO: 1, or an N-terminal or C-terminal side, or an internal position of a sequence that is at least 50% identical to the sequence of SEQ ID NO: 1, Alternatively, it refers to a polypeptide lacking one or more amino acid residues on the N-terminal side or C-terminal side.

  The term `` sequence identity '', as used herein, is a published technique that is aligned to obtain the highest order match and organized in a computer program such as, for example, BLASTP, BLASTN, FASTA, or A method can be used to calculate (Altschul 1990, J MoI Biol 215: 403), which refers to the determination of identity between a reference amino acid sequence and a query sequence. Percent identity values are, in one embodiment, calculated over the entire amino acid sequence. In another embodiment, the sequence identity is up to 50aa residue, up to 100aa residue, up to 150aa residue, 250aa residue, 300aa residue, 350aa residue, 400aa residue, 450aa residue, 500aa residue, or Calculate over sequence lengths up to 550aa residues. In another embodiment, sequence identity is calculated over at least 50 aa residues, at least 100 aa residues, at least 150 aa residues, or at least 250 aa residues. In a preferred embodiment, sequence identity is determined over the entire length of SEQ ID NO: 1 or 2, ie over a length of 333aa or 581aa, respectively. One skilled in the art can utilize a series of programs based on various algorithms to compare different sequences. In this context, the Needleman and Wunsch or Smith and Waterman algorithms give particularly reliable results. In order to perform sequence alignment and calculate the sequence identity values described herein, version 7.1.0 of the commercial program DNASTAR Lasergene MegAlign based on the algorithm Clustal W was run over the entire sequence region with the following settings: Used: Pairwise Alignment parameter: Gap penalty: 10.00, Gap length penalty: 0.10, Protein weight matrix Gonnet 250. This setting will always be used as the standard setting for sequence alignment unless specified otherwise.

  The term “at least 50% sequence identity” as used herein is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85. %, At least 90%, at least 95%, or 100%.

  The proteolytically active polypeptide of the present invention may have the same number of amino acids as the reference polypeptide sequence as shown in SEQ ID NO: 1. Polypeptides having additional amino acid residues or few amino acid residues are also included in the present invention. In one aspect, the proteolytically active polypeptide of the present invention is a truncated variant of SEQ ID NO: 1 or 2, or a truncated variant of a polypeptide having at least 50% sequence identity with the sequence of SEQ ID NO: 1 or 2. Or include it. The shortened variant of SEQ ID NO: 2 may have, for example, one or more amino acid residues deleted at the N-terminal side at amino acid position 249. The truncation mutant may be a proteolytically active N-terminal or C-terminal truncation mutant and / or an internal truncation mutant. In one aspect, the truncated variant of SEQ ID NO: 2 lacks amino acid positions 1-248 of SEQ ID NO: 2. In another embodiment, the truncation mutant of SEQ ID NO: 2 is a C-terminal truncation mutant. In one aspect, the truncation mutant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 150, or 170 consecutive amino acid residues. Is deleted. In another embodiment, the proteolytically active polypeptides of the invention have an amino acid length of at least 200 aa residues, at least 250 aa residues, at least 300 aa residues, or at least 333 aa residues. In another aspect, the proteolytically active polypeptides of the invention have up to 333aa residue, up to 350aa residue, up to 573 residue, up to 581aa residue, up to 592aa residue, up to 600aa residue, or 617aa residue Have up to.

  In another embodiment, the proteolytically active polypeptide of the invention is N-terminal or C-terminal to the polypeptide chain of SEQ ID NO: 1 or a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. And / or polypeptides containing additional amino acid residues at internal positions thereof. These additional amino acid residues may comprise up to 5, up to 10, or even up to 200, 300, or 400 consecutive amino acid residues. In one aspect, the additional amino acid residue functions as an inhibitor of proteolytic activity. In another aspect, additional amino acid residues can be removed by proteases. In another aspect, additional residues that inhibit the proteolytic activity of the polypeptides of the invention are eliminated. The additional amino acid residue may be adjacent to one or more protease cleavage sites. In another aspect, the additional amino acid sequence functions as a detectable tag and / or allows binding to a solid support.

  In another embodiment, the polypeptide chain of SEQ ID NO: 1 or a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1 has been modified by the exchange of one or more amino acid residues. The term “exchanging” as used herein means replacing one amino acid with another. For example, up to 1aa, up to 2aa, up to 3aa, up to 4aa, up to 5aa, up to 6aa, up to 7aa, up to 8aa, up to 9aa, up to 10aa, up to 15aa, up to 20aa, or up to 50aa within this polypeptide sequence It may be done. For example, for the purpose of increasing or decreasing the substrate binding activity or proteolytic activity of the polypeptide of the present invention, this exchange may involve conservative or non-conservative amino acid changes.

  In one aspect, the proteolytically active polypeptides of the present invention include polypeptides that can hydrolyze a substrate into two or more natural cleavage product (s). In another embodiment, the polypeptides of the invention hydrolyze a substrate into two or more cleavage products that are different from the natural cleavage products. The terms `` native cleavage products '' or `` native products '' as used herein are products that arise in and are derived from a culture of wild-type cells. Refers to a product that is identical in amino acid sequence when compared to a product resulting from the same substrate. In one aspect, the cleavage product is a botulinum or tetanus neurotoxin double-stranded neurotoxin, and in another aspect, the double-stranded neurotoxin is serotype A, B, C1, D, E, A neurotoxin isolated from F or G botulinum. In another aspect, the double-stranded neurotoxin is a natural double-stranded neurotoxin.

  It is to be understood that the definitions and explanations of the terms made above and below apply mutatis mutandis to all aspects described herein, unless otherwise indicated.

  The proteolytically active polypeptides of the present invention are suitable for a variety of uses. A commercially relevant application is its use in the production of therapeutic neurotoxins, eg isolated from Clostridium botulinum. Currently, cell cultures of Clostridium botulinum used to prepare commercial formulations of botulinum neurotoxins are contaminated with significant amounts of partially processed and / or unprocessed neurotoxins. Both of which negatively impair, ie reduce, the specific activity of these pharmaceutical compositions. For example, after lysis of Clostridium botulinum, unprocessed neurotoxins and / or partially processed nerves using the polypeptides of the present invention that are proteolytically active or activated It is now possible to process compositions containing poisons to convert these contaminants into fully processed neurotoxins. As a result, a product with increased specific activity of the neurotoxin can be provided, in which case the total amount of bacterial protein can be reduced and the patient risk of antibody formation is also reduced.

  In another aspect, the invention relates to a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide of the invention and optionally a regulatory element. The term “regulatory element” as used herein refers to a regulatory element of gene expression, including transcription and translation, which includes a TATA box, promoter, enhancer, ribosome binding site, Shine-Dalgarno sequence, IRES region. , Elements such as polyadenylation signals, end-capping structures. The regulatory element may comprise one or more heterogeneous regulatory elements or one or more cognate regulatory elements. A “homologous regulatory element” is a regulatory element of a wild type cell that is involved in the regulation of gene expression of the nucleic acid molecule or polypeptide in the wild type cell from which the nucleic acid molecule of the invention is derived. The invention also encompasses nucleic acid molecules comprising heterologous regulatory elements. The term “heterologous regulatory element” is a regulatory element that is not involved in the regulation of gene expression of the nucleic acid molecule or polypeptide in the wild type cell. Also included are regulatory elements for inducible expression, such as inducible promoters. The nucleic acid molecule can be, for example, hnRNA, mRNA, RNA, DNA, PNA, LNA, and / or a modified nucleic acid molecule. The nucleic acid molecule may be circular, linear, integrated into the genome or episomal. Also included are chains that encode fusion proteins comprising 3, 4, 5, 6, 7, 8, 9, or 10 polypeptides of the invention. In addition, the nucleic acid molecule may contain a sequence encoding a signal sequence for intracellular transport, such as a signal for transport to an intracellular compartment or a signal for transport across a cell membrane.

  In another aspect, the invention relates to a vector comprising a nucleic acid molecule according to the nucleic acid molecule of the invention. The vector may be suitable for in vitro and / or in vivo expression of a polypeptide of the invention. This vector may be a vector for transient and / or stable gene expression. In one embodiment, the vector further comprises regulatory elements and / or selectable markers. In one embodiment, the vector is derived from a virus. In another embodiment, the vector is derived from a phage. In another embodiment, the vector is derived from a bacterium.

  In another aspect, the invention relates to a cell comprising a nucleic acid molecule or vector of the invention. The term “cell”, as used herein, includes the nucleic acid molecule or the vector, especially prokaryotic and / or eukaryotic cells suitable for expressing the polypeptide of the invention. The cell may be a host cell that does not express the polypeptide of the present invention or a homologue thereof. The term “homolog” as used herein refers to a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. However, cells expressing the polypeptide of the present invention or homologues thereof, particularly wild type cells, are also encompassed by the present invention. In one particular embodiment, the cells of the invention are selected from Clostridium botulinum, C. butyricum, C. baratii, and tetanus. In a preferred embodiment, the cell is serotype A, B, or F Clostridium botulinum. In another embodiment, the cell is the Clostridium botulinum Hall strain (ATCC3502). In another embodiment, the cell is the BoNT / A producing strain ATCC19397, also known as Clostridium botulinum NCTC4587 and NCTC7272. In another embodiment, the cell is the BoNT / A producing strain NCTC2916 of Clostridium botulinum. In another embodiment, the cells are the BoNT / A2 producing strains of Clostridium botulinum, Kyoto-F and Mauritius / NCTC9837. In another embodiment, the cell is the BoNT / A3 producing strain A254 Loch Maree / NCTC2012 of Clostridium botulinum. In another embodiment, the cell is the Clostridium botulinum BoNT / A4 and B production strain CDC657. In another embodiment, the cell is the BoNT / A5 and B3 'producing strain H04402 065 of Clostridium botulinum. In another embodiment, the cell is the BoNT / B1 producing strain Okra / NCTC7273 of Clostridium botulinum. In another embodiment, the cell is the Clostridium botulinum BoNT / B and F production strain CDC4013 / NCTC12265. In another embodiment, the cell is the BoNT / F1 producing strain Langeland / NCTC10281 of Clostridium botulinum. In another embodiment, the cells are Clostridium sporogenes, Clostridium perfringens, Clostridium acetobutylicum, B. cereus, B. thuringiensis, Bacillus thuringiensis, Bacillus.・ B. mycoidis, B. thermoproteolyticus, B. anthracis, B. megaterium, B. subtilis, E. coli Or a yeast cell. In one aspect, the polypeptides of the invention are modified inside the cell (ie, glycosylation, phosphorylation, processing by proteases, etc.). Modification also includes the addition of non-protein cofactors including metal ions. Cells comprising the above proteolytically inactive polypeptides, any intermediate polypeptide products, and the final proteolytic active polypeptides disclosed herein are encompassed by the present invention. A cell containing an inducer of expression of the polypeptide of the present invention is also encompassed by the present invention. Such inducers of expression are chemistry of nucleic acid molecules, or polypeptides, or small molecules that have the effect of increasing the amount or activity of the proteolytically active polypeptides of the invention in cell culture or lysates thereof. It may be a chemical component including the component. An inducer of expression can, for example, increase transcription or translation of a nucleic acid molecule encoding a polypeptide of the invention. Alternatively, the inducer of expression activates a polypeptide comprising a proteolytically inactive polypeptide SEQ ID NO: 2 or a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 2. It may be a compound capable of In one aspect, the cell comprises an inducer that is a proteolytically active polypeptide capable of removing inhibitory amino acid residues from the N-terminus of the polypeptide. This inducer can be expressed, for example, by recombinant means known to those skilled in the art. Alternatively, the inducer can be isolated from a cell, such as a clostridial cell.

  The invention also relates to the use of the nucleic acid molecules of the invention for producing the proteolytically active polypeptides of the invention.

  In a related aspect, the invention provides a method for producing a proteolytically active polypeptide comprising (a) a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. A method comprising chemically synthesizing a peptide or translating from a nucleotide sequence; and (b) purifying the polypeptide of step (a).

  The term “chemically synthesized” means synthesizing a polypeptide by chemical means. Such methods are reviewed, for example, in Nilsson et al., Ann. Rev. Biophys. Biomol. Struct. 2005. 34: 91-118. The term “purify polypeptide” means to remove compounds other than the above polypeptide from the mixture containing the polypeptide of the present invention. The term also refers to removing the polypeptide of the present invention from a mixture containing compounds other than the polypeptide. In one particular aspect, the term means separating the proteolytically active polypeptide from its proteolytically inactive precursor.

  The nucleic acid can be translated in a cell or cell-free system. One skilled in the art can utilize a variety of systems for cell-free translation. The present invention relates to a cell-free protein translation system comprising, for example, a rabbit reticulocyte lysate, a wheat malt lysate, an Escherichia coli lysate, or another cell lysate such as a lysate derived from Clostridium botulinum. Includes translation. Translation of a polypeptide of the invention from a nucleotide sequence of the invention or a vector of the invention is also encompassed. Transcription can be regulated or controlled by one or more heterologous regulatory elements or by cognate regulatory elements. Translations in wild type cells, ie cells isolated from nature, such as any known isolates of Clostridium botulinum, butyric acid bacteria, Clostridium balati, and tetanus are also encompassed by this aspect of the invention. In one particular embodiment, the cell is a Clostridium botulinum Hall strain (ATCC3502). One skilled in the art can utilize a variety of standard means and methods to place a nucleic acid molecule or vector into a cell and express the polypeptide of the invention as a recombinant protein in the cell. In addition, those skilled in the art are aware of many standard techniques for isolating polypeptides from cells or cell lysates, or cell-free expression systems (see, for example, Recombinant DNA Principles and Methodologies, J. Green, Marcel Dekker Inc., 1998; The Condensed Protocols: From Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory, 2006; Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory, 2000). Any of these means and methods can be used in the methods of the present invention.

  The first polypeptide of the present invention can be translated from a nucleic acid molecule encoding a proteolytically active polypeptide. SEQ ID NO: 26 is an example of such a nucleic acid molecule. Alternatively, the nucleic acid molecule is proteolytically inactive, but may encode a precursor polypeptide that can be converted to the proteolytically active polypeptide of the invention. SEQ ID NO: 27 is an example of such a nucleic acid molecule. This proteolytically inactive precursor is also referred to as “inactive BoNTHydrolase”, iBH for short. This proteolytically inactive polypeptide can be used during or after translation, for example, by converting the proteolytically inactive polypeptide to the N-terminus of the proteolytically inactive polypeptide. It can be activated by contact with a protease that can remove certain inactivated amino acid residues. An example of a proteolytically inactive polypeptide is the polypeptide represented by SEQ ID NO: 2. Another example is a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 2. The term “inactivated amino acid residue at the N-terminus” relates in one aspect to the first 248aa residue of the polypeptide. In another embodiment, the term refers to a fragment of the polypeptide comprising up to 10aa, 50aa, 100aa, 150aa, 200aa, 250aa residues. Any of these polypeptides are useful in the methods of the invention for producing proteolytically active polypeptides. In one aspect, proteases that can remove inactivated amino acid residues from the N-terminus of the polypeptide are isolated from, for example, Clostridium botulinum, Butyric acid bacteria, Clostridium balati, and Tetanus. In another aspect, a protease capable of removing the inactivated amino acid is provided by obtaining a solubilized solution of the cell that is fractionated or not fractionated. Inactivated amino acid residues are incubated until the proteolytically inactive polypeptide is contacted with the lysate and the proteolytically inactive polypeptide is converted to a proteolytically active polypeptide. Can be removed.

  In another embodiment of the method of the invention, the polypeptide is translated intracellularly. The cell may be a prokaryotic cell or a eukaryotic cell. In one aspect, the cell is selected from E. coli, Bacillus subtilis, or yeast. Translation of the polypeptide of the invention in wild-type cells, ie cells isolated from nature such as Clostridium botulinum, butyrate, Clostridium balati, and any known isolate of tetanus is also encompassed by the present invention. Is done. In one particular embodiment, the cell is a Clostridium botulinum Hall strain (ATCC3502). In another specific embodiment, the cell is a cell of the invention as described herein above.

  All translation products obtained by the methods of the present invention can be purified by various means known to those skilled in the art (e.g., Recombinant DNA Principles and Methodologies, J. Green, Marcel Dekker Inc., 1998; The Condensed Protocols: From Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory, 2006; Molecular Cloning: A Laboratory Manual, Sambrook et al., Cold Spring Harbor Laboratory, 2000). Typical methods for purifying the polypeptides of the present invention include cell lysate spin, protein ammonium sulfate precipitation, protein resuspension, resuspended protein centrifugation, ion exchange chromatography, size exclusion chromatography. May be accompanied by hydrophobic interaction chromatography and the like. Some of the combinations of such steps in various orders may be useful for purifying the polypeptides of the present invention. Preferred methods for purifying the polypeptides of the invention are described in the Examples that illustrate the invention.

  In one aspect, the purification step comprises binding a polypeptide of the invention to a solid support. The term “solid support” refers to a matrix including, for example, silica, cross-linked dextran, cross-linked polyacrylamide, or cross-linked agarose. Others include, among others, polypeptides, glass, polystyrene, polypropylene, polyethylene, polyethylene glycol (PEG), dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. In one aspect of the invention, the solid support is a polysaccharide matrix selected from the group consisting of sepharose, sephadex, agarose, sephacell, microcellulose, and alginate beads. In another embodiment, the solid support may consist of glass beads and / or a polypeptide matrix.

  In one aspect, the solid support is linked to an antibody of the invention. The term “linked” in one aspect means stably linked or stably linked. In another aspect, “linked” includes interactions such as indirect or direct bonds, irreversible or reversible bonds, physical and chemical bonds, electrostatic bonds, and / or covalent bonds. It is. In one aspect, the antibody is covalently attached to the solid support, either directly or via a linker molecule. The antibody may be bound to the solid support via a linker including a small molecule compound and a peptide (or polypeptide) linker molecule. This solid support can have virtually any possible structural configuration or configuration as long as the coupled antibody can bind to its antigen. Thus, the matrix or solid support may be spherical, as in the bead, or cylindrical, as in the inner surface of the test tube or the outer surface of the rod. Alternatively, the surface may be irregular or flat, such as a sheet or specimen.

  The above antibody linked to a solid support can be used, for example, in the production method of the present invention or in a diagnostic method. In one aspect, the method of manufacture may comprise an affinity chromatography step based on an antibody linked to a solid support. In one embodiment, the antibody is an antibody that specifically binds to a proteolytically active polypeptide of the invention. In another embodiment, the antibody is an antibody that specifically binds to a proteolytically inactive polypeptide of the invention.

  In another aspect, the method for producing a proteolytically active polypeptide of the present invention comprises the step of purifying the polypeptide of the present invention from a mixture containing additional components. Purification can be based on, for example, polarity, charge, and size. Thus, this method in one aspect included normal phase HPLC, reverse phase HPLC, hydrophilic interaction chromatography (HILIC), hydrophobic interaction chromatography (HIC), anion exchange chromatography and cation exchange chromatography. One or more separation steps selected from the group consisting of ion exchange chromatography (IEC), size exclusion chromatography (SEC), gel permeation chromatography (GPC) may be included.

  In another embodiment, the purification is performed by (a) separation by anion exchange chromatography; (b) separation by size exclusion chromatography; (c) separation by hydrophobic interaction chromatography; and (d) by size exclusion chromatography. Including a separation step.

  One or more fractions collected from the chromatography column can be concentrated by precipitation or ultrafiltration.

  In one aspect, the invention relates to a composition comprising a proteolytically active polypeptide of the invention. By using the methods disclosed herein, it is possible to produce the proteolytically active polypeptides of the present invention that are substantially free of proteolytically inactive polypeptides. In other words, the methods of the present invention provide compositions that are free of substantial contamination with proteolytically active polypeptides, and inactive precursor proteins of the polypeptides of the present invention. If less than 5% of proteolytically inactive precursors can be detected by using a Western blot based detection method, the composition does not contain substantial contaminants or protein It is believed that it is substantially free of degradable inactive precursor polypeptides, wherein the 5% is proteolytically inactive relative to the sum of proteolytic active polypeptide and inactive polypeptide. Relates to the amount of precursor. In another embodiment, the composition is substantially pure and comprises at least 50% of a proteolytic active polypeptide of the invention, wherein the 50% is the protein contained in the composition. Relates to the amount of proteolytically active precursor relative to the total amount. In another embodiment, the substantially pure composition comprises at least 75%, 80%, 90%, or at least 98% proteolytically active polypeptide.

In another aspect, the invention also relates to a polypeptide obtainable from a method for producing a proteolytically active polypeptide as described herein above and as exemplified in the examples. In one embodiment, the proteolytic active polypeptide is a proteolytic active polypeptide having the polypeptide sequence of SEQ ID NO: 1. In another embodiment, the proteolytically active polypeptide is a polypeptide having at least 50% sequence identity with the sequence of SEQ ID NO: 1. In yet another embodiment, the proteolytically active polypeptide is a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1. The term “obtainable polypeptide” as used herein, in one aspect, refers to a polypeptide that is translated from a nucleic acid of the invention. Following this polypeptide, post-translational modifications such as acylation, alkylation, amidation, amino acid addition, amino acid deletion, glycosylation, oxidation, S-glutathione addition, phosphorylation, sulphatation, protein processing, etc. Can be applied. In addition, the polypeptide can be Li ⁺ , Na ⁺ , K ⁺ , Ag ⁺ , Cs ⁺ , Mg ²⁺ , Ca ²⁺ , Co ²⁺ , Ni ²⁺ , Mn ²⁺ , Cu ²⁺ , or Zn ^2+. Can bind to metal ions such as Preferably, the metal ion is Zn ²⁺ , Mn ²⁺ , or Co ²⁺ .

  The invention also in one aspect relates to an antibody that specifically binds to a polypeptide of the invention. The term “antibody” as used herein includes monoclonal antibodies, polyclonal antibodies, single chain antibodies, human antibodies, humanized antibodies, primatized antibodies, chimerized antibodies, bispecific antibodies, synthetic antibodies, Included are chemically or enzymatically modified derivatives, any fragments of the above antibodies, or aptamers consisting of naturally occurring and / or chemically modified nucleic acids. Such antibody fragments include F (ab ') 2, F (ab), Fv, or scFv fragments, or any chemically or enzymatically modified derivative of these fragments.

  In one embodiment, the antibody of the present invention specifically binds to the proteolytically active polypeptide of the present invention or a proteolytically inactive precursor thereof. In one aspect, an antibody specific for a proteolytically active polypeptide of the present invention cross-reacts with a proteolytically inactive polypeptide described herein. In another aspect, the antibody can distinguish between a proteolytically active polypeptide of the invention and its inactive precursor. In another embodiment, the epitope to which the antibody is specific is located in an amino acid region that is present in a proteolytically inactive polypeptide but not in a proteolytically active polypeptide. For example, the epitope may be an epitope of a polypeptide region consisting of amino acid residues 1-248 of a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 2.

  In another embodiment, the epitope is formed by amino acid residues located N-terminal to amino acid 249 of a polypeptide comprising a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 2. In another aspect, the epitope is removed from the proteolytically inactive polypeptide described herein by protein processing.

  In another embodiment, the epitope specific for an antibody of the invention is an epitope located at the N-terminus of a polypeptide comprising a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 1. The term “N-terminus” as used in this aspect of the invention refers to a region of a polypeptide comprising the N-terminal 50 amino acid residues of the polypeptide sequence, preferably the N-terminal 25 amino acid residues of the polypeptide sequence. Point to. In one particular embodiment, the term refers to the N-terminal 14 amino acid residues. The term “epitope” as used herein relates to an antigenic determinant recognized by an antibody of the invention. In one aspect, the epitope is a linear epitope, and in another aspect, the epitope is a conformational epitope. In one particular embodiment, the antigenic determinant consists of a peptide having the N-terminal amino acid sequence of the proteolytically active polypeptide of the present invention, wherein the peptide comprises 7 to 14 amino acid residues, preferably 8 , 9, 10, 11, 12, 13, or 14 amino acid residues.

The term “specifically binds” or “specifically binds” in one aspect indicates that the antibody of the present invention is generally of significant significance on other polypeptides on the polypeptide of the present invention or on other polypeptides. Means no cross reaction. Epitope specificity is an important feature of the antibodies of the present invention. The specificity of the antibody is at least 95%, at least 96%, at least 97%, at least 98%, at least, in one aspect, relative to a proteolytically inactive polypeptide relative to the proteolytically active polypeptide 99%. Specific binding can be tested by various well-known techniques including, for example, competition studies. Another important feature is antibody sensitivity. Sensitivity is in one aspect of the invention such that at least 70%, at least 80%, at least 90%, at least 95% of the epitopes contained in the sample bind. Sensitivity can be tested by well-known techniques. One skilled in the art will be able to determine the effective and optimal assay conditions for each determination by using routine experimentation. Conventional techniques for binding studies include radioimmunoassay, ELISA, equilibrium dialysis, isothermal microcalorimetry, BIACORE® assay (surface plasmon resonance, SPR), or other surface adsorption methods. The BIACORE® SPR system measures antibody-antigen interactions. The SPR response reflects the change in mass concentration at the detector surface as the analyte binds or dissociates. Based on SPR, real-time BIACORE® measurements monitor interactions directly as they occur (BIAapplications Handbook, version AB (reprinted 1998), BIACORE® code No: BR-1001-86; BIAtechnology Handbook, version AB (reprinted 1998), see BIACORE® code No: BR-1001-84). Binding properties, such as sensitivity, of the antibodies of the invention can basically be determined by binding studies using immobilized antigens (ligands) provided on the sensor surface. The antibody (analyte) to be tested will be supplied in the mobile phase, ie in solution. In some cases, the antigen is indirectly bound to the surface via binding to another immobilized molecule called a capture molecule. When an antibody is injected in a single pulse onto a surface with immobilized antigen, essentially three phases can be subdivided: (i) Binding of antibody and antigen during sample injection (ii) the equilibrium or steady state during sample injection, where the rate of antibody binding is balanced with the dissociation from the antibody-antigen complex; (iii) the amount of antibody from the surface while the buffer is flowing; dissociation. It will be appreciated that such assays can alternatively be performed using the immobilized antibody to be investigated and the antigen-containing solution as the mobile phase. The binding and dissociation phases provide information about the kinetics of the analyte-ligand interaction (k _a and k _d , the rate of complex formation and dissociation, k _d / k _a = K _D ). The equilibrium phase provides information about the affinity of the analyte-ligand interaction (K _D ). In one aspect of the present invention, the antibody of the present invention is less than 0.5 [mu] M, in another embodiment, less than 0.05 [mu] M, and in another embodiment, have a K _D of less than 0.02 uM.

  Antibodies as referred to in the present invention can be obtained, for example, by using the methods described in Harlow and Lane, 1988 (Harlow and Lane, “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988). Can be manufactured. Monoclonal antibodies can be prepared by the techniques originally described in Kohler & Milstein, 1975 (Kohler & Milstein 1975, Nature 256: 495) and Galfre & Milstein, 1981 (Galfre & Milstein 1981, Meth Enzymol 73: 3). it can. The technique involves fusing mouse myeloma cells to spleen cells derived from the immunized mammal. Antibodies can be further improved by techniques well known in the art. For example, surface plasmon resonance as used in the BIACORE® system can be used to increase the efficiency of phage antibodies that bind to the aforementioned epitopes within the polypeptides of the invention (Schier et al., 1996 , Human Antibodies Hybridomas 7: 97; see Malmborg et al., 1995, J. Immunol Methods 183: 7).

  In one aspect of the invention, the antibody is produced by using a peptide comprising or consisting of the epitope described above. This peptide can be produced, for example, synthetically or by recombinant expression. Alternatively, the antibodies of the present invention can be produced by applying the naturally occurring proteolytic active or inactive polypeptides of the present invention. In the latter case, it should be understood that the resulting antibody will be further tested for specificity with the polypeptides of the invention. In a further aspect of the invention, the monoclonal antibodies of the invention are produced by using a polypeptide of the invention that can be treated with a surfactant to make its epitope immunologically available. However, it will be understood that such detergent treatment should not be performed if the antibody is directed against a conformational epitope. In a further aspect, immunostimulants such as keyhole limpet hemocyanin (KLH) can also be applied to such processes, particularly when using synthetic peptides.

  The antibodies of the invention are used, for example, for affinity chromatography, immunoprecipitation and immunolocalization of the polypeptides of the invention, and for monitoring the presence of the polypeptides in a sample or recombinant organism. can do. Furthermore, the antibodies of the present invention can be used in detection methods or diagnostic methods. In one particular embodiment, the antibodies of the invention are used in a Western blot or ELISA. In addition, the antibodies of the present invention can be used in therapeutic applications. In particular, this antibody can be used to inhibit the activity of the proteolytically active polypeptides of the present invention. Accordingly, the antibodies of the present invention also have various therapeutic uses as described herein below.

  The invention also relates to the use of the proteolytically active polypeptides of the invention in a method for proteolytically processing a polypeptide. In one aspect, the invention provides a method for producing a proteolytically processed polypeptide comprising: (a) a first polypeptide that is a polypeptide of the invention, (b) the first Contacting with a second polypeptide that is susceptible to proteolysis by said polypeptide, wherein said contacting results in protein processing of said second polypeptide into at least two cleavage products.

  The invention also relates to the use of Lys-N and / or Lys-C and / or arginyl endopeptidase (endoproteinase Arg-C, LeR) from Lysobacter enzymogenes (ATCC 29487) (Wright DS, Graham LD, Jennings PA. Biochim Biophys Acta. 1998 Dec 22; 1443 (3): 369-74). Furthermore, in methods for proteolytic processing of CNTs such as BoNT / A, membrane binding that cleaves with plasmin and / or omptin (OmpT), ((Arg / Lys)-(Arg / Lys) motifs The use of type serine proteases (K. Sugimura and T. Nishihara. J. Bacteriol. 170 (1988), pages 5625-5632)) is also encompassed. In one aspect, the invention provides a method for producing a proteolytically processed polypeptide comprising: a) a first polypeptide that is Lys-C or Lys-N, (b) Contacting with a second polypeptide that is susceptible to proteolysis by one polypeptide, wherein said contacting results in protein processing of said second polypeptide into at least two cleavage products, and second Wherein the polypeptide is a single chain of BoNT / A. The term `` Lys-C '' refers to the 33 kDa serine endoproteinase Lys-C (lysyl endopeptidase, LeK, Genbank accession number Q7M135) derived from Lysobacter enzymogenes that specifically cleaves peptide bonds at the C-terminal side of lysine or Refers to its homolog with at least 60% sequence identity. The term “Lys-N” refers to the metalloendopeptidase Lys-N isolated from Grifola frondosa and Pleurotus ostreatus (Nonaka T et al., 1997, J Biol Chem. 272: 30032-30039; Nonaka T et al., 1998, J Biochem. 1998 124: 157-162; Hori T et al., 2001, Acta Crystallogr D Biol Crystallogr. 57: 361-368). The term also encompasses homologs of the above proteases that have at least 60% sequence identity.

  This method can be used, for example, to produce proteolytically processed neurotoxins (CNT) or botulinum neurotoxins (BoNT). The term “BoNT” as used throughout the present invention means botulinum neurotoxin and can be obtained from Clostridium botulinum, such as serotype A, B, C1, D, E, F, or G BoNT. Refers to neurotoxin. The terms “CNT” and “BoNT” also encompass recombinant and modified neurotoxins that include one or more modifications, including chemical or genetic modifications. The term “genetic modification” refers to the deletion, substitution, or addition of one or more amino acid residues. By using the method of the present invention, it is now possible to obtain a neurotoxin composition that is not significantly contaminated by unprocessed or partially processed neurotoxins, since This is because these contaminants are efficiently processed into double-stranded neurotoxins. In one aspect, the double-stranded neurotoxin is a natural, wherein the C-terminus of the light chain and the N-terminus of the heavy chain are identical to the corresponding fully processed double-stranded neurotoxin isolated from the wild type Clostridium genus. It is a double-stranded neurotoxin.

  The term “contacting” as used herein refers to at least two different compounds in physical proximity to allow physical and / or chemical interaction of the compounds. In the method of the present invention, the two different compounds are, in one embodiment, first and second polypeptides contained in a solution. The contacting is performed for a sufficient amount of time under conditions sufficient to allow the first and second polypeptides to interact. The term “proteolytically processed polypeptide”, as used herein, in one aspect, refers to a polypeptide whose polypeptide chain is hydrolyzed or cleaved at one or more peptide bonds. . In another aspect, the term refers to a polypeptide that has been proteolytically cleaved by an endoproteinase or endopeptidase. In another aspect, the term refers to a polypeptide that is cleaved to at least as much as 50%. In another embodiment, the proteolytically processed polypeptide is a second polypeptide. In another embodiment, at least 60%, 70%, 80%, 90%, or 95% is proteolytically processed.

  The term “first polypeptide” as used herein refers to a polypeptide of the invention, ie, a proteolytically active or activated polypeptide, also referred to as “active BoNT hydrolase”. Since the active BoNT hydrolase can be obtained from the supernatant of Clostridium botulinum, it was originally named native BoNT hydrolase, abbreviated “nBH”. However, the terms “first polypeptide” and “nBH” also refer to BoNT hydrolases that can be obtained from other sources. The term “second polypeptide” as used herein refers to a substrate for the first polypeptide. The term “proteolytically sensitive” refers to a characteristic or requirement of a second polypeptide, wherein the second polypeptide can be proteolytically cleaved by the first polypeptide. Used to mean. In other words, the term “proteolytically sensitive” means that the second polypeptide contains a protease recognition and cleavage site that allows it to function as a substrate for the first polypeptide. A “second polypeptide” is a substrate for a first polypeptide that is proteolytically processed into two or more cleavage products. Using the assays described hereinabove, one of ordinary skill in the art will know that a given polypeptide is a substrate for a first polypeptide and is therefore a “second polypeptide” according to the definition of the present invention. Can be tested. The term “at least two cleavage products” includes, for example, up to 2, 3, 4, 5, and 6 cleavage products.

  This method can be used, for example, to prepare a pharmaceutical composition comprising a Clostridial neurotoxin or to produce polypeptide fragments used in mass spectrometry. The first polypeptide and the second polypeptide can be contacted at various steps in the production process of the proteolytically processed polypeptide. In one aspect, the step of contacting the first polypeptide and the second polypeptide can be intracellular. In one particular aspect of this embodiment, the first and second polypeptides are expressed in the cell.

In another aspect, the contacting step is in a cell lysate or a purified cell lysate. This embodiment includes adding the first polypeptide to the cell lysate or the purified cell lysate. The first polypeptide can be added at various steps during the purification of the second polypeptide from the cell lysate. For example, the first polypeptide can be added before or after protein precipitation, ion exchange chromatography, hydrophobic interaction chromatography, and / or size exclusion chromatography. Further included is adding a first polypeptide to the pharmaceutical composition. In this aspect, the polypeptide of the present invention activates a second polypeptide that is a therapeutic agent, eg, in a pharmaceutical composition, eg, to proteolytically cleave the second polypeptide. Used for. It is also contemplated that the first polypeptide is administered to the subject in order to proteolytically process the second polypeptide in the subject. Administration also includes simultaneous administration of the first and second polypeptides. The method also includes incubating for a sufficient amount of time under conditions sufficient to cleave the second polypeptide. In one aspect, the conditions can include adding a buffer selected from the group consisting of 100 mM Tris-HCl, pH 8.0 or PBS (50 mM Na ₂ HPO ₄ , 150 mM NaCl, pH 7.4). Preferred buffer conditions include 100 mM Tris-HCl, pH 8.0. “Sufficient time to cleave” can be determined using the assays described hereinabove. In one aspect, the “enough time for cleavage” depends on the degree of cleavage that the proteolytically processed polypeptide or composition comprising it should exhibit. In one aspect, the method comprises incubating the first and second polypeptides for at least 30 minutes, 60 minutes, 120 minutes, or at least 240 minutes. In another embodiment, the first and second polypeptides are incubated for 30 minutes, 60 minutes, 120 minutes, 240 minutes, up to 480 minutes, or up to 600 minutes. In another embodiment, the method comprises incubating the first and second polypeptides at 4 ° C. or at 37 ° C. In another embodiment, the method comprises incubating for up to 1 hour, 2 hours, 4 hours, 6 hours, 10 hours, or 16 hours.

In one aspect, the polypeptide chain of the second polypeptide comprises a sequence selected from any one of SEQ ID NOs: 4-25. In a more particular aspect, the polypeptide chain of the second polypeptide comprises a sequence selected from any one of SEQ ID NOs: 4-25, wherein the second polypeptide is SEQ ID NO: It is cleaved at the C-terminal side of the basic amino acid residue in any one of the above sequences of 4-25. The above sequence represents the amino acid sequence of a known substrate of the proteolytically active polypeptide of the present invention. As shown herein, the substrate is cleaved on the C-terminal side of basic amino acid residues contained in the sequence. Compare the LC and H _N columns in Table 1. In a preferred embodiment, the second polypeptide comprises a sequence selected from SEQ ID NOs: 4-10. In another preferred embodiment, the second polypeptide is BoNT / A or a derivative thereof, including, for example, the polypeptide of SEQ ID NO: 3 and derivatives thereof. The term “derivative” as used in this and other aspects of the invention includes amino acid variations such as addition, substitution, deletion or truncation of one or more amino acid residues.

  In one aspect, the second polypeptide comprises a derivative of any one of SEQ ID NOs: 4-25 or SEQ ID NO: 3, wherein the derivative comprises one or more point mutations and / or one or more Has additional amino acid residues. In another embodiment, the derivative is up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 15, Has a point mutation. By using an activity assay to determine protease activity as described herein, one of skill in the art can determine whether a given derivative is processed by a proteolytically active polypeptide of the present invention. Can be determined. In another aspect, the derivative comprises a point mutation that changes a basic amino acid residue to a non-basic amino acid residue. In another embodiment, the derivative has at least 50% sequence identity with any one of SEQ ID NOs: 4-25. In another embodiment, the derivative or a polypeptide comprising the derivative is a substrate for the first polypeptide and can be proteolytically cleaved by the first polypeptide. A typical example is a derivative of SEQ ID NO: 3 containing, for example, one or more point mutations in the light or heavy chain.

In another embodiment, the second polypeptide comprises (a) a polypeptide sequence having at least 30% sequence identity with the sequence of SEQ ID NO: 3 [(ATCC3502 BoNT / A, Genbank accession number AAA23262)]; or (b) a polypeptide sequence selected from the group consisting of tetanus neurotoxin, coagulation cascade factor X or prothrombin (factor II) protein, pancreatic digestive enzymes such as trypsin, chymotrypsin, pepsin, and papain. At least 30% means at least 30%, at least 40%, at least 50%, at least 85%. In one particular embodiment, the sequence identity of the second polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 3 is determined based on amino acid positions 420-466 of SEQ ID NO: 3, In an embodiment, the sequence identity is determined based on any of SEQ ID NOs: 4-25. In other words, the above embodiment provides, for example, at least 30% sequence identity to the polypeptide sequence present between amino acid positions 420 to 466 of SEQ ID NO: 3, or any one of the sequences of SEQ ID NOs: 4-25. A second polypeptide comprising a polypeptide sequence having at least 30% sequence identity to the peptide sequence. A polypeptide according to this definition can be obtained, for example, from Clostridium botulinum, tetanus, or Sporogenes. The second polypeptide is a naturally occurring neurotoxin such as, for example, BoNT / A, B, C1, D, E, F, or G, or addition, substitution of one or more amino acid residues, It may be a derivative thereof containing one or more amino acid mutations such as deletion or truncation. For example, a derivative lacking the _HC domain of natural neurotoxin or part thereof, or a derivative containing other amino acid residues that replace the _HC domain of neurotoxin, and further fused N-terminally to the light chain of BoNT Such as a derivative having an additional light chain or another protein cargo molecule.

  In another embodiment, the second polypeptide may contain additional amino acid residues at the N-terminus or C-terminus, or at an internal position. This additional amino acid residue may be adjacent to one or more protease cleavage sites. In another embodiment, this additional amino acid sequence functions as a detectable tag and / or allows binding to a solid support. An example is a his tag or a GST tag. Another example is the amino acid sequence VPPTPGSAWSHPQFEK, preferably appended to the C-terminus, containing a Strep tag.

  In one particular embodiment, the second polypeptide comprises GenBank numbers: CBZ04958.1, YP_002805603.1, ZP_02994746.1, YP_001788403.1, YP_001782718.1, ZP_02616437.1, ZP_02614241.1, YP_001392361.1, YP_001255575 A polypeptide comprising a polypeptide sequence as shown in .1, or a homolog thereof having at least 50% sequence identity.

  In another embodiment, the biological activity of the second polypeptide is modulated by protein cleavage. It is well known to those skilled in the art that protein processing can modulate the function of many polypeptides. “Modulated” as used herein means increased or decreased, activated or inactivated. For example, many clostridial neurotoxin organisms by proteolytically processing single-chain neurotoxins into double-chain neurotoxins composed of light and heavy chain polypeptides covalently linked through disulfide bridges. The biological activity is increased or induced. The biological activity of a neurotoxin includes at least three distinct activities: The first activity is the “proteolytic activity” present in the light chain of the neurotoxin, which regulates cell membrane fusion. It is responsible for the hydrolysis of the peptide bonds of one or more related polypeptides. The second activity is the “translocation activity” present in the N-terminal part of the processed neurotoxin heavy chain, which involves transporting the light chain across the lysosomal membrane into the cytoplasm. ing. The third activity is the “receptor binding activity” present in the C-terminal part of the processed neurotoxin heavy chain, which is related to the binding and uptake of the neurotoxin to the target cells. In one preferred aspect, the term biological activity as used herein means proteolytic activity. In a more preferred embodiment, the term refers to increased proteolytic activity.

The biological activity of a clostridial neurotoxin can be measured by various tests, all of which are known to those skilled in the art. These tests make it possible to determine one or more of the above mentioned activities. For example, Pearce et al., 1994 (Pearce LB, Borodic GE, First ER, MacCallum RD (1994), Toxicol Appl Pharmacol 128: 69-77) and Habermann et al., 1980 (Habermann E, Dreyer F, Bigalke H. (1980), Mouse LD ₅₀ assay or ex vivo mouse phrenic hemidiaphragm (MPN) assay as described by Naunyn Schmiedebergs Arch Pharmacol. 311: 33-40) is a living organism or an isolated neuromuscular preparation. Makes it possible to determine the toxic effects of a given neurotoxin formulation. In order to establish a toxic effect in the LD ₅₀ assay, this neurotoxin must be biologically active for each of the above three activities. In addition, various other assays can be utilized that allow, for example, determining whether a neurotoxin or the light chain of that neurotoxin is proteolytically active. Such an assay is based on, for example, contacting BoNT / A with SNAP-25. Alternatively, a peptide representing the cleavage site of SNAP-25 can be used, where the peptide can be labeled to facilitate detection. In a preferred embodiment, biological activity is determined by using the MPN assay described herein above.

  In another embodiment, the first polypeptide is active by proteolytically processing an inactive precursor polypeptide comprising a polypeptide sequence having at least 60% sequence identity with the sequence of SEQ ID NO: 2. It becomes. This embodiment is based on the observation that the polypeptide having the polypeptide sequence of SEQ ID NO: 2 is proteolytically inactive, but its N-terminal truncation is proteolytically active. The use of proteolytically inactive polypeptides in the methods described herein is also contemplated by the present invention. The proteolytically inactive polypeptides described herein can be activated, for example, by removing a fragment from the N-terminus or by removing the entire N-terminus including amino acid residues 1-248 of SEQ ID NO: 2. Can do. In one aspect, the N-terminus is removed by a protease, and in another aspect, the N-terminus is removed by autoproteolysis of SEQ ID NO: 2. 60% sequence identity relates to sequence alignment with full length NT02CB1447.

  In another embodiment, the method of the invention for producing a proteolytically processed polypeptide comprises a proteolytically processed second polypeptide, or at least one or more cleavage products thereof. A step of purifying. Purification of BoNT / A expressed by Clostridium botulinum can be performed, for example, as described mainly in the prior art (DasGupta 1984, Toxicon 22, 415; Sathyamoorthy 1985, J Biol Chemistry 260, 10461). In particular, the purification of the neurotoxin can include one or more precipitation-extraction steps, one or more concentration steps, and even a separate chromatography step. Recombinant single chain BoNT / A and its purification have been described in the prior art (Rummel et al., 2004, Mol Microbiol. 51: 631-43).

  In a preferred embodiment, the Clostridium strain is, for example, Clostridium botulinum that produces BoNT / A, or a derivative thereof. For fermentation, the process described by DasGupta B. R. et al. (Toxicon, vol. 22, No. 3, pages 414-424, 1984) can be used. Therefore, 0.5% yeast extract and 0.6% autoclaved yeast paste are added to 2% NZ amine type A medium and 4N NaOH is used to adjust the pH to 7.2, and the medium so prepared is then It will be autoclaved. To this medium, separately autoclaved glucose (20% by weight with respect to the volume) can be added so that the final glucose concentration in the medium is 0.5%. Incubation can be carried out, for example, without stirring at 37 ° C., with the fermentation being stopped after, for example, 96 hours. In addition to the batch fermentation described above, it is within the scope of the present invention that semi-batch fermentation, repeated batch fermentation, or continuous fermentation can also be performed.

After the actual fermentation and separation of the fermentation medium from the cells, the fermentation medium can be subjected to a first precipitation in order to remove large proteins. This precipitation is preferably an acid precipitation. Reaction conditions for such acid precipitation are known to those skilled in the art. Typically, 1.5 MH ₂ SO ₄ can be used to acidify the supernatant to a pH of 3.5. Centrifugation is usually performed at 2400 × g for 20 minutes at 4 ° C. The pellet obtained by centrifugation can be washed with water, preferably repeatedly. Subsequently, the pellet can be extracted with 0.1 M citrate-trisodium citrate buffer, pH 5.5, for example for 1 hour. Subsequently, further centrifugation steps can be performed for 20 minutes at, for example, 9800 × g, 4 ° C. The pellets thus obtained can then optionally be extracted again as described above. Then, the supernatant of the extraction, and if the extraction is repeated, both supernatants can be subjected to protamine sulfate precipitation. This precipitation can be continued, for example, at 8 ° C. overnight. Subsequently, the precipitate can be centrifuged, for example, at 4 ° C. and 12,000 × g for 20 minutes. The centrifugation supernatant can be subjected to precipitation, such as ammonium sulfate precipitation, thereby removing other relatively large proteins. After the ammonium sulfate precipitation step, another centrifugation step can be added, followed by redissolving the pellets so obtained and optionally dialysis. Preferably, the dialyzed and re-centrifuged extract can be subjected to successive chromatographic steps for the purpose of purifying the neurotoxin. Each of these chromatographic steps serves to remove protamine sulfate, residual DNA, relatively small protein portions, and contaminants such as medium protein, as well as hemagglutinin in the botulinum neurotoxin protein complex. For this purpose, in a preferred embodiment, one or more chromatographic steps can be used. Optionally, the eluate of the last chromatography step can be filtered, for example, to reduce microorganisms. Optionally, the eluent can be diluted prior to filtration and a suitable adjuvant can be added. During further steps, another sterile filtration can be performed after adding the adjuvant. In one aspect, this filtration can be performed in a reaction vessel, which can then be subjected to a lyophilization step.

  The invention also relates to a composition obtainable by the method of the invention for producing a proteolytically processed polypeptide. In one aspect, the composition comprises a mixture of a processed second polypeptide and an unprocessed second polypeptide, wherein the mixture comprises 5%, 4%, 3%, 2% Or may contain less than 1% unprocessed second polypeptide. In one embodiment of the composition, the second polypeptide is BoNT or a derivative thereof. The BoNT can be selected from the group consisting of, for example, serotypes A, B, C, D, E, F, and G BoNTs including derivatives thereof. The composition may be, for example, a liquid composition or a solid composition, and may contain one or more carriers, adjuvants, and / or additives.

  In another aspect, the present invention also provides a method for producing a medicament, i.e. a pharmaceutical composition, comprising the steps of the above-described method and the further step of formulating the purified double-stranded neurotoxin as a medicament. And a method comprising: In one aspect, the medicament comprises a mixture of a processed second polypeptide and an unprocessed second polypeptide, wherein the mixture comprises less than 5% unprocessed second polypeptide. Contains the polypeptide. In preferred embodiments, the mixture contains less than 4%, 3%, 2%, or 1% unprocessed second polypeptide.

  The invention also relates to various medical uses of the compounds disclosed herein:

  In one aspect, the invention relates to a proteolytically active polypeptide according to the invention for use as a medicament or in a pharmaceutical composition.

  In another aspect, the invention relates to a composition according to the invention for use as a pharmaceutical or in a pharmaceutical composition.

  In another aspect, the invention relates to an antibody according to the invention for use as a medicament or in a pharmaceutical composition.

  In yet another aspect, the invention relates to an inhibitor according to the invention for use as a medicament or in a pharmaceutical composition.

  In particular, the invention relates to a pharmaceutical composition comprising a polypeptide of the invention, an antibody of the invention, a composition of the invention, or an inhibitor of the invention.

  The term “composition” as used herein refers to any composition formulated into a solid, liquid, aerosol (or gaseous) form, or the like. Such compositions comprise, for example, a therapeutically active compound of the present invention, optionally together with a suitable auxiliary compound such as an excipient or carrier, or further ingredients. In one aspect, the therapeutically active compound is a proteolytically active polypeptide of the present invention. In another embodiment, the therapeutic compound is a second proteolytically processed polypeptide as described herein above, such as a double-chain neurotoxin. In another aspect, the therapeutically active compound is an antibody of the invention. In another aspect, the therapeutically active compound is an inhibitor of the proteolytically active polypeptide of the present invention.

  In this context, the present invention relates to auxiliary compounds, i.e. compounds that do not contribute to the action elicited by the compounds of the invention, and further ingredients, i.e. further effects, in applying the composition for its desired purpose. Are distinguished from compounds that cause or modulate the action of the compounds of the invention. Suitable excipients and / or carriers depend on the purpose for which the composition is to be used and other ingredients. One skilled in the art can determine such suitable excipients and / or carriers without further mess.

  The carrier (s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The pharmaceutical carrier employed can include a solid, gel, or liquid. Exemplary solid carriers are lactose, gypsum, sucrose, talc, gelatin, agar, pectin, gum arabic, magnesium stearate, stearic acid and the like. Exemplary liquid carriers are phosphate buffered saline, syrup, oil, water, emulsions, various types of wetting agents, and the like. Similarly, the carrier or excipient may include a time delay material well known in the art, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. Suitable carriers include those described above and others well known in the art. See, for example, Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

  The excipient (s) are selected so as not to affect the biological activity of the combination. Examples of such excipients are distilled water, saline, Ringer's solution, dextrose solution, and Hank's solution, and in addition, the pharmaceutical composition or formulation may contain other carriers, adjuvants, or non-toxic, non-toxic Therapeutic, non-immunogenic stabilizers and the like may be included.

  In one aspect, the pharmaceutical composition, as used herein, comprises a biologically active neurotoxin obtained by the method of the invention, optionally one or more pharmaceutically acceptable carriers. Including. This active neurotoxin can be present in liquid or lyophilized form. In one aspect, the compound may be present with glycerol, a protein stabilizer (eg, human serum albumin (HSA)), or a non-protein stabilizer such as polyvinylpyrrolidone or hyaluronic acid. This pharmaceutical composition, in one aspect, is administered topically. Conventionally used drug administration is intramuscular and subcutaneous (near the gland). However, depending on the nature and mechanism of action of the compound, the pharmaceutical composition can be administered by other routes. The double-chain neurotoxin polypeptide is the active ingredient of the composition and, in one aspect, is administered in a conventional dosage form prepared by combining the drug with a standard pharmaceutical carrier by conventional procedures. . These procedures may involve mixing, granulating, compressing, or dissolving the ingredients as appropriate to the desired formulation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or excipient will be dictated by the amount of active ingredient to be mixed therewith, the route of administration, and other well-known variables.

A therapeutically effective dose refers to the amount of compound, neurotoxin, used in the pharmaceutical composition of the invention that prevents, ameliorates or treats symptoms associated with the disease or condition referred to herein. The therapeutic potency and toxicity of this compound is determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, ED50 (therapeutically effective dose in 50% of the population) and LD ₅₀ (in 50% of the population). The fatal dose). The dose ratio between therapeutic and toxic effects is the therapeutic index and it can be expressed as the ratio, LD ₅₀ / ED ₅₀ .

  The dosage regimen should be determined by the attending physician and other clinical factors. As is well known in the medical field, the dosage for any patient is determined by the patient's physique, body surface area, age, specific compound to be administered, gender, time and route of administration, general health, and other It depends on many factors, including drugs. Progress can be monitored by periodic assessment. To treat or ameliorate or prevent the diseases or conditions listed herein, the pharmaceutical compositions and formulations referred to herein are administered at least once. However, the pharmaceutical composition can be administered more than once.

  In a further aspect of the invention, the composition described above is a pharmaceutical or cosmetic composition. In one aspect, the medicament comprising a biologically active neurotoxin can be used to prevent and / or treat at least one of the following diseases and disorders: voluntary muscle stiffness, localized dystonia ( Cervical, cerebral dystonia), and benign idiopathic blepharospasm, unilateral facial convulsions, and focal spasms, gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction; in a further aspect, Maxillofacial dystonia (jaw opening type, jaw closing type), bruxism, Meige syndrome, tongue dystonia, inability to bleed, open neck dystonia, cervical anterior flexion, cervical retroflexion, cervical side Flexion, torticollis, pharyngeal dystonia, laryngeal dystonia, convulsive dysphonia / adductor type, convulsive dysphonia / abductor muscular type, convulsive dyspnea, extremity dystonia, arm dystonia, motion-specific dystonia, writer's cramp, music Home cramps, golfer cramps, leg dystonia, thigh adduction, thigh addendum knee flexion, knee extension, ankle flexion, ankle extension, clubfoot, deformed foot dystonia, thumb extension (striatal toe), toe Flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, unilateral dystonia, unilateral dystonia, systemic dystonia, dystonia in lubag disease, dystonia in cerebral cortex basal ganglia degeneration, in lebug disease Dystonia, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallelfolden Spats disease, dopa-induced dyskinesia / dopa-induced dystonia, tardive dyskinesia / late Dystonia, paroxysmal dyskinesia / dystonia, luck Inducible / non-motor-induced / motion-induced palatal myoclonus, myoclonus, myokemia, stiffness, benign muscle spasm, hereditary jaw tremor, bizarre jaw muscle activity, unilateral masticatory muscle spasm, hypertrophic bowel muscle disease, Masseter muscle hypertrophy, anterior tibial muscle hypertrophy, nystagmus, swaying vision, supranuclear gaze palsy, epilepsy, persistent partial epilepsy, spastic slant neck surgery plan, vocal fold abductor palsy, refractory variant dysphonia, upper esophageal sphincter function Disorder, vocal fold granuloma, stuttering, Gilles de la Tourette syndrome, middle ear myoclonus, protective laryngeal closure, post-pharyngectomy language disorder, protective ptosis, entropion, dysphincter dysfunction, Pseudoachalasia, non-achalasia, esophageal movement disorder, vaginal spasticity, postoperative fixed tremor, bladder dysfunction, detrusor / sphincter dysfunction, bladder sphincter spasm, unilateral facial spasm, dyskinesia due to nerve regeneration, Purpose, fine lines on the corners of the eyes, astringent facial asymmetry, stinging of the stiff person syndrome, stiff person syndrome, hypertrophy, prostatic hypertrophy, adiposity, cerebral palsy treatment, retinal detachment surgery, after cataract surgery, aphakic myositis strabismus With mixed paralysis, myopathic strabismus, alternation of superior oblique position associated with strabismus surgery, esotropia, exotropia, achalasia, anal fissure, exocrine activity, Frey's syndrome, crocodile tear syndrome, axilla, palm, foot Hyperhidrosis of the bottom, rhinorrhea, stroke, Parkinson's disease, associated salivary hypertrophy in amyotrophic lateral sclerosis, encephalitis and myelitis, autoimmune process, multiple sclerosis, transverse myelitis, Devic syndrome, virus Hereditary spastic paraplegia, syndrome after stroke, hemisphere infarction, brainstem infarction, spinal cord infarction, migraine, central in infection, bacterial infection, parasitic infection, fungal infection Transsystem trauma, cerebral hemisphere lesion, brain stem lesion, spinal cord lesion, central nervous system hemorrhage, intracerebral hemorrhage, subarachnoid hemorrhage, subdural hemorrhage, intrathecal hemorrhage, neoplasm, cerebral hemisphere tumor, brain stem tumor, spinal cord tumor Convulsions, snoring (WO2000 / 033863). For details and symptoms, see Jost 2007, Drugs 67 (5), 669 or Dressier 2000 in Botulinum Toxin Therapy, Thieme Verlag, Stuttgart, New York.

  In another aspect of the invention, the composition is a cosmetic composition that can be formulated as described for the pharmaceutical composition above. Similarly, in cosmetic compositions, it is contemplated that the compounds of the invention, in one aspect, are used in substantially pure form. In a further aspect, the cosmetic composition is applied intramuscularly. In yet a further aspect of the invention, a cosmetic composition comprising a neurotoxin can be formulated as a wrinkle removal solution.

  In another aspect, the pharmaceutical composition comprises an antibody or inhibitor of the invention. Since the polypeptides of the present invention are responsible for the activation of Clostridial neurotoxins, this antibody would be useful for reducing the toxic effects observed during infection with Clostridium. Accordingly, the antibodies of the present invention, in one aspect, include, It can be used to treat infections caused by Clostridium, including Clostridium novyi, and Clostridium oedematiens. Furthermore, the antibodies of the present invention can in another aspect be used to treat symptoms associated with the above infections. Furthermore, the antibody can be used in treating a condition or symptom associated with a condition selected from botulism, tetanus, pseudomembranous colitis, gangrene, food poisoning and the like.

  In another aspect, the pharmaceutical composition comprises a proteolytically active polypeptide of the present invention. The pharmaceutical composition, in one aspect, is used for proteolytically cleaving a polypeptide associated with coaglutination, especially for treating patients with hypocoaglutination. be able to. In another aspect, the pharmaceutical composition may be used as a fibrinolytic agent to treat, inter alia, patients with myocardial infarction, pulmonary embolism, deep vein thromboembolism, i.e., to remove a thrombus. it can. In addition, the use of the pharmaceutical composition in the treatment of stroke is contemplated. Furthermore, in other embodiments, the pharmaceutical composition can be used to supplement any of trypsin, chymotrypsin, or pepsin in the treatment of exocrine pancreatic insufficiency. Furthermore, in another aspect, the pharmaceutical composition proteolytically cleaves tumor antigens exposed on the surface, particularly in the treatment of cancer patients, when treating patients suffering from an inflammatory response. Can be used for. Furthermore, in another aspect, the pharmaceutical composition can be used in the treatment of papillomas.

  The present invention is also a method of screening for inhibitors, comprising: (a) contacting a proteolytically active polypeptide of the invention with a known substrate, and optionally with a putative inhibitor; Determining the effect of the putative inhibitor on converting the substrate to a cleaved product, wherein a reduction in the amount of cleaved product indicates the inhibitory effect of the putative inhibitor Regarding the method. In one aspect, the putative inhibitor comprises an amino acid sequence selected from any one of SEQ ID NOs: 4-25, but at least one basic amino acid in the amino acid sequence is replaced with a non-basic amino acid. Peptide. In a further aspect, the peptide comprises one or more chemical modifications. In another embodiment, the inhibitor is a peptidomimetic of the peptide. In another aspect, the putative inhibitor is a chemical compound microarray, ie, part of a collection of organic chemical compounds. In another aspect, the inhibitor is an antibody of the invention. This method is useful for identifying compounds capable of inhibiting the proteolytically active polypeptides of the present invention. The initial screen can be based on, for example, a peptide comprising an amino acid sequence selected from any one of SEQ ID NOs: 4-25. In order to increase inhibition, peptides capable of inhibiting the polypeptides of the invention may be modified. Modifications include amino acid substitutions or chemical modifications. Typically, the method is performed by contacting a polypeptide of the invention with a known substrate (step (a) of the method) and cleaving from the substrate in the presence and absence of putative inhibitors. This is done by comparing the effect of the putative inhibitor on the conversion to product. A reduction in conversion rate in the presence of a putative inhibitory substance indicates an inhibitory effect.

  The present invention also includes an inhibitor of the proteolytically active polypeptide of the present invention, comprising (a) an amino acid sequence as shown in any one of SEQ ID NOs: 4 to 25, Inhibitors wherein one basic amino acid is replaced by a non-basic amino acid; or (b) an inhibitor which is an antibody of the present invention.

  All references cited in this specification are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

It is a figure which shows the activity test of the fraction collected from HiPrep 16/10 Q FF. Fractions collected from the HiPrep 16/10 Q FF run were analyzed for enzyme activity by incubation with 2 μg scBoNTA (lane 2) for 1 hour at 37 ° C. followed by 10% SDS-PAGE. . Lane 1: Low molecular weight marker (LMW): 116 kDa, 66 kDa, 45 kDa, 35 kDa. FIG. 6 shows analysis of fractions collected from SEC (HiLoad 16/60 Superdex 75) for content of nBH having a molecular weight of about 37.3 kDa by 12.5% SDS-PAGE. Fractions 9-11 contain the majority of nBH. (Lane 1: LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, 14.4 kDa) FIG. 2 shows 12.5% SDS-PAGE analysis to determine the purity and protein concentration of three purification batches of nBH. Lane 1, LMW (116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa, 18.4 kDa, 14.4 kDa); Lane 2, nBH lot TE311206 (192 ng / μl mature NT02CB1446 / CBO1444, Genbank accession numbers CAL82987.1 amino acids 254 to 594, Lane 3, nBH lot TIK301009 (130 ng / μl mature NT02CB1447 / CBO1445, SEQ ID NO: 1, amino acids 249-581 of Genbank accession number CAL82988.1, MW: 37.3 kDa); Lane 4, nBH lot TIK280509 (114 ng / μl mature NT02CB1447 / CBO1445, SEQ ID NO: 1, amino acids 249-581 of Genbank accession number CAL82988.1, MW: 37.3 kDa). It is a figure which shows an ESI-MS / MS spectrum analysis report. The 38.6 kDa protein band of nBH lot TE311206 was identified as NT02CB1446 / CBO1444 with a Mascot score of 725 and a peptide MS / MS sequence coverage of 29.6% over the entire open reading frame (ORF). None of the peptides (gray box, identified MS peptide; red square, amino acid y ion / b ion of the peptide identified after MS / MS degradation) were identified as originating from the N-terminal 253 amino acids. MS / MS analysis of lot TE311206 showed 52% sequence coverage at the C-terminal amino acids 254-594 forming nBH. It is a figure which shows an ESI-MS / MS spectrum analysis report. The 37.3 kDa protein band of nBH lot TIK301009 was identified as NT02CB1447 / CBO1445 with a Mascot score of 555 and a peptide MS / MS sequence coverage of 28.4% over the entire open reading frame (ORF). Except one, all identified peptides (gray box, identified MS peptide; red square, amino acid y ion / b ion of peptide identified after MS / MS degradation) are C-terminal 333 Derived from amino acids. MS / MS analysis of lot TIK301009 showed 49.5% sequence coverage at the C-terminal amino acids 249-581 forming nBH. FIG. 6 shows a comparison of nBH concentration-dependent proteolytic activity from three purification batches. A: Activity test to analyze nBH from batches TIK301009, TIK280509, and TE311206 using the following dilutions of concentrated nBH: 1:10, 1:30, 1: 100, 1: 300, 1: 1000 12.5% SDS-PAGE. This assay was performed by incubating 1 μg scBoNT / A and 2 μl dH ₂ O and corresponding diluted 1 μl nBH at 37 ° C. for 60 minutes. For SDS-PAGE analysis, 3 μl of reducing 4 × SDS Laemmli buffer was added to a final volume of 10 μl. The 150 kDa scBoNT / A was cleaved into a 100 kDa heavy chain and a 50 kDa light chain. B: The optical density of the heavy, light chain, and scBoNT / A protein bands was quantified and the sum of the light and heavy chain product bands was divided by the sum of the LC, HC, and scBoNT / A protein bands. The higher the dilution of the first polypeptide, the lower the cleavage rate. The specific proteolytic activity of the three different batches is nearly identical. Figure 3 shows time-dependent cleavage of mutants containing wild-type and modified loops of scBoNT / A by nBH. A: Modification of the loop sequence. In scBoNTAS Throm, all lysine residues have been removed and the thrombin recognition sequence LVPRGS has been inserted, while in scBoNT Res, the loop lacks any basic amino acids. Shortening the loop to 8 small residues or 5 amino acids with bulky side chains yields scBoNTAS (GGSG) ₂ and scBoNTAS FQWYI, respectively. In scBoNTAS CGS-C, the entire loop is deleted and cysteines that form disulfide bridges are replaced by glycine and serine. B: SDS-PAGE analysis of time-dependent cleavage of scBoNT / A wild type and mutants. C: scBoNTAS wild type is activated by nBH to light and heavy chains within 120 minutes in a time-dependent manner. Deletion of lysine and insertion of a single arginine residue prolongs the loop break (scBoNTAS Throm). Loops lacking all basic residues can also be cleaved (scBoNTAS Res). Shortening the loop to the octamer peptide, introducing 5 amino acids with bulky side chains, or deleting the entire loop results in an uncleavable scBoNT / A. It is a figure which shows the MS / MS analysis of 50 kDa and 100 kDa cleavage product when scBoNT / A is digested with nBH. A: Analysis of a 50 kDa cleavage product identified as a BoNT / A light chain with a Mascot score of 1460. The most C-terminally assigned peptide includes amino acids G433-K438, which corresponds to the physiologically observed C-terminus of BoNT / A LC. B: Analysis of a 100 kDa cleavage product identified as a BoNT / A heavy chain with a Mascot score of 96. The most N-terminally assigned peptide includes amino acids A449-K456, which corresponds to the physiologically observed N-terminus of BoNT / A HC. A: The protein content (mg / ml) of anti-nBH-IgY in three consecutive pools was analyzed by 12.5% SDS-PAGE. B: ELISA: Nunc Maxisorp F96 microtiter plates were coated overnight at 4 ° C with various lots of nBH (500 ng / mL) in PBS, then 0.1% Tween-20 and 2% non-fat skim milk Blocking was carried out for 1 hour with PBS-containing blocking buffer. After washing, IgY dilution of each pool (10 μg / ml in blocking buffer) is added over 1 hour, biotin-labeled donkey anti-chicken IgY, streptavidin-horseradish peroxidase (both Dianova, Hamburg, Germany), and Detection was performed using 3,3 ′, 5,5′-tetramethylbenzidine (Sigma). A: Recombinant expression of inactive BH 1-581 (63 kDa) and isolation by Talon IMAC. 10% SDS-PAGE analysis of Talon IMAC fraction (LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa; SS34, clear lysate; TD, flow through fraction; W, wash fraction; E1-E7, imidazole elution Fractions 1-7). B: After 1 hour at 37 ° C, recombinant iBH (SEQ ID NO: 2; `` E ''; 63 kDa) does not observe endo-proteolysis of scBoNT / A to LC (50 kDa) and HC (100 kDa) (lanes). 6) (LMW: 116 kDa, 66 kDa, 45 kDa, 35 kDa, 25 kDa). FIG. 5 shows the use of purified active BoNT hydrolase (nBH) to obtain proteolytically processed polypeptides. A: 200 μg of purified recombinant scBoNT / A is incubated for 12 minutes at 37 ° C. with 350 ng of purified active BoNT hydrolase. To stop the reaction, nBH was removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min) Are analyzed by 10% SDS-PAGE. B: Fraction 1 (1800 μl) containing approximately 40% processed BoNT / A is incubated with 350 ng of purified active BoNT hydrolase at 37 ° C. for 15 minutes and concentrated to 300 μl by ultrafiltration. To finally stop the reaction, nBH was removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min). The amount of cleavage is analyzed by 10% SDS-PAGE. C: Fractions 1 and 2 (1800 μl) containing approximately 80% processed BoNT / A are combined, incubated with 120 ng of purified active BoNT hydrolase for 25 minutes at 37 ° C. and concentrated to 300 μl by ultrafiltration To do. To finally stop the reaction, nBH was removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min). The amount of cleavage is analyzed by 10% SDS-PAGE. > 95% processed BoNT / A (SEQ ID NO: 3) is obtained.

  The sequence list is as follows:

SEQ ID NO: 1: Proteolytic active polypeptide of GenBank accession number “CAL82988.1” lacking 248 N-terminal amino acid residues derived from Botulinum strain ATCC3502 SEQ ID NO: 2: Derived from Botulinum strain ATCC3502 , GenBank accession number “CAL82988.1” proteolytically inactive polypeptide SEQ ID NO: 3 ATCC3502, Genbank accession number “AAA23262” BoNT / A
SEQ ID NO: 4: BoNT / A1 loop SEQ ID NO: 5: BoNT / A2 / A6 loop SEQ ID NO: 6: BoNT / A3 loop SEQ ID NO: 7: BoNT / A3 loop SEQ ID NO: 8: BoNT / A4 loop SEQ ID NO: 9 : BoNT / A5 loop SEQ ID NO: 10: BoNT / A7 loop SEQ ID NO: 11: BoNT / B1 / B4bv / B6 loop SEQ ID NO: 12: BoNT / B2 / B3 loop SEQ ID NO: 13: BoNT / B5np loop SEQ ID NO: 14: BoNT / C / CD loop SEQ ID NO: 15: BoNT / D loop SEQ ID NO: 16: BoNT / DC loop SEQ ID NO: 17: BoNT / E1 to E5 loop SEQ ID NO: 18: BoNT / E6 loop SEQ ID NO: 19 : BoNT / F1 / F6 loop SEQ ID NO: 20: BoNT / F2 / F3 loop SEQ ID NO: 21: BoNT / F4 loop SEQ ID NO: 22: BoNT / F5 loop SEQ ID NO: 23: BoNT / F7 loop SEQ ID NO: 24: BoNT / G loop SEQ ID NO: 25: TeNT loop SEQ ID NO: 26: nucleic acid sequence encoding SEQ ID NO: 1 SEQ ID NO: 27: nucleic acid sequence encoding SEQ ID NO: 2

  The invention is illustrated in the following examples, which should in no way be construed as limiting the scope of the invention.

  Example 1 Purification and characterization of a natural BoNT hydrolase (nBH) that specifically cleaves single-chain BoNT / A into its active double-stranded form.

  (1) Readout system / activity test: Botulinum neurotoxin A (BoNT / A) in 50 kDa light chain (LC) and 100 kDa heavy chain (HC) in culture supernatant of Clostridium botulinum and between chromatography steps In order to specifically detect and purify the enzymatic activity that hydrolyzes, the present inventors expressed 150 kDa BoNT / A as a single chain, sc polypeptide in E. coli. Incubation of recombinant scBoNT / A with appropriate enzyme activity (nBH) should yield a 50 kDa LC and a 100 kDa HC visualized by reducing 10-13% SDS-PAGE.

  (2) Expression of Clostridium protease: A single colony of Botulinum strain ATCC3502 was inoculated into 100 ml of brain heart exudate (BHI) medium and the culture was incubated overnight at 37 ° C. under anaerobic conditions. 10 ml of the overnight culture was inoculated into 1 L of BHI medium and incubated anaerobically for 48-72 hours.

  (3) Ammonium sulfate precipitation: 1 L of culture supernatant was collected by centrifugation (4 ° C., 6500 × g, 25 minutes). Ammonium sulfate was added to a final concentration of 85% (in this case 575 g) and the suspension was stirred at 4 ° C. for 6 hours, followed by centrifugation (4 ° C., 6500 × g, 30 minutes). The pelleted ammonium sulfate precipitate was dissolved in a small volume (in this case 5 ml) of 50 mM NaP pH 7.5 and dialyzed against 50 mM NaP, 150 mM NaCl pH 7.5. Finally, the dialysate was centrifuged (4 ° C., 40000 × g, 60 minutes) and the supernatant was used for IEC.

  (4) Ion exchange chromatography (IEC, column HiPrep 16/10 Q FF): The supernatant from (3) (Figure 1, lane 3) is applied to the equilibrated HiPrep 16/10 Q FF anion exchange column. Applied and flushed with buffer containing 50 mM NaP pH 7.5, 150 mM NaCl. This flow was performed at a flow rate of 1 ml / min. Activity tests were performed by incubating every other 5 μl fraction with 2 μg scBoNTA for 1 hour at 37 ° C. followed by analysis by SDS-PAGE (FIG. 1). Fractions 6-24 were combined and the volume was concentrated to 3.5 ml by using ultrafiltration (Amicon-Ultra MWCO 10,000).

  (5) Size exclusion chromatography (SEC, HiLoad 16/60 Superdex 200): Subsequently, the concentrated protein solution of (4) was equilibrated with 50 mM NaP pH 7,5, 150 mM NaCl. A Superdex 200 column was packed. Separation was performed at a flow rate of 1 ml / min. Fractions with a retention volume between 80 ml and 100 ml are analyzed using activity test (1), the appropriate fractions with enzyme activity (nBH) are combined (about 10 ml) and concentrated to 3 ml by ultrafiltration did. Subsequently, ammonium sulfate was added to a final concentration of 12.5% = 500 mM (+0.2 g).

  (6) Hydrophobic interaction chromatography (HIC, HiTrap Phenyl Sepharose): nBH was bound to Phenyl Sepharose in buffer A (50 mM NaP pH 7.5, 500 mM ammonium sulfate). Bound nBH was eluted by reducing the amount of ammonium sulfate by a linearly increasing gradient with buffer B (50 mM NaP pH 7.5) at a flow rate of 1 ml / min. All protein-containing fractions were analyzed using activity test (1) and the appropriate fractions were combined and concentrated to 3.5 ml by ultrafiltration. The solution buffer was adjusted to 50 mM NaP pH 7.5; 150 mM NaCl.

  (7) SEC (HiLoad 16/60 Superdex 75): Finally, nBH was purified by SEC using a HiLoad 16/60 Superdex 75 column at 50 mM NaP pH 7.5, 150 mM NaCl, and a flow rate of 1 ml / min. Fractions with a retention volume between 70 ml and 80 ml were analyzed by 12.5% SDS-PAGE (Figure 2) and fractions 8-12 containing nBH migrating to approximately 37.3 kDa were combined (approximately 10 ml) and limited. Concentrated to 1 ml by external filtration.

  (8) A prominent protein (nBH) migrating to approximately 37.3 kDa was analyzed by N-terminal peptide sequencing according to the Edman degradation protocol. The sequence of the identified peptide is V Q G Q S V K G V G, which corresponds to the first 10 residues of SEQ ID NO: 1.

  (9) Two lots of nBH (NT02CB1447, 37.3 kDa, FIG. 3, lane 3: TIK301009, lane 4: TIK280509) were reproducibly isolated according to the procedure as described above. The isolation procedure is modified as follows to obtain nBH isoform NT02CB1446 (38.6 kDa, FIG. 3, lane 2, lot TE311206): (i) Growth of Clostridium botulinum culture: instead of 48-72 hours (Ii) Chromatographic step change: IEC → SEC Superdex 200 → HIC Phenyl Sepharose → SEC Superdex 75 instead of IEC → SEC Superdex 75 → HIC Phenyl Sepharose

  [Example 2] Sequence identification of nBH derived from Clostridium botulinum by mass spectrometry (MS)

(1) Trypsin digestion: A protein band that migrates to approximately 38 kDa (nBH) in SDS-PAGE is excised for trypsin digestion and gently shaken in 50 mM NH ₄ HCO ₃ , 50% acetonitrile at 37 ° C. for 30 minutes. By de-staining. Destaining was repeated until the gel spot became transparent. Acetonitrile (100%) was added and removed after 3 minutes. Subsequently, the spots were dried on a Speed Vac system (Eppendorf, Germany). Trypsin (10 ng / μl) in 50 mM NH ₄ HCO ₃ was added and incubated on ice for 1 hour. The remaining trypsin solution was then removed and a small volume of 50 mM NH ₄ HCO ₃ was added and degradation was carried out overnight at 37 ° C. The supernatant was collected and the gel pieces were extracted twice using 5% TFA, 10% acetonitrile. All fluids were combined, dried on Speed Vac, and the extracted peptide was stored at 4 ° C.

  (2) Matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF / TOF) MS: Samples were analyzed on a MALDI-TOF / TOF mass spectrometer (Ultraflex1 Bruker Daltonik GmbH) in linear mode with an acceleration voltage of 25 kV . Mass was detected from 700 m / z to 4,500 m / z. Samples (2 μl) were co-crystallized directly with 2 μl of sinapinic acid solution containing 50% acetonitrile and 0.2% trifluoroacetic acid (TFA) on a stainless steel MALDI target plate. 500 laser shots were collected for each sample.

  (3) Peptide separation by reverse phase chromatography: Peptide separation was performed by reverse phase chromatography using a nano HPLC system (Agilent Technologies, Waldbronn, Germany) consisting of an autosampler and a gradient pump. Dissolve the sample in buffer A (5% acetonitrile, 0.1% formic acid) and aliquot up to 10 μl onto a C18 column (Zorbax SB-C18, 5 μm, 300 A, 0.5 mm ID, length 15 cm) at a flow rate of 5 μl / min. Injected. After loading, the column was washed with buffer A for 15 minutes and the peptide was eluted from 0% eluent B to 100% eluent B in 75 minutes with eluent A and eluent B (0.1% (v / v) Elution was performed using a gradient of 70% (v / v) acetonitrile in formic acid.

(4) Electrospray ionization (ESI) interface ion trap mass spectrometry: The HPLC outlet was directly connected to the nano ESI source of the ion trap mass spectrometer and an Agilent coaxial sheath liquid nebulizer was used (Agilent Technologies). The exit capillary was held by a surrounding steel needle and was visible from 0.1 to 0.2 mm therefrom. The spray was stabilized with N ₂ as nebulizer gas (5 L / min). The ionization voltage was set to 4,500 V and dry gas was applied at 5 psi and 250 ° C. Spectra were collected on an Esquire 3000+ ion trap mass spectrometer (Bruker Daltonik) at a scanning speed of 13,000 m / z per second. Using ESI in positive mode, mass spectra were obtained at 50-1600 m / z, in scan mode, and MS-dependent and MS / MS-analyzed switching. To improve the quality of the MS / MS spectrum, only two precursor ions from one spectrum are selected for MS / MS analysis and active exclusion is set to 2 minutes and already measured. Excluded precursor ions.

  (4) Data processing: Data processing was performed with Data Analysis (version 3.0) and BioTools (version 3.0) software package (Bruker Daltonik). Protein identification was performed using MASCOT software (version 2.1) and MSDB database (Matrix Science, London, UK).

  (5) Results:

  The 38.6 kDa protein band in lane 2 (nBH lot TE311206) was identified as NT02CB1446 / CBO1444 with a Mascot score of 725 and 29.6% peptide MS / MS sequence coverage over the entire open reading frame (ORF). A peptide derived from the N-terminal 253 amino acids was not identified (FIG. 4). MS / MS analysis of lot TE311206 showed 52% sequence coverage at the C-terminal amino acids 254-594 forming nBH.

  The 37.3 kDa protein band in lane 3 (nBH lot TIK301009) and lane 4 (nBH lot TIK280509) was identified as NT02CB1447 / CBO1445 with Mascot scores of 555 and 609, respectively. Except for one, all identified peptides are derived from the C-terminal 333 amino acids (FIG. 5). MS / MS analysis of lot TIK301009 showed 49.5% sequence coverage at the C-terminal amino acids 249-581 forming nBH.

  [Example 3] Determination of enzyme specificity of nBH

  (1) The concentration-dependent proteolytic activity of nBH derived from three purification batches was compared (FIG. 6). Activity tests analyzing nBH from batches TIK301009, TIK280509, and TE311206 using various dilutions of nBH demonstrate that the higher the dilution, the lower the cleavage rate. The proteolytic activities of these three different batches are nearly identical, indicating that the mature isoform NT02CB1446 (TE311206) exhibits specific activity similar to mature NT02CB1447 (SEQ ID NO: 1) .

(2) Time-dependent cleavage of scBoNT / A wild type and mutants by nBH was analyzed using an activity test (FIG. 7). scBoNTAS wild type is over 95% activated by nBH to light and heavy chains within 120 minutes in a time-dependent manner. The loop sequence was modified to characterize the cleavage site. In scBoNTAS Throm, all lysine residues were removed and the thrombin recognition sequence LVPRGS was inserted, which extended the cleavage rate. In scBoNT Res, the loop lacks all basic amino acids, which dramatically delays complete hydrolysis, which is a basic residue such as lysine and arginine at the cleavage site. It shows the strong recognition priority of nBH about the group. Furthermore, nBH accessibility to the loop is impaired by shortening the loop to 5 amino acids with 8 small residues or bulky side chains (scBoNTAS (GGSG) ₂ and scBoNTAS FQWYI).

  (3) MS / MS analysis of 50 kDa cleavage product of scBoNT / A digested with nBH shows that the most C-terminally assigned peptide corresponds to the physiologically observed C-terminal of BoNT / A LC It was shown to encompass amino acids G433-K438 (FIG. 8A). Analysis of the 100 kDa cleavage product identified as the heavy chain of BoNT / A includes the amino acids A449 to K456, the most N-terminally assigned peptide corresponding to the physiologically observed N-terminus of BoNT / A HC (FIG. 8B). Thus, isolated nBH yields physiologically processed BoNT / A, preferentially hydrolyzing peptide bonds on the C-terminal side of lysine and arginine residues.

  [Example 4] Evolutionary conservation of BoNT hydrolase and its isoforms

  Sequence analysis of the protein of SEQ ID NO: 2 (Genbank accession number CAL82988.1 / YP_001253958.1) revealed three conserved domains. Residues 18-573 have a Blast score of 738 and correspond to zinc metalloprotease (elastase) or LasB involved in amino acid transport and metabolism. Residues 148-212 correspond to the peptidase propeptide and the YPEB domain or PepSY (Blast score 97). Residues 336-573 are part of the peptidase M4 family (Blast score 803), including thermolysin, protealysin, aureolysin, and neutral protease.

  Genomic sequencing of Clostridium botulinum ATCC3502 reveals the presence of six ORFs encoding iBH isoforms (Sebaihia et al., 2007, Genome Res. 17 (7): 1082-11092). Additional genomic data is available for 10 Group I botulinum strains, as well as non-BoNT-secreting Sporogenes that contain any of the 5-7 ORFs encoding iBH. nBH (SEQ ID NO: 1) shares at least 64% amino acid sequence identity with the other 63 isoforms.

  [Example 5] Production of antibodies specific to BoNT hydrolase

  (1) IgY production: 16-week-old chickens [ISA Brown and Lohmann Selected Leghorn (LSL), Spreenhagener Vermehrungsbetrieb fuer Legehennen GmbH, Bestensee, Germany] in individual cages specially built for chicken rearing (Ebeco, Castrop-Rauxel, Germany). Feed (ssniff Legehuehner-Zucht 1 and 2; ssniff Spezialitaeten GmbH, Soest, Germany) and water were freely available, and these chickens began to lay eggs between 23-25 weeks of age. Eggs were collected daily, labeled and stored at 4 ° C. until further processing. All animal care and experiments were performed according to local authority guidelines Berlin (No. H0069 / 03). Chickens were immunized and boosted via the intramuscular route (left and right pectoral muscles) a total of 10 times at 4-8 week intervals over a year. The interval used was based on previous studies that did not show demonstrable memory cells until at least 3 weeks after immunization (Pei and Collisson, 2005). The antigen concentration used was approximately 20 μg (nBH) per injection. Less than 500 μl of antigen solution was injected per immunization. Freund's complete adjuvant was used for the first immunization and FIA was used for subsequent booster injections. The method for IgY purification was arranged from the method of Polson et al. (1980). Briefly, egg yolk was diluted 1: 2 with sterile PBS (pH 7.4, Roche, Mannheim, Germany). To remove lipids and lipoproteins, 3.5% (w / v) polyethylene glycol (PEG) 6000 (Roth, Karlsruhe, Germany) was added. After gentle shaking followed by centrifugation (10,000 × g, 20 minutes at 4 ° C.), the supernatant was decanted and solid PEG 6000 was added to a final concentration of 12% (w / v) . The mixture was then centrifuged as described above. The precipitate was dissolved in 10 ml PBS, PEG was added to 12% (wt / vol) and the solution was centrifuged. Finally, the precipitate was dissolved in 1.2 ml PBS, transferred to a microdialysis device (QuixSep, Roth, Germany) and dialyzed against PBS at 4 ° C. Protein content (mg / ml) was analyzed by 12.5% SDS-PAGE (FIG. 9A), measured photometrically at 280 nm, and calculated for IgY with an extinction coefficient of 1.33 according to the Lambert-Beer law.

  (2) ELISA: Nunc Maxisorp F96 microtiter plates (VWR International GmbH, Darmstadt, Germany) were coated overnight at 4 ° C with various lots of nBH (500 ng / mL) in PBS, then 0.1% Blocked with PBS blocking buffer containing Tween-20 and 2% non-fat skim milk (Merck, Darmstadt, Germany) for 1 hour. After washing, IgY dilution (10 μg / ml in blocking buffer) was added over 1 hour, biotin-labeled donkey anti-chicken IgY, streptavidin-horseradish peroxidase (both Dianova, Hamburg, Germany) and 3,3 ′ , 5,5'-tetramethylbenzidine (Sigma) was used for detection. The detected nBH is illustrated in FIG. 9B.

  (3) Western blot: nBH was separated by 12.5% SDS-PAGE and transferred to a polyvinylidene fluoride membrane (Invitrogen GmbH, Karlsruhe, Germany) using standard immunoblotting techniques. The membrane was blocked overnight at 4 ° C. and incubated with IgY (1: 5,000 in blocking buffer) for 1 hour. After washing, the membrane was probed with biotin-labeled donkey anti-chicken IgY for 30 minutes and developed using alkaline phosphatase and CDP-Star (Perkin Elmer, Waltham, Mass.).

  [Example 6] Recombinant expression of BoNT hydrolase

  Plasmid construction: The gene portion encoding native BH (SEQ ID NO: 1) and its propeptide (SEQ ID NO: 2) is amplified by PCR using the appropriate oligonucleotide and genomic DNA of Clostridium botulinum ATCC3502 and His6 tag It was fused to the encoding oligonucleotide and inserted into pQE3 (Qiagen) (resulting in expression plasmids pQ-BH1445H6-249-581 and pQ-BH1445H6-1-581, respectively). The nucleotide sequence was confirmed by DNA sequencing.

  Purification of recombinant protein: nBH and iBH fused to the carboxyl-terminal His6 tag were generated during 10 hours incubation at room temperature using E. coli strain M15pREP4 (Qiagen) and Talon Sepharose beads (Clontech Inc. And purified according to the manufacturer's instructions. Fractions containing the desired protein were pooled, frozen in liquid nitrogen and stored at -70 ° C. iBH was isolated as a recombinant protein with a MW of 63 kDa (FIG. 10A). An activity test was used to demonstrate iBH inactivity. That is, after 1 hour at 37 ° C., wild type scBoNT / A was not hydrolyzed to LC and HC (FIG. 10B).

  [Example 7] Inhibition of BoNT hydrolase

  (1) Screening for peptide inhibitors of BH: Peptides based on SEQ ID NOs: 4 to 25 are synthesized by deleting one or more basic residues. Each peptide is added to the activity test mixture. Peptides that can reduce the amount of processed scBoNT / A, extend the time required for complete processing of scBoNT / A, or block scBoNT / A processing are inhibitors of nBH Judge.

  (2) Antibody-based inhibitor screening: Antibodies raised against epitopes derived from nBH, such as IgY of Example 5, are incubated with nBH and subsequently subjected to activity testing. Antibodies that can reduce the amount of processed scBoNT / A, extend the time required for complete processing of scBoNT / A, or block scBoNT / A processing are inhibitors of nBH Judge.

  Example 8 Use of Purified Active BoNT Hydrolase (nBH) to Obtain Proteolytically Processed Polypeptide

  (1) 200 μg of purified recombinant scBoNT / A is incubated with 350 ng of purified active BoNT hydrolase at 37 ° C. for 12 minutes. To stop the reaction, nBH was removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min) Are analyzed by 10% SDS-PAGE (FIG. 11A).

  (2) Fraction 1 (1800 μl) containing approximately 40% processed BoNT / A is incubated with 350 ng of purified active BoNT hydrolase at 37 ° C. for 15 minutes and concentrated to 300 μl by ultrafiltration. To finally stop the reaction, nBH was removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min). The amount of cleavage is analyzed by 10% SDS-PAGE (FIG. 11B).

  (2) Combine fractions 1 and 2 (1800 μl) containing approximately 80% processed BoNT / A, incubate with 120 ng of purified active BoNT hydrolase for 25 minutes at 37 ° C. and ultrafiltration to 300 μl Concentrate. To finally stop the reaction, nBH was removed by SEC (column Superdex 200 10/300 GL, buffer: 50 mM NaP pH 7.5, 150 mM NaCl, sample volume = 0.3 ml, flow rate = 0.25 ml / min). The amount of cleavage is analyzed by 10% SDS-PAGE (FIG. 11C). > 95% processed BoNT / A (SEQ ID NO: 3) is obtained. When the second polypeptide was processed in one step for 50 minutes at 37 ° C. (200 μg scBoNT / A incubated with 350 ng nBH), the same fully processed second polypeptide (> 95 % Processed BoNT / A). After an incubation time of 1 hour at 37 ° C, more than 97% of BoNT / A is processed.

item
[Item 1]
A proteolytically active polypeptide comprising a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 1.
[Item 2]
A nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide according to item 1 and optionally regulatory elements.
[Item 3]
A vector comprising the nucleic acid molecule according to item 2.
[Item 4]
A cell comprising the nucleic acid molecule according to item 2 or the vector according to item 3.
[Item 5]
A method for producing a proteolytically active polypeptide comprising the steps of:
(a.) chemically synthesizing or translating from a nucleotide sequence a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1;
(b.) purifying the polypeptide of step (a.).
[Item 6]
6. A polypeptide obtainable from the method according to item 5.
[Item 7]
An antibody that specifically binds to the polypeptide according to item 1 or 6.
[Item 8]
Use of the antibody according to item 7 in a method for purifying the polypeptide according to item 1 or 6.
[Item 9]
A method for producing a proteolytically processed polypeptide comprising:
(a) a first polypeptide selected from the polypeptide of item 1 or item 6, Lys-N, Lys-C, arginyl endopeptidase, plasmin, or omptin,
(b) contacting a second polypeptide that is susceptible to proteolysis by the first polypeptide, wherein the contacting results in protein processing of the second polypeptide to at least two cleavage products. How to bring.
[Item 10]
The second polypeptide comprises an amino acid sequence having at least 50% sequence identity with a polypeptide sequence selected from any one of SEQ ID NOs: 3-25;
Preferably, the first polypeptide is a C-terminus of a basic amino acid residue (e.g., His, Lys, Arg) within the sequence of any one of SEQ ID NOs: 3-25 as the second polypeptide. Item 10. The method according to Item 9, wherein proteolytic cleavage is performed at a side position.
[Item 11]
11. The method of item 9 or item 10, wherein the second polypeptide is a clostridial neurotoxin (eg, BoNT / A).
[Item 12]
The clostridial neurotoxin lacks a functional binding domain (H _CC ) and is therefore unable to bind to the native clostridial neurotoxin receptor (e.g., the LH _{N of} clostridial neurotoxin) A polypeptide comprising or consisting of a fragment), a clostridial neurotoxin polypeptide having a modified clostridial neurotoxin binding domain (H _CC ) that binds to a native clostridial neurotoxin receptor, or a clostridial neurotoxin non-natural clostridial A clostridial neurotoxin polypeptide having a non-clostridial binding domain that binds to a neurotoxin receptor (the polypeptide is optionally functionally linked to minimize the binding of the clostridial neurotoxin to the native clostridial neurotoxin receptor. Is selected from the main lacking (H _CC)), The method of claim 11.
[Item 13]
13. The method of item 12, wherein the light and heavy chain components of the clostridial neurotoxin are derived from the same or different clostridial neurotoxin serotypes and / or subtypes.
[Item 14]
14. The method according to any one of items 9 to 13, wherein the second polypeptide is a single-chain clostridial neurotoxin prepared by recombinant expression in E. coli.
[Item 15]
The clostridial neurotoxin wherein the second polypeptide is a single-chain clostridial neurotoxin, and the contact between the first polypeptide and the second polypeptide is covalently bound to the clostridial neurotoxin H chain component by a disulfide bond 15. A method according to any one of items 9 to 14, resulting in a clostridial neurotoxin double-chain polypeptide comprising a light chain component.
[Item 16]
The proteolytically processed second polypeptide is a clostridial neurotoxin double-stranded polypeptide, and the C-terminus of the L chain and the N-terminus of the H chain are the same single-chain Clostridial in the wild type Clostridium genus 16. The method according to any one of items 9 to 15, wherein the method is identical to the corresponding end of the corresponding double-stranded Clostridial neurotoxin resulting from the neurotoxin polypeptide.
[Item 17]
The proteolytically processed second polypeptide has the same amino acid sequence when compared to the corresponding clostridial neurotoxin double-chain polypeptide derived from the same single-chain clostridial neurotoxin polypeptide in the wild-type Clostridium genus Item 17. The method according to any one of Items 9 to 16, which is a clostridial neurotoxin double-chain polypeptide having:
[Item 18]
Use of the method according to any one of items 9 to 17 for assessing product quality or in producing a medicinal product.
[Item 19]
Any of items 9-18, comprising a mixture of a processed second polypeptide and an unprocessed second polypeptide, wherein said mixture contains less than 5% unprocessed second polypeptide A composition obtainable by the method according to item 1.
[Item 20]
A method of screening for inhibitors,
(a) contacting the polypeptide of item 1 or 6 with a known substrate, and optionally with a putative inhibitor;
(b) determining the effect of the putative inhibitor on converting the substrate into a cleavage product;
In this case, a method in which the amount of the cleavage product is reduced indicates the inhibitory effect of the putative inhibitor.
[Item 21]
(a.) an inhibitor comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4-25, wherein a basic amino acid contained therein is replaced with a non-basic amino acid; or
(b.) The inhibitor of the proteolytically active polypeptide according to item 1 or 6, which is the antibody according to item 7.
[Item 22]
A pharmaceutical composition comprising the polypeptide according to item 1 or 6, the antibody according to item 7, the composition according to item 19, or the inhibitor according to item 21.

Figure 1
C.bot.SN (precipitated + dialysed): Clostridium botulinum supernatant (precipitation + dialysis)
Figure 2
pooled + conc. HIC Fractions: Pool + concentrated HIC fraction Figure 6
Activation: Activation
Dilutions: Dilution
Linear: Fig. 7
% Abbau scCNT:% decomposition scCNT
t [min]: hour [minute]
FIG.
anti-nBH-IgY H110 Pool 1: Anti-nBH-IgY H110 Pool 1
anti-nBH-IgY H110 Pool 2: anti-nBH-IgY H110 Pool 2
anti-nBH-IgY H110 Pool 3: anti-nBH-IgY H110 Pool 3
preimmun .: before immunization
H110 pool 1: H110 pool 1
H110 pool 2: H110 pool 2
H110 pool 3: H110 pool 3

Claims (22)

  A proteolytically active polypeptide comprising a polypeptide sequence having at least 50% sequence identity to the sequence of SEQ ID NO: 1.   A nucleic acid molecule comprising a nucleic acid sequence encoding the polypeptide of claim 1 and optionally regulatory elements.   A vector comprising the nucleic acid molecule according to claim 2.   A cell comprising the nucleic acid molecule according to claim 2 or the vector according to claim 3. A method for producing a proteolytically active polypeptide comprising the steps of:
(a.) chemically synthesizing or translating from a nucleotide sequence a polypeptide comprising a polypeptide sequence having at least 50% sequence identity with the sequence of SEQ ID NO: 1;
(b.) purifying the polypeptide of step (a.).   A polypeptide obtainable from the method of claim 5.   An antibody that specifically binds to the polypeptide of claim 1 or 6.   Use of the antibody according to claim 7 in a method for purifying the polypeptide according to claim 1 or 6. A method for producing a proteolytically processed polypeptide comprising:
(a) a first polypeptide selected from the polypeptide of claim 1 or claim 6, Lys-N, Lys-C, arginyl endopeptidase, plasmin, or omptin,
(b) contacting a second polypeptide that is susceptible to proteolysis by the first polypeptide, wherein the contacting results in protein processing of the second polypeptide to at least two cleavage products. How to bring. The second polypeptide comprises an amino acid sequence having at least 50% sequence identity with a polypeptide sequence selected from any one of SEQ ID NOs: 3-25;
Preferably, the first polypeptide is a C-terminus of a basic amino acid residue (e.g., His, Lys, Arg) within the sequence of any one of SEQ ID NOs: 3-25 as the second polypeptide. 10. The method according to claim 9, wherein proteolytic cleavage is performed at the side position.   11. The method of claim 9 or claim 10, wherein the second polypeptide is a clostridial neurotoxin (eg, BoNT / A). The clostridial neurotoxin lacks a functional binding domain (H _CC ) and is therefore unable to bind to the native clostridial neurotoxin receptor (e.g., the LH _{N of} clostridial neurotoxin) A polypeptide comprising or consisting of a fragment), a clostridial neurotoxin polypeptide having a modified clostridial neurotoxin binding domain (H _CC ) that binds to a native clostridial neurotoxin receptor, or a clostridial neurotoxin non-natural clostridial A clostridial neurotoxin polypeptide having a non-clostridial binding domain that binds to a neurotoxin receptor (the polypeptide is optionally functionally linked to minimize the binding of the clostridial neurotoxin to the native clostridial neurotoxin receptor. Is selected from the main lacking (H _CC)), The method of claim 11.   13. The method of claim 12, wherein the light and heavy chain components of the clostridial neurotoxin are derived from the same or different clostridial neurotoxin serotypes and / or subtypes.   14. The method according to any one of claims 9 to 13, wherein the second polypeptide is a single chain clostridial neurotoxin prepared by recombinant expression in E. coli.   The clostridial neurotoxin wherein the second polypeptide is a single-chain clostridial neurotoxin, and the contact between the first polypeptide and the second polypeptide is covalently bound to the clostridial neurotoxin H chain component by a disulfide bond 15. A method according to any one of claims 9 to 14 resulting in a clostridial neurotoxin double-chain polypeptide comprising a light chain component.   The proteolytically processed second polypeptide is a clostridial neurotoxin double-stranded polypeptide, and the C-terminus of the L chain and the N-terminus of the H chain are the same single-chain Clostridial in the wild type Clostridium genus 16. The method according to any one of claims 9 to 15, wherein the method is identical to the corresponding end of the corresponding double-stranded Clostridial neurotoxin resulting from the neurotoxin polypeptide.   The proteolytically processed second polypeptide has the same amino acid sequence when compared to the corresponding clostridial neurotoxin double-chain polypeptide derived from the same single-chain clostridial neurotoxin polypeptide in the wild-type Clostridium genus The method according to any one of claims 9 to 16, wherein the polypeptide is a clostridial neurotoxin double-chain polypeptide.   Use of a method according to any one of claims 9 to 17 for assessing product quality or in producing a medicinal product.   21. Any of claims 9 to 18, comprising a mixture of a processed second polypeptide and an unprocessed second polypeptide, said mixture containing less than 5% unprocessed second polypeptide. A composition obtainable by the method according to claim 1. A method of screening for inhibitors,
(a) contacting the polypeptide of claim 1 or 6 with a known substrate, and optionally with a putative inhibitor;
(b) determining the effect of the putative inhibitor on converting the substrate into a cleavage product;
In this case, a method in which the amount of the cleavage product is reduced indicates the inhibitory effect of the putative inhibitor. (a.) an inhibitor comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 4-25, wherein a basic amino acid contained therein is replaced with a non-basic amino acid; or
(b.) The proteolytically active polypeptide inhibitor according to claim 1 or 6, which is the antibody according to claim 7.   A pharmaceutical composition comprising the polypeptide according to claim 1 or 6, the antibody according to claim 7, the composition according to claim 19, or the inhibitor according to claim 21.

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