EP1874803A2

EP1874803A2 - Carrier for targeting nerve cells

Info

Publication number: EP1874803A2
Application number: EP06724595A
Authority: EP
Inventors: Andreas Rummel; Tanja Weil; Aleksandrs Gutaits
Original assignee: Merz Pharma GmbH and Co KGaA; Toxogen GmbH
Current assignee: Merz Pharma GmbH and Co KGaA; Toxogen GmbH
Priority date: 2005-04-26
Filing date: 2006-04-26
Publication date: 2008-01-09
Also published as: EA200702323A1; HUE044200T2; WO2006114308A2; EP3181578A1; ES2723723T3; MX2007013284A; DK3181578T3; US20150038401A1; EP3511338A2; US10266816B2; US20130315888A1; DE102005019302A1; EP3181578B1; US20090311275A1; KR20080014754A; EP2345666A1; EP3511338A3; ZA200709166B; BRPI0610252A2; AU2006239506A1

Abstract

The present invention relates to a transport protein which can be obtained by modifying the heavy chain of the neurotoxin formed by Clostridium botulinum wherein (i) the protein binds specifically to nerve cells with a higher or lower affinity as the native neurotoxin; (ii) the protein has an increased or reduced neurotoxicity compared to the native neurotoxin, the neurotoxicity being preferably determined in the hemidiaphragma assay; and/or (iii) the protein comprises a lower affinity against neutralizing antibodies compared to the native neurotoxin. The invention also relates to methods for producing the same and the use thereof in cosmetic and pharmaceutical compositions.

Description

Carrier for targeting nerve cells

The present invention relates to a transport protein that binds to higher or lower affinity neurons than the neurotoxin produced by Clostridium botulinum. The transport protein is preferably taken up by receptor-mediated endocytosis. This protein finds utility as a transporter that translocates other chemical substances (e.g., proteases) from the acidic endosomal compartment into the cytosol of neurons that can not penetrate physiologically through the plasma membrane into the cytosol of nerve cells. In particular, the present invention relates to the use of a transport protein for the introduction of inhibitors of neurotransmitter secretion.

Nerve cells release transmitter substances through exocytosis. Exocytosis refers to the fusion of the membranes of intracellular vesicles with the plasma membrane. In this process, the vesicular content is simultaneously released into the synaptic cleft. The fusion of the two membranes is regulated by calcium, which reacts 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 vesicle membranes, SNAP-25 is only weakly bound to the plasma membrane. As the intracellular calcium concentration increases, the three proteins bind to each other, with both membranes approaching and then fusing together. Cholinergic neurons release acetylcholine, which causes muscle contractions, sweating, and other cholinergic-mediated reactions. •. The above-mentioned fusion proteins are the target molecules (substrates) of the light chain (LC) of the clostridial neurotoxins formed by the bacteria C. botulinum, C. butyricum, C. baratii and C. tetani.

The anaerobic, gram-positive bacterium C. botulinum produces seven different serotypes of clostridial neurotoxins. These are referred to as the botulinum neurotoxins (BoNT / A to BoNT / G). Of these, BoNT / A and BoNT / B in particular cause a neuroparalytic disorder in humans and animals called botulism. The spores of C. botulinum are found in the soil, but may develop into improperly sterilized and sealed home-grown canned foods, which are responsible for many of the botulism cases.

BoNT / A is the most active of all known biological substances. Only about 5-6 pg of purified BoNT / A represents an MLD (median lethal dose). A Unit (U) of BoNT / A is defined as the MLD, which after intraperitoneal injection kills half of female Swiss Webster mice weighing 18-20 g each. Seven immunologically different BoNTs were characterized. They bear the names BoNT / A, B, Cl, D, E, F and G and can be distinguished by neutralization with serotype-specific antibodies. The different serotypes of BoNT differ in affected species with regard to the severity and duration of the caused paralysis. So z. B. in the rat with regard to the paralysis BoNT / A 500 times more effective than BoNT / B. In addition, BoNT / B has been found to be non-toxic in primates at a dose of 480 U / kg body weight. The same amount of BoNT / A is equivalent to 12 times the lethal dose of this substance in primates. On the other hand, in mice the paralysis time after injection of BoNT / A is 10 times longer than after injection of BoNT / E.

The BoNTs are used to treat neuromuscular disorders caused by hyperactivity in skeletal muscle caused by pathologically overactive peripheral nerves are characterized. BoNT / A is approved by the US Food and Drug Administration for the treatment of blepharospasm, strabismus, hyperhidrosis, wrinkles and hemifacial spasm. Compared with BoNT / A, the remaining BoNT serotypes apparently have a lower potency and shorter duration of action. Clinical effects of peripheral intramuscularly administered BoNT / A usually occur within one week. The duration of symptom suppression by a single intramuscular injection of BoNT / A is usually about three to six months.

10 The clostridial neurotoxins specifically hydrolyse different proteins of the fusion apparatus. BoNT / A, Cl and E cleave SNAP-25, while BoNT / B, D, F, G and tetanus neurotoxin (TeNT) attack the vesicle-associated membrane protein (VAMP) 2 - also called synaptobrevin 2. BoNT / Cl also splits syntaxin IA.

! 5

The Clostridia bacteria release the neurotoxins as single-chain polypeptides, each with 1251 to 1315 amino acids. Subsequently, endogenous proteases of each of these proteins cleave at a given site in two chains ('nicking'), but the two chains remain connected by a disulfide bridge. These two-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 segment, the light chain (LC), represents a Zn ²⁺ -dependent endoprotease, the heavy chain (HC) is the light chain transporter. Treatment of the HC 5 with endopeptidases gave rise to two 50 kDa fragments (see Gimenez et al., 1993, J. Protein Chem. 12, 351-363). The amino-terminal half (H _N fragment) integrates into membranes at low pH and translocates the LC into the cytosol of the nerve cell. The carboxyl-terminal half (Hc fragment) binds to complex polysialogangliosides which occur only in nerve cell membranes, and to protein receptors which have hitherto been only partially identified (Halpern et al., 1993, Curr Top Microbiol Immunol 195, 221-241 ). This explains the high neuroselectivity of clostridial neurotoxins. Crystal structures confirm that BoNT / A has three domains that can be reconciled with the three steps of the mechanism of action (see Lacy et al., 1998, Nat. Struct. Biol. 5: 898-902). Furthermore, these data suggest that within the Hc fragment there are two autonomous subunits (subdomains) of 25 kDa each. The first evidence for the existence of the two functional subdomains was provided by the amino-terminal (H _CN ) and carboxyl-terminal half (Hcc) of the TeNT Hc fragment, which were expressed in a recombinant fashion and revealed that, although the Hcc but not the Hc _N domain binds to neurons (see Herreros et al (2000), Biochem J. 347, 199-204). At a later date, a single ganglioside binding site was located and characterized within the Hcc domains of BoNT / A and B (see Rummel et al., (2004), Mol. Microbiol. 51, 631-643). The site for the binding of the synaptotagmines I and II identified as protein receptor for BoNT / B and G could also be limited to the range of the Hcc domains of BoNT / B and G (see Rummel et al., 2004, J. Biol. Chem. 279, 30865-70). However, the document does not disclose the amino acids relevant to the binding pocket of BoNT / B and G.

Under physiological conditions, the HC binds with the Hc fragment to neuronal gangliosides, is taken up by the receptor-mediated endocytosis into the cell interior and reaches the natural vesicle circulation via the endosomal compartment. In the acidic environment of the early endosomes, the H _N fragment invades the vesicle membrane and forms a pore. Any substance (X) that is linked to the HC through a disulfide bridge will be separated from the HC by intracellular redox systems that gain access to and reduce the disulfide bond. Ultimately, X will appear in the cytosol.

In the case of clostridial neurotoxins, the HC is the carrier of an LC, which in the final step cleaves its specific substrate in the cytosol. The cycle of complex formation and dissociation of the fusion proteins is interrupted and thus inhibited the release of acetylcholine. As a result, striped muscles are paralyzed, and sweat glands cease their secretion. The duration of action of the individual BoNT serotypes varies and depends on the presence of intact LC in the cytosol. Since all neurons have receptors for clostridial neurotoxins, it is not only the release of acetylcholine that may be affected but also potentially the release of substance P, norepinephrine, GABA, glycine, endorphin, and other transmitters and hormones.

The preferential blocking of cholinergic transmission can be explained by the HC entering the neuron at the periphery. Central synapses are protected by the blood-brain barrier, which proteins can not overcome.

In a ligand-receptor study, certain amino acid residues within the Hcc domain of BoNT / B and G were exchanged to identify and characterize the binding pocket of the protein receptor so as to alter the affinity to the protein receptor. The affinity of the mutated Hc fragments of BoNT / B and G was determined in glutathione S-transferase (GST) pulldown experiments with synaptotagmin. Then the HC was coupled with the same mutations to LC-B and LC-G, respectively. The potency of these constructs was analyzed with the aid of the mouse isolated nerve-muscle preparation (HDI). This preparation contains the phrenic nerve, which consists of cholinergic motor neurons and is the most important physiological target of clostridial neurotoxins. Subsequently, single amino acids were exchanged in the Hcc domain of BoNT / A in a well located analogously to the protein receptor binding pockets in BoNT / B and G. The full-length BoNT / A single mutants were then also analyzed for altered potency in HDA, indicating evidence for altered ligand-protein receptor interactions. The BoNT / A complex, also called progenitor toxin A, has been used in the recent past to treat motor dystonia, as well as to attenuate excessive sympathetic activity (see Benecke et al., 1995, Act Neurol., 22, 209ff) and for alleviation 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 in a few minutes. The resulting neurotoxin is the only component of the complex that is therapeutically relevant and causes symptom relief. Since the underlying neurological disease is not cured, the complex must be re-injected at intervals of three to four months. Depending on the amount of injected foreign protein, some patients will form specific BoNT / A antibodies. These patients become resistant to the neurotoxin. Once antigen-sensitive cells have recognized the neurotoxin and antibodies have been generated, the memory cells in question will remain for years. Therefore, it is important to treat the patients with drugs of highest activity in the lowest possible dosage. The preparations should also contain no further proteins of bacterial origin, since they can act as immune adjuvants. Such substances attract macrophages that recognize both the immune adjuvants and the neurotoxins and present to the lymphocytes, which then respond to the formation of immunoglobulins. Consequently, only products of the highest purity, which do not contain foreign proteins, should be used for therapy. The resistance of the patients to the neurotoxin is based, on a molecular level, predominantly on the presence of neutralizing antibodies.

With the present invention, a transport protein (Trapo) is now presented, which can overcome the above-described problems of the previously known methods. This object has been achieved with a novel transport protein obtainable by modifying the heavy chain of the neurotoxin produced by Clostridium botulinum, wherein

(i) the protein binds to higher or lower affinity nerve cells than the native neurotoxin;

(ii) the protein has increased or decreased neurotoxicity compared to the native neurotoxin; preferably, the neurotoxicity is determined in the hemidiaphragm assay; and or

(iii) the protein has a lower affinity for neutralizing antibodies compared to the native neurotoxin.

According to a preferred embodiment of the present invention, there is provided a transport protein which binds to nerve cells with higher or lower affinity than the native neurotoxin produced by C. botulinum.

According to another preferred embodiment of the present invention, there is provided a transport protein obtained by modifying the HC of the neurotoxin produced by C. botulinum, wherein the protein having higher or lower affinity than the native neurotoxin binds specifically to nerve cells. Preferably, the transport protein from these cells is taken up by endocytosis.

According to a further preferred embodiment, a transport protein is also obtained which is obtained by modifying the HC of the neurotoxin formed by C. botulinum, the protein no longer being accessible by exchanging surface-exposed amino acids, in particular at the ganglioside and protein receptor binding pockets of the binding neutralizing antibodies is. Hereinafter, terms are defined as they are to be understood in the context of the present application.

"Native neurotoxin is the native neurotoxin of C. botulinum, preferably the botulinum neurotoxin A and / or botulinum neurotoxin B and / or botulinum neurotoxin G from C. botulinum The recombinantly produced botulinum neurotoxin from E. coli, which contains, among other things, the amino acid sequence identical to the native botulinum neurotoxin, behaves pharmacologically identically to the native botulinum neurotoxin and is called recombinant botulinum neurotoxin wildtype Preferably, the transport protein specifically binds to plasma membrane associated molecules, transmembrane proteins, synaptic vesicle proteins, a synaptotagin family protein or synaptic vesicle glycoproteins 2 (S V2), preferably synaptotagmin I and / or synaptotagmin II and / or SV2A, SV2B or SV2C, more preferably human synaptotagmin I and / or human synaptotagmin II and / or human SV2A, SV2B or SV2C. The binding is preferably determined in vitro. Most preferably, the determination is made by using a GST pull-down assay detailed in the Examples.

"The protein has increased or decreased neurotoxicity compared to the native neurotoxin. The native neurotoxin is the native novotoxin of C. botulinum. The native neurotoxin is preferably the botulinum neurotoxin A and / or botulinum neurotoxin B and / or botulinum neurotoxin G from C. botulinum. The recombinantly produced botulinum neurotoxin from E. coli, which contains, among other things, the amino acid sequence identical to the native botulinum neurotoxin, behaves pharmacologically identically as the native botulinum neurotoxin and is called recombinant botulinum neurotoxin wild type. The nerve cells mentioned here are cholinergic motor neurons. The neurotoxicity is preferably determined by the hemi-diaphragm assay (HDA) known in the art. The neurotoxicity of the muteins can preferably be determined as described by Habermann et al., Naunyn Schmiedeberg's Arch. Pharmacol. 31 1 (1980), 33-40.

"Neutralizing Antibodies" Botulinum neurotoxin-targeting neutralizing antibodies are known (Göschel H, Wohlfarth K, Frevert J, Dengler R, Bigalke H. Botulinum A toxin therapy: neutralizing and non-neutralizing antibodies-therapeutic consequences, Exp. Neurol 1997 Sep; 147 (1997); l): 96-102.) Neurotoxin neutralizing antibodies have been found to interact particularly with the active sites such as the ganglioside and protein receptor binding pockets within the Hcc domain of the neurotoxin, and in the neurotoxin, the surfaces surrounding the binding pockets are altered by amino acid substitutions Negatively affecting functionality, the neutralizing antibodies lose their binding sites and the mutated neurotoxin is no longer neutralized.

The term "modification of the heavy chain of the neurotoxin produced by C. botulinum." The amino acid and / or nucleic acid sequence of the heavy chain (HC) of the neurotoxin produced by C. botulinum are generally available from publicly available databases for each of the known serotypes A. to G (see also Table 1). Modification here comprises that at least one amino acid is deleted, added, inserted into the amino acid sequence, or at least one amino acid of the native neurotoxin is substituted by another natural or non-naturally occurring amino acid and / or Post-translational modifications include glycosylations, acetylations, acylations, deaminations, phosphorylations, isoprenylations, glycosylphosphatidylinositolations, and other modifications known to the person skilled in the art. The HC of the neurotoxin produced by C. botulinum comprises three subdomains, namely the 50 kDa amino terminal translocation domain H _N , the subsequent 25 kDa Hc _N domain and the carboxyl terminal 25 kDa Hcc domain. In summary, the H _CN and H _C c domains are referred to as the Hc fragment. The corresponding amino acid sections of the respective subdomains can be seen for the individual serotypes and their variants from Table 1.

"Ganglioside Receptor" The HCs of the botulinum neurotoxins have a high affinity for peripheral nerve cells, which are predominantly mediated by the interaction with complex polysial gangliosides - these are glycolipids consisting of more than one sialic acid (Halpern et al. (1995), Curr., Top Microbiol Immunol 195, 221-41, WO 2006/02707) Thus, the LCs bound to them only reach this cell type and become effective only in these cells BoNT / A and B bind only one molecule of ganglioside GTIb.

In the case of BoNT / B and BoNT / G, the protein receptors are synaptotagmin I and synaptotagmin II. In the case of BoNT / A, the protein receptors are the synaptic vesicles glycoproteins 2 (SV2), preferably SV2A, SV2B and SV2C.

At present, 13 isoforms from the family Synaptotagmine known. All are characterized by two carboxyl-terminal Ca ²⁺ binding C2 domains, a median transmembrane domain (TMD) anchoring the synaptotagmin in the synaptic vesicle membrane, and a different amino terminus. After Ca ²⁺ influx, the fusion of the synaptic vesicle with the plasma membrane is initiated, whereupon the intraluminal amino terminus of synaptotagmine is extracellularly presented and used as a receptor anchor for BoNT / B and G is available. Similarly, the fourth luminal domain of SV2 isoforms after exocytosis is available extracellularly for interaction with BoNT / A.

By targeting mutagenesis, individual amino acids of the binding pocket have been altered in their character such that binding to a protein receptor is impeded or prevented. For this purpose, the Hc fragments of BoNT / B and BoNT / G were recombinantly expressed as wild type or with single amino acid substitutions (mutations / substitutions) in the postulated binding pocket in E. coli and isolated. For a GST pull-down assay to study the in vitro interaction between BoNT / B and BoNT / G as well as Synaptotagmin I and Synaptotagmin II, the respective GST-synaptotagmin fusion protein with different amounts of the respective Hc fragment of BoNT / B or BoNT / G incubated and carried out a phase separation. Free Hc fragment remained in the separated supernatant while bound BoNT Hc fragment was detectable in the solid phase along with GST-synaptotagmine fusion protein. Replacement of the full-length Hc fragments with BoNT / B and G in the GST pull-down assay gave the same results.

It was found that the BoNT / B wild type binds only in the presence of complex gangliosides and synaptotagmin I with transmembrane domain, while synaptotagmin II is bound both with or without transmembrane domain as well as in the presence or absence of complex gangliosides. Targeted substitution of amino acids within the protein receptor binding site of BoNT / B significantly increased or decreased the interaction with both synaptotagmin molecules (Figure 1).

Furthermore, it has been demonstrated for the BoNT / G wild type that both in the presence and in the absence of complex gangliosides binding to Synaptotagmin I and Synaptotagmin II takes place each with or without transmembrane domain. By targeted substitution of BoNT / B homologous amino acids Within the protein receptor binding site of BoNT / G, the interaction with both synaptotagmin molecules could be significantly increased or attenuated (FIG. 2).

The potency of the full-length form of BoNT / A, B and G wild types was determined in the HDA by means of a dose-response curve (FIGS. 3 and 6). Subsequently, the potency of the various full-length forms of BoNT / A, B and G single mutants in the HDA was determined (FIG. 6) and related to the potency of the BoNT / B and G wild types by means of an applied power function (FIGS. 4 and 5). , By way of example, the replacement of the amino acids valine 1118 by aspartate or lysine 1192 by glutamate in BoNT / B leads to a drastic reduction of the potency to <2%. In contrast, the mutation of tyrosine 1 183 in leucine and arginine, respectively, produces a marked enhancement of the potency of BoNT / B (FIG. 4). Modification of tyrosine 1256 to phenylalanine in BoNT / G also results in an increase in potency while the glutamine 1200 mutation in glutamate, lysine or tyrosine causes a large decrease in the potency of BoNT / G (Figure 5). In the case of BoNT / A, the modification of serine 1207 to arginine or tyrosine produces an increase in potency while the mutation lysine 1260 to glutamate causes a drastic reduction in the potency of BoNT / A (Figure 6).

In a preferred embodiment, the transport protein has at least 15% higher affinity or at least 15% lower affinity than the native neurotoxin. Preferably, the transport protein has at least 50% higher or lower, more preferably at least 80% higher or lower, and especially at least 90% higher or lower affinity than the native neurotoxin.

According to a preferred embodiment, the modification of the HC takes place in the region of the Hc fragment of the given neurotoxin. If the modification comprises a substitution, deletion, insertion or addition, this can be carried out for example by targeted mutagenesis, method These are known to the skilled person. The amino acids present in the native neurotoxin are hereby altered either by naturally occurring or non-naturally occurring amino acids. Basically, amino acids are divided into different physicochemical groups. The negatively charged amino acids include aspartate and glutamate. The positively charged amino acids include histidine, arginine and lysine. The polar amino acids include asparagine, glutamine, serine, threonine, cysteine and tyrosine. The non-polar amino acids include glycine, alanine, valine, leucine, isoleucine, methionine, proline, phenylalanine and tryptophan. Aromatic side groups are found in the amino acids histidine, phenylalanine, tyrosine and tryptophan. Generally, it is preferred that one amino acid be replaced with another amino acid belonging to another physicochemical group.

According to a preferred embodiment of the invention, the transport protein is a botulinum neurotoxin serotype A to G. Here, the amino acid sequences of the native neurotoxins are obtainable from publicly available databases as follows:

Table 1: Database numbers of the amino acid sequences and subdivision of the seven botulinum neurotoxins.

With respect to the Hc fragment of these botulinum neurotoxins, the amino acids in the amino acid positions from 867 to 1296 of C. botulinum neurotoxin serotype A, 866 to 1291 of C. botulinum neurotoxin serotype B, 864 to 1291 and 1280, respectively, are preferred for modification C. botulinum neurotoxin serotype Cl, 860 to 1276 or 1285 of C. botulinum neurotoxin serotype D, 843 to 1251 and 1252, respectively, of the C. botulinum and C. butyricum neurotoxin serotype E, respectively 861 to 1274, 862 to 1280 and 1278 and 854 to 1268 of the C. botulinum or

C. baratii neurotoxin serotype F,

861 to 1297 of C. botulinum neurotoxin serotype G.

It is therefore preferred that at least one amino acid in the aforementioned positions be post-translationally modified, and / or added, and / or deleted, and / or inserted, and / or substituted by an amino acid which is either naturally occurring or of non-natural origin.

According to a preferred embodiment, the neurotoxin botulinum neurotoxin is serotype A. Preferably, at least one amino acid in the positions threonine 1 195, asparagine 1196, glutamine 1199, lysine 1204, isoleucine 1205, leucine 1206, serine 1207, leucine 1209, aspartate 1213, leucine No. 1217, phenylalanine 1255, asparagine 1256, isoleucine 1258 and / or lysine 1260 the botulinum neurotoxin serotype A, protein sequences post-translationally modified, and / or added, and / or deleted, and / or inserted and / or substituted by an amino acid, either naturally occurring or not of natural origin. Particularly preferred are the positions asparagine 1196, glutamine 1199, serine 1207, phenylalanine 1255, isoleucine 1258 and / or lysine 1260 of the botulinum neurotoxin serotype A protein sequences. In particular, the positions serine 1207 substituted by arginine or tyrosine and lysine 1260 substituted for glutamate are preferred.

According to a preferred embodiment, the neurotoxin is botulinum neurotoxin serotype B. Preferably, at least one amino acid in the positions lysine 1113, aspartate 1114, serine 1116, proline 1117, valine 1118, threonine 1182, tyrosine 1183, phenylalanine 1186, lysine 1188, glutamate 1191 , Lysine 1192, leucine 1193, phenylalanine 1194, phenylalanine 1204, phenylalanine 1243, glutamate 1245, lysine 1254, aspartate 1255 and tyrosine 1256 the botulinum neurotoxin serotype B protein sequences post-translationally modified, and / or adrenaline. diert, and / or deleted, and / or inserted and / or is substituted by an amino acid, which either occurs naturally or is not of natural origin. Particularly preferred are the positions valine 1 1 18, tyrosine 1 183, glutamate 1 191, lysine 1 192, glutamate 1245 and tyrosine 1256 of the botulinum neurotoxin serotype B protein sequences. In particular, the positions tyrosine 1183 and glutamate 1 191, which are replaced by leucine, are preferred.

According to another preferred embodiment, the neurotoxin is botulinum neurotoxin serotype G. Preferably in this case at least one amino acid in the positions phenylalanine 1121, lysine 1123, alanine 1124, serine 1125, methionine 1 126, valine 1190, leucine 1191, serine 1194, glutamate 1196, threonine 1199, glutamine 1200, leucine 1201, phenylalanine 1202, phenylalanine 1212, phenylalanine 1248, lysine 1250, aspartate 1251 and tyrosine 1262 the botulinum neurotoxin serotype G protein sequences post-translationally modified, and / or added, and / or deleted, and / or inserted and / or substituted by an amino acid which is either naturally occurring or of non-natural origin. Particularly preferred are the positions methionine 1126, leucine 1191, threonine 1199, glutamine 1200, lysine 1250 and tyrosine 1262 of the botulinum neurotoxin serotype G protein sequences. In particular, the position tyrosine 1262, which is replaced by phenylalanine, is preferred.

The transport protein provided in the present invention has an increased or decreased specific affinity of its protein binding domain, in particular to molecules of the synaptotagmine or synaptic vesicle glycoproteins 2 family.

A further embodiment of the present invention relates to a composition which contains a transport protein according to the invention and at least one intervening molecule (X). The intervening molecule may be a small organic molecule, a peptide or a protein; preferably covalently by a peptide bond, ester bond, ether bond, sulfide bond, Disulfide bond or carbon-carbon bond bound to the transport protein.

The intervening molecule additionally comprises all known therapeutically active substances. Preference is given here cytostatics, antibiotics, antivirals, but also immunoglobulins.

In a preferred embodiment, the protein is a protease which cleaves one or more proteins of the neurotransmitter release mechanism, the protease being selected from the group of neurotoxins selected from the LC of C. botulinum neurotoxins, in particular serotypes A, B, Cl, D, E, F and G or a proteolytically active fragment of the LC of a C. botulinum neurotoxin, in particular a neurotoxin of the serotype A, B, Cl, D, E, F and G, the fragment being at least 0, 01% of the proteolytic activity of the native protease, preferably at least 5%, more preferably at least 50%, especially at least 90%. Preferably, the transport protein and the protease are derived from the same C. botulinum neurotoxin serotype, more preferably the H _t M domain of the transport protein and the protease are derived from the C. botulinum neurotoxin serotype A. The sequences of the proteases are generally accessible from databases and the database numbers are shown in Table 1. The proteolytic activity of the proteases is determined by means of a substrate cleavage kinetics (see Binz et al. (2002), Biochemistry 41 (6), 1717-23).

According to another embodiment of the present invention, there is provided a process for producing the transport protein. In a first step, a nucleic acid encoding the transport protein is provided here. The coding nucleic acid may be RNA, DNA or mixtures thereof. The nucleic acid may also be modified with regard to its nuclease resistance, such as, for example, B. by incorporation of phosphorothioate bonds. The nucleic acid can be prepared from an initial nucleic acid be, wherein the starting nucleic acid z. B. is accessible by cloning from genomic or cDNA libraries. Furthermore, the nucleic acid can be prepared directly by solid phase synthesis. Suitable methods are known to the person skilled in the art. If it is assumed that an initial nucleic acid, z. B. by site-directed mutagenesis a targeted change can be performed, which leads to at least one addition, insertion, deletion and / or substitution at the amino acid level. The nucleic acid is then operably linked to a suitable promoter. Suitable promoters for expression in known expression systems are known to the person skilled in the art. The choice of the promoter depends on the expression system used for the expression. In general, constitutive promoters are preferred, but inducible promoters are also useful. The construct thus produced comprises at least part of a vector, in particular regulatory elements, the vector being selected, for example, from λ derivatives, adenoviruses, baculoviruses, vaccinia viruses, SV40 viruses and retroviruses. The vector is preferably capable of expressing the nucleic acid in a given host cell.

Further, the invention provides host cells that contain the vector and that are suitable for expression of the vector. Numerous prokaryotic and eukaryotic expression systems are known in the prior art, the host cells being selected, for example, from prokaryotic cells such as E. coli or B. subtilis, from eukaryotic cells such as S. cerevisiae and P. pastoris. Although higher eukaryotic cells can be used, such as insect cells or mammalian cells, host cells are preferred which, like C. botulinum, have no glycosylation apparatus.

According to a preferred embodiment, the nucleic acid encodes the Hc fragment of C. botulinum neurotoxin. This nucleic acid contains endonuclease cleavage sites flanking the Hc fragment-encoding nucleic acid, which endonuclease sites are compatible with those of other Hc fragments of C. botulinum neurotoxins to facilitate their easy modular exchange in the permitting the gene encoding the transport protein while retaining the similarity of the amino acid sequence.

If a composition according to the invention is provided which contains, in addition to the transport protein, at least one intervening molecule, and this intervening molecule is functionalized with either a carboxy-terminal cysteine or a mercapto group, then it can be carried out in an analogous manner as before described the peptide or the protein are produced recombinantly, for example using binary vectors or by different host cells. If the same host cell is used for the expression of both the transport protein and the peptide or protein, preferably an intermolecular disulfide bond is formed in situ. For more efficient production in the same host cell, the nucleic acid encoding the peptide or protein can also be translated with that of the transport protein in the same reading frame to produce a single-chain polypeptide. In this case, an intramolecular disulfide bond then preferably forms in situ. For simple hydrolysis of the likewise present peptide linkage between transport protein and peptide or protein, an amino acid sequence, which is either recognized and cleaved specifically by the protease thrombin or a specific endoprotease of the host cell, is inserted at the amino terminus of the transport protein.

Surprisingly, it has been found that the insert sequence CXXXZKTKSLVPRGSKBXXC (SEQ ID NO: 1), wherein X is any amino acid and Z and B are independently selected from alanine, valine, serine, threonine and glycine, from an endogenous protease of a bacterial host, preferably E. coli is efficiently cleaved in vivo. The insertion of the insert sequence between the amino acid sequence of the transport protein and a further peptide or protein therefore has the advantage that subsequent post-processing, such as thrombin, for example, is not required. Particularly preferred is the E. co / ϊ strain E. coli Kl 2. The insert sequence is preferably part of a loop containing 18-20, preferably, amino acids.

If this is not possible, after separate purification of the transport protein and the protein, a corresponding intermolecular disulfide bond can subsequently be brought about by oxidation processes known to the person skilled in the art. The peptide or protein can also be obtained directly by synthesis or fragment condensation. Corresponding methods are known to the person skilled in the art.

The transport protein and the peptide or protein are subsequently purified. Here, the skilled worker known methods are used, such. As chromatography method or electrophoresis.

Another embodiment of the present invention relates to the pharmaceutical composition containing the transport protein or a composition and optionally a pharmaceutically acceptable carrier, diluent and / or additive.

The pharmaceutical composition is suitable for oral, intravenous, subcutaneous, intramuscular and topical administration. Intramuscular administration is preferred. A dosage unit of the pharmaceutical composition contains about 0.1 pg to 1 mg of transport protein and / or the composition according to the invention.

The pharmaceutical composition is useful for the treatment of disorders of neurotransmitter release and diseases such as, e.g. As hemifacial spasm, spasmodic torticollis, blepharospasm, spasticity, dystonia, migraine, pain, diseases, the cervical and lumbar spine, strabismus, hyper-salivation, wound healing, snoring and depression suitable. Another embodiment of the present invention includes a cosmetic composition containing a transport protein and a cosmetically acceptable carrier, diluent and / or additive. The cosmetic composition is suitable for the treatment of hyperhidrosis and facial wrinkles.

FIG. 1: Investigation of the in vitro binding of the wild-type and mutated BoNT / B Hc fragments on truncated GST-Syt I and GST-Syt II fusion proteins in the presence or absence of complex gangliosides by means of GST pull-down assay.

FIG. 2: Investigation of the in vitro binding of the wild-type and mutated BoNT / G Hc fragments on truncated GST-Syt I and GST-Syt II fusion proteins in the presence or absence of complex gangliosides by means of GST pull-down assay.

FIG. 3: Dose-response curve of the BoNT / B and G wild types in the HDA. The applied power functions allow a relative comparison of the paralysis times of the single mutants with those of the associated wild types.

FIG. 4: Decrease and increase of the neurotoxicity of the BoNT / B single mutants in FIG

Comparison to the wild type in HDA.

FIG. 5: Decrease and increase of the neurotoxicity of the BoNT / G single mutants in FIG

Comparison to the wild type in HDA.

FIG. 6: Dose-response curves of the BoNT / A wild type and the BoNT / A single mutants in the HDA. In detail, the present invention involves a transport protein (Trapo) which results from modifying the HC of the C. botulinum-produced neurotoxin, preferentially binds specifically to neurons, and is preferentially acquired intracellularly by receptor-mediated endocytosis and from the acidic endosomal compartment into the cytosol of Neurons is translocated. This protein is used as a transporter to infect cell-bound proteases and other substances that can not physiologically penetrate the plasma membrane and enter the cytosol of nerve cells. The substrates of the proteases are intracellularly localized proteins and peptides involved in transmitter release. After cleavage of the substrates, the specific functions of the neurons are blocked, whereby the cells themselves are not damaged. One of these functions is exocytosis, which effects neurotransmitter release. If the release of transmitters is inhibited, the transmission of signals from cell to cell is blocked. For example, striped muscles are paralyzed when the release of acetylcholine is inhibited at the neuromuscular junction. This effect can be used therapeutically when the transport protein is applied to nerve endings of spastic or dystonic muscles. Other active substances include agents with antiviral activity. Conjugated with the transport protein, they are useful for the treatment of viral infections of the nervous system. The present invention also relates to the use of a transport protein to inhibit the release of neurotransmitters.

Lower affinity transport proteins bind to, but are not taken up by the nerve cells. These transport proteins are therefore suitable as specific transporters to the nerve cell surface.

When patients are treated with C. botulinum progenitor toxins A and B, the injection of these non-human proteins, despite the low dose, causes the formation of antibodies, so that the therapy is ineffective and therefore must be discontinued, ultimately leading to anaphylactic shock to avoid. By applying a substance of the same mechanism of action with a higher transport efficiency of the enzymatic activity, the dose can be drastically reduced and no antibodies will be produced. These properties are attributed to the transport protein described here.

Although examples of the application are given, generally the appropriate mode of administration and dosage will be determined individually by the attending physician. Such decisions are routinely made by any physician skilled in the art. So z. For example, the mode of application and dosage of the neurotoxin according to the invention set forth herein may be selected based on criteria such as the solubility of the selected neurotoxin or the intensity of the pain to be treated.

At present, the treatment interval for C. botulinum native progenitor toxins A and B averages three to four months. Prolonging this interval would reduce the risk of antibody formation and allow longer duration of treatment with BoNT. The increase in LC in the cytosol would extend their degradation time and thus extend the duration of action. The transport protein described here has a higher affinity and uptake rate than the native HC.

The following example is for the purpose of illustration only and should not be construed as limiting.

material and methods

Plasmid construction and production of recombinant proteins

Plasmids for the E. coli expression of recombinant Hc fragments of BoNT / B and BoNT / G and the full-length form of BoNT / A, B and G with carboxyl-terminal StrepTag for affinity purification were analyzed by PCR Method with suitable primers, BoNT / A (AAA23262) BoNT / B (AAA23211) and BoNT / G (CAA52275) encoding chromosomal DNA and the expression vector pQe3 (Qiagen AG) generated as the starting vector. Truncated variants of rat synaptotagmin I (syt I) (amino acids 1-53, amino acids 1-82) and rat synaptotagmin II (syt II) (amino acids 1-61, amino acids 1-90) were transfected into the GST-encoding vector pGEX- 2T (Amersham Biosciences AB). The nucleic acid sequences of all plasmids were confirmed by DNA sequencing. The recombinant Hc fragments and the VoIl-length form of BoNT were prepared in E. coli strain Ml 5 [pRep4] (Qiagen) during a 10-hour induction at room temperature and on a StrepTactin matrix (IBA GmbH) according to cleaned according to the manufacturer's recommendations. The GST fusion proteins obtained from E. coli BL21 were isolated using glutathione immobilized on sepharose beads. Fractions containing the desired proteins were pooled and dialysed against Tris-NaCl-Triton buffer (20 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100, pH 7.2).

GST pull-down Assav

GST fusion proteins (0.12 nmol each) immobilized on 10 μl GT-Sepharose beads were incubated with Hc fragments (0.1 nmol) in the absence or presence of a bovine brain ganglioside mixture (18% GM1, 55% GDIa, 10% GTIb, 2% other ganglooside, Calbiochem, 20 μg each) in a total volume of 180 μl Tris-NaCl-Triton buffer for 2 h at 4 ° C. The beads were collected by centrifugation, the supernatant removed and the separated beads each washed three times with 400 μl of the same buffer. The washed pellet fractions were boiled in SDS sample buffer and assayed along with the supernatant fractions by SDS-PAGE and Coomassie blue staining.

The BoNT / B wild type only binds in the presence of complex gangliosides and synaptotagmin I with transmembrane domain, whereas synaptotagmin II binds to the transmembrane domain. probably bound with or without transmembrane domain as well as in the presence or absence of complex gangliosides. By targeted substitution of amino acids within the protein receptor binding site of BoNT / B, the interaction with both synaptotagmin molecules could be significantly increased (E1191L, Yl 183L) and attenuated (V1118D, Kl 192E) (Figure 1).

For the BoNT / G wildtype it has been shown that both in the presence and in the absence of complex gangliosides binding to synaptotagmin I and synaptotagmin II takes place respectively with or without transmembrane domain. By targeted substitution of BoNT / B homologous amino acids within the protein receptor binding site of BoNT / G, interaction with both synaptotagmin molecules could be significantly enhanced (YI 262F) and attenuated (Q 1200E), respectively (Figure 2).

By detecting the binding of the recombinant H _c fragments of BoNT / B and G to isolated, immobilized gangliosides, damage to the function of the adjacent ganglioside binding pocket could be excluded by the mutations introduced in the syt binding pocket and sufficient conclusions could be drawn for an intact tertiary structure of Hc Fragments are closed. These results are supported by CD spectroscopic studies as well as thermal denaturation experiments, which also show intact tertiary structures of the mutated Hc fragments of BoNT / B and G.

Mouse Hemidiaphragm Assay (HDA)

The neurotoxicity of the BoNT / A, B and G muteins was determined as described by Habermann et al., Naunyn Schmiedeberg's Arch. Pharmacol. 311 (1980), 33-40. The potency of the full-length form of BoNT / A, B and G wild types was determined in the HDA by means of a dose-response curve (FIGS. 3 and 6). Subsequently, the potency of the various full-length forms of BoNT / A, B and G single mutants in the HDA was determined (FIG. 6) and related to the potency of the BoNT / B and G wild types by means of an applied power function (FIGS. 4 and 5). , Thus, the replacement of the amino acids valine 1 118 against aspartate or lysine 1192 against glutamate in BoNT / B leads to a drastic reduction of the potency to <2%. In contrast, the mutation of tyrosine 1 183 in leucine and arginine, respectively, significantly enhances the potency of BoNT / B (Figure 4). Modification of tyrosine 1256 to phenylalanine in BoNT / G also results in an increase in potency while the glutamine 1200 mutation in glutamate, lysine or tyrosine causes a large decrease in the potency of BoNT / G (Figure 5). In the case of BoNT / A, the modification of serine 1207 to arginine or tyrosine produces an increase in potency while the mutation lysine 1260 to glutamate causes a drastic reduction in the potency of BoNT / A (FIG. 6).

Claims

claims

1. A transport protein obtainable by modifying the heavy chain of the neurotoxin produced by Clostridium botulinum, wherein

2. The transport protein according to claim 1, wherein the neutralizing antibody prevents binding of the native neurotoxin to the protein receptor or ganglioid receptor and / or prevents uptake of the neurotoxin into the nerve cell.

3. The transport protein according to claim 1 or 2, wherein the transport protein is taken up by endocytosis of the cells.

The transport protein according to any one of claims 1 to 3, wherein said protein is specifically associated with plasma membrane molecules, transmembrane proteins, synaptic vesicle proteins, synaptotagmin family proteins or synaptic vesicle glycoproteins 2 (SV2) and / or synaptotagmin I and / or synaptotagmin II (cholinergic motor neuron) and / or SV2A, SV2B or SV2C, preferably human synaptotagmin I and / or human synaptotagmin II and / or human SV2A, SV2B or SV2C.

The transport protein according to any one of claims 1 to 4, wherein the protein has at least 15% higher affinity or at least 15% lower affinity than the native neurotoxin, preferably at least 50%, more preferably at least 80%, most preferably at least 90%. ,

6. The transport protein according to any one of claims 1 to 5, wherein the Hc fragment of the transport protein at least one substitution and / or deletion and / or insertion and / or addition and / or post-translational modifications of amino acids, either naturally occurring or not of natural origin are and who are the affinity to the native

Neurotoxin increase or decrease.

The transport protein of any one of claims 1 to 6, wherein the neurotoxin is Botulinum neurotoxin serotype A to G.

8. The transport protein of claim 7, wherein at least one amino acid in the amino acid positions

867 to 1296 of Clostridium botulinum neurotoxin serotype A, 866 to 1291 of Clostridium botulinum neurotoxin serotype B, 864 to 1291 and 1280 of Clostridium botulinum neurotoxin serotype Cl, respectively

860 to 1276 and 1285, respectively, of Clostridium botulinum neurotoxin serotype D, 843 to 1251 and 1252 of Clostridium botulinum and Clostridium butyrimus neurotoxin serotype E, respectively;

861 to 1274, 862 to 1280 and 1278 and 854 to 1268 of Clostridium borulinum and Clostridium baratii neurotoxin serotype F, respectively;

861 to 1297 of Clostridium botulinum neurotoxin serotype G. post-translationally modified, and / or added, and / or deleted, and / or inserted and / or substituted by amino acids that are either naturally occurring or non-natural.

9. The transport protein according to any one of the preceding claims, wherein the native neurotoxin neurotoxin serotype A and, preferably, the transport protein to the synaptic vesicle glycoproteins 2 (SV2), particularly preferably SV2A, SV2B or SV2C binds.

10. The transport protein according to claim 9, wherein at least one amino acid in the positions threonine 1195, asparagine 1196, glutamine 1199, lysine 1204, isoleucine 1205, leucine 1206, serine 1207, leucine 1209, aspartate 1213, leucine 1217, phenylalanine 1255, asparagine 1256 , Isoleucine 1258 and / or lysine 1260 of the botulinum neurotoxin serotype A protein sequences posttranslationally modified, and / or added, and / or deleted, and / or inserted and / or substituted by an amino acid that is either naturally occurring or not of natural origin is.

11. The transport protein according to claim 10, wherein at least one amino acid in the positions asparagine 1196, glutamine 1199, serine 1207, phenylalanine 1255, isoleucine 1258 and / or lysine 1260 post-translationally modified, and / or added, and / or deleted, and / or inserted and / or substituted by an amino acid which is either naturally occurring or of non-natural origin.

12. The transport protein according to any one of claims 10 or 11, wherein the amino acid serine at position 1207 is replaced by arginine or tyrosine.

The transport protein of any one of claims 10 or 11, wherein the amino acid lysine at position 1260 is replaced by glutamate.

14. The transport protein according to any one of claims 1 to 8, wherein the neurotoxin is botulinum neurotoxin serotype B and, preferably, binds the transport protein to synaptotagmin.I or II.

15. The transport protein according to claim 14, wherein at least one amino acid in the positions lysine 1113, aspartate 1 114, serine 1116, proline 1117, valine 11 18, threonine 1182, tyrosine 1 183, phenylalanine 1186, lysine 1188, glutamate 1191, lysine 1192 , Leucine 1193, phenylalanine 1194, phenylalanine 1204, phenylalanine 1243, glutamate 1245, lysine 1254, aspartate 1255 and tyrosine

1256 the botulinum neurotoxin serotype B protein sequences are post-translationally modified, and / or added, and / or deleted, and / or inserted and / or substituted by an amino acid that is either naturally occurring or not of natural origin.

16. The transport protein according to claim 15, wherein at least one amino acid in the positions valine 118, tyrosine 1183, glutamate 1191, lysine 1192, glutamate 1245 and / or tyrosine 1256 of the botulinum neurotoxin serotype B protein sequences posttranslationally modified, and / or added, and / or deleted, and / or inserted and / or substituted by an amino acid which either occurs naturally or is not of natural origin.

The transport protein of claim 16, wherein the amino acid tyrosine at position 1 183 is substituted by leucine.

18. The transport protein according to claim 16, wherein the amino acid glutamate at position 1 191 is substituted by leucine.

19. The transport protein according to any one of claims 1 to 8, wherein the neurotoxin botulinum neurotoxin serotype G is.

20. The transport protein according to claim 19, wherein at least one amino acid in the positions phenylalanine 1 121, lysine 1 123, alanine 1 124, serine 1 125, methionine 1126, valine 1 190, leucine 1 191, serine 1194, glutamate 1 196, Thre - onin 1199, glutamine, 1200, leucine 1201, phenylalanine 1202, phenylalanine

1212, phenylalanine 1248, lysine 1250, aspartate 1251 and tyrosine 1262 of Botulinum neurotoxin serotype G protein sequences are posttranslationally modified, and / or added, and / or deleted, and / or inserted and / or substituted by an amino acid that is either naturally occurring or not of natural origin.

21. The transport protein according to claim 20, wherein at least one amino acid in the positions methionine 1126, leucine 1191, threonine 1199, glutamine 1200, lysine 1250 and tyrosine 1262 of the botulinum neurotoxin serotype G protein posttranslationally modifies, and / or adds, and / or dele and / or inserted and / or substituted by an amino acid which is either naturally occurring or of non-natural origin.

The transport protein of claim 21, wherein the amino acid tyrosine at position 1262 is substituted by phenylalanine.

23. A composition containing a transport protein according to any one of claims 1 to 22 and at least one intervening molecule.

24. The composition of claim 23, wherein the intervening molecule is covalently bound to the transport protein by a peptide bond, ester bond, ether bond, sulfide bond, disulfide bond, or carbon-carbon bond.

The composition of claim 23 or 24, wherein the intervening molecule is either a small organic molecule, a peptide or a protein.

26. The composition of claim 25, wherein the small organic molecule is a virustatic, cytostatic, antibiotic or immunoglobulin.

27. The composition of claim 25, wherein the protein is a protease.

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

29. The composition of claim 27, wherein the protease contains a proteolytically active fragment derived from the light chain (LC) of the serotypes A, B, Cl, D, E, F and / or G of the Clostridium botulinum neurotoxin and thereby characterized in that it has at least 0.01% of the proteolytic activity of the native protease, preferably at least 50%.

The composition of claims 28 and 29, wherein the protease specifically cleaves certain substrates within the cholinergic motoneuron.

31. The assembly of claim 30, wherein the substrates are selected from proteins involved in the release of neurotransmitters into nerve cells and proteins capable of catalytic reactions within the nerve cell.

The composition of claims 28 and 29, wherein the protease and the transport protein are covalently linked by an amino acid sequence that is specifically recognized and cleaved by an endopeptidase.

33. The composition of claim 32, wherein the amino acid sequence comprises the sequence CXXXZKTKSLVPRGSKBXXC, wherein X is any amino acid and Z and B are independently selected from alanine, valine, serine, threonine and glycine.

34. The composition of claim 32, wherein after cleavage by the endopeptidase a disulfide bridge links the protease and the transport protein together, which in turn leads to the formation of an active holotoxin.

35. A pharmaceutical composition containing the transport protein of any one of claims 1 to 22 or the composition of any of claims 23 to 34, and optionally a pharmaceutically acceptable carrier, diluent and / or additive.

36. Use of the pharmaceutical composition according to claim 35 for the treatment of disorders and diseases for which therapy with botulinum neurotoxin is indicated.

The use of claim 36, wherein the disorder or disease is any of the following: hemifacial spasm, spasmodic torticollis, blepharospasm, spasticities, dystonia, migraine, pain, cervical and lumbar spine disorders, strabismus, hypersalivation, snoring, wound healing and depressive disorders.

38. A cosmetic composition containing the transport protein according to any one of claims 1 to 22 or the composition according to any one of claims 23 to 34, and optionally a cosmetically acceptable carrier, diluent and / or additive.

39. Use of a cosmetic composition according to claim 38 for the treatment of the cosmetic indications Hyperhydrose and facial wrinkles.

40. A method for producing a transport protein according to claims 1 to 22 or a composition according to claims 23 to 34 by recombination according to known methods.

41. A method for producing the transport protein according to claim 40, wherein the gene of the Hc fragment is flanked by restriction endonuclease cleavage sites containing two nucleic acids, wherein the restriction endonuclease cleavage sites interfaces are compatible with those of the other Hc fragments of Clostridium botulinum neurotoxins to allow for their easy modular exchange while retaining the similarity of the amino acid sequence.

42. A method of making the composition of claim 40, wherein the gene of the protease is flanked by restriction endonuclease cleavage sites containing two nucleic acids, wherein the restriction endonuclease sites are compatible with those of the other protease domains of Clostridium botulinum neurotoxins to facilitate their easy modular exchange while retaining the similarity of the amino acid sequence.

43. A host cell containing a recombinant expression vector, wherein the expression vector encodes a transport protein according to claims 1 to 22 or a composition according to any one of claims 23 to 34.

44. The host cell according to claim 43, wherein the host cell can be a cell of Escherichia coli, in particular E. coli K 12, Saccharomyces cerevisiae, Pichia pastoris or Bacillus megaterium.

45. An expression vector, wherein the vector comprises a nucleic acid encoding a transport protein according to any one of claims 1 to 22 or a composition according to any one of claims 23 to 34.