WO2009015840A2

WO2009015840A2 - Polypeptide for targeting of neural cells

Info

Publication number: WO2009015840A2
Application number: PCT/EP2008/006151
Authority: WO
Inventors: Tanja Weil; Andreas Rummel
Original assignee: Merz Pharma Gmbh & Co. Kgaa
Priority date: 2007-07-27
Filing date: 2008-07-25
Publication date: 2009-02-05
Also published as: WO2009015840A3

Abstract

The present invention provides a polypeptide having an increased or decreased affinity with respect to its receptor on neural cells, an increased or decreased neurotoxicity and/or a reduced affinity for neutralising antibodies against botulinum neurotoxin A. Further, active fragments or derivatives are provided. In addition, pharmaceutical and cosmetic uses thereof are indicated. Also, methods for producing and using them are disclosed.

Description

Polypeptide for targeting of neural cells

The present invention relates to a polypeptide which binds with an increased af- finity to its receptor on neural cells, has an increased or decreased toxicity in comparison to native neurotoxin, and/or has a reduced affinity for neutralizing antibodies in comparison with a native neurotoxin. Further, active fragments and derivatives theτeof are provided. In addition, compositions and pharmaceutical and cosmetic uses thereof are provided. Further, methods for producing and using them are indicated.

Nerve cells release transmitter substances by exocytosis. The fusion of the membranes of intracellular vesicles with the plasma membrane is referred to as exocytosis. In the course of this process the vesicular content is simultaneously dis- charged into the synaptic gap. The fusion of the two membranes is regulated by calcium, reacting with the protein synaptotagmin. Together with other cofactors synaptotagmin controls the status of three so-called fusion proteins, SNAP-25, synaptobrevin 2 and syntaxin IA. While syntaxin IA and synaptobrevin 2 are integrated into the plasma and/or vesicle membrane, SNAP -25 binds via its palmi- toyl side chain > to the plasma membrane. New studies have identified the receptor for BoNT A as the synaptic vesicle protein SV2 (Dong et al., Science 2006: 312(5773): 592-596; Mahrhold et al., FEBS Lett 2006; 580 (8): 2011-2014). To the extent that the intracellular calcium concentration increases, the three proteins form the SNARE-complex, both membranes are approaching one another, and subsequently fusing together. In the case of cholinergic neurons acetylcholine is released, causing muscle contractions, perspiration and other cholinergically provoked reactions. The above mentioned fusion proteins are the target molecules (substrates) of the light chains of the clostridial neurotoxin, formed by the bacteria C. botulinum, C. butyricum, C. baratii and C. tetani.

The anaerobic, gram-positive bacterium Clostridium botulinum produces seven different serotypes of proteinaceous neurotoxins. The latter are referred to as the botulinum neurotoxins (BoNT/A to BoNT/G). Among these, in particular BoNT/A and BoNT/B cause a neuroparalytic disorder in humans and animals, referred to as botulism. The spores of Clostridium botulinum can be found in the soil, but may also develop in incorrectly sterilized and sealed home-made food preserves, to which many cases of botulism are attributed.

BoNT/A is the most lethal of all known biological substances. As little as 5-6 pg of purified BoNT/A represents an MLD (median lethal dose). One unit (Unit, U) of BoNT is defined as the MLD, killing half of the female Swiss Webster mice, each weighing 18 - 2O g, after intraperitoneal injection. Seven immunologically different BoNTs were characterized. They are denoted as BoNT/A, B, Ci, D, E, F and G and may be distinguished by neutralization with serotype-specific antibodies. The seven primary sequences show an overall amino acid identity between 32 to 68 % including wide variations within their four structural domains. Furthermore, several closely related subtypes of each BoNT serotype have been explored, displaying the same neural receptor and substrate specificity, but dissimilarities in binding affinity of their receptors and cleavage efficiency of their substrates as well as significantly different antibody-binding properties. Currently, four sub- types of BoNT/A are defined, termed BoNT/Al to A4. The different serotypes of BoNTs differ in affected animal species with regard to severity and duration of the paralysis caused. Thus, with regard to paralysis, BoNT/A is 500 times more potent in rats for example, than BoNT/B. In addition, BoNT/B has proved to be non-toxic in primates at a dosage of 480 U/kg of body weight. The same quantity of BoNT/ A corresponds to 12 times the MLD of this substance in primates. On the other hand, the duration of paralysis after BoNT/A injection in mice is ten times longer than after injection of BoNT/E.

BoNTs have been used clinically for treating neuromuscular disorders, character- ized by hyperactivity in skeleton muscles, caused by pathologically overactive peripheral nerves. BoNT/ A has been approved by the U.S. Food and Drug Administration for treating blepharospasm, strabism, hemifacial spasm, cervical dystonia, excessive sweating and frown lines. Compared with BoNT/A the remaining BoNT serotypes are evidently less efficacious and manifest a shorter du- ration of efficacy. Clinical effects of BoNT/ A administered peripheral- intramuscularly are usually noticeable within a week. The duration of symptom suppression by one single intramuscular injection of BoNT/ A is normally about 3 to 6 months, but may also be significantly longer.

The clostridial neurotoxins specifically hydrolyse different proteins of the fusion apparatus. BoNT/ A, Ci and E cleave SNAP-25, while BoNT/B, D, F, G as well as tetanus neurotoxin (TeNT) cleave the vesicle-associated membrane protein (VAMP) 2 - also referred to as synaptobrevin 2. BoNT/Ci furthermore cleaves syntaxin IA.

The Clostridium bacteria release the neurotoxins as single-chain polypeptides each having 1251 to 1315 amino acids. Thereafter endogenous proteases cleave each of these proteins at a defined location into 2 chains ('nicking¹), the two chains, however, remaining interlinked by a disulphide-bridge. These dual-chain proteins are referred to as holotoxins (see Shone et al. (1985), Eur J Biochem 151, 75-82). The two chains have different functions. While the smaller fragment, the light chain (light chain = LC), represents a Zn^-dependent endoprotease, the larger unit (heavy chain = HC) represents the transporting means of the light chain. By treating the HC with endopeptidases two 50 kDa fragments were brought a- bout (see Gimenez et al. (1993), J Protein Chem 12, 351-363). The amino- terminal half (Hs-fragment) integrates into membranes at a low pH-value and enables the LC to penetrate into the cytosol of the nerve cell. The carboxy- terminal half (Hc- fragment) binds to complex polysialogangliosides, occurring exclusively in nerve cell membranes and to synaptic vesicle protein receptors like synaptotagmin I and II and SV2A, B & C (Rossetto O and Montecucco C. (2007) ACS Chem Biol. 2(2), 96-8.). The latter explains the high neuroselectivity of the clostridial neurotoxins. Crystalline structures confirm that BoNT/A disposes of three domains, which may be harmonized by the three steps of the mechanism of action (see Lacy et al. (1998) Nat Struct Biol 5, 898-902). Moreover, these data give rise to the conclusion that within the He-fragment two autonomous subunits (domains) exist of 25 kDa each. The first proof for the existence of the two functional sub-domains was brought about by the amino terminal half (H_CN) and the carboxy-terminal domain (Hcc) of the He-fragment of the TeNT, which were expressed in recombinant form and which revealed that the Hcc-, but not the Hc_N-domain binds to neural cells (see Herreros et al. (2000) Biochem J 347, 199- 204). The ganglioside binding site was identified in the Hcc-domains of BoNT/ A and B (see Rummel et al. (2004) MoI. Microbiol. 51(3), 631-43) and also the binding site of the protein receptor synaptotagmin II was discovered inside the Hcc- domains of BoNT/B and G, proving their separate functionality (see Rummel et al. (2007) PNAS, 104(l):359-64).

Under physiological conditions the HC binds to neural gangliosides and a protein receptor and is received inside the cell presumably by receptor-mediated endocy- tosis. In the acidic medium of the early endosomes, HN, the amino-terminal half of HC is thought to penetrate into the vesicle membrane and to form a pore. Each substance (X) linked to HC via a disulphide bridge, will be split off the HC by intracellular redox systems which gain access to the disulphide bridge and reduce it. X will ultimately appear in the cytosol.

In the case of the clostridial neurotoxins the HC is the carrier of an LC, finally hydrolysing its specific substrate in the cytosol. The cycle of SNARE complex formation and dissociation of the fusion proteins is interrupted and the release of acetylcholine is consequently inhibited. As a result thereof, e.g. muscles are paralyzed and glands cease their secretion. The active period of the individual BoNT serotypes differs and depends on the presence of intact LC in the cytosol. As all neural cells possess receptors for clostridial neurotoxins, it is not only the release of acetylcholine which may be affected, but potentially also the release of the substance P, of noradrenalin, GABA, glycine, endorphin and other transmitters and hormones.

The BoNT/A complex, also designated progenitortoxin A was used in the more recent past for treating motor dystonia as well as for attenuating excessive sympathetic activity (see Benecke at al. (1995), Akt. Neurol. 22, 209ff) and for alleviating pain and migraine (see Sycha et al. (2004), J. Neurol. 251, 19-30). This complex consists of the neurotoxin, various hemagglutinins and a non-toxic, non- hemagglutinating protein. Under physiological pH the complex dissociates within minutes. The resulting neurotoxin is the sole ingredient of the complex, which is therapeutically active. Since the underlying neurological disease is generally not curable, the complex needs to be injected at repeating intervals of three to four months. Depending on the quantity of injected foreign protein, some patients develop antibodies specific for BoNT/A. As some of these antibodies can neutralize the effect of BoNT/A, they are considered to be neutralizing ("neutralizing antibodies")- Patients that develop neutralizing antibodies become resistant to the specific serotype. Once antigen-sensitive cells have recognized the neurotoxin and neutralizing antibodies have been formed, the relevant memory cells are conserved over years. For this reason, it is important to treat the patients with prepara- tions of the highest possible activity at a lowest possible dosage. Preferably, the preparation does not contain any further proteins of bacterial origin, since they may act as immune adjuvants. Consequently, it is desirable to use products of highest purity for therapy.

The resistance of patients in view of neurotoxin is on the molecular level based primarily on the presence of neutralizing antibodies. To solve these problems, the present invention now provides polypeptides, active fragments and derivates thereof, which overcome the above-mentioned problems.

The present invention provides a polypeptide, or an active fragment or derivative thereof, wherein the polypeptide comprises a stretch of amino acids having an amino sequence corresponding to the amino acid sequence of botulinum neurotoxin of type A at positions 1092 to 1296, wherein at least one amino acid position selected from the group consisting of 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103,1104,1105,1106,1107, HOS, 1109, 1110, 111 I, 1112, 1113,1114,1115, 1116,1117,1118,1119, 1120,1121,1122, 1123,1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150 1151, 1152, 1153, 1154, 1155, 1156, 1157,1158, 1159, 1160, 1161, 1162,1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295 and/or 1296 of said sequences is added, deleted, inserted and/or substituted by a naturally or non-naturally occurring amino acid, wherein the naturally occurring amino acid is selected from glycine, alanine, valine, leucine, iso- leucine, methionine, proline, phenylalanine, tryptophane, serine, threonine, aspar- agine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid, wherein (i) the polypeptide or the fragment or the derivative binds with an increased affinity to its receptor;

(ii) the polypeptide or the fragment or the derivative has an increased or decreased neurotoxicity in comparison to the native neurotoxin, preferably the neurotoxicity is determined in the hemidiaphragm assay; and/or

(iii) the polypeptide or the fragment or the derivative has a reduced affinity for neutralising antibodies in comparison with the native neurotoxin.

in a ligand-receptor-study specific amino acid residues were thus characterized according to the invention in the ganglioside binding pocket and protein receptor binding pocket of BoNT/A and modified in order to modify the affinity to the receptor, the neurotoxicity and/or the reduction for binding to neutralising antibodies. The affinity of the mutated Hc- fragment was determined La in ganglioside and synaptosome binding assays.

According to a preferred embodiment of the present invention a polypeptide, an active fragment or derivative thereof is provided having an increased affinity to its receptor.

According to a further preferred embodiment of the present invention a polypeptide, an active fragment or derivative thereof is provided having an increased or decreased neurotoxicity in comparison to the native neurotoxin.

According to a further preferred embodiment a polypeptide, an active fragment or derivative thereof is provided having a reduced affinity for neutralizing antibodies in comparison to the native neurotoxin.

Subsequently, the terms are defined how they should be understood in the context of the present invention.

"Binding with an increased affinity to its receptor" Binding affinity may be determined in comparison to a native neurotoxin, i.e. a neurotoxin derived from C. botulinum and having a wild-type amino acid sequence. Alternatively, binding assays may be performed with a fragment of said neurotoxin. Preferably said neurotoxin is obtainable from C. botulinum. An in- creased affinity means that the neurotoxin according to the invention has a lower dissociation constant in comparison to the non-modified neurotoxin. Preferably, the native neurotoxin is botulinum neurotoxin of serotype A including any subtype A, which is defined in detail below. A recombinantly produced botulinum neurotoxin of serotype A, which amino acid sequence is identical to a botulinum neurotoxin obtained from C. botulinum, behaves pharmacologically identical or similar to the native botulinum neurotoxin obtained from C. botulinum. Such a recombinant neurotoxin may be produced in e.g. E. coli and is commonly referred to as "recombinant botulinum neurotoxin". Binding assays may be performed with neurotoxin isolated from C. botulinum or neurotoxin obtained by recombi- nant protein expression. Preferably, the polypeptide, the active fragment or derivative according to the present invention binds specifically to plasma membrane associated molecules, transmembrane proteins, synaptic vesicle proteins, a protein of the synaptotagmin family or the synaptic vesicle glycoproteins 2 (SV2), preferably synaptotagmin I and/or synaptotagmin II and/or SV2A, SV2B or SV2C, particularly preferred human synaptotagmin I and/or human synaptotagmin II and/or human SV2A, SV2B or SV2C. The binding is preferably determined in vitro. The skilled person knows various assays for determining binding affinities between a first protein (the neurotoxin) and a second protein (the receptor). Any such assay may be useful for determining the effect of a mutation on receptor binding. One such assay is a GST-pull-down-assay, which is preferred in accordance with the teaching of the present invention. This assay is described in the examples of the present invention. Surface plasmon resonance may also be used to study the binding affinity. Experimental conditions therefore are e.g. described in Yowler et al., Biochemistry 43(2004), 9725-9731. In addition, the binding af- finity may be assessed using isothermal microcalorimetry. "The polypeptide, the active fragment or the derivative has an increased or decreased neurotoxicity in comparison to the native neurotoxin" The "native neurotoxin" is botulinum neurotoxin A purified from of C. botulinum including any subtype thereof. As pointed out herein above, recombinantly pro- duced botulinum neurotoxin may be generated in E. coli. Recombinantly expressed neurotoxin, which is of identical amino acid sequence, behaves pharmacologically identical to native botulinum neurotoxin and is designated "recombinant botulinum neurotoxin wild type". Nerve cells used herein are e.g. cholinergic motoneurons. Neurotoxicity is preferably determined using the mouse phrenic nerve- hemidiaphragm-assay (HDA) known in the art. Neurotoxicity of polypeptides, the active fragments or derivatives according to the invention is preferably determined as described by Habermann et al., Naunyn Schmiedeberg's Arch. Pharmacol. 31 1 (1980), 33-40.

"Neutralizing antibodies" against botulinum neurotoxin have been described in the art (Gδschel H et al, (1997) Exp. Neurol. 147(1), 96-102). It is believed that such antibodies are directed against e.g. receptor binding sites such as e.g. the gan- glioside and protein receptor binding pockets within the Hcc-domain of the neurotoxin. As a consequence, if the surfaces surrounding the binding pockets of the neurotoxin are modified, preferably without negatively affecting their functionality, the neutralizing antibodies loose their binding sites and the mutated neurotoxin is no longer neutralized. Patients treated with such a modified neurotoxin will therefore respond to therapy, even if neutralizing antibodies directed against the native neurotoxin (i.e. neurotoxin of wild-type sequence) have been devel- oped.

"Botulinum neurotoxin A"

Up to now, four different subtypes of botulinum neurotoxin A are known, i.e. Al, A2, A3 and A4. The teaching of the present invention refers to any subtype, which is classified by the skilled person as a serotype A subtype, including the aforementioned subtypes. The amino acid sequences of the different botulinum neurotoxin A. subtypes are accessible using the accession numbers of table 1.

The term "polypeptide" refers to a polymer of at least 10 amino acid residues. The polypeptide, the active fragment or derivative may be non-narurally occurring.

"Active fragment"

The term "active fragment" means any fragment of the polypeptide according to the invention, which exhibits biological activity. The term "biological activity" refers to an activity such as receptor binding, receptor mediated uptake and/or proteolytic activity. Also included are e.g. activities related to signaling and intracellular transport.

For example, a fragment of the polypeptide of the present invention may be a neurotoxin fragment. As used herein, said fragment is biologically active, if it is ca- pable of mediating transport across a membrane. A corresponding example of an active fragment of the polypeptide of the present invention is a polypeptide, comprising the He-fragment of botulinum neurotoxin, binding to complex ganglioside receptors and being endocytosed upon protein receptor binding. The He-fragment of the botulinum neurotoxin A typically comprises the residues 867 to 1296. An- other example of an active fragment of the polypeptide is the heavy chain (HC) typically comprising residues 449 to 1296 which binds to the neural double receptors, is endocytosed and forms a membrane pore for translocation of cargo molecules. A further example of an active fragment of the polypeptide of the present invention is a polypeptide fragment with proteolytic activity. Typically such fragment would comprise residues 2 to 438 of the light chain of botulinum neurotoxin A. Another example of an active fragment is the LH>i-fragment comprising residues 1 to 866. The LH_N-fragment penetrates acidic endosomal membranes and releases the light chain for proteolytic cleavage of its substrates under reductive conditions. Carboxyl-terminal fusion of the LHN- fragment to cell binding domains of other proteins allows the retargeting of botulinum neurotoxins. Preferably, the fragment of the polypeptide of the present invention is fully active, i.e. in comparison with a full-length, wild-type polypeptide 100% activity is observed. Polypeptide fragments with more or less biological activity may, however, also be useful and are thus also an object of the present invention. Such polypep- tide fragments with increased biological activity may, for example, have a biological activity of up to 1 10%, up to 120%, up to 130%, up to 140%, up to 150% or even up to 200%. In exceptional cases the biological activity maybe even higher.

As pointed out above, the present invention also refers to polypeptide fragments with reduced biological activity. Such polypeptide fragments with reduced biological activity may, for example, have a biological activity of up to 90%, up to 80%, up to 70%, up to 60%, up to 50% or only up to 20%. The skilled person is aware of various assays to determine biological activity. Such assays will typically involve comparing the biological activity of a full- length, wild-type polypeptide with the activity observed with the fragment. A first example is an assay involving a measurement of the proteolytic activity of the polypeptide fragment of the present invention. Such an assay might, for example, involve mixing the polypeptide fragment of the present invention and SNAP-25. After incubation at suitable conditions, the amount of SNAP-25 cleavage product might be determined.

A second example is an assay involving a measurement of the receptor binding activity. Typically, the active polypeptide fragment and brain synaptosomes, tissue containing the natural composition of gangliosides and the protein receptor, are mixed and the amount of formed complex is determined. An example measuring the direct binding affinity of an active fragment to its protein receptor is performed employing a GST-pull-down-assay, which is preferred in accordance with the teaching of the present invention. The full biological activity is preferably determined using the hemidiaphragm assay (HDA) known in the art. The active fragment may consist of up to 50, preferably up to 100, more preferably up to 200, in particular up to 400 or up to 900 amino acids. Also preferred are active polypeptides fragments of the full-length neurotoxin, which only lack a few N- or C-terminal or internal amino acids. A typical example is a polypeptide fragment lacking the first amino acid such as methionine. More preferably, such polypeptide fragments lack consecutive amino acid residues. In particular, the polypeptide fragment of the present invention may lack up to 10, up to 20, up to 30, up to 40, up to 50, up to 60, up to 100, up to 200 or even up to 500 amino acid residues. Preferably, the active fragment comprises the complete ganglioside binding pocket and/or the complete protein receptor binding pocket.

"Derivative"

The term derivative as used herein denotes any naturally or non-naturally occurring modification of a polypeptide or the polypeptide fragment of the present in- vention. For example, the modification includes N- and/or C- terminal modifications such as pyroglutamic acid. Further, side chain modifications are also encompassed by the term derivative. Typical side chain modifications include gly- cosylation, acetylation, acylation. deamidation, phoshorylation, isoprenylations, glycosyl-phosphatidyl-inositylation. Further, suitable derivatives may include the production of dimers, oligomers or polymers by the cross-linking of side chains such as e.g. reaction with formaldehyde or oxidation of free thiol groups under formation of a disulphide linkage. The modification further includes glycosylation or introduction of protective groups or anchor groups. The term "protective group" means any protective group used in organic chemistry to avoid undesired terminal or side chain modifications before the intended use. Typical examples include Fmoc, tButyl and further protective groups known to the person skilled in the art. The term "anchor group" means any group which may confer binding to another compound. Typical examples include avidin, biotin, antibodies, antigens, ferridoxin. The modifications may be introduced naturally in the producing cell in case of recombinant production or may artificially be produced by methods known in the art. Further, dimers, oligomers or polymers of the neurotoxins may be produced by methods known in the art such as cross-linking. Cross-linking may be effected by disulphide bridge formation.

,,Neural cell" as used herein, means any mammalian nerve cell capable of interact- ing with botuϋnum neurotoxin A. in particular cholinergic neurons, particularly preferred cholinergic motoneurons.

"Binding to its receptor"

The term "binding to its receptor" refers to binding of the polypeptide of the in- vention to its receptor. Said receptor may be the ganglioside receptor or the protein receptor. According to the teaching of the present invention, the affinity of said polypeptide to any of said receptors may be modified. In one embodiment modified means increased, in another embodiment modified means decreased. Modified binding affinities may be determined as outlined herein above.

The HC of the neurotoxin A formed by C. botulinum comprises three sub- domains, i.e. arnino-terminal 50 kDa translocation domain HN with the subsequent 25 kDa Hc_N-domain and the carboxy-terminally located 25 kDa Hcc-domain. Taken together, the HCN - and H_Cc-domains are designated as He-fragment. The re- spective amino acid ranges of the respective domains are shown for the different BoNT/A serotypes and its variations in table 1.

Table 1: Database accession numbers of the amino acid sequences of botulinum neurotoxin A subtypes 1-4 and amino acid ranges of the respective domains.

Regarding the term "ganglioside binding pocket" the HC of botulinum neurotoxins has a high affinity with respect to peripheral neural cells, which is particularly mediated by the interaction with complex polysialogangliosides, i.e. glycolipids consisting of more than one sialic acid - (Halpern et al. (1995), Curr. Top. Microbiol. Immunol. 195, 221-41; WO 2006/02707). Accordingly, the LC attached to the HC only reaches this cell type and become effective only in those cells. BoNT/A only binds one molecule of ganglioside GTIb. Regarding the term "protein receptor binding pocket", the protein receptor of BoNT/A are synaptic glycoproteins 2 (SV2), preferably SV2A, SV2B and SV2C wherein SV2C is most preferred. Receptor binding may be determined by cell-based assays or in vitro. Such assays generally involve determining the affinity of the polypeptide of the invention to its receptor, wherein said receptor may be the ganglioside receptor, the protein recep- tor or both. In such assays involving the protein receptor, said protein receptor may be, e.g. full-length synaptic glycoprotein 2 or a fragment thereof comprising the binding site to which the polypeptide of the invention binds. Receptor binding assays may involve full-length polypeptide and/or full-length receptor. Alternatively, either the polypeptide of the invention and/or the receptor maybe repre- sented by a fragment. This fragment may also be fused to other proteins such as an immunoglobulin, GST or the transferrin receptor or a fragment thereof. These other proteins may be useful in receptor binding assays as they may be used to immobilise one of the binding partners (e.g. the polypeptide or the receptor).

Such assays generally involve the wild-type neurotoxin as reference, wherein its degree of receptor binding may be used as an indicator or measure for 100% binding. Alternatively, said reference may only comprise a fragment of the wild-type neurotoxin, wherein the fragment may comprise a single or both receptor binding sites. Analysis of binding affinity of an active fragment to isolated gangliosides is performed by immobilising the gangliosides on the surface of a microtiter plate and immunochemically determining the amount of bound active fragment. Analysis of binding affinity of an active fragment to its protein receptor is performed by expressing a GST-fusion protein containing the protein receptor or its active domain which is immobilized to glutathion-sepharose beads. Mixing of the immobilized protein receptor with the polypeptide or active fragment of the boru- linum neurotoxin, subsequent phase separation and determination of the protein amount in the solid phase via SDS-PAGE reveals the level of binding affinity compared to an active fragment with wild-type sequence. Analysis of binding affinity of an active fragment to neural membranes which contains the physiological receptor composition is performed by preparation and subsequent mixing of brain synaptosomes and radioactively labeled active fragments. The amount of bound radioactive active fragment is determined by SDS- PAGE and autoradiography.

Preferably, the reference polypeptide differs from the polypeptide of the invention only within said stretch of amino acids corresponding to positions 1092 to 1296, wherein said sequence of the reference polypeptide is identical to the sequence of the native or wild type botulinum neurotoxin of type A and wherein the sequence outside said stretch is preferably not altered.

Individual amino acids of the binding pockets have been modified by site-directed mutagenesis such that a binding to the protein receptor and/or to the ganglioside receptor has been increased or decreased.

It was found that on the basis of site-directed modification, in particular deletion or substitution of amino acids within the protein receptor binding pocket and/or ganglioside binding pocket of BoNT, the interaction of BoNT/ A with its receptors was significantly increased or decreased. Further, the neurotoxicity of polypeptide, the active fragment or derivative could be modified. In addition, it was found that for some modifications the polypeptide, the active fragment or the derivative had a reduced affinity for neutralizing antibodies in comparison with the native neurotoxin.

He-fragments of BoNT/A were expressed recombinantly as wild-type or with in- dividual amino acid modifications in E. coli and isolated. To investigate the in vitro interaction between the recombinantly produced BoNT/A with its protein receptor SV2, in particular SV2C, the respective GST-SV2C fusion protein was incubated with different amounts of the respective He-fragment and a phase separation was performed. The He-fragment remained in the separated supernatant while bound BoNT He-fragment in the solid phase together with GST-SV2C fu- sion protein was detectable. The replacement of the respective He-fragments by the full length BoNT/A in GST-pull-down-assay yielded the same results.

The potency of full-length form of BoNT/ A was assessed in HDA using a dose- response-curve.

According to a preferred embodiment in said amino acid stretch of the polypeptide not more than 50, preferably not more than 25, more preferably not more 10, particularly preferred not more than 5, in particular 4, 3, 2 or 1 amino acids are added, deleted, inserted and/or substituted. Said amino acids may be contiguous or non-contiguous amino acids. Preferably, the polypeptide is altered by recombinant techniques.

According to a further preferred embodiment at least one amino acid at the posi- tion selected from group consisting of 1 117. 1202, 1203, 1204, 1252, 1253, 1254, 1262, 1263, 1264, 1265, 1266, 1267, 1270, 1278 and/or 1279 of the botulinum neurotoxin A protein sequences are added, deleted, inserted and/or substituted by a naturally or non-naturally occurring amino acid.

According to a particular preferred embodiment the amino acid phenylalanin at position 1252 is substituted by histidine and/or the amino acid phenylalanine at the position 1278 is substituted by histidine or tyrosine and/or the amino acid leucine at position 1278 is substituted by histidine, phenylalanine or tyrosine.

According to a preferred embodiment two to four amino acids at an amino acid position selected from the group consisting of 1 1 17, 1252, 1253 and 1278 of the botulinum neurotoxin A protein sequences are substituted, preferably the double substitutions are selected from the group consisting of

Y1 1 17F/L1278F, F 1252H/L1278F, Y11 17A/F1252H, Y1 1 17A/L1278F, YI l 17A/L1278Y, Yl 117C/F1252H, Yl 117C/L1278F, Yl 117C/L1278Y,

Yl 1 17V/F1252H, YI l 17V/L1278F and Yl 1 17V/L 1278Y, preferably the triple substitutions are selected from the group consisting of YIl 17F/F1252H/L1278F, Yl 117A/F1252H/H1253K, Ylll7A/F1252H/L1278F,Y1117A/F1252H/U278H, Yl 117A/F1252H/L1278Y, Yl 117A/F1252Y/L1278F, Ytll7A/F1252Y/L1278H,Y1117A/F1252Y/Ll278Y,

Y1117C/F1252H/H1253K,Y1117C/F1252H/L1278F, Yl 117C/F1252H/L1278H, Yl 117C/F1252H/L1278Y, Yl 117C/F1252Y/L1278F, Yl 117C/F1252Y/L1278H, Y1117C/F1252Y/L1278Y,Y1H7V/F1252H/H1253K, Y1117V/F1252H/L1278F,Y1117V/FΪ252H/L1278H,

Yl 117V/F 1252H/L1278Y, Yl 117V/F1252Y/H1253K, Y1117V/F1252Y/L1278F,Y1117V/F1252Y/L1278H, Yl 117V/F1252Y/L1278Y and Yl 117V/V1262I/L1278F, preferably the quadruple substitutions are selected from Y1117A/F1252H/H1253K/L1278F,Y1117A/F1252H/H1253K/L1278H,

Y1117A/F1252H/H1253K/L1278Y,Y1117A/F1252Y/H1253K/L1278F, Y1U7A/F1252Y/H1253K/L1278H,Y1117A/F1252Y/H1253K/L1278Y, Y1117C/F1252H/H1253K/L1278F,Y1117C/F1252H/Hl253KyLl278H, Yl 117C/F1252H/H1253K/L1278Y, Yl 117C/F1252Y/H1253K/L1278F, Y1117C/F1252Y/H1253K/L1278H,Y1117C/F1252Y/H1253K/L1278Y

Yl 117V/F1252H/H1253K/L1278F, Yl 117V/F1252H/H1253K/L1278H, Y1117V/F1252H/H1253K/L1278Y,Y1117V/F1252Y/H1253K/L1278F, Y1117V/F1252Y/H1253K/L1278H and Yl 117V/F1252Y/H1253K/L1278Y, particularly preferred are the following combination of substitutions:

Yl 117V/F1252H, Yl 117V/L1278F, Yl 117V/F1252H/H1253K, Y1117V/F1252H/L1278F,Y1117V/F1252H/L1278H, Yl 117V/F1252H/L1278Y, YIl 17V/F1252Y/H1253K, Y1117V/F1252Y/L1278F,Y1117V/F1252Y/L1278H, Y1117V/F1252Y/L1278Y,Y1117V/F1252H/H1253K/L1278F, Yl 1 l7V/F l252Y/H1253K/L1278F_and Yl 1 17V/F1252Y/H1253K/L127SH.

According to another preferred embodiment at least one amino acid of the poly- peptide at an amino acid position selected from the group consisting of 1 126, 1 127, 1 129, 1130, 1 131, 1133, 1 135, 1137, 1 195, 1 196, 1 199, 1200, 1201, 1204, 1205, 1206, 1207, 1209, 1213, 1215, 1216, 1217, 1255, 1256, 1258 and/or 1260 of the botulinum neurotoxin A protein sequences are added, deleted, inserted, and/or substituted by a naturally or non-naturally occurring amino acid.

It is particularly preferred that the amino acid is selected from the group consisting of 1 126, 1127, 1129, 1 130, 1131, 1 133, 1 135, 1137, 1200, 1201, 1215 and/or 1216 of the botulinum neurotoxin A protein sequences is deleted and/or substituted by a naturally or non-naturally occurring amino acid.

According to a further aspect of the present invention the polypeptide, the fragment or the derivative has at least one mutation in the ganglioside binding pocket together with at least one mutation in the protein receptor binding site.

It is preferred that (i) at least one amino acid at an amino acid position selected from 1117, 1202, 1203, 1204, 1252, 1253, 1254, 1262, 1263, 1264, 1265, 1266, 1267, 1270, 1278 and/or 1279 of the botulinum neurotoxin A protein sequences is added, deleted, inserted and/or substituted by a naturally or non-naturally occurring amino acid, and (ii) at least one amino acid at an amino acid position selected from the group consisting of 1 126, 1127, 1129, 1130, 1131, 1 133, 1135, 1137, 1195, 1196, 1 199, 1200, 1201, 1204, 1205, 1206, 1207, 1209, 1213, 1215, 1216, 1217, 1255, 1256, 1258 and/or 1260 of the botulinum neurotoxin A protein sequences is added, deleted, inserted and/or substituted by a naturally or non- naturally occurring amino acid. It is particularly preferred that at least one amino acid at an amino acid position selected from the group consisting of 1117, 1252, 1253 anάVor 1278 of the botulinum neurotoxin A protein sequences is deleted and/or substituted by a naturally or non-naturally occurring amino acid, and (ii) at least one amino acid at an amino acid position selected from 1195, 1196, 1 199, 1204, 1205, 1206, 1207, 1209, 1213, 1217, 1255, 1256, 1258 and/or 1260 of the botulinum neurotoxin A protein sequences is deleted and/or substituted by a natu- rally or non-naturally occurring amino acid. Preferably, the position (ii) is selected from 1 196, 1199, 1205, 1207, 1255, 1258 and/or 1260.

According to a preferred embodiment of the present invention the neutralising antibody inhibits the binding of the native neurotoxin to the protein receptor or to the ganglioside receptor and/or inhibits the uptake of the neurotoxin into the neural cell.

According to a preferred embodiment of the present invention the polypeptide binds specifically to molecules associated with the plasma membrane, trans- membrane proteins, synaptic vesicle proteins, proteins of the synaptotagmin family or synaptic vesicle glycoproteins 2 (S V2) and/or synaptotagmin I and/or synaptotagmin II and/or SV2 A, SV2B or SV2C, preferably to human synaptotagmin I and/or human synaptotagmin II and/or human SV2A, SV2B or SV2C.

According to a preferred embodiment the polypeptide of the invention or the active fragment or the derivative has an increase in affinity for the neural cells of at least 15 % compared to the native neurotoxin, preferably at least 50 %, particularly preferred at least 80 %, in particular at least 90 %.

According to a further preferred embodiment the binding of the polypeptide, the active fragment or the derivative to its receptor is increased by at least 15 %, preferably at least 50 %, particularly preferred at least 80 %, in particular at least 90 %.

According to a further preferred embodiment the affinity for neutralising antibodies compared to the native neurotoxin of the polypeptide, the active fragment or the derivative is reduced by at least 15 %, preferably at least 50 %, particularly preferred at least 80 %, in particular at least 90 %.

The active fragment or the derivative of the polypeptide according to the inven- tion consists according to one embodiment of the invention of up to 10, preferably up to 20, more preferably up to 50, in particular up to 100 or up to 200 amino acids.

It is particularly preferred that the active fragment comprises the complete gan- glioside binding pocket and/or the complete protein receptor binding pocket. Preferably, said ganglioside binding pocket consists/is composed of aa 1117 and aa 1202 to aa 1279 and said protein receptor binding pocket consists of aa 1 126 to 1260.

The derivative according to the invention is preferably modified by cross-linking or side-chain modifications.

According to another aspect of the present invention a composition is provided comprising the polypeptide, the active fragment and/or the derivative according to the invention and optionally an intervening molecule. The intervening molecule may be a small organic molecule, a peptide or a protein. The intervening molecule may be linked to the polypeptide, the active fragment or the derivative, covalently by a peptide bond, ester bond, ether bond, sulphide bond, disulphide bond or a carbon-carbon bond. The small organic molecule may be a virostatic agent, cy- tostatic agent, an antibiotic or an immunoglobulin.

The intervening molecule may also be a therapeutically effective agent.

According to a further preferred embodiment the protein is a protease which may specifically hydrolyse one or more proteins of the release apparatus for neurotransmitters, wherein the protease is selected from the group consisting of LC of C. botulinum neurotoxins, in particular of serotypes A. B, Cl, D, E, F and G or a proteolytically active fragment of LC of C. botulinum neurotoxin, in particular a neurotoxin of the serotypes A, B, C 1 , D, E, F and G, wherein the fragment has at least 0.01 % of the proteolytic activity of the native protease, preferably at least 5 %. particularly preferred at least 50 %, in particular at least 90 %. Preferably, the polypeptide and the protease are derived from the same C. botulinum neurotoxin serotype, in particular it is derived from the He-domain of the polypeptide and protease of C. botulinum neurotoxin serotype A. The sequences of the proteases are publicly available in databases and the database numbers are accessible from table 1. The proteolytic activity of the proteases is determined using the substrate cleavage kinetics (see Binz et al. 2002, Biochemistry 41(6), 1717-23).

According to a preferred embodiment of the invention the protease cleaves specifically particular substrates in the cholinergic motoneuron. The substrates are preferably selected from proteins being involved in the release of neurotransmitters in neural cells and proteins being capable of catalytic reactions in a neural cell. Also preferred is that the protease and the polypeptide are covalently bound via an amino acid sequence which is recognized and cleaved specifically by an endopeptidase. Upon cleavage by the endopeptidase a disulphide bridge links the protease and the polypeptide which leads to the formation of an active holotoxin.

According to a further aspect of the present invention a nucleic acid is provided which encodes the polypeptide, the active fragment or the derivative according to the invention. The encoding nucleic acid may be RNA, DNA or mixtures thereof. The nucleic acid may comprise an open reading frame encoding a selectable marker such as ampicillin, kanamycin, neomycin, hygromycin or puromycin. The nucleic acid may be obtainable e.g. by cloning from genomic or cDNA libraries. Further, the nucleic acid can directly be synthesized by solid phase synthesis. Sui- table methods are known to the person skilled in the art. Insofar a starting nucleic acid is used, it is possible to perform a site-specific modification by site-directed mutagenesis which results on amino acid level in at least an addition, insertion, deletion and/or substitution.

A popular general approach for site-directed mutagenesis involves cloning the gene or cDNA into an M 13 or phagemid vector which permits recovery of single- stranded recombinant DNA, a mutagenic oligonucleotide primer is then designed whose sequence is perfectly complementary to the gene sequence in the region to be mutated, but with a single difference: at the intended mutation site it bears a base that is complementary to the desired mutant nucleotide rather than the origi- nal. The mutagenic oligonucleotide is then allowed to prime new DNA synthesis to create a complementary full-length sequence containing the desired mutation. The newly formed heteroduplex is used to transform cells, and the desired mutant genes can be identified by screening for the mutation. Phagemid vectors are plas- mids which have been artificially manipulated so as to contain a small segment of the genome of a filamentous phage, such as Ml 3, fd or fl . The selected phage sequences contain all the c/5-acting elements required for DNA replication and assembly into phage particles. They permit successful cloning of inserts several kilobases long (unlike M 13 vectors in which such inserts tend to be unstable). Following transformation of a suitable E. coli strain with a recombinant phage- mid, the bacterial cells are superinfected with a filamentous helper phage, such as fl, which is required to provide the coat protein. Phage particles secreted from the superinfected cells will be a mixture of helper phage and recombinant phagemids. The mixed single-stranded DNA population can be used directly for DNA sequencing because the primer for initiating DNA strand synthesis is designed to bind specifically to a sequence of the phagemid vector adjacent to the cloning site. Commonly used phagemid vectors include the pEMBL series of plasmids and the pBluescript family.

The present invention further provides a recombinant expression vector compris- ing the nucleic acid capable of encoding the polypeptide, the active fragment or the derivative. The nucleic acid is preferably linked in an operative manner with a suitable promoter. Suitable promoters for expression in known expression systems are known to the person skilled in the art. The use of a promoter depends on the expression system to be used in expression. Generally, constitutive promoters are preferred, however, also inducible promoters may be used. The recombinant ex- pression vector comprises at least a part of a vector, in particular regulatory elements wherein the vector may be e.g. selected from λ-derivatives, adenoviruses, baculoviruses, vaccinia viruses, SV40-viruses and retroviruses. The vector is preferably suitable for expression of the nucleic acid in a suitable host cell. It is particularly preferred that the expression vector is a linear sequence, a transposon, a plasmid based expression vector, a virus or a phage.

The present invention further provides a recombinant host cell comprising the expression vector being capable of expressing the polypeptide, the active fragment or the derivative under suitable conditions. In the state of the art numerous prokaryotic and eukaryotic expression systems are known wherein the host cell is e.g. selected from prokaryotic cells such as E. coli or B. subtilis, Bacillus megate- rhim, Clostridium butyricum, Clostridium barati, Clostridium botulinum, Clostridium tetani and Clostridium histolyticum, from eukaryotic cells such S. cere- visiae, P. pastoris.

Although also cells from higher eukaryotic organisms may be used such as insect or mammalian cells, host cells are preferred which do not possess a glycosylation apparatus as C. botulinum.

The present invention also provides a method for producing the polypeptide, the active fragment of the derivative according to invention comprising the steps of

(a) providing the host cell;

(b) culturing the cell under conditions suitable for recombinant expression; and (c) recovering and optionally purifying the polypeptide, the active fragment or the derivative from the cell of the supernatant.

Numerous methods are known in the art for purifying the designed polypeptide from the culture medium such as chromatographic procedures or electrophoresis. Chromatographic procedures comprise ion exchange, affinity chromatography and reversed phase chromatography.

The present invention further provides a method for producing the polypeptide, the active fragment or the derivative comprising the steps of preparing the polypeptide, the active fragment or the derivative by chemical methods; and recovering and optionally purifying the polypeptide, the active fragment or the derivative. The preparation by chemical methods includes Merrifield-synthesis of oligo- or polypeptides and optionally fragment condensation.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition comprising the polypeptide, the active fragment and/or the derivative according to the invention or comprising the composi- tion as mentioned above or the peptide obtainable from the host cell or product of the methods as described above and optionally a pharmaceutically acceptable carrier, diluent, and/or additive. Where desired, toxicity adjusting agents such as sodium chloride, glycerol and various sugars can be added. Stabilizers such as human serum albumin may also be included. The formulation may be preserved by means of a pharmaceutically acceptable preservative such as a paraben, although preferably it is unpreserved.

The pharmaceutical composition is suitable for oral, intravenous, subcutaneous, intramuscular and topical administration. The intramuscular administration is pre- ferred. The dose of the drug administered to the patient will depend on the severity of the pathological condition; e.g. the number of muscle groups requiring treatment, the age and size of the patient and the potency of the drug. The potency of the drug is expressed in MLD (mouse), i.e. as a multiple of the MLD value. One MLD is de- fined as being the equivalent amount of the drug that kills 50 % of a group of female Swiss-Webster mice, weighing 18 to 20 grams each.

A dosage unit for a pharmaceutical composition comprises about 0.1 pg up to I mg of the polypeptide, the active fragment and/or the derivative according to the invention and/or the composition according to the invention.

In a preferred embodiment, said a pharmaceutical composition contains a single therapeutically active component (i.e. the polypeptide of the invention). In another preferred embodiment, the pharmaceutical composition contains one or more ad- ditional therapeutically active components. Such additional active component may be e.g. a neurotoxin of a different serotype such as BoNT/B or a modified neurotoxin. The additional active component may also be an analgesic.

The pharmaceutical composition is suitable for the treatment of disorders and dis- eases for which a therapy with botulinum neurotoxin is indicated. Such diseases comprise in particular spastic conditions and focal dystonias, but the pharmaceutical composition of the present invention may also be administered in order to treat associated symptoms. In particular, the pharmaceutical composition of the present invention may be administered to patients affected by e.g. hemifacial spasm, spasmodic torticollis (cervical dystonia), blepharospasm, migraine, pain, diseases of the cervical and lumbar spine, strabism, hypersalivation, snoring, wound healing, depression, hyperhidrosis, low back pain and strabismus. Furthermore, the pharmaceutical composition of the present invention might be used for treating urological conditions rooted in a spastic dysfunction of the sacral reflex arcs. Ex- amples of such conditions include pelvic pain (e.g., interstitial cystitis, endometriosis, prostatodynia, urethral instability syndromes, hyperactive bladder), pelvic myofascial elements (e.g., levator sphincter, dysmenorrhea, anal fistula, hemorrhoid), urinary incontinence (e.g., unstable bladder, unstable sphincter), prostate disorders (e.g., BPK prostatitis, prostate cancer), recurrent infection (secondary to sphincter spasticity), and urinary retention (secondary to spastic sphincter, hyper- trophied bladder neck) and neurogenic bladder dysfunction (e.g., Parkinson's Disease, spinal cord injury, stroke, multiple sclerosis, spasm reflex). The present invention's composition may also be used in the treatment of spasmodic dysphonia, oromandibular dystonia, achalasia, anal fissures, vaginismus, focal hand dystonia, tremor, anal fissures, post-stroke spasticity, cerebral palsy or spastic bladder.

The pharmaceutical composition according to the invention can be used to treat one or more autoimmune disorders. Autoimmune disorders can be categorized into two general types: (1) systemic autoimmune diseases (i.e., disorders that damage many organs or tissues), and (2) localized autoimmune diseases (i.e., disor- ders that damage only a single organ or tissue). However, the effect of localized autoimmune diseases, can be systemic by indirectly affecting other body organs and systems

The autoimmune disorders treatable by the present methods include either or both of system autoimmune diseases or localized autoimmune diseases. Examples of autoimmune disorders treated by the present methods include one or more of any of the following rheumatoid arthritis which can affect joints, and possibly lung and skin; lupus, including systemic lupus erythematosus (SLE), which can affect skin, joints, kidneys, heart, brain, red blood cells, as well as other tissues and or- gans; scleroderma, which can affect skin, intestine, and lungs; Sjogren's syndrome, which can affect salivary glands, tear glands, and joints; Goodpasture's syndrome, which can affect lungs and kidneys; Wegener's granulomatosis, which can affect sinuses, lungs, and kidneys; polymyalgia rheumatica, which can affect large muscle groups; temporal arteritis/giant cell arteritis, which can affect arteries of the head and neck; Type 1 Diabetes Mellitus, which affects pancreas islets; Hashimoto's thyroiditis and Graves' disease, which affect the thyroid; celiac dis- ease, Crohn's diseases, and ulcerative colitis, which affect the gastrointestinal tract; multiple sclerosis (MS) and Guillain-Barre syndrome, which affect the central nervous system; Addison's disease, which affects the adrenal glands; primary biliary sclerosis, sclerosing cholangitis, and autoimmune hepatitis, which affect the liver; Raynaud's phenomenon, which can affect the fingers, toes, nose, ears; pernicious anemia; Addison's disease; dermatomyositis; myasthenia gravis (MG); Reiter's syndrome; Pemphigus vulgaris; scleroderma or CREST syndrome; autoimmune hemolytic anemia; autoimmune thrombocytopenic purpura; ankylosing spondylitis; vasculitis; and amyotrophic lateral schlerosis (Lou Gehrig's disease).

According to a further aspect of the present invention, the present invention provides a composition comprising the polypeptide, the active fragment and/or the derivative according to the invention or the composition according to invention or the peptide obtainable by the host cell as described above or as a product of the methods described above and optionally a cosmetically acceptable carrier, diluent and/or additive. Cosmetically acceptable carriers, diluents and/or additives are known to the person skilled in the art. The cosmetic indications include, but are not limited to hyperhydrosis and wrinkles. In fact, the compositions of the present invention may be used to relax any facial muscle.

In a preferred embodiment, the cosmetic composition is used to treat nasolabial folds:

These folds extend from the alae nasi to the lateral aspect of the lips. They can become deep, aging, or unattractive as a result of loss of subcutaneous tissue with age or lipoatrophy. Patients can benefit from injection of small amounts of the cosmetic composition of the present invention (such as 1 U) to each Hp elevator group of muscles — levator labii superiors alaeque nasi or zygomaticus major and minor. The position of the injections may be determined by examination of the facial movements of the patients and can be assisted by EMG-guided injections. Overdosing may lead to flattening of the lip and also lower facial asymmetry if injections are not placed into symmetrical positions. These problems can last for several weeks or months.

In another preferred embodiment, the cosmetic composition of the present invention is used to treat perioral lines:

Again, there are a number of causes of perioral lines, including photoaging, smoking, loss of subcutaneous tissue from the lower face, and reflective action of obi- cularis oris muscles. According to the teaching of the present invention, a treat- ment of the aforementioned causes is a treatment of perioral lines. The present invention's composition is particularly useful for patients with deep perioral lines worsened by lip "pursing." Preferably, a dose of up to 2 U is injected into each half of the upper lip. The total dose and position of injection is usually varied by patient examination before and during perioral muscle injection. The dose may also be slightly lower, i.e. 1 to 1.5 units per side into lower lip rhytides. Patients who have, and wish to retain a cupid bow of the upper lip should not be injected in the middle upper lip.

Perioral injection of the present invention's composition can be successfully used to improve the effects and duration of laser resurfacing, nonablative laser, light rejuvenation and perioral dermal fillers. Accordingly, the combination may be used in combination with said agents/techniques.

In another preferred embodiment, the cosmetic composition of the present inven- tion is used for mentalis (chin) puckering and mental creases:

The present invention's composition is very successful at decreasing these particular lower facial movements. In most patients, a single injection into the center of the mentalis muscle of 4 to 6 U will be effective.

In another preferred embodiment, the cosmetic composition of the present invention is used for neck lines and platysmal bands: In particular, vertical platysmal bands are very successfully treated with the present invention's composition. For administration, the patient is usually asked to hyperextend the neck. Either the injections can be made into the extended platysmal bands which has the advantage of being grasped between finger and thumb and the needle inserted vertically into the muscle band. The dose may be e.g. 2 to 4 U^" spaced 4 cm apart. Some patients cannot hyperextend for sufficient time and here the bands are marked with skin marker at the sites for injection and the composition is injected into the muscles at rest.

In another preferred embodiment, the cosmetic composition of the present invention is used to perform "Lower facial contouring" — masseteric injections: Recent observations have shown that the contour of the lateral cheek area can be flattened and the jaw line defined by injections of BOTOX to the Masseter muscle (M. Y. Park, Y. A. Ki and SJ. Duck, Botulinum toxin type A treatment for con- touring of the lower face. Dermatol Surg 29 (2003), pp. 477^83). This seems to be esthetically useful in the rounder-faced individuals and was described initially in a group of 45 Korean patients. Accordingly, lower facial contouring is a preferred application of the present invention's composition.

In another preferred embodiment, the present invention's composition is used to to treat glabellar frown lines:

For treatment of the glabella, 20 to 40 U may be injected into the procerus and/or the corrugator supercilli, often in a "V" distribution. The first injections are often placed directly above the medial canthus, 1 cm above each orbital rim, directly into the bellies of the corrugator supercilli muscles. The second injections are generally performed around 2 cm lateral to these and slightly superior, again at least 1 cm above the orbital rim.

The corrugators are strong muscles, and their activity can be eliminated without any adverse events. They can be especially strong in men and may require more toxin activity. Besides eliminating or reducing visible dynamic glabellar wrinkles, the present invention's polypeptide smoothes this area, reduces a stern or angry appearance, and elevates or "opens" the medial eye area by lifting the medial brow by as much as one or two millimeter, because all muscles in this location are depressors. Further elevation of the lateral eyebrows can be achieved by injecting 2 to 4 U at the lateral two thirds of each eyebrow. This injection is usually not performed in men, who typically have flatter, more horizontal eyebrows than women, as it would feminize their appearance.

In another preferred embodiment, the present invention's composition is used to to treat horizontal forehead lines: For treating horizontal forehead lines or wrinkles, 10 to 20 U of the present invention's composition may be divided into four to six evenly distributed injections, which is usually sufficient, especially if the glabella is also treated. The pattern may be varied for the shape and size of the brow. A simple technique for the standard forehead is to perform four injections horizontally across the middle of the forehead, extending laterally to the midpupillary line or just beyond it. Injecting 1 to 4 U lateral to the midpupillary line in the mid height of the forehead drops the lateral two thirds of the eyebrow, may eliminate or prevent a "BOTOX eyebrow," and is desirable in men because of their naturally flatter brows. Wider foreheads may require more than 4 injections across. Taller foreheads may be treated in more of a V-shaped pattern, or additional injections may be administered to the upper forehead. Applying pressure to the sites after injection can distribute the toxin further and is safe to do on the forehead.

In another preferred embodiment, the present invention's composition is used to to treat crow's feet:

Injections into this area are usually performed superficially, because the skin is extremely thin and deeper injections are especially prone to ecchymosis. A total of 12 to 40 U, divided equally between the two sides of the face, is usually effective in eliminating crow's feet wrinkles. A variety of techniques exist. A simple method is to divide the toxin among 3 injection sites, each 1 cm from the lateral orbital rim, the first into the midst of the wrinkles accentuated by smiling, then approximately 1 cm above and below this site.

In another preferred embodiment, the present invention's composition is used to treat lower eyelid orbicularis hypertrophy:

Lower eyelid orbicularis hypertrophy produces a prominence and elevation of the lower eyelid, resulting in an infraorbital crease with wide smiling. It closes the aperture of the eyelids, resulting in a diminished appearance of the eyes. A total of 3 to 15 U of the present invention's composition may be injected, preferably su- perficially, preferably divided among 1 or 2 sites along the lateral half of the lower eyelids 1 to 2 millimeter below the lid margin. Digital pressure may help to distribute the toxin.

Lower eyelid treatment also rounds and opens the eyes. Most patients actually do not mind the rounding of the eyes and like the eye-opening effect, long thought in the blepharoplasty literature to be an unwanted side effect. One study demonstrated that injection into the lateral orbital areas in combination with the lower eyelid was superior at widening the eye and reducing infraorbital wrinkles than injections in the lower eyelid alone.

In another preferred embodiment, the present invention's composition is used to treat overactive zygomaticus muscles:

When the zygomaticus major and minor muscles contract, such as with smiling, they contribute to the formation of crow's foot wrinkles and deepen the melolabial creases. Preferably a total of 5 to 15 U of the present invention's composition is divided into two injections at the upper lateral aspect of each cheek near the origin of these muscles. The result is softened infraorbital creases and melolabial folds.

In another preferred embodiment, the present invention's composition is used to treat lip pucker lines. In another preferred embodiment, the present invention's composition is used to treat down-turned mouth:

Patients who have strong depressor anguli oris muscles have down-turned oral commissures. The problem is accentuated with smiling. A total of 5 to 15 U of the present invention's composition, divided, may be injected into a site at each lower rim of the mandible, approximately 1 cm lateral to the oral commissures.

Any of the treatments described herein above may be combined with an injection with one or more filler substances. Filler substances are injected into or below the skin to replace lost volume or increase existing volume. The injection of filler agents is less invasive than the placement of implants, which may, however, also be used in combination with the present invention's composition. Most filler substances are not permanent Many different agents exist, all of which are in accordance with the present invention's teaching. In general, the most "physiologic" solutions tend to give the most natural and longest-lasting results; for example, fat is ideal for deep contour defects, and collagen or hyaluronic acid works well for more superficial defects. Ideal qualities of filler substances include the following: good correction, long-lasting effects, a natural look and feel, propensity not to migrate, ease of implantation, cost-effective, readily available, inert, noncarcino- genie, nonimmunogenic, sterile, and easy removable. No one substance has all of these qualities. As a result, many exist, and the physician must choose the ones best suited to the task at hand. For the purpose of the present invention, preferred fillers are made of protein (i.e. dermal collagen, fascia lata), polysaccharides (i.e. hyaluronic acid), fat, and synthetic agents. Each individual filler substance has unique applications, benefits, complications, and duration of action.

Typical fillers include: AlloDerm (LifeCell Corporation, The Woodlands, Tex), Artecoll (Rofil Medical International, Breda, The Netherlands), Cymetra (Obagi, Long Beach, Calif), Dermalogen (Collagenesis, Beverly, Mass), Fascian (Fascia Biosystems, Beverly Hills, Calif (processed by Medical Aesthetics International, Redmond, Wash), Gore-Tex (ePTFE) (WL Gore, Flagstaff, Ariz), Hylan B, HyIa- form (Biomatrix Incorporated, Ridgefield, NJ), Restylane (Q-Med Incorporated. Uppsala, Sweden) SoftForm (Kinamed Incorporated, Newbury Park, Calif), Zy- deππ I and II, Zyplast (McGhan Medical. Santa Barbara, Calif). Restylane®, Per- lane®, Belotero®, Teosyal®, Hydrafill® Softline and Softline Max® are further examples of fillers which may be used in accordance with the present invention.

This applications include wound healing, cosmetic surgery, orthopedic surgery, medical surgery and gynecologic surgery.

Further, the present invention provides a method for selecting a compound having (i) an increased or decreased affinity to its receptor compared to botulinum neurotoxin A produced by Clostridium botulinum;

(ii) an increased or decreased neurotoxicity in comparison to the native neurotoxin, preferably the neurotoxicity is determined in the hemidiaphragm assay; and/or (iii) a reduced affinity for neutralizing antibodies in comparison to the native neurotoxin, the method comprising the steps of:

(a) contacting the polypeptide, the active fragment or the derivative according to the invention with neural cell or a fragment thereof or isolated gan- glioside or synaptic vesicle protein or the phage according to the invention; and

(b) selecting the polypeptide having the desired activity and

(c) optionally repeating steps (a) and (b).

The method for selection of a compound displaying altered interactions with its receptors or neutralizing antibodies can be directed evolution which is a powerful tool for protein optimization. The most efficient methods combine multiple rounds of diversity generation and gene recombination with functional screening to identify improved variants. The iterative nature of this approach results in step- wise improvements in overall function, yielding substantial improvements in desired enzyme properties. Directed evolution can be performed in a variety of ways, which are distinguished, for example, by the approach to generating libraries from available diversity. The field of in vitro recombination-based directed evolution yields alternative gene libraries. E.g. a gene diversity library can be used to increase the affinity of the active fragment to its receptors. An SV2-active fragment based yeast display library can be generated by using spiked oligonucleotides to introduce mutations into the receptor binding sites of the active fragment and flow cytometry is employed for detection of increased interaction with its protein receptor.

Claims

1. A polypeptide, or an active fragment or derivative thereof, wherein the poly- peptide comprises a stretch of amino acids having an amino sequence corresponding to the amino acid sequence of botulinum neurotoxin of type A at positions 1092 to 1296, wherein at least one amino acid position selected from the group consisting of 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1 100, 1101, 1 102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 11 13, 1114, 1 115, 11 16, 11 17, 1 118, 1119, 1120, 1121, 1122, 1 123, 1124, 1 125, 1126, 1127, 1 128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1 136, 1 137, 1 138, 1139, 1 140, 1141, 1 142, 1143, 1144, 1 145, 1 146, 1 147, 1 148, 1 149, 1 150 1 151, 1152, 1 153, 1 154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1 164, 1165, 1 166, 1 167, 1168, 1169, 1 170, 1 171, 1 172, 1173, 1 174, 1175, 1 176, 1177, 1178, 1 179, 1 180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1 189, 1 190, 1191, 1192, 1193, 1194, 1195, 1 196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295 and/or 1296 of said sequences is added, deleted, inserted and/or substituted by a naturally or non-naturally occurring amino acid, wherein the naturally occurring amino acid is selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophane, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid, wherein (i) the polypeptide or the fragment or the derivative binds with an increased affinity to its receptor;

(ii) the polypeptide or the fragment or the derivative has an increased or decreased neurotoxicity in comparison to the native neurotoxin, preferably the neurotoxicity is determined in the hemidiaphragma assay; and/or

2. The polypeptide according to claim I, wherein the botulinum neurotoxin A is selected from neurotoxin Al, A2, A3 or A4, preferably the botulinum neurotoxin

A is Al (Genbank accession number AAA23262).

3. The polypeptide according to claim 1 or 2, wherein in said stretch not more than 50, preferably not more than 25, more preferably not more than 10, particu- larly preferred not more than 5, in particular one, two, three or four amino acids, are added, deleted, inserted and/or substituted.

4. The polypeptide according to any one of claims I to 3, wherein at least one amino acid at a position selected from the group consisting of 1117, 1202, 1203, 1204, 1252, 1253, 1254, 1262, 1263, 1264, 1265, 1266, 1267, 1270, 1278 and/or 1279 of the botulinum neurotoxin A protein sequences is added, deleted, inserted and/or substituted by a naturally or non-narurally occurring amino acid and the naturally occurring amino acid is selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophane, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid.

5. The polypeptide according to claim 4, wherein the amino acid phenylalanine at position 1252 is substituted by histidine and/or the amino acid phenylalanine at position 1278 is substituted by histidine or tyrosine and/or the amino acid leucine at position 1278 is substituted by histidine, phenylalanine or tyrosine.

6. The polypeptide according to claim 4, wherein two, three or four amino acids at amino acid positions selected from the group consisting of 1117, 1252.1253 and 1278 of the botulinum neurotoxin A protein sequences are substituted, preferably the double substitutions are selected from the group consisting of

Yl 117F/L1278F, F1252H/L1278F, Yl 117A/F1252H, Yl 117A/L1278F, Y1117A/L1278Y,Y1117C/F1252H,Y1117C/L1278F,Y1117C/L127SY, Yl 117V/F1252H, Yl 117V/L1278F and Yl 117V/L1278Y, preferably the triple substitutions are selected from the group consisting of Yl 117F/F1252H/L1278F, Yl 117A/F1252H/H1253K,

Y1117A/F1252H/L1278F,Y1117A/F1252H/L1278H, YIl 17A/F1252H/L1278Y, Yl 117A/F1252Y/L1278F, Yl 117A/F1252Y/L1278H, Yl 117A/F1252Y/L1278Y, Yl 117C/F1252H/H1253K, Yl 117C/F1252H/L1278F, YIl 17C/F1252H/L1278H, Yl 117C/F1252H/L1278Y,

Y1117C/T1252Y/L1278F,Y1117C/F1252Y/L1278H, Y1117C/F1252Y/L1278Y,Y1117V/F1252H/H1253K, Y1117V/F1252H/L1278F,Y1117V/F1252H/L1278H, Y1117V/F1252H/L1278Y,Y1117V/F1252Y/H1253K, Yl 117V/F1252Y/L1278F, Yl 117V/F1252Y/L1278H,

Y1117V/Fl252Y/L1278YandY1117V/V1262I/L1278F, preferably the quadruple substitutions are selected from Yl 117A/F1252H/H1253K/L1278F, Yl 117A/F1252R/H1253K/L1278H, Y1117A/F1252PLΗ1253K/L1278Y₎Y1117A/F1252Y/H1253K/L1278F, Y1117A/F1252Y/H1253K/L1278H, Y1117A/F1252Y/H1253K/L1278Y,

Y1117C/F1252H/H1253K/L1278F₍Y1117C/F1252H/H1253K/L1278H, Y1117C/F1252H/H1253K/L1278Y,Y1117C/F1252Y/H1253K/L1278F, Y1117C/F1252Y/H1253K/L1278H, YU17C/F1252Y/Η1253K/L1278Y Y1117V/F1252H/H1253K/L1278F,Y1117V/F1252H/H1253K/L1278H, Y1117V/F1252H/H1253K/L1278Y,Y1117V/F1252Y/H1253K/L1278F, Yl 117V/F1252Y/H1253K/L1278H and

Yl 1 17V/F1252Y/H1253K7L1278Y, particularly preferred are the following combination of substitutions:

Yl 1 17V/F1252H, Yl 1 17V/L1278F, Yl 117V/F1252H/H1253K, Yl 1 17V/F1252H/L1278F, Yl 117V/F1252H/L1278H,

Yl 1 17WF1252H/L1278Y, Yl 117V/F1252Y/H1253K, Y11 17V/F1252Y/L1278F, Y1 117V/F1252Y/L1278H, Yl 1 17V/F1252Y/L1278Y, Yl 117V/F1252H/H1253K/L1278F, Yl 1 17V/F1252Y/H1253K/L1278F_and Y1 1 17WF1252Y/H1253K/L1278H.

7. The polypeptide according to any one of claims 1 to 3, wherein at least one amino acid at an amino acid position selected from the group consisting of 1126, 1127, 1129, 1130, 1131, 1 133, 1 135, 1 137, 1 195, 1 196, 1 199, 1200, 1201, 1204, 1205, 1206, 1207, 1209, 1213, 1215, 1216, 1217, 1255, 1256, 1258 and/or 1260 of the botulinum neurotoxin A protein sequences is added, deleted, inserted and/or substituted by a naturally or non-naturally occurring amino acid and wherein the naturally occurring amino acid is selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophane, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid.

8. The polypeptide according to claim 7, wherein at least one amino acid at an amino acid position selected from the group consisting of 1 126, 1127, 1129, 1130, 1 131, 1133, 1135, 1137, 1200, 1201, 1215 and/or 1216 of the botulinum neurotoxin A protein sequences is deleted and/or substituted by a naturally or non- naturally occurring amino acid and wherein the naturally occurring amino acid is selected from glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tryptophane, serine, threonine, asparagine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid.

9. The polypeptide according to any one of claims 1 to 3, wherein the polypeptide has at least one mutation in the ganglioside binding pocket and at least one mutation in the protein receptor binding pocket.

10. The polypeptide according to claim 9, wherein (i) at least one amino acid at an amino acid position selected from 1 1 17, 1202, 1203, 1204, 1252, 1253, 1254, 1262, 1263, 1264, 1265, 1266, 1267, 1270, 1278 and/or 1279 of the botulinum neurotoxin A protein sequences is added, deleted, inserted and/or substituted by a naturally or non-narurally occurring amino acid, and (ii) at least one amino acid at an amino acid position selected from the group consisting of 1 126, 1 127, 1 129, 1130, 1131, 1133, 1135, 1137, 1195, 1196, 1199, 1200, 1201, 1204, 1205, 1206, 1207, 1209, 1213, 1215, 1216, 1217, 1255, 1256, 1258 and/or 1260 of the borulinum neurotoxin A protein sequences is added, deleted, inserted and/or substituted by a naturally or non-narurally occurring amino acid, wherein the natu- rally occurring amino acid is selected from glycine, alanine, valine, leucine, iso- leucine, methionine, proline, phenylalanine, tryptophane, serine, threonine, aspar- agine, glutamine, tyrosine, cysteine, lysine, arginine, histidine, aspartic acid and glutamic acid.

11. The polypeptide according to claim 10, wherein (i) at least one amino acid at an amino acid position selected from the group consisting of 1117, 1252, 1253 and/or 1278 of the borulinum neurotoxin A protein sequences is deleted and/or substituted by a naturally or non-narurally occurring amino acid, and (ii) at least one amino acid at an amino acid position selected from 1195, 1196, 1199, 1204, 1205, 1206, 1207, 1209, 1213, 1217, 1255, 1256, 1258 and/or 1260 of the borulinum neurotoxin A protein sequences is deleted and/or substituted by a naturally or non-narurally occurring amino acid.

12. The polypeptide according to claim 11 , wherein (i) at least one amino acid at an amino acid position selected from 11 17, 1252, 1253 and/or 1278 of the botulinum neurotoxin A protein sequences is deleted and/Or substituted by a natu- rally or non-naturally occurring amino acid, and (ii) at least one amino acid at an amino acid position selected from 1196, 1 199, 1205, 1207, 1255, 1258 and/or 1260 of the botulinum neurotoxin A protein sequences is deleted and'or substituted by a naturally or non-naturally occurring amino acid. 5

13. The polypeptide according to any one of claims 1 to 12, wherein the neutralising antibody inhibits the binding of the native neurotoxin to the protein receptor or to the ganglioside receptor and/or inhibits the uptake of the neurotoxin into the neural cell.

I O

14. The polypeptide according to any one of claims 1 to 13, wherein the polypeptide is taken up by the cells via endocytosis.

15. The polypeptide according to any one of claims 1 to 14, wherein the polypep- 15 tide binds specifically to molecules associated with the plasma membrane, transmembrane proteins, synaptic vesicle proteins, proteins of the synaptotagmin family or synaptic vesicle glycoproteins 2 (SV2) and/or synaptotagmin I and/or synaptotagmin II and/or SV2A, SV2B or SV2C, preferably to human synaptotagmin I and/or human synaptotagmin II and/or human SV2A, SV2B or SV2C.

20

16. The polypeptide according to any one of claims 1 to 15, wherein the polypeptide or the active fragment or the derivative has an increase in affinity for the neural cells of at least 15 % compared to the native neurotoxin, preferably at least 50 %, particularly preferred at least 80 %, in particular at least 90 %.

25

17. The polypeptide according to any one of claims 1 to 15, wherein the binding of the polypeptide, the active fragment or the derivative to its receptor is increased by at least 15 %. preferably at least 50 %, particularly preferred at least 80 %, in particular at least 90 %.

30

18. The polypeptide according to any one of claim I to 15, wherein the affinity for neutralising antibodies compared to the native neurotoxin of the polypeptide, the active fragment or the derivative is reduced by at least 15 %. preferably at least 50 %, particularly preferred at least 80 %, in particular at least 90%.

19. The polypeptide according to claim 1 , wherein the active fragment or the derivative consists of up to 10, preferably up to 20, more preferably up to 50. in particular up to 100 or up to 200 amino acids.

20. The polypeptide according to claim 19, wherein the active fragment comprises the complete ganglioside binding pocket and/or the complete protein receptor binding pocket, preferably, said ganglioside binding pocket is composed of aa 11 17 and aa 1202 to aa 1279 and said protein receptor binding pocket consists of aa 1 126 to 1260.

21. The polypeptide according to claim 1, wherein the derivative is modified by crosslinking or side-chain modifications.

22. A composition comprising the polypeptide, the active fragment and/or the derivative according to any one of claims 1 to 21 and optionally an intervening molecule.

23. The composition according to claim 22, wherein the intervening molecule is covalently bound to the polypeptide, the active fragment or the derivative by a peptide bond, an ester bond, an ether bond, a sulphide bond, a disulphide bond, a carbon-carbon bond.

24. The composition according to claim 22 or 23, wherein the intervening molecule is a small organic molecule, a peptide or a protein.

25. The composition according to any of claims 22 or 24, wherein the small organic molecule is a virostatic agent, cytostatic agent, an antibiotic or an immunoglobulin.

26. The composition according to claim 25, wherein the protein is a protease.

27. The composition according to claim 26, wherein the protease comprises one or more of the light chains (LC) of the serotypes A, B, Cl, D, E, F and/or G of Clostridium botulinum.

28. The composition according to claim 27, wherein the protease comprises a pro- teolytically active fragment which is derived from the light chain (LC) of the serotypes A, B, Cl, D, E, F and/or G of the Clostridium botulinum neurotoxin and characterized in that it has at least 0.01 % of the proteolytic activity of the native protease, preferably at least 50 %.

29. The composition according to any of claims 26 to 28, wherein the protease cleaves specifically particular substrates in a cholinergic motoneuron.

30. The composition according to claim 29, wherein the substrates are selected from proteins being involved in the release of neurotransmitters in neural cells and proteins being capable of catalytic reactions in a neural cell.

31. The composition according to claim 30, wherein the protease and the polypeptide are covalently bound via an amino acid sequence which is recognized and cleaved specifically by an endopeptidase.

32. The composition according to claim 31, wherein upon cleavage by the endopeptidase a disulphide bridge links the protease and the polypeptide which leads to the formation of an active holotoxin.

33. A recombinant expression vector comprising a nucleic acid capable of encoding the polypeptide, the active fragment or the derivative according to any one of claims 1 to 21, preferably the expression vector is a linear sequence, a transposon, a plas- mid-based expression vector, a virus or a phage.

34. A recombinant host cell comprising the expression vector according to claim 33 being capable of expressing the polypeptide, the active fragment or the derivative under suitable culruring conditions.

35. The recombinant host cell according to claim 34, wherein the host cell may be selected from the group consisting of Escherichia coli, in particular E. coli Kl 2, Sac- choromyces cerevisiae, Pichia pastoris, Bacillus megaterium, Clostridium bu- tyricum, Clostridium barati, Clostridium botulinum, Clostridium tetani and Clostridium hystolyticum.

36. A method for producing the polypeptide, the active fragment or the derivative according to any one of claims 1 to 21 comprising the steps of

(a) providing the cell of claim 34 or 35;

(b) culturing the cell under conditions suitable for recombinant expression; and (c) recovering and optionally purifying the polypeptide, the active fragment or the derivative from the cell or the supernatant.

37. A method for producing the polypeptide, the active fragment or the derivative according to any one of claims 1 to 21 comprising the steps of (a) preparing the polypeptide, the active fragment or the derivative by chemical methods; and

(b) recovering and optionally purifying the polypeptide, the active fragment or the derivative.

38. A pharmaceutical composition comprising the polypeptide, the active fragment and/or the derivative according to any of claims 1 to 21 or the composition according to any of claims 22 to 32 or the polypeptide obtainable from the cell of claim 34 or 35 or the product of the method of claim 36 or 37 and optionally a pharmaceutically acceptable carrier, diluent and/or additive.

39. Use of the polypeptide, the active fragment and/or the derivative according to anyone of claims 1 to 21 or the composition according to any one of claims 22 to 32 or the peptide obtainable from the cell of claim 34 or 35 or the product of method of claim 36 or 37 for the preparation of a pharmaceutical composition for the treatment of disorders and diseases for which a therapy with botulinum neurotoxin is indicated.

40. Use according to claim 39, wherein the disorder or disease is selected from the group consisting of hemifacial spasm, spasmodic torticollis (cervical dystonia), blepharospasm, migraine, pain, diseases of the cervical and lumbar spine, stra- bism, hypersalivation, snoring, wound healing, depression, hyperhidrosis, low back pain, strabismus, urological conditions rooted in a spastic dysfunction of the sacral reflex arcs such as pelvic pain (e.g., interstitial cystitis, endometriosis, prostatodynia, urethral instability syndromes, hyperactive bladder), pelvic myofascial elements (e.g., levator sphincter, dysmenorrhea, anal fistula, hemorrhoid), urinary incontinence (e.g., unstable bladder, unstable sphincter), prostate disorders (e.g., BPK prostatitis, prostate cancer), recurrent infection (secondary to sphincter spasticity), and urinary retention (secondary to spastic sphincter, hypertrophied bladder neck), neurogenic bladder dysfunction (e.g., Parkinson's Disease, spinal cord injury, stroke, multiple sclerosis, spasm reflex), spasmodic dysphonia, oro- mandibular dystonia, achalasia, anal fissures, vaginismus, focal hand dystonia, tremor, anal fissures, post-stroke spasticity, cerebral palsy or spastic bladder.

41. Use according to claim 39, wherein the disease is an autoimmune disorder including either or both of system autoimmune diseases or localized autoimmune diseases, preferably the autoimmune disorder includes one or more of any of the following rheumatoid arthritis which can affect joints, and possibly lung and skin; lupus, including systemic lupus erythematosus (SLE), which can affect skin, joints, kidneys, heart, brain, red blood cells, as well as other tissues and organs; scleroderma, which can affect skin, intestine, and lungs; Sjogren's syndrome, which can affect salivary glands, tear glands, and joints; Goodpasture's syndrome, which can affect lungs and kidneys; Wegener's granulomatosis, which can affect sinuses, lungs, and kidneys; polymyalgia rheumatica, which can affect large muscle groups; temporal arteritis/giant cell arteritis, which can affect arteries of the head and neck; Type I Diabetes Mellitus, which affects pancreas islets; Hashimoto's thyroiditis and Graves' disease, which affect the thyroid; celiac disease, Crohn's diseases, and ulcerative colitis, which affect the gastrointestinal tract; multiple sclerosis (MS) and Guillain-Barτe syndrome, which affect the central nervous system; Addison's disease, which affects the adrenal glands; primary biliary sclerosis, sclerosing cholangitis, and autoimmune hepatitis, which affect the liver; Raynaud's phenomenon, which can affect the fingers, toes, nose, ears; pernicious anemia; Addison's disease; dermatomyositis; myasthenia gravis (MG); Reiter's syndrome; Pemphigus vulgaris; scleroderma or CREST syndrome; autoimmune hemolytic anemia; autoimmune thrombocytopenic purpura; ankylosing spondylitis; vasculitis; and amyotrophic lateral schlerosis (Lou Gehrig's disease).

42. A cosmetic composition comprising the polypeptide, the active fragment and/or the derivative according to any of claims 1 to 21 or the composition according to any of claims 22 to 32 or the polypeptide obtainable from the cell of claim 34 or 35 or the product of the method of claim 36 or 37 and optionally a cosmetically acceptable carrier, diluent and/or additive.

43. Use of the composition of claim 38 for cosmetic treatment.

44. Use according to claim 43 for the treatment of the cosmetic indications hyperhy- drosis and wrinkles.

45. A method for selecting a compound having (i) an increased affinity to its receptor neural cells compared to botulinum neurotoxin A produced by Clostridium botulinum; (ii) an increased or decreased neurotoxicity in comparison to the native neurotoxin, preferably the neurotoxicity is determined in the hemidiaphragm assay; and/or (iii) a reduced affinity for neutralising antibodies in comparison with the native neurotoxin, the method comprising the steps of (a) contacting the polypeptide, the active fragment or the derivative according to any one of claims 1 to 21 with a neural cell or a fragment thereof or an isolated gan- glioside or synaptic vesicle protein or the phage set forth in claim 33; and

(b) selecting the polypeptide having the desired activity, and

(c) optionally repeating steps (a) and (b).