METHOD FOR THE MANUFACTURING OF DI-CHAIN PROTEINS FOR USE IN HUMANS

Abstract

This invention relates to a novel method for producing di-chain proteins for use in humans from single-chain precursors, including di-chain clostridial neurotoxins. The method comprises the step of expressing a nucleic acid sequence encoding a single-chain precursor comprising a thrombin-cleavage site and the step of cleaving the single-chain precursor with a human factor Xa or a human thrombin, particularly a human thrombin drug product authorized for human therapeutic use. The invention further relates to novel di-chain clostridial neurotoxins and nucleic acid sequences encoding such novel di-chain clostridial neurotoxins.

Claims

1. A method for the generation of a disulfide-linked di-chain clostridial neurotoxin, comprising the step of: (i) treating a disulfide-linked single-chain precursor clostridial neurotoxin molecule, which comprises a cleavage signal for human thrombin in the loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain, with human factor Xa or human thrombin.

2. The method of claim 1, wherein the human thrombin is a recombinant human thrombin.

3. The method of claim 1, wherein the human thrombin is comprised in a human thrombin-containing drug product authorized for human therapeutic use.

4. The method of claim 3, wherein the human thrombin-containing drug product is selected from RECOTHROM and EVICEL.

5. The method of claim 1, wherein the cleavage signal for human thrombin comprises a Xxx-Arg-Yyy or Xxx-Lys-Yyy tripeptide motif, wherein Xxx is selected from Pro, Gly, Ala, Leu and Val, and wherein Yyy is independently selected from Ser, Ala, Gly, Thr, Arg and Leu.

6. The method of claim 5, wherein the tripeptide motif is Pro-Arg-Yyy or Pro-Lys-Yyy.

7. The method of claim 1, wherein the cleavage signal for human thrombin comprises a Pro-Arg-Gly-Ser (SEQ ID NO: 7) tetrapeptide motif.

8. The method of claim 5, wherein the loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain of the clostridial neurotoxin comprises one of a KSLVPRGS (SEQ ID NO: 9), a NKSLVPRGS (SEQ ID NO: 10), or a ENKSLVPRGS (SEQ ID NO: 11) polypeptide motif.

9. The method of claim 5, wherein a loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain of a clostridial neurotoxin does not comprise the dipeptide motif Lys-Ser at the N-terminus of a thrombin cleavage signal sequence LVPRGS (SEQ ID NO: 8).

10. The method of claim 9, wherein the loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain of the clostridial neurotoxin comprises one of a TSLVPRGS (SEQ ID NO: 12) or a GGLVPRGS (SEQ ID NO: 13) polypeptide motif.

11. The method of claim 1, wherein the step (i) is performed in a buffer solution selected from the group consisting of: (i) Tris pH 7.6-8.0 and 70-150 mM NaCl; (ii) Tris pH 7.6-8.0 and 300-500 mM NaCl; (iii) Tris pH 7.8-8.2 and 20 mM NaCl; (iv) Phosphate buffer pH 7.6-8.0 and 70-150 mM NaCl; (v) Phosphate buffer pH 7.6-8.0 and 300-500 mM NaCl; (vi) Phosphate buffer pH 7.6-8.0 and 20 mM NaCl; (vii) HEPES pH 7.6-8.0 and 70-150 mM NaCl; (viii) HEPES pH 7.6-8.0 and 300-500 mM NaCl; and (ix) HEPES pH 7.6-8.2 and 20 mM NaCl.

12. The method of claim 1, wherein the clostridial neurotoxin is selected from Clostridium botulinum neurotoxin serotype A, B, C, D, E, F, and G.

13. The method of claim 1, wherein the clostridial neurotoxin is Clostridium botulinum neurotoxin serotype A or E.

14. The method of claim 1, wherein the clostridial neurotoxin is Clostridium botulinum neurotoxin serotype E or a modified Clostridium botulinum neurotoxin serotype E.

15. The method of claim 1, further comprising a step of: (ii) isolating the disulfide-linked di-chain clostridial neurotoxin by chromatography on an ion exchange matrix, a hydrophobic interaction matrix or a multimodal chromatography matrix.

16. The method of claim 15, wherein the ion exchange matrix is a strong ion exchange matrix.

17. The method of claim 16, wherein the strong ion exchange matrix is a strong cation exchange matrix.

18. The method of claim 15, wherein step (ii) further comprises the step(s) of: (iia) conditioning to obtain a low-salt solution at a pH of between about 7.9 and about 8.1; (iib) applying a low-salt solution containing the disulfide-linked di-chain clostridial neurotoxin on a sulfopropyl-substituted chromatography matrix; (iic) washing the sulfopropyl-substituted chromatography matrix with a buffer solution at a pH of between about 7.9 and about 8.1 and containing about 20 mM salt; and (iid) eluting the disulfide-linked di-chain clostridial neurotoxin from the sulfopropyl-substituted chromatography matrix by applying to the sulfopropyl-substituted chromatography matrix the buffer solution of step (iic) which has been modified to exhibit a salt concentration between about 50 and about 500 mM salt.

19. A single-chain precursor clostridial neurotoxin molecule comprising (i) a functionally active clostridial neurotoxin light chain, (ii) a functionally active clostridial neurotoxin heavy chain, and (iii) a loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain, wherein the loop region comprises a cleavage signal for human thrombin.

20. The single-chain precursor clostridial neurotoxin molecule of claim 19 comprising the sequence of SEQ ID NO: 6.

21. A nucleic acid encoding the single-chain precursor clostridial neurotoxin molecule of claim 19, wherein the nucleic acid exhibits the sequence of SEQ ID NO: 5.

22. A method for obtaining the nucleic acid of claim 21, comprising a step of inserting a cleavage signal for human thrombin into a nucleic acid sequence encoding a single-chain precursor clostridial neurotoxin molecule.

23. A vector comprising the nucleic acid of claim 21.

24. A recombinant host cell comprising the nucleic acid of claim 21.

25. A recombinant host cell comprising the vector of claim 23.

26. A method for producing a single-chain precursor clostridial neurotoxin molecule, comprising the step of expressing the nucleic acid of claim 21 under conditions that result in the expression of the nucleic acid.

27. A disulfide-linked di-chain clostridial neurotoxin, wherein the C-terminus of the light chain ends with the amino acid sequence VPR.

28. The disulfide-linked di-chain clostridial neurotoxin of claim 27, wherein the C-terminus of the light chain ends with an amino acid sequence selected from KSLVPR (SEQ ID NO: 14) and Xaa-Xaa-LVPR (SEQ ID NO: 17) provided that the dipeptide sequence Xaa-Xaa is not K-S, TSLVP (SEQ ID NO: 15) or GGLVP (SEQ ID NO: 16), and the N-terminus of the heavy chain begins with the amino acid sequence GSK.

29. A pharmaceutical composition comprising the disulfide-linked di-chain clostridial neurotoxin of claim 28.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0075] The present invention may be understood more readily by reference to the following detailed description of the invention and the examples included therein.

[0076] Thus, in one aspect, the present invention relates to a method for the generation of a disulfide-linked di-chain protein, comprising the step of (i) treating a disulfide-linked single-chain precursor protein, which comprises a cleavage signal for human thrombin in a linking peptide linking the C-terminus of a first chain with the N-terminus of a second protein chain, with a human factor Xa or a human thrombin.

[0077] In the context of the present invention, the term comprises or comprising means including, but not limited to. The term is intended to be open-ended, that specify the presence of any stated features, elements, integers, steps, or components, but do not preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. The term comprising thus includes the more restrictive terms consisting of and consisting essentially of.

[0078] In the context of the present invention, the term disulfide-linked di-chain protein refers to a protein comprising at least two chains elements, where one end of the first chain element is linked to one end of the second chain element not via a peptide bond, but via a disulfide bond.

[0079] In the context of the present invention, the term disulfide-linked single-chain precursor protein refers a precursor for a disulfide-linked di-chain protein, where the ends of at least two chain elements are linked by a polypeptide linker.

[0080] In the context of the present invention, the term cleavage signal for human thrombin relates to a polypeptide sequences that is recognized and cleaved by human thrombin. Cleavage signals for human thrombin are well known to anyone of ordinary skill in the art (see, for example, Schilling & Overall, Nature Biotechnology 26 (2008) 685-694).

[0081] Human factor Xa is known to cleave proteins C-terminally of the arginine residue in the factor Xa recognition sequence IDGR or IEGR. However, in certain instances factor Xa may also cleave proteins having a thrombin recognition sequence. Surprisingly, it was found that factor Xa very effectively cleaves the disulfide-linked single-chain precursor proteins in the methods of the present invention.

[0082] In the context of the present invention, the term human thrombin-containing drug product authorized for human therapeutic use refers to a drug product that contains a human thrombin, which product has been approved by a regulatory authority for use as a therapeutic in humans. In particular embodiments, the regulatory authority is selected from the European Medicines Agency (EMA), the U.S. Food and Drug Administration (FDA), and the Japanese Pharmaceuticals and Medical Devices Agency (PMDA).

[0083] In particular embodiments, cleavage of the precursor single-chain protein at a defined cleavage site is near-complete.

[0084] In the context of the present invention the term near-complete is defined as more than about 95% cleavage, particularly more than about 97.5%, more particularly more than about 99% as determined by SDS-PAGE or reversed phase chromatography.

[0085] Thus, in particular embodiments of the method of the present invention, the precursor single-chain protein is cleaved at the defined cleavage site to more than about 97.5%, more particularly more than about 99% as determined by SDS-PAGE or reversed phase chromatography.

[0086] In particular embodiments, cleavage of the precursor single-chain protein at a defined cleavage site is without accidental cleavage at other sites.

[0087] In the context of the present invention the term without accidental cleavage is defined as less than about 1%, particularly less than about 0.1%, more particularly less than about 0.01% of cleavage products other than the desired di-chain protein resulting from cleavage of the precursor single-chain protein, as determined by liquid chromatographymass spectrometry (LC-MS) or mass spectrometry, than those resulting from the defined and intended cleavage site.

[0088] Thus, in particular embodiments of the method of the present invention, less than about 1%, particularly less than about 0.1%, more particularly less than about 0.01% of cleavage products other than the desired di-chain protein are resulting from cleavage of the precursor single-chain protein, as determined by LC-MS or mass spectrometry, than those resulting from the defined and intended cleavage site.

[0089] In certain embodiments, the disulfide-linked di-chain protein is a disulfide-linked di-chain clostridial neurotoxin, the single-chain precursor protein is a disulfide-linked single-chain precursor clostridial neurotoxin, and the linking peptide linking the C-terminus of a first chain with the N-terminus of a second protein chain is the loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain of a clostridial neurotoxin.

[0090] In the context of the present invention, the term clostridial neurotoxin refers to a natural neurotoxin obtainable from bacteria of the class Clostridia, including Clostridium tetani and Clostridium botulinum, or to a neurotoxin obtainable from alternative sources, including from recombinant technologies or from genetic or chemical modification. Particularly, the clostridial neurotoxins have endopeptidase activity.

[0091] In the context of the present invention, the term recombinant clostridial neurotoxin refers to a composition comprising a clostridial neurotoxin, that is obtained by expression of the neurotoxin in a heterologous cell such as E. coli, and including, but not limited to, the raw material obtained from a fermentation process (supernatant, composition after cell lysis), a fraction comprising a clostridial neurotoxin obtained from separating the ingredients of such a raw material in a purification process, an isolated and essentially pure clostridial neurotoxin, and a formulation for pharmaceutical and/or aesthetic use comprising a clostridial neurotoxin and additionally pharmaceutically acceptable solvents and/or excipients.

[0092] In the context of the present invention, the term functionally active refers to the property of a recombinant clostridial neurotoxin to perform the biological functions of a naturally occurring Clostridium botulinum neurotoxin to at least about 50%, particularly to at least about 60%, to at least about 70%, to at least about 80%, and most particularly to at least about 90%, where the biological functions include, but are not limited to, association of light and heavy chain to form the two-chain neurotoxin protein, entry of the neurotoxin into a neuronal cell, release of the light chain from the two-chain neurotoxin, and endopeptidase activity of the light chain. Methods for determining a neurotoxic activity can be found, for example, in WO 95/32738, which describes the reconstitution of separately obtained light and heavy chains of tetanus toxin and botulinum toxin.

[0093] In the context of the present invention, the term about or approximately means within 20%, alternatively within 10%, including within 5% of a given value or range. Alternatively, especially in biological systems, the term about means within about a log (i.e., an order of magnitude), including within a factor of two of a given value.

[0094] In the context of the present invention, the term clostridial neurotoxin light chain refers to that part of a clostridial neurotoxin that comprises an endopeptidase activity responsible for cleaving one or more proteins that is/are part of the so-called SNARE-complex involved in the process resulting in the release of neurotransmitter into the synaptic cleft: In naturally occurring clostridial neurotoxins, the light chain has a molecular weight of approx. 50 kDa.

[0095] In the context of the present invention, the term clostridial neurotoxin heavy chain refers to refers to that part of a clostridial neurotoxin that is responsible for entry of the neurotoxin into the neuronal cell: In naturally occurring clostridial neurotoxins, the heavy chain has a molecular weight of approx. 100 kDa.

[0096] In certain embodiments, the human factor Xa is recombinant human factor Xa.

[0097] In certain embodiments, the human thrombin is recombinant human thrombin.

[0098] The use of recombinant human factor Xa and/or human thrombin provides the advantage that animal products, such as bovine serum albumin or the like can be avoided during production of the protease, which may simplify the regulatory path for producing pharmaceutical products using such proteases.

[0099] In certain embodiments, the human thrombin is a human thrombin-containing drug product authorized for human therapeutic use, or is a human thrombin-containing drug substance which is also contained in a drug product authorized for human therapeutic use.

[0100] In the context of the present invention, a human thrombin-containing drug product authorized for human therapeutic use is a human thrombin that is regarded as safe by regulatory authorities for use in the production of drug product authorized for human therapeutic use.

[0101] In certain embodiments, the human thrombin for a thrombin-containing drug product authorized for human therapeutic use is isolated from human plasma cells, produced according to GMP regulations, and/or is used as pharmaceutical agent in a human drug product used as a Fibrin sealant.

[0102] In certain embodiments, the human thrombin is manufactured according to the Good Manufacturing Practice (GMP) for human use.

[0103] In certain embodiments, the human thrombin-containing drug product is selected from RECOTROM and EVICEL. In other embodiments, the human thrombin-containing drug product is BERIPLAST P.

[0104] In certain embodiments, the thrombin cleavage signal comprises a tripeptide motif Xxx-Arg-Yyy or Xxx-Lys-Yyy, preferably Pro-Arg-Yyy or Pro-Lys-Yyy, wherein Xxx is selected from Pro, Gly, Ala, Leu and Val, and Yyy is independently selected from Ser, Ala, Gly, Thr, Arg and Leu.

[0105] In particular embodiments, the thrombin cleavage signal comprises the tetrapeptide motif Pro-Arg-Gly-Ser (SEQ ID NO: 7), preferably in the form of Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 8).

[0106] In a particular embodiment, the loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain of a clostridial neurotoxin comprises one of the polypeptide motifs

TABLE-US-00001 (SEQIDNO:9) KSLVPRGS; (SEQIDNO:10) NKSLVPRGS; or (SEQIDNO:11) ENKSLVPRGS.

[0107] Surprisingly, it has been found that removal of the dipeptide motif Lys-Ser from the N-terminus of the thrombin cleavage signal sequence LVPRGS (SEQ ID NO: 8) in the loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain of a clostridial neurotoxin results in a highly effective, i. e. near-complete cleavage of the precursor single-chain protein at the defined cleavage site, i. e. without accidental cleavage at other sites. Accidental cleavage was particularly observed during expression of the precursor single-chain protein in E. coli host cells (see WO 2006/076902 as discussed in Section [0016] above).

[0108] Thus, in a particular embodiment, the loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain of a clostridial neurotoxin does not comprise the dipeptide motif Lys-Ser at the N-terminus of the thrombin cleavage signal sequence LVPRGS (SEQ ID NO: 8). Thus, in a particular embodiment, the loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain of a clostridial neurotoxin comprise a dipeptide motif Xaa-Yaa N-terminally of the thrombin cleavage signal sequence LVPRGS (SEQ ID NO: 8), wherein Xaa and Yaa are independently selected from the 20 naturally occurring amino acid residues, provided that the dipeptide sequence Xaa-Yaa is not K-S.

[0109] In particular such embodiments, the loop region comprises one of the polypeptide motifs

TABLE-US-00002 (SEQIDNO:12) TSLVPRGS or (SEQIDNO:13) GGLVPRGS.

[0110] In particular embodiments, between 1 and 1000 international units (IU) of said human thrombin-containing drug product, particularly between 5 and 500 IU, more particularly between 10 and 200 IU, are used per mg of said disulfide-linked single-chain precursor protein (as determined with the Bradford reagent, Sigma-Aldrich article no. B6916, according to the colorimetric method of Bradford (Bradford, M. M. (1976): A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. Vol. 72, 248-254)).

[0111] In particular embodiments, step (i) is performed with crude host cell lysates containing said single-chain precursor protein or with partially purified single-chain precursor protein, particularly after a first chromatographic enrichment step.

[0112] In particular embodiments, where step (i) is performed with crude host cell lysates containing said single-chain precursor protein, between 2.5 and 900 international units (IU) of said human thrombin, particularly between 10 and 500 IU, more particularly between 50 and 300 IU, are used per mg of soluble total protein in the crude host cell lysate.

[0113] In a particular embodiment, step (i) is performed after a first chromatographic enrichment step, particularly using a multimodal chromatography matrix, particularly Capto MMC (GE Healthcare).

[0114] In particular embodiments, where step (i) is performed with partially purified single-chain precursor protein, particularly after a first chromatographic enrichment step, between 1 and 500 international units (IU) of said human thrombin, particularly between 5 and 200 IU, more particularly between 10 and 40 IU, are used per mg of single-chain precursor protein in the partially purified fraction.

[0115] In particular embodiment, step (i) is performed at a temperature between about 0 C. and about 15 C., particularly between 0 C. and about 12 C.

[0116] In particular such embodiments, step (i) is performed for between about 1 hour and 24 hours, particularly for between about 2 hours and about 18 hours, more particularly for 142 hours.

[0117] In a particular embodiment step (i) is performed at a temperature between about 0 C. and about 12 C. for between about 20 min and about 5 h.

[0118] In another embodiment, step (i) is performed at a temperature between about 15 C. and about 40 C., particularly between about 15 C. and about 30 C., more particularly at 20 C.2 C.

[0119] In particular such embodiments, step (i) is performed for between about 1 minute and about 2 hours, particularly for between about 5 minutes and about 1 hour, more particularly for between about 5 and about 20 minutes.

[0120] In particular embodiments, step (i) is performed in a buffer solution selected from the list of:

[0121] (i) Tris pH 7.6-8.0 and 70-150 mM NaCl;

[0122] (ii) Tris pH 7.6-8.0 and 300-500 mM NaCl;

[0123] (iii) Tris pH 7.8-8.2 and 20 mM NaCl;

[0124] (iv) Phosphate buffer pH 7.6-8.0 and 70-150 mM NaCl;

[0125] (v) Phosphate buffer pH 7.6-8.0 and 300-500 mM NaCl;

[0126] (vi) Phosphate buffer pH 7.6-8.0 and 20 mM NaCl;

[0127] (vii) HEPES pH 7.6-8.0 and 70-150 mM NaCl;

[0128] (viii) HEPES pH 7.6-8.0 and 300-500 mM NaCl; and

[0129] (ix) HEPES pH 7.6-8.2 and 20 mM NaCl.

[0130] In particular embodiments, the buffer substance is present at a concentration of between about 10 mmol/l and about 100 mmol/l, particularly of between about 30 mol/l and about 70 mmol/l, more particularly of about 50 mmol/l.

[0131] In particular embodiments, step (i) is performed at a pH value of between about 6.8 and about 8.8, particularly between about 7.6 and about 8.0, more particularly at about 7.8.

[0132] In particular embodiments, where the di-chain protein is a clostridial neurotoxin, the clostridial neurotoxin is selected from Clostridium botulinum neurotoxin serotype A, B, C, D, E, F, and G, particularly Clostridium botulinum neurotoxin serotype A and E, particularly Clostridium botulinum neurotoxin serotype E, more particularly a modified Clostridium botulinum neurotoxin serotype E.

[0133] In one embodiment of the present invention, the recombinant clostridial neurotoxin is a functional homologue of a Clostridium botulinum neurotoxin taken from the list of: Clostridium botulinum neurotoxin serotype A, B, C, D, E, F, and G.

[0134] In the context of the present invention, the term Clostridium botulinum neurotoxin serotype A, B, C, D, E, F, and G refers to neurotoxins obtainable from Clostridium botulinum. Currently, seven serologically distinct types, designated serotypes A, B, C, D, E, F and G, are known, including certain subtypes (e.g. A1, A2, and A3).

[0135] In a particular embodiment, the recombinant clostridial neurotoxin is a functional homologue of a Clostridium botulinum neurotoxin serotype E.

[0136] In the context of the present invention, the term functional homologue of a Clostridium botulinum neurotoxin refers to a neurotoxin that differs in the amino acid sequence, or the nucleic acid sequence encoding the amino acid sequence, from a Clostridium botulinum neurotoxin but is still functionally active. On the protein level, a functional homologue will maintain key features of the corresponding Clostridium botulinum neurotoxin, such as key residues for the endopeptidase activity in the light chain, or for the attachment to the neurotoxin receptors in the heavy chain, but may contain one or more mutations comprising a deletion of one or more amino acids of the parental Clostridium botulinum neurotoxin, an addition of one or more amino acids to the parental Clostridium botulinum neurotoxin and/or a substitution of one or more amino acids of the parental Clostridium botulinum neurotoxin. Preferably, said deleted or added amino acids are consecutive amino acids. According to the teaching of the present invention, any number of amino acids may be added or deleted, as long as the neurotoxin is biologically active. For example, 1, 2, 3, 4, 5, up to 10, up to 15, up to 25, up to 50, up to 100, up to 200, up to 400, up to 500 amino acids or even more amino acids may be added or deleted. In certain aspects, the present invention refers to neurotoxins, with an addition of more than 500 amino acids, such as for example up to 600 or up to 800 additional amino acids, or even more additional amino acids. Accordingly, a derivative of the neurotoxin may be a biologically active fragment of a naturally occurring neurotoxin. This neurotoxin fragment may contain an N-terminal, C-terminal and/or one or more internal deletion(s). Biologically active, as used in this context, means, said derivative is taken up into the nerve cell and is capable of denervating said nerve from the muscle or gland, to which it is connected. Methods for designing and constructing homologues of a Clostridium botulinum neurotoxin and for testing of such homologues for functionality are well known to anyone of ordinary skill in the art.

[0137] In particular embodiments, the functional homologue has a sequence identity of at least about 40%, at least about 50%, at least about 60%, at least about 70% or most particularly at least about 80%, and a sequence homology of at least about 60%, at least about 70%, at least about 80%, at least about 90%, or most particularly at least about 95%. Methods and algorithms for determining sequence identity and/or homology, including the comparison of variants having additions and/or insertions relative to a parental sequence, are well known to the practitioner of ordinary skill in the art. On the DNA level, the nucleic acid sequences encoding the functional homologue and the parental Clostridium neurotoxin may differ to a larger extent due to the degeneracy of the genetic code. It is known that the usage of codons is different between prokaryotic and eukaryotic organisms (see, for example, Table 1 in WO 2006/017749). Thus, when expressing a prokaryotic protein such as a Clostridium neurotoxin, in a eukaryotic expression system, it may be necessary, or at least helpful, to adapt the nucleic acid sequence to the codon usage of the expression host cell, meaning that sequence identity or homology may be rather low on the nucleic acid level.

[0138] In particular embodiments, the functional homologue of the Clostridium neurotoxin is a derivative of the Clostridium neurotoxin.

[0139] In the context of the present invention, the term derivative and the term variant or synthetic analogue each individually refer to a neurotoxin that is a chemically, enzymatically or genetically modified derivative of a parental Clostridium neurotoxin, including chemically or genetically modified neurotoxin from C. botulinum, particularly of C. botulinum neurotoxin serotype A. A chemically modified derivative may be one that is modified by pyruvation, phosphorylation, sulfatation, lipidation, pegylation, glycosylation and/or the chemical addition of an amino acid or a polypeptide comprising between 2 and about 100 amino acids, including modification occurring in the eukaryotic host cell used for expressing the derivative. An enzymatically modified derivative is one that is modified by the activity of enzymes, such as endoor exoproteolytic enzymes, including by modification by enzymes of the eukaryotic host cell used for expressing the derivative. As pointed out above, a genetically modified derivative is one that has been modified by deletion or substitution of one or more amino acids contained in, or by addition of one or more amino acids (including polypeptides comprising between 2 and about 100 amino acids) to, the amino acid sequence of said Clostridium neurotoxin. Methods for making such chemically or genetically modified derivatives, and methods for identifying whether such derivatives maintain neurotoxic activity, are well known to anyone of ordinary skill in the art.

[0140] In particular embodiments, the method further comprises the step of [0141] (ii) isolating the disulfide-linked di-chain clostridial neurotoxin from the human thrombin-containing drug product by chromatography on an ion exchange matrix or a hydrophobic interaction matrix or a multimodal chromatography matrix, particularly a strong ion exchange matrix, more particularly a strong cation exchange matrix.

[0142] In particular such embodiments, step (ii) comprises the step(s) of: [0143] (iia) a conditioning step to obtain a low-salt about 20 mM salt) solution at a pH of between about 7.9 and about 8.1, e.g. by appropriate dilution, diafiltration or dialysis; [0144] (iib) application of said low-salt solution containing the disulfide-linked di-chain clostridial neurotoxin on a sulfopropyl-substituted chromatography matrix; [0145] (iic) washing the sulfopropyl-substituted chromatography matrix with a buffer solution at a pH of between about 7.9 and about 8.1 and containing about 20 mM salt (e. g. NaCl) [0146] (iid) elution of the disulfide-linked di-chain clostridial neurotoxin from the sulfopropyl-substituted chromatography matrix by increasing the salt concentration/ionic strength of the buffer solution of step (iic) to between about 50 and about 500 mM salt.

[0147] In another aspect, the present invention relates to a single-chain precursor molecule of a disulfide-linked di-chain protein comprising (i) a functionally active first chain, (ii) a functionally active second chain, and (iii) a loop region linking the first and the second chain, wherein the loop region comprises a cleavage signal for human thrombin.

[0148] In certain embodiments, the single-chain precursor molecule is a single-chain precursor clostridial neurotoxin molecule comprising (i) a functionally active clostridial neurotoxin light chain, (ii) a functionally active clostridial neurotoxin heavy chain, and (iii) a loop region linking the C-terminus of the light chain with the N-terminus of the heavy chain, wherein the loop region comprises a cleavage signal for human thrombin.

[0149] In a particular embodiment of the present invention, the light chain has the amino acid sequence as found in SEQ ID NO: 1.

[0150] In a particular embodiment of the present invention, the heavy chain has the amino acid sequence as found in SEQ ID NO: 2.

[0151] In a further particular embodiment of the present invention, the light chain has the amino acid sequence as found in SEQ ID NO: 1, and the heavy chain has the amino acid sequence as found in SEQ ID NO: 2.

[0152] In another embodiment of the present invention, the recombinant clostridial neurotoxin is a functional homologue of a Clostridium tetani neurotoxin.

[0153] In the context of the present invention, the term functional homologue of a Clostridium tetani neurotoxin refers to a neurotoxin that differs in the amino acid sequence, or the nucleic acid sequence encoding the amino acid sequence, from a Clostridium tetani neurotoxin but is still functionally active. On the protein level, a functional homologue will maintain key features of the corresponding Clostridium tetani neurotoxin, such as key residues for the endopeptidase activity in the light chain, or for the attachment to the neurotoxin receptors in the heavy chain, but may contain one or more mutations comprising a deletion of one or more amino acids of the parental Clostridium tetani neurotoxin, an addition of one or more amino acids to the parental Clostridium tetani neurotoxin and/or a substitution of one or more amino acids of the parental Clostridium tetani neurotoxin. Preferably, said deleted or added amino acids are consecutive amino acids. According to the teaching of the present invention, any number of amino acids may be added or deleted, as long as the neurotoxin is biologically active. For example, 1, 2, 3, 4, 5, up to 10, up to 15, up to 25, up to 50, up to 100, up to 200, up to 400, up to 500 amino acids or even more amino acids may be added or deleted. In certain aspects, the present invention refers to neurotoxins, with an addition of more than 500 amino acids, such as for example up to 600 or up to 800 additional amino acids, or even more additional amino acids. Accordingly, a derivative of the neurotoxin may be a biologically active fragment of a naturally occurring neurotoxin. This neurotoxin fragment may contain an N-terminal, C-terminal and/or one or more internal deletion(s). Biologically active, as used in this context, means, said derivative is taken up into the nerve cell and is capable of denervating said nerve from the muscle or gland, to which it is connected. Methods for designing and constructing homologues of a Clostridium tetani neurotoxin and for testing of such homologues for functionality are well known to anyone of ordinary skill in the art.

[0154] In particular embodiments, the functional homologue has a sequence identity of at least about 40%, at least about 50%, at least about 60%, at least about 70% or most particularly at least about 80%, and a sequence homology of at least about 60%, at least about 70%, at least about 80%, at least about 90%, or most particularly at least about 95%. Methods and algorithms for determining sequence identity and/or homology, including the comparison of variants having additions and/or insertions relative to a parental sequence, are well known to the practitioner of ordinary skill in the art. On the DNA level, the nucleic acid sequences encoding the functional homologue and the parental Clostridium neurotoxin may differ to a larger extent due to the degeneracy of the genetic code. It is known that the usage of codons is different between prokaryotic and eukaryotic organisms (see, for example, Table 1 in WO 2006/017749). Thus, when expressing a prokaryotic protein such as a Clostridium neurotoxin, in a eukaryotic expression system, it may be necessary, or at least helpful, to adapt the nucleic acid sequence to the codon usage of the expression host cell, meaning that sequence identity or homology may be rather low on the nucleic acid level.

[0155] In a particular embodiment of the present invention, the light chain has the sequence as found in SEQ ID NO: 3, and the heavy chain has the sequence as found in SEQ ID NO: 4.

[0156] In another aspect, the present invention relates to a nucleic acid sequence encoding the single-chain precursor molecule of a disulfide-linked di-chain protein, particularly of a disulfide-linked di-chain clostridial neurotoxin molecule, of the present invention.

[0157] In particular embodiments, the single-chain precursor molecule of a disulfide-linked di-chain protein, particularly of a clostridial neurotoxin molecule, has the sequence as found in SEQ ID NO: 6.

[0158] In certain embodiments, the present invention relates to a nucleic acid sequence encoding the recombinant clostridial neurotoxin of the present invention, comprising (i) a nucleic acid sequence encoding a functionally active clostridial neurotoxin light chain, (ii) a nucleic acid sequence comprising a sequence encoding a human thrombin cleavage site, and (iii) a nucleic acid sequence encoding a functionally active clostridial neurotoxin heavy chain.

[0159] In particular embodiments, the nucleic acid sequences (i), (ii) and (iii) encode a single contiguous amino acid sequence.

[0160] In the context of the present invention, the term single contiguous amino acid sequence refers to an amino acid sequence that would, when expressed from the corresponding nucleic acid sequence in an appropriate host cell in the absence of protease activity, be formed as a single chain protein.

[0161] In a particular embodiment, the nucleic acid sequence has the sequence as found in SEQ ID NO: 5.

[0162] In another aspect, the present invention relates to a nucleic acid sequence encoding the recombinant clostridial neurotoxin of the present invention as described elsewhere herein in detail, wherein the coding sequence encoding said neurotoxin comprises a coding sequence for a signal peptide. Usually, said coding sequence encoding the signal peptide will be located upstream of the coding sequence encoding the neurotoxin. Many such signal sequences are known in the art and are comprised by the present invention. In particular, the signal sequence is a signal peptide resulting in transport of the neurotoxin across a biological membrane, such as the membrane of the endoplasmic reticulum, the golgi membrane or the plasma membrane of a eukaryotic or prokaryotic cell. It has been found that signal sequences, when attached to the neurotoxin, will mediate secretion of the neurotoxin into the supernatant of the cells. In certain embodiments, the signal peptide will be cleaved off in the course of, or subsequent to, such secretion, so that the secreted protein lacks the N-terminal signal peptide, is composed of separate light and a heavy chains, which are covalently linked by SS bridges and which are proteolytically active.

[0163] In another aspect, the recombinant clostridial neurotoxin according to the present invention comprises an N-terminal elongation of 5 to 50 amino acid residues, said elongation comprising a signal peptide capable of mediating transport across a biological membrane. In a more specific aspect of the present invention, this signal peptide is removable by a protease.

[0164] In another aspect, the present invention relates to a method for obtaining the nucleic acid sequence of the present invention, comprising the step of inserting a cleavage signal for human thrombin into a nucleic acid sequence encoding the single-chain precursor molecule of a disulfide-linked di-chain protein, particularly of a disulfide-linked di-chain clostridial neurotoxin molecule, of the present invention.

[0165] In a particular embodiment, the insertion is achieved by inserting the cleavage signal for human thrombin into the nucleic acid sequence encoding wild-type clostridial neurotoxin. In another particular embodiment, the insertion results in the deletion and/or modification of one or more amino acid residues of said wild-type clostridial neurotoxin.

[0166] In another aspect, the present invention relates to a vector comprising the nucleic acid sequence of the present invention, or the nucleic acid obtainable by the method of the present invention.

[0167] A further aspect of the present invention relates to a recombinant host cell comprising a nucleic acid sequence or a vector of the present invention.

[0168] In another aspect, the present invention relates to a method for producing the single-chain precursor molecule of a disulfide-linked di-chain protein, particularly of a disulfide-linked di-chain clostridial neurotoxin molecule, of the present invention, comprising the step of expressing the nucleic acid sequence of the present invention, the nucleic acid sequence obtainable by the method of the present invention, or the vector of the present invention in a recombinant host cell, or cultivating the recombinant host cell of the present invention under conditions that result in the expression of said nucleic acid sequence.

[0169] Yet another aspect of the invention relates to a method for producing the single-chain precursor molecule of a disulfide-linked di-chain protein, particularly of a disulfide-linked di-chain clostridial neurotoxin molecule, of the present invention, comprising the step of expressing a nucleic acid sequence or vector of the present invention in a recombinant host cell, or cultivating a recombinant host cell of the present invention under conditions that result in the expression of the nucleic acid sequence.

[0170] In another aspect, the present invention relates to a disulfide-linked di-chain protein, wherein the C-terminus of the first chain ends with the sequence VPR, and the N-terminus of the second chain begins with the sequence GSK.

[0171] In certain embodiments, the disulfide-linked di-chain protein is a disulfide-linked di-chain clostridial neurotoxin, wherein the C-terminus of the light chain ends with the sequence VPR, particularly a sequence selected from KSLVPR (SEQ ID NO: 14) and Xaa-Xaa-LVPR (SEQ ID NO: 17), wherein Xaa-Xaa are independently selected from the 20 naturally occurring amino acid residues, provided that the dipeptide sequence Xaa-Xaa is not K-S, particularly Xaa-Xaa-LVPR (SEQ ID NO: 17), particularly selected from TSLVP (SEQ ID NO: 15) and GGLVP (SEQ ID NO: 16), and the N-terminus of the heavy chain begins with the sequence GSK.

[0172] In a further aspect, the present invention relates to a pharmaceutical composition comprising the disulfide-linked di-chain protein, particularly the disulfide-linked di-chain clostridial neurotoxin of the present invention.

[0173] In one embodiment, the pharmaceutical composition comprises a disulfide-linked di-chain clostridial neurotoxin of the present invention, and is for use in the treatment of a disease or condition taken from the list of: cervical dystonia (spasmodic torticollis), blepharospasm, severe primary axillary hyperhidrosis, achalasia, lower back pain, benign prostate hypertrophy, chronic focal painful neuropathies, migraine and other headache disorders, and cosmetic or aesthetic applications.

[0174] Additional indications where treatment with Botulinum neurotoxins is currently under investigation and where the pharmaceutical composition of the present invention may be used, include pediatric incontinence, incontinence due to overactive bladder, and incontinence due to neurogenic bladder, anal fissure, spastic disorders associated with injury or disease of the central nervous system including trauma, stroke, multiple sclerosis, Parkinson's disease, or cerebral palsy, focal dystonias affecting the limbs, face, jaw, or vocal cords, TMJ pain disorders, diabetic neuropathy, wound healing, excessive salivation, vocal cord dysfunction, reduction of the Masseter muscle for decreasing the size of the lower jaw, treatment and prevention of chronic headache and chronic musculoskeletal pain, treatment of snoring noise, assistance in weight loss by increasing the gastric emptying time.

[0175] In a further aspect, the invention relates to a method of treating a disease or condition taken from the list of: cervical dystonia (spasmodic torticollis), blepharospasm, severe primary axillary hyperhidrosis, achalasia, lower back pain, benign prostate hypertrophy, chronic focal painful neuropathies, migraine and other headache disorders, and cosmetic or aesthetic applications; comprising the administration of a recombinant clostridial neurotoxin, or a pharmaceutical composition comprising a disulfide-linked di-chain clostridial neurotoxin, according to the present invention.

Examples

Example 1: Cloning and Expression of a Recombinant Botulinum Neurotoxin Serotype E Comprising a Thrombin Cleavage Site

[0176] A first nucleic acid molecule encoding amino acids 1 to 412 of wild type Botulinum neurotoxin serotype E, followed by a modified Botulinum neurotoxin serotype E interchain region comprising a thrombin cleavage site (nucleotides 1-1308 of SEQ ID NO: 5) and with flanking restriction endonuclease sites suitable for cloning said nucleic acid molecule into a pMEx expression vector (proprietary vector, Merz Pharmaceuticals) is synthesized. A second nucleic acid molecule encoding amino acids 426 to 1252 of wild type Botulinum neurotoxin serotype E, preceded by a modified Botulinum neurotoxin serotype E interchain region comprising a thrombin cleavage site (nucleotides 1234-3789 of SEQ ID NO: 5) and with flanking restriction endonuclease sites suitable for cloning said nucleic acid molecule into a pMEx expression vector (proprietary vector, Merz Pharmaceuticals) is synthesized, wherein the restriction endonuclease site flanking the 5end is identical to the restriction endonuclease site flanking the 3end of the first DNA molecule. The first synthetic nucleic acid molecule is digested with restriction enzymes cutting the flanking restriction endonuclease sites. After digestion of the pMEx vector with the same restriction enzyme combination, followed by enzymatic dephosphorylation, the digested first synthetic nucleic acid molecule is directionally cloned into the linearized vector using a T4 DNA ligase reaction mix. The ligation mixture is transformed into chemically competent E. coli using a heat shock method, plated on Luria-Bertani agar plates containing 30 g/ml Kanamycin, and placed in a 37 C. incubator for overnight growth. Bacteria containing vector constructs are identified as Kanamycin-resistant colonies, of which several are used to inoculate separate small-scale liquid cultures containing 30 g/ml Kanamycin, which are then incubated in a shaker at 37 C. for overnight growth. After preparation of plasmid DNA using an alkaline lysis method, clones containing the desired plasmid construct pMEx-NTE-LC are identified by restriction analysis and subsequent DNA sequencing. The second nucleic acid molecule is digested with restriction enzymes cutting the flanking restriction endonuclease sites, and directionally cloned into pMEx-NTE-LC following the procedure described above. The resulting plasmid construct pMEx-NTE contains an open reading frame encoding, from N- to C-terminus, amino acids 1 to 412 of wild type Botulinum neurotoxin serotype E, followed by a modified Botulinum neurotoxin serotype E interchain region comprising a thrombin cleavage site, followed by amino acids 426 to 1252 of wild type Botulinum neurotoxin serotype E (SEQ ID NO: 5), so that transcription of the open reading frame from the tac promoter of the vector is enabled.

[0177] The expression of the modified BoNT/E is achieved as follows. The prokaryotic expression plasmid pMEx-NTE encoding Botulinum neurotoxin serotype E with a modified interchain region (SEQ ID NO: 6) is introduced into chemically competent bacterial cells suitable for expression of the pMEx-NTE expression construct using a standard transformation protocol, such as, e.g., a heat-shock transformation protocol. The transformation reactions are plated onto Luria-Bertani agar plates containing suitable antibiotics and placed in a 37 C. incubator for overnight growth. A single antibiotic-resistant colony of transformed cells containing pMEx-NTE is used to inoculate a 10 mL tube containing 5 mL Luria-Bertani medium supplemented with 30 g/ml Kanamycin that is then placed in a 37 C. incubator, shaking at 250 rpm, for overnight growth. Approximately 3 mL of the resulting overnight starter culture is used to inoculate a 2000 mL baffled culture flask containing 800 mL Luria-Bertani medium supplemented with 30 g/ml Kanamycin that is then placed in a 37 C. incubator, shaking at 250 rpm, for approximately 4-5 hours. After inducing the expression of the modified BoNT/E protein by adding IPTG to a final concentration of 0.5-1.0 mM, the culture is transferred to a 18 C. incubator shaking at 170 rpm for overnight expression. Cells are harvested by centrifugation (4,000 rpm at 4 C. for 20-30 minutes), and the pellet is resuspended in 200 mM Tris buffer pH 7.5 containing 100 mM NaCl. After cell lysis by high pressure homogenization at 2000 bar, the insoluble fraction is removed by centrifugation (20 min at 12000g, 4 C.) or by cross-flow filtration using a 0.1 m membrane. Culture samples taken before IPTG induction, at various time-points after induction, and after cell harvest are used to evaluate expression by SDS polyacrylamide gel electrophoresis and Western blot analysis using anti-BoNT/E antibodies.

Example 2: Cleavage of Single-Chain Botulinum Neurotoxin Precursor Molecule Using Human Thrombin

[0178] The product of example 1 in phosphate buffer pH 7.8 and 150 mM NaCl is treated for 90 min with 25 IU (international units) human thrombin (RECOTHROM) per mg BoNT/E at 4-8 C. Thereafter, the amount of cleavage to the di-chain molecule is evaluated by evaluate expression by SDS polyacrylamide gel electrophoresis of the reaction under both reducing and non-reducing conditions, followed by Western blot analysis using anti-BoNT/E antibodies. The disulfide-bonded heterodimeric BoNT/E molecule applied onto SDS-PAGE under non-reducing conditions results in a protein band of approx. 150 kDa, while the same sample applied under reducing conditions results in two proteins of 100 kDa and 50 kDa, corresponding to the heavy and light chain, respectively.

Example 3: Cleavage of Single-Chain Botulinum Neurotoxin Precursor Molecule Using Human Factor Xa

[0179] Purified single-chain Botulinum neurotoxin type E (30 g) generated and expressed in accordance with the protocol of Example 1 was treated and incubated with 40 IU (international units) human factor Xa (Novagen, Cat. No. 69036-3, Lot D00134035) for 24 h at 20 C. in phosphate buffer pH 7.8 and 150 mM NaCl. Thereafter, it could be shown by SDS-PAGE that a two-chain molecule had been formed. Examination by nano-CL-ESI-FT MS/MS showed that the C-terminus of the light chain ended in the sequence . . . LVPR (amino acids 424-427 of SEQ ID NO:3), while Edman degradation of the heavy chain identified an N-terminus having the sequence GS . . . . Thus, it could be shown that factor Xa is cleaving the single-chain precursor molecule C-terminally of the arginine residue in the thrombin recognition sequence.