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WO2013011055A1 - Moyens et procédés pour déterminer la présence de neurotoxines clostridiales - Google Patents

Moyens et procédés pour déterminer la présence de neurotoxines clostridiales Download PDF

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Publication number
WO2013011055A1
WO2013011055A1 PCT/EP2012/064068 EP2012064068W WO2013011055A1 WO 2013011055 A1 WO2013011055 A1 WO 2013011055A1 EP 2012064068 W EP2012064068 W EP 2012064068W WO 2013011055 A1 WO2013011055 A1 WO 2013011055A1
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WIPO (PCT)
Prior art keywords
neurotoxin
fragment
liposome
cleavage
vamp
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PCT/EP2012/064068
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English (en)
Inventor
Oliver WEINGART
Marc AVONDET
Martin Loessner
Andreas Rummel
Frank Gessler
Original Assignee
ETH Zürich
Bundesamt Für Bevölkerungsschutz Und Sport Im Eidgenössischen Departement Für Verteidigung, Bevölkerungsschutz Und Sport
Toxogen Gmbh
Miprolab Gmbh
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Application filed by ETH Zürich, Bundesamt Für Bevölkerungsschutz Und Sport Im Eidgenössischen Departement Für Verteidigung, Bevölkerungsschutz Und Sport, Toxogen Gmbh, Miprolab Gmbh filed Critical ETH Zürich
Priority to US14/233,740 priority Critical patent/US20140287433A1/en
Priority to EP12735564.2A priority patent/EP2734635A1/fr
Publication of WO2013011055A1 publication Critical patent/WO2013011055A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/5432—Liposomes or microcapsules
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/33—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Clostridium (G)

Definitions

  • This invention relates to a method of determining presence, amount and/or activity of a clostridial neurotoxin in a sample, the method comprising or consisting of the following steps: (a) bringing said sample into contact with a liposome, said liposome comprising (aa) at least one receptor on its outer surface, said receptor being capable of binding said neurotoxin and comprising or consisting of (i) a glycolipid and (ii) a peptide or protein; and (ab) a substrate in its interior, said substrate (i) being cleavable by the peptidase comprised in said neurotoxin and (ii) generating a detectable signal upon cleavage, said detectable signal preferably being generated by (1 ) the donor of a FRET pair, said donor exhibiting increased fluorescence upon cleavage by said peptidase, (2) a luminescent compound formed upon said cleavage, or (3) an enzyme formed upon said cleavage; and (b) determining whether an increase
  • Clostridial neurotoxins include various serotypes of botulinum neurotoxin and tetanus neurotoxin.
  • BoNT Botulinum neurotoxin
  • HC Heavy Chain
  • LC Light Chain
  • BoNT specifically binds via the C-terminal part of its HC (H c ) to receptors on the motoneuron's membrane, typically SV2C and the trisialoganglioside GT1 b in case of BoNT/A, and Synaptotagmin-ll (Sytll) and GT1 b in case of BoNT/B [3,4].
  • the toxin is internalised into the motoneuron via receptor-mediated endocytosis [5].
  • the pH in the endosome is typically lowered from pH 7.2 to pH 5.3, and the HC N-terminal part (H N ) refolds into a transmembrane channel crossing the endosomal membrane, allowing for translocation of the LC into the cytosol of the motoneuron [6].
  • the LC specifically cleaves distinct SNARE-proteins (soluble N-ethylmaleimide-sensitive fusion protein attachment receptor).
  • the LC of BoNT/A specifically cleaves SNAP-25 (synaptosome associated protein of 25 kDa), and the LC of BoNT/B specifically cleaves Synaptobrevin/VAMP-2 (vesicle associated membrane protein) [7-9]. If one or more SNARE proteins are cleaved, then the SNARE complex cannot assemble anymore, release of neurotransmitter is inhibited, and hence, the following muscle cell is paralysed. The amount of toxin needed to cause severe symptoms of botulism and even death is extremely low. From studies in mice and monkeys it was calculated that already 1 ng per kg body weight via the intravenous route might be lethal in humans [10-12].
  • the neurotoxin is used in modern biomedical applications [13- 15]. If applied in minute doses, the toxin exerts a locally constrained paralysing effect, which is used for the treatment of a large variety of diseases, such as strabismus, hyperhidrosis, or chronic pain. Moreover, treatment of wrinkles, frown lines, or facial asymmetries and other applications of aesthetical surgery, present a large application area [16]. However, to guarantee patients' safety, it is crucial that the toxic activity in all batches of pharmaceutical and cosmetic BoNT preparations is consistent. Accordingly, strict controls apply, where potency in each batch is measured with the mouse LD50 test.
  • mice are injected intraperitoneally with defined volumes of different dilutions of BoNT containing preparations and observed for typical botulism symptoms for up to 96 hours.
  • the injected amount where 50% of mice die is considered the LD50, which is defined as 1 Unit of BoNT [17].
  • LD50 which is defined as 1 Unit of BoNT [17].
  • the mouse LD50 test is still the only detection method officially validated and accepted for testing the toxic activity of BoNT in pharmaceutical products [20].
  • the heavy binding is translocation chain to generally and cleavage receptor independent
  • Endo- determines 3-24 h applicable to no depeptidase cleavage all serotypes termination of test activity of the to the extent the activity of
  • botulinum endplate which neuroneurotoxin; is the botulinum transmitter depending neurotoxin release on the cell target cell
  • Mouse readout via 1 -3 h + + + determines low throughput; diadecrease of complete labor intensive; phragm diaphragm mechanism consumes premuscle of action of animals
  • a further example of a botulinum neurotoxin assay, which fails to determine the entire mechanism of action of the neurotoxin is described in US 201 1/0033866.
  • This document describes neurotoxin substrates suitable for a FRET assay.
  • the vesicles merely serve as a carrier for such substrate.
  • the vesicle is not equipped with receptors and accordingly does not provide for determining receptor binding and/or translocation.
  • FRET is not used as detection scheme. The technical problem underlying the present invention can therefore be seen in the provision of alternative or improved means and methods for determining clostridial neurotoxins.
  • the present invention provides a method of determining presence, amount and/or activity of a clostridial neurotoxin in a sample, the method comprising or consisting of the following steps: (a) bringing said sample into contact with a liposome, said liposome comprising (aa) at least one receptor on its outer surface, said receptor being capable of binding said neurotoxin and comprising or consisting of (i) a glycolipid and (ii) a peptide or protein; and (ab) a substrate in its interior, said substrate (i) being cleavable by the peptidase comprised in said neurotoxin and (ii) generating a detectable signal upon cleavage, said detectable signal preferably being generated by (1 ) the donor of a FRET pair, said donor exhibiting increased fluorescence upon cleavage by said peptidase, (2) a luminescent compound formed upon said cleavage
  • Said determining may be a merely qualitative determining, i.e. determining presence or absence of the clostridial neurotoxin.
  • said determining may be quantitatively, i.e. the determining of amount and/or activity of a clostridial neurotoxin.
  • Amount and activity to be determined may be relative or absolute amounts and/or activities. This will depend inter alia on how the method is calibrated and/or which controls and standards are used. Such specifics of the assay design are within the abilities of the skilled person. An exemplary illustration is comprised in the examples enclosed herewith. Generally speaking, amount and activity will be proportional to each other across a significant range of values.
  • a defined amount of a clostridial neurotoxin may exhibit different activity depending on presence or absence of accessory non- toxin proteins, also referred to as neurotoxin associated proteins (NAPs).
  • the neurotoxin as such consists of light and heavy chain as discussed in the introductory section herein above.
  • serotype A of botulinum toxin said neurotoxin has an approximate molecular weight of 150 kDa.
  • Clostridium botulinum typically produces botulinum type A toxin complexes, wherein said complexes comprise said neurotoxin on the one hand and one or more non-toxin proteins on the other hand.
  • botulinum type A toxin complexes are found which have molecular weights of about 900 kDa, 500 kDa or 300 kDa. Similar findings, although different in terms of molecular weight, apply to the other botulinum toxins, which is well-known in the art.
  • the presence of non-toxin proteins may have a stabilizing effect, the consequence being that a given amount of neurotoxin, when provided in complex form, may have a different, for example higher activity as compared to the same amount of neurotoxin in the absence of non-toxin proteins.
  • the stabilizing effect of non-toxin proteins as well as conditions where non-toxin proteins are dispensable are known to the skilled person.
  • formulations of clostridial neurotoxins are known or at the skilled person's disposal, which, instead of said non-toxin proteins or in addition thereto, contain further stabilizing agents such as human serum albumin, sucrose and/or gelatine.
  • said neurotoxin as comprised in said sample may be associated with NAPs, it is preferred that a sample comprising neurotoxin and being free of NAPs is subjected to the methods of the invention.
  • presence of heavy chain and light chain is necessary, noting that the method probes for the concomitant occurrence of receptor binding, translocation and cleavage.
  • clostridial neurotoxin refers to an entity comprising, preferably consisting of heavy and light chain (see also the introductory section above), either fused as single chain form or as di-chain form, and providing the activities required for said receptor binding, translocation and cleavage.
  • the neurotoxin has an approximate molecular weight of 150 kD.
  • neurotoxic component is used in the art to designate the entity consisting of heavy and light chain.
  • amount includes concentration, mass, weight, and amount of substance, and preferably is expressed in terms of a concentration. To the extent molecular weights are known, mass, weight and concentration on the one hand and amount of substance on the other hand can be interconverted.
  • the third step of the mechanism of action of clostridial neurotoxins is an enzymatic activity, more specifically a proteolytic activity (herein also referred to as peptidase activity), activity may be expressed in terms of the number of cleaved substrates per unit of time.
  • the measured activity results from the efficacy of all three steps of the mechanism of action of the neurotoxin, i.e. receptor binding, translocation and substrate hydrolysis, herein also referred to as cleavage of the substrate.
  • Receptor binding is governed inter alia by the affinity of the respective neurotoxin for the receptor according to the invention.
  • Translocation i.e. formation of a transmembrane channel and transport of the peptidase into the lumen of the liposome according to the invention, is triggered or enhanced by a pH shift (for details see below).
  • the methods of the invention when used for quantitative determination of neurotoxins, yield activities in the first step. If the specific activity of the sample is known or can be determined, activity may be converted into amounts. Alternatively, the amount, in particular in case of samples, which are concentrated solutions of neurotoxin, may be determined by means such as SDS-PAGE, photometry and ELISA. Upon determination of activity by the method of the invention and of the amount by such means, the specific activity may be calculated.
  • a further common measure of the activity of neurotoxins, in particular of botulinum neurotoxins, are units.
  • a Unit is defined by reference to the above-mentioned mouse LD50 assay.
  • a Unit is the median intraperitoneal lethal dose (LD50) in mice.
  • a reference sample may be used, said reference sample comprising a defined activity of clostridial neurotoxin, said defined activity preferably being expressed in terms of units. In such a case, the activity of sample can then be specified in terms of units as well.
  • Clostridial neurotoxins are neurotoxins produced by bacteria of the genus Clostridium. Toxin producing species include Clostridium botulinum, Clostridium butyricum, Clostridium baratii, Clostridium argentinense as well as Clostridium tetani. Clostridial neurotoxins are further detailed below. Clostridial neurotoxins are typically characterized by a similar mechanism of action, which, as detailed above, comprises binding to receptors on the target nerve cell, translocation through the membrane and cleavage of the target protein by the peptidase component of the neurotoxin. Receptors and peptidase substrate may be different for the various toxins.
  • the receptor is an integral or peripheral membrane protein, which consists of or comprises one or more extracellular domains, thereby being accessible to the neurotoxin heavy chain for binding.
  • these are typically SNARE proteins.
  • SNARE proteins Several serotypes of botulinum neurotoxin cleave the same SNARE protein, however, at different sites.
  • a substrate to be cleaved not only the cleavage site, but also one or more recognition sites as present in the natural substrate must be present in an artificial substrate which may be used in the methods of the present invention (for details see below).
  • the clostridial neurotoxins share a common mechanism of action, wherein all three steps of said common mechanism of action are assayed by the methods according to the present invention.
  • a sample according to the invention is known to comprise or suspected of comprising at least one clostridial neurotoxin. Depending on its origin, said sample may comprise further constituents. Preferred samples are the subject of a preferred embodiment described in detail below.
  • the sample to be subjected to the method of determining according to the present invention may not only comprise further constituents owing to its origin, but also as a consequence of the deliberate addition of such further constituents.
  • Such deliberately added constituent may be a test compound or a plurality of test compounds, wherein it is to be determined whether such compounds are capable of modulating the activity of clostridial neurotoxins.
  • the primary sample as it might be obtained from, for example, a patient might be known to comprise or suspected of comprising neutralizing antibodies against one or more clostridial neurotoxins, but otherwise free of neurotoxins as such. In that case, it is envisaged to deliberately add one or more neurotoxins to such primary sample, thereby obtaining the sample to be subjected to the method according to the present invention.
  • Such preferred aspects are the subject of preferred embodiments discussed in more detail below.
  • Said sample is to be brought into contact with a liposome, the liposome being further characterized by items (i) and (ii) as recited in step (a) of the method according to the first aspect of the invention. It is understood that said bringing into contact is to be effected under conditions, which allow binding of said neurotoxin, if present, to said receptor.
  • the skilled person can readily establish such conditions, such conditions including, for example, buffered solutions. Further preferred/exemplary conditions are specified in the examples enclosed herewith.
  • said determining according to (b) is effected at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 24, 36 or 48 hours after said bringing into contact according to (a). These preferred periods of time refer to the total incubation time.
  • said liposome meets at least the requirements (i) and (ii).
  • Such liposome may either be obtained from natural sources such as nerve tissue or nerve cell cultures, or, in the alternative, by preparation starting from defined constituents. Preference is given to the latter approach.
  • a method of preparing a liposome is also the subject of the present invention; see the fourth aspect discussed further below.
  • the use of defined constituents and defined procedures of manufacture of the liposomes provides for a reproducible method of determining according to the first aspect.
  • the liposome according to the invention which is also subject of the third aspect described in more detail below, comprises a receptor and a substrate.
  • a liposome is, as known in the art, a closed compartment formed by a lipid bilayer.
  • liposomes are artificially prepared. It is understood that the term "liposome”, as used herein, extends to liposomes, which are prepared from naturally occurring cells or tissues.
  • the receptor is defined in both structural and functional terms. In structural terms, the receptor comprises or consists of a glycolipid on the one hand and a peptide or protein on the other hand. In functional terms, said receptor is required to be capable of binding said neurotoxin. As a consequence, the skilled person understands that said receptor is intended to mimic the neurotoxin receptor, as it is present on the surface of nerve cells.
  • said glycolipid is preferably to be chosen from a glycolipid which naturally occurs in the membrane of nerve cells or a modified version thereof, said modified version being capable of acting as a constituent of said receptor being capable of binding said neurotoxin.
  • Preferred glycolipids are described further below.
  • the second, proteinaceous component of said receptor is a protein naturally occurring in nerve cells and known to be involved in neurotoxin binding, or a fragment thereof (herein also referred to as peptide), or a modified version of said protein or fragment, wherein said fragment, i.e. said peptide as well as said modified version are capable of acting as a component of a receptor being capable of binding said neurotoxin.
  • the skilled person in view of the present knowledge on targets and mechanism of action of clostridial neurotoxins, and furthermore provided with the teaching of the present invention is in a position to devise receptors meeting both the structural and functional requirements according to item (i) of the first aspect.
  • various truncated, deleted and/or modified versions of the respective naturally occurring protein may be prepared and tested for their capability of binding a clostridial neurotoxin or a heavy chain thereof.
  • the testing of receptors is also described in Nishiki et al. [76], Nishiki et al. [48], Dong et al. [50] Dong et al. [77], Dong et al.
  • the substrate according to item (ii) may either comprise the cognate substrate of the respective clostridial neurotoxin to be assayed, or a subsequence thereof, such subsequence being cleavable by the peptidase comprised in the neurotoxin. Again, both substrates as well as several subsequences are known to the skilled person and further detailed herein below.
  • the substrate further generates a detectable signal upon cleavage.
  • any means may be used which generate a detectable signal upon cleavage.
  • the signal may be emission of light, be it by fluorescence or luminescence. Light may be emitted by the substrate itself once it is cleaved, or may be generated as a result of a downstream event triggered by cleavage of the substrate. Preferred options are detailed below.
  • the substrate comprises a FRET pair, such FRET pair consisting of a fluorescence donor and a fluorescence acceptor (option (1 )).
  • FRET is well-known in the art and stands for fluorescence resonance energy transfer.
  • FRET Fluorescence Reduction reaction mediated endometrial cleavage of the substrate by the peptidase.
  • Said FRET pair is to be positioned such that fluorescence increases upon cleavage of the substrate by the peptidase. This implies positioning of donor and acceptor (i) on different sides of the substrate's cleavage site, and furthermore (ii) that donor and acceptor have an average spatial distance in the substrate that provides for sufficient quenching of the donor fluorescence by the acceptor in the uncleaved substrate. Distances between donor and acceptor for quenching to occur are typically in the order of up to 10 nM. Regardless thereof, suitable positions of donor and acceptor within a given substrate can be determined by the skilled person without further ado by simple tests.
  • the acceptor does not (only) act as a quencher, but is capable of fluorescence emission, in particular when energy is transferred from a donor in sufficient spatial proximity. In that case, little or no donor emission, but significant acceptor emission will be measured in the uncleaved state of the substrate, while upon cleavage, the acceptor emission is decreased and the donor emission is increased.
  • Suitable donor/acceptor pairs are known in the art and at the skilled person ' s disposal.
  • a luminescent compound is formed upon cleavage (option (2)).
  • Said luminescent compound may be, for example, oxyluciferin, which is formed from luciferin in the presence of the enzyme luciferase.
  • Luciferase in turn may be provided in the form of two fragments of luciferase, said fragments being generated by said cleavage and, while being enzymatically inactive themselves, assemble into a functional enzyme having luciferase activity upon cleavage.
  • a substrate which, upon cleavage, assembles to a yield a functional enzyme, wherein the activity of said enzyme is detected (option (3)).
  • a functional enzyme wherein the activity of said enzyme is detected (option (3)).
  • Such enzyme complementation system on the one hand and the generation of a luminescent compound on the other hand may characterize one and the same read-out scheme; see, for example, the luciferase system described above.
  • Preferred substrates are SNARE proteins comprising such FRET pair or otherwise modified to generate a signal upon cleavage, or fragments of SNARE proteins comprising FRET pairs or otherwise modified to generate a signal upon cleavage.
  • Step (b) provides for a comparison of signal generated by the liposome with and without sample, but otherwise preferably under the same conditions.
  • substrates with FRET pairs the following applies.
  • a preferred wavelength is the wavelength of maximum fluorescence emission which is a characteristic known for most commercially available fluorophor or can be determined without further ado, for example by performing a spectral scan.
  • Said receptor is located on the outer surface of the liposome, and the substrate in the interior of the liposome, preferably only in the interior of the liposome.
  • Preferred means of ensuring occurrence of the substrate only in the interior of the liposome include the following: (i) purification by means of size exclusion chromatography (as shown in Figure 2 or alternatively in FPLC or HPLC format), (ii) purification by means of dialysis using a molecular weight cutoff which is above the molecular weight of the substrate, (iii) ultrafiltration with a molecular cut-off which is above the molecular weight of the substrate, and (iv) separation of liposomes by ultracentrifugation. These methods serve to separate the liposomes (with substrate molecules in their interior) from substrate molecules present in the mixture obtained from liposome preparation, the latter substrate molecules not being encapsulated in the liposomes.
  • the substrate is soluble and dissolved in the aqueous medium in the lumen of the liposome
  • membrane-bound substrates alternative or additional means of ensuring that the substrate is pointing only inwards, i.e. into the lumen of the liposome, may be used.
  • substrate may be incorporated into the liposomes prior to incorporation of the receptor, at least of the peptide or protein component of the receptor.
  • substrate molecules may be digested by a suitable enzyme such as proteinase K or the light chain (LC) of the neurotoxin/serotype to be determined by the methods of the invention.
  • the receptors are incorporated, thereby obtaining a liposome according to the present invention with membrane-bound substrate pointing only into the interior of the liposome.
  • aqueous medium in the lumen of the liposome is such that cleavage of said substrate by said neurotoxin can take place.
  • aqueous medium can be chosen by the skilled person without further ado.
  • a particularly preferred aqueous medium comprises or consists of HEPES buffer, preferably between 10 and 100 mM such as 20 mM, with a pH between 7 and 8, preferably 7.4, preferably supplemented with ZnS0 4 , preferably at a concentration between 0.01 and 0.1 mM such as 0.05 mM, and furthermore preferably supplemented with TCEP, preferably at a concentration between 1 and 10 mM such as 2 mM.
  • said liposome mimics the characteristics of a nerve cell which are essential for the entire mechanism of action of a clostridial neurotoxin to be monitored, said mechanism of action comprising receptor binding, translocation and proteolytic cleavage of the target protein, see above.
  • the known neurotoxin assays either fail to test the entire mechanism of action or, to the extent they do, suffer from other deficiencies (see Table 1 herein above).
  • the assay according to the first aspect of the present invention does not require mice (be it whole animals or diaphragm preparations obtained therefrom) nor are cultures of nerve cells needed.
  • the assay is robust, reproducible, amenable to standardization and capable of being effected in high throughput format. Sterile conditions are not required. Accordingly, a high throughput screen can be effected fast.
  • the assay according to the first aspect wherein instead of said clostridial neurotoxins other bacterial toxins are to be determined.
  • Such other toxins include the Bacillus anthracis toxin and diphtheria toxin.
  • a commercially available substrate MAPKKide
  • diphtheria toxin the enzymatic reaction occurring in the liposome is ADP- ribosylation of eukaryotic elongation factor 2 instead of proteolytic cleavage.
  • said clostridial neurotoxin is a botulinum neurotoxin, preferably type A, B, C1 , D, E, F or G botulinum neurotoxin or tetanus neurotoxin.
  • the various serotypes of botulinum toxin are known in the art and well characterized.
  • Pharmaceutical or cosmetic compositions typically comprise botulinum neurotoxin type A (BoNT/A) or BoNT/B as active pharmaceutical ingredient.
  • said sample is selected from a pharmaceutical composition, a diagnostic composition, a cosmetic composition, a clinical or patient sample, a food or feed sample, a beverage sample, a sample taken from a biotechnological process, a sample obtained from an animal and an environmental sample.
  • a variety of botulinum neurotoxin formulations are presently approved for medical and cosmetic uses. In the course of manufacture of these compositions, their testing is indispensable, in particular in view of the high toxicity of the active agent as well as its fragility and easy denaturation.
  • Clinical samples, patient samples and samples obtained from an animal include samples taken from subjects or animals suspected to suffer from botulism.
  • samples may be from subjects suspected to contain neutralizing antibodies directed to one or more clostridial neurotoxins.
  • Food, feed and beverage as well as environmental samples may be assayed for the presence of neurotoxin activity in order to determine whether any threat to the health of humans or animals is present.
  • Samples taken from biotechnological processes include samples taken from biological processes for the manufacture of clostridial neurotoxins. For example, such a sample may be taken from a fermenter containing a bacterial culture expressing clostridial neurotoxins.
  • said liposome comprises or consists of the following constituents: (a) (i) one or more liposome-forming lipids, preferably at least one phosphatidylcholine and cholesterol, said phosphatidylcholine preferably being selected from the group consisting of SPC, DOPC, and POPC; (ii) optionally tocopherol; (b) said at least one receptor, wherein said receptor preferably comprises or consists of (i) a glycolipid, preferably selected from the tri-sialo ganglioside GT1 b, the di-sialo ganglioside GD1 b and the di-sialo ganglioside GD1 a; and (ii) a peptide or protein selected from (1 ) SV2C or a fragment thereof, wherein said fragment is capable of binding to said neurotoxin and preferably comprises or consists of the luminal domain 4 and at least one transmembrane domain; (2) synaptotagmin I or II or
  • said one or more liposome formic lipids are selected from SPC, DOPC, POPC, Lecithin (egg yolk or soy bean), Asolectin (soy bean), posphatidylinositol, posphatidylserin, phosphatidic acid, phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, DOPG (di-oleoyl-phosphatidylglycerol), DOPE (di-oleoyl- phosphatidylethanolamine) and POPG (palmitoyl oleoyl phosphatidylglycerol).
  • Lecithin and Asolectin are lipid mixtures from natural sources. The remainder of lipids are preferably single molecular species. Moreover, also other amphiphilic molecules, either alone or in combination with any one of the above, can be used for the purpose of generating liposomes. The skilled person is aware of such further amphiphilic molecules. The lipids, lipid mixtures and amphiphilic molecules described above can be used in conjunction with any embodiment of the present invention. Also provided is a liposome comprising or consisting of constituents (a), (b) and (c) as defined above as well as liposomes comprising or consisting of constituents (a) and (c); or constituents (a) and (b).
  • said liposome may further comprise or further consist of constituent (d).
  • constituent (d) provides the lipid bilayer forming compounds
  • constituent (b) provides the compounds forming the receptor
  • constituent (c) is the substrate.
  • each of said constituents may - either optionally or compulsory - comprise or consist of more than one compound.
  • phosphatidylcholine As well as cholesterol are preferred.
  • phosphatidylcholines there is soy phosphatidylcholine (SPC), dioleylglycerol phosphocholine (DOPC) and palmitoyl oleoyl phosphocholine (POPC).
  • SPC soy phosphatidylcholine
  • DOPC dioleylglycerol phosphocholine
  • POPC palmitoyl oleoyl phosphocholine
  • DOPC dioleylglycerol phosphocholine
  • POPC palmitoyl oleoyl phosphocholine
  • PA phosphatidic acid
  • Acidic lipids such as PA may have a beneficial effect on the activity of the peptidase of said neurotoxin.
  • compositions forming constituent (a) are apparent from the examples enclosed herewith.
  • the liposome forming lipids as used in the examples as well as their relative amounts are generally applicable for the purposes of the present invention.
  • Receptors for clostridial neurotoxins as they naturally occur in nerve cells are typically made up by two components, namely a glycolipid and a protein. Accordingly, the receptor present on the liposomes according to the present invention, said receptor being capable of binding said neurotoxin, mimics such naturally occurring neurotoxin receptors.
  • Preferred glycolipids are gangliosides and particularly preferred are the gangliosides according to (b)(i) of the present preferred embodiment. Gangliosides are glycosphingolipids.
  • gangliosides i.e. GT1 b, GD1 b and GD1 a
  • GT1 b Gal[33NAcGai 4(NAcNeua8NAcNeu 3)Gal[1 ⁇ 4Glc Cer
  • GD1 a NAcNeua3Gal 3NAcGal( (N AcNeuoc3)Galp4GlcpCer
  • GT1 b NAcNeucx3Gal[33NAcGal
  • the proteinaceous component of the receptor is defined in part (b)(ii) of this embodiment.
  • Either the full length naturally occurring proteins may be used or fragments thereof, the fragments being capable of binding to the respective neurotoxin.
  • SV2A, SV2B and SV2C are forms of synaptic vesicle glycoprotein 2.
  • Synaptic vesicle proteins are membrane trafficking proteins comprising 12 transmembrane segments in case of SV2 but only one in case of Syt- II and Syt-I.
  • SEQ ID NOs: 1 to 12 Preferred peptides and proteins comprised in said receptor are provided in SEQ ID NOs: 1 to 12. More specifically, SEQ ID NO: 1 is a fusion protein of human SV2C, including the transmembrane domain, with glutathione S transferase (GST). The SV2C component of said fusion protein are residues 454 to 603 of human SV2C. SEQ ID NO: 2 consists of residues 454 to 603 of human SV2C. SEQ ID NO: 3 is a GST fusion protein with the luminal and transmembrane domain of rat Synaptotagmin I (residues 1 to 88).
  • SEQ ID NO: 4 consists of residues 1 to 82 of rat Synaptotagmin I and accordingly comprises the luminal domain and the transmembrane domain.
  • SEQ ID NO: 5 consists of residues 35 to 82 of rat Synaptotagmin I which is the minimal luminal domain including the transmembrane domain.
  • SEQ ID NO: 6 is a fusion protein of GST with the luminal domain and the transmembrane domain of rat Synaptotagmin II (residues 1 to 90).
  • SEQ ID NO: 7 consists of luminal and transmembrane domain of rat Synaptotagmin II (residues 1 to 90).
  • SEQ ID NO: 8 consists of the minimal luminal domain and the transmembrane domain of rat Synaptotagmin II (residues 44 to 90).
  • SEQ ID NO: 9 is a fusion protein of GST with the luminal domain 4 and the transmembrane domain of rat SV2A.
  • SEQ ID NO: 10 consists of luminal domain 4 and transmembrane domain of rat SV2A (residues 469 to 619).
  • SEQ ID NO: 1 1 is a fusion protein of GST with the luminal domain 4 and the transmembrane domain of rat SV2B.
  • SEQ ID NO: 12 consists of luminal domain 4 and transmembrane domain of rat SV2B (residues 413 to 560).
  • said substrate is located in the interior of the liposome. Preferably, it is exclusively located in the interior of the liposome. Moreover, preference is given to a soluble substrate such that the substrate is located in the lumen of the liposome. Alternatively or in addition, it is envisaged that the substrate may comprise a transmembrane segment and/or a membrane anchor, such membrane anchor being provided, for example, by covalently attached hydrophobic molecules such as fatty acids. While generally preference is given to soluble substrates, it is noted that, in conjunction with serotype C of botulinum neurotoxin, preference is given to a membrane attached substrate, said substrate still being located inside the liposome.
  • said bringing into contact is effected at a pH between 6 and 8, preferably between 7 and 7.4, more preferably at about 7.2.
  • the pH is changed to a value between 4 and 6, preferably between 5 and 5.4, more preferably about 5.2.
  • Further preferred pH values are 3.8, 3.9, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.1 , 5.3, 5.5, 5.6, 5.7, 5.8 and 5.9.
  • said change of pH is effected at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 24, 36 or 48 hours after said bringing into contact. These periods of time specify the binding incubation time.
  • buffer e.g. HEPES buffer at pH 7.2
  • Another way to adjust the pH may be performed as follows: After incubation of botulinum neurotoxin and liposomes, preferably both with as little dilution as possible, preferably at 4°C (on ice), preferably for 5-120 minutes, the mixture is transferred, for example into the cavities of black 96-well microplates (preferably pre-heated to 37°C) and buffer, preferably HEPES buffer, with the appropriate pH (preferably between pH 4 to 6, preferably preheated to 37°C) is added to give a desired pH as well as a specific concentration of liposomes and botulinum neurotoxin in the final reaction volume.
  • buffer preferably HEPES buffer
  • Desired pH values ar preferably selected from 3.8, 3.9, 4.0, 4.1 , 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1 , 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 and 6.0.
  • These preferred embodiments provide for a pH shift when performing the methods according to the present invention. While not strictly necessary, such shift in pH facilitates the formation of a membrane channel by a portion of the heavy chain of neurotoxin, said membrane channel providing for translocation of peptidase comprised by the light chain across the membrane. If said bringing into contact is effected at a pH of 6, it is preferred that after step (a) and prior to step (b) the pH is changed to a value below 6.
  • said determining according to (b) is effected at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 1 5, 20, 24, 36 or 48 hours after said change of pH. These periods of time specify the translocation and cleavage incubation time.
  • said liposome has a mean diameter between 50 and 500 nm, preferably between 100 and 200 nm, most preferred about 150 nm. Any size within these intervals, such as 250, 300, 350, 400 and 450 nm as well as larger diameters such 600, 700, 800 and 900 nm or 1 pm are deliberately envisaged.
  • liposomes may be prepared in such a manner that they exhibit a defined, preferably narrow size distribution. The means for ensuring such size distribution as defined in the examples are generally applicable to any liposome according to the present invention.
  • said liposome comprises or consists of: (i) DOPC and/or POPC; (ii) cholesterol; (iii) GT1 b, GD1 b and/or GD1 a; (iv) (1 ) SV2C or said fragment thereof; (2) synaptotagmin I or I I or said fragment thereof; (3) SV2A or said fragment thereof; and/or (4) SV2B or said fragment thereof; (v) said substrate; (vi) an aqueous medium in the interior of said liposome; and (vii) optionally tocopherol.
  • the aqueous medium is preferably as defined herein above as well as further below.
  • the present invention provides the use of a liposome as defined in any one of the preceding claims for determining presence, amount and/or activity of a clostridial neurotoxin.
  • the present invention furthermore relates in a third aspect to a liposome as defined in any one of the preceding claims.
  • Such liposomes may either be obtained from nerve cells or nerve tissues via procedures known in the art (synaptosome preparations). Alternatively, the liposomes are prepared by the methods of the invention as described further below.
  • said sample is known to comprise or suspected of comprising neutralising antibodies against said neurotoxin, wherein said sample, prior to subjecting it to said method, is combined with a known amount or activity of said neurotoxin, and wherein a decreased amount or activity of said neurotoxin as determined by said method in comparison to a control sample is indicative of the presence of said neutralising antibodies, wherein said control sample comprises said known amount or activity of said neurotoxin but is free of said neutralising antibodies.
  • neutralizing antibodies may interfere with the effects triggered by the neurotoxin and may prevent any beneficial effect altogether. In such a case it might be considered, for example, to switch serotype. In order to properly decide when such measures should be considered, monitoring of presence and amount of neutralizing antibodies may be useful.
  • test sample and a control sample, wherein both samples comprise the same known amount or activity of neurotoxin.
  • said sample comprises a test compound and a known amount or activity of said neurotoxin, wherein a decreased or increased amount or activity of said neurotoxin as determined by said method in comparison to a control sample is indicative of the test compound being an inhibitor or activator, respectively, of said neurotoxin, wherein said control sample comprises said known amount or activity of said neurotoxin but is free of said test compound.
  • This embodiment provides for a screening method for neurotoxin modulators, preferably inhibitors. Given the robustness and simplicity of the assay according to the present invention, it readily can be effected in high throughput format.
  • the readout may be effected in an automatic manner, for example by means of a CCD camera coupled to a data processing unit. Since liposomes may be kept in solution, the assay may be performed in wells of microtiter plates. Robotic systems for the handling of microtiter plates as known in the art may be employed.
  • the present invention provides a method of preparing a liposome, said method comprising or consisting of the following steps: (a) dissolving (i) liposome-forming lipid(s), preferably at least one phosphatidylcholine and cholesterol, said phosphatidylcholine preferably being selected from the group consisting of SPC, DOPC, and POPC; (ii) GT1 b; and optionally (iii) tocopherol in a suitable organic solvent; (b) evaporating said organic solvent; (c) resuspending the residue of step (b) in an aqueous medium, said aqueous medium comprising (ca) at least one receptor, said receptor being capable of binding a clostridial neurotoxin and comprising or consisting of a glycolipid and a peptide or protein, wherein preferably (i) said glycolipid is selected from the tri-sialo ganglioside GT1 b, the di- sialo ganglioside
  • Suitable organic solvents to be used in step (a) include polar solvents.
  • Polar protic as well as polar aprotic solvents may be used, wherein preference is given to mixtures thereof.
  • a preferred protic solvent is methanol and preferred aprotic solvents are dichloromethane and chloroform. Particularly preferred solvents are mixtures of methanol and dichloromethane and mixtures of methanol and chloroform, preferably 1 : 1 mixtures; see also Example 1 enclosed herewith.
  • the aqueous medium to be used in step (c) may comprise, in addition to the constituents recited in step (c) one or more buffers and/or salts.
  • HEPES (4-(2-hydroxyethyl)-1 -piperazine- ethanesulfonic acid) buffer, preferably at a concentration between 10 and 100 mM, and preferably at a pH between 7 and 8 may be used. Particularly preferred is 20 mM HEPES buffer at a pH of 7.4 and optionally 150 mM K-Glu or NaCI.
  • a particularly preferred aqueous medium comprises or consists of HEPES buffer, preferably between 10 and 100 mM such as 20 mM, with a pH between 7 and 8, preferably 7.4, preferably supplemented with ZnS0 4 , preferably at a concentration between 0.01 and 0.1 mM such as 0.05 mM, and furthermore preferably supplemented with TCEP, preferably at a concentration between 1 and 10 mM such as 2 mM.
  • buffers with a pH between 7 and 8 preferably a pH of 7.4 may be used, for example phosphate buffered saline (for example 137 mM NaCI, 2.7 mM KCI, 8.1 mM Na 2 HP0 4 , 1.76 mM KH 2 P0 4 ; also referred to as PBS) or Tris buffered saline (50 mM Tris.HCI and 150 mM NaCI; also referred to as TBS).
  • PBS phosphate buffered saline
  • TBS Tris buffered saline
  • said aqueous medium is free of any detergent, in particular free of Tween-20.
  • said aqueous medium is free of dithiothreitol (DDT).
  • Suitable membranes to be used for preparing liposomes by means of extruding as recited in step (d) are known to the skilled person.
  • Preferred membranes are polycarbonate membranes, which are available with various pore sizes.
  • Purification according to optional step (e) is a means of (i) removing components from the preparation obtained in step (d) which are not constituents of the formed liposomes, and (ii) obtaining a more uniform or narrow size distribution of liposomes.
  • Other preferred means of purification are described further above.
  • the present invention in addition provides the following methods.
  • a method of preparing a liposome comprising or consisting of the following steps: (a) dissolving a substrate in an aqueous medium; (b) adding liposome-forming lipids; (c) extruding the mixture obtained in (b) through a suitable membrane; (d) adding lactose or trehalose; (e) cooling the obtained mixture to a temperature between -50°C and -100°C. preferably -80°C; (f) subjecting the frozen mixture to freeze- drying; and (g) reconstituting the lyophilisate with aqueous medium.
  • said liposome solution is cooled in a first step to a temperature between 0 and 10°C, preferably 4°C, and in a second step to a temperature between 0 and -50°C, preferably -20°C.
  • Said reconstituting according to (g) is preferably effected with 10 times concentrated aqueous medium, wherein after shaking, centrifuging and again shaking, distilled water is added until the desired 1 -fold concentration is reached.
  • the receptor according to the invention is added concomitantly with said substrate.
  • a method of preparing a liposome comprising or consisting of the following steps: (a) diluting a substrate in an aqueous medium; (b) adding liposome-forming lipids; (c) cooling the emulsion obtained in (b) to about 0°C; (d) sonicating said solution.
  • the receptor according to the invention is added concomitantly with said substrate.
  • said substrate consists of or comprises a compound of the following formula (I): X - L - Y; wherein L is a peptide or protein comprising or consisting of a sequence which is deavable by said peptidase; "— " denotes a covalent bond, X-L-Y is preferably soluble in aqueous medium and/or free of any transmembrane domain or membrane anchor; and (a) X is moiety comprising or consisting of a FRET donor or acceptor; and Y is a moiety comprising or consisting of a FRET acceptor if X comprises or consists of a donor, or a FRET donor if X comprises or consists of an acceptor; or (b) X is a fragment of an enzyme, said enzyme preferably being luciferase; and Y is another fragment of said enzyme, said enzyme preferably being luciferase, where
  • L is a peptide or protein
  • moieties X and Y are attached to the N- and C-terminus of said peptide or protein L, respectively.
  • donor and acceptor are placed on different sides of the cleavage site.
  • the cleavage site defines an N-terminal and a C-terminal portion of the peptide or protein, said N- and C-terminal portions being connected by the scissile bond, donor and acceptor may be placed anywhere in said N- and C-terminal portion, respectively, provided that they are in sufficient spatial proximity for fluorescence resonance transfer to occur in the uncleaved form of the substrate.
  • X - L - Y is preferably soluble in aqueous medium. Also, it is preferred that X - L - Y is free of any transmembrane domain or membrane anchor, such membrane anchor being, for example, a hydrophobic covalently attached moiety (for example palmitoyl side chains as present in naturally occurring SNAP-25).
  • L comprises or consists of (i) SNAP-25 or a fragment thereof, said fragment being cleavable by the peptidase comprised in said neurotoxin and preferably being selected from a sequence comprising or consisting of residues 93 to 206, 146 to 203, or 156 to 184 of SNAP-25; (ii) VAMP-2, VAMP-1 , VAMP-3 or a fragment thereof, said fragment being cleavable by the peptidase comprised in said neurotoxin and preferably being selected from a sequence comprising or consisting of residues 30 to 62, 30 to 86, 38 to 62, 47 to 96, or 62 to 86 of VAMP-2; and/or (iii) Syntaxin-1 , -2, -3 or a fragment thereof, said fragment being cleavable by the peptidase comprised in said neurotoxin and preferably being a sequence comprising or
  • SEQ ID NOs: 13 to 21 provide sequences of preferred L moieties according to the present invention.
  • SEQ ID NO: 13 is the sequence of residues 1 to 206 of rat SNAP-25.
  • SEQ ID NO: 14 is a subsequence of SEQ ID NO: 13 consisting of residues 140 to 206 of SNAP-25.
  • SEQ ID NO: 15 is the sequence of human VA P-2/Synaptobrevin II.
  • SEQ ID NO: 16 is the sequence of rat VAMP-2 Synaptobrevin II.
  • SEQ ID NO: 17 is the sequence of rat VAMP1 .
  • SEQ ID NO: 18 is the sequence of rat VAMP-3.
  • SEQ ID NO: 19 is the sequence of human Syntaxin-1.
  • SEQ ID NO: 20 is the sequence of rat Syntaxin-1.
  • SEQ ID NO: 21 is the sequence of mouse Syntaxin-1 .
  • subsequences of the natural substrates are also substrates of the respective clostridial neurotoxins. The subsequence preferences may vary between serotypes or between botulinum neurotoxins and tetanus toxin.
  • the skilled person can determine by using simple tests whether a given fragment of any one of SNAP-25, VAMP-2, VAMP-1 , VAMP-3 and Syntaxin qualifies as a substrate.
  • said substrate is selected from SNAPtide, SNAP Etide, VAMPtide, and SYNTAXide.
  • This embodiment refers to commercially available botulinum neurotoxin substrates.
  • SNAPtide as described in US 6,504,006 is a SNAP- derived BoNT/A substrate
  • SNAP Etide is a BoNT/E substrate
  • VAMPtide is a VAMP-derived BoNT/B substrate
  • SYNTAXide is a Syntaxin-derived BoNT/CI substrate.
  • said neurotoxin is botulinum neurotoxin type A; said receptor comprises or consists of (i) GT1 b and (ii) SV2C or said fragment thereof; and L comprises or consists of SNAP-25 or said fragment thereof, said fragment preferably consisting of residues 146 to 203 of SNAP-25;
  • said neurotoxin is botulinum neurotoxin type B; said receptor comprises or consists of (i) GT1 b and (ii) synaptotagmin II or I or said fragment thereof; and L comprises or consists of VAMP-2, VAMP, VAMP-3 or said fragment of any of these, said fragment preferably consisting of residues 62 to 86 of VAMP-2;
  • said neurotoxin is botulinum neurotoxin type C1 ; said receptor comprises or consists of GT1 b; and L comprises or consists of SNAP-25, said fragment thereof, said fragment thereof preferably consisting of residues 62 to 86 of VAMP-2;
  • said neurotoxin is bot
  • VAMP-2 is also known as synaptobrevin 2.
  • VAMP is also known as synaptobrevin 1 .
  • VAMP-3 is also referred to as cellubrevin.
  • both BoNT/B and BoNT/G bind to synaptotagmins I and II, wherein the affinities for binding are generally as follows: BoNT/B - synaptotagmin II » BoNT/G - synaptotagmin I > BoNT/G - synaptotagmin II > BoNT/B - synaptotagmin I.
  • the recited substrates to the extent they are fragments defined in terms of specific residue ranges, are generally susceptible to cleavage by the neurotoxin peptidase to the same extent as the protein they originate from.
  • This embodiment provides preferred neurotoxin/receptor/substrate combinations, wherein the substrate is defined in terms of L as recited in herein above. Accordingly, it is understood that the preferred fragments are to be modified by introduction of fluorophor A and B in order to obtain substrates according to the present invention.
  • X is o-aminobenzoyl (o-Abz) or fluorescein isothiocyanate (FITC) and/or Y is 2,4-dinitrophenyl (DNP) or DABCYL.
  • o-Abz o-aminobenzoyl
  • FITC fluorescein isothiocyanate
  • DNP 2,4-dinitrophenyl
  • a large variety of FRET pairs available from several manufacturers, is at the skilled person ' s disposal.
  • the present invention provides a kit comprising or consisting of (a) at least one receptor, said receptor being capable of binding a clostridial neurotoxin and comprising (1 ) a glycolipid and (2) a peptide or protein; (b) a substrate which (1 ) is cleavable by the peptidase comprised in said neurotoxin and (2) comprises a FRET pair, the donor of said FRET pair exhibiting increased fluorescence upon cleavage by said peptidase; and (c) optionally one or more liposome-forming lipids.
  • the kit according to the present invention further comprises or further consist of one or more of the following (a) a manual containing instructions for performing the method of determining according to the invention; and/or (b) a manual containing instructions for performing the method of a liposome according to the invention.
  • constituents (a), (b) and (c) of said kit are as defined in conjunction with any of the other aspects of the present invention as disclosed herein above.
  • Figure 1 Illustration of the method according to the first aspect of the invention.
  • Binding BoNT binds via its HC (oval) to nerve cell receptors (short and long bars) immobilized in the liposome membrane; translocation: conformational change of BoNT HC and insertion into a transmembrane channel upon pH-shift and translocation of BoNT LC (dark oval) into the liposome lumen; cleavage: enzymatic cleavage of the peptide reporter (acceptor fluorophor dark grey, donor fluorophor light grey) in the liposome lumen by the BoNT LC (dark oval with triangle opening). H + represents free protons present upon acidification of the milieu surrounding the liposomes.
  • Figure 2 Assembly of SEC-columns and illustration of collected fractions.
  • Figure 3 Structure of lipids used for liposome production and extrusion of liposome emulsions.
  • B Liposome suspension after consecutive extrusion through PC membranes with different pore sizes; numbers represent the respective extrusion cycles.
  • Figure 4 Immunological detection of GT1 b and GST-Synaptotagmin II (Sytll) fusion protein.
  • Figure 5 Binding of BoNT/B to GST-Sytll under different pH conditions.
  • Figure 6 Immunological detection of GT1 b and GST-Sytll integrated in liposomes.
  • A Dot blot analysis of liposomes with and without GT1 b and GST-Sytll in comparison to diluted GT1 b and GST-Sytll standards as positive control and empty liposomes and buffer as negative control.
  • B ELISA detection of GST-Sytll and GT1 b integrated into dialysed receptor liposomes (triangles and inverted triangles), in comparison to isolated GST-Sytll (squares) and GT1 b (circles). Results for PBS (negative control) are shown as dotted black line.
  • Liposome were composed of 20 mM SPC/Cholesterol/D,L-alpha-tocopherol (77.1 : 19.5:0.63), 0.2 mM GT1 b (1 mM GT1 b corresponds to 2.129 mg/mL), and 0.45 mg/mL GST-Sytll.
  • FIG. 7 Separation of receptor liposomes by SEC.
  • A Dot blot of serially diluted non-separated receptor liposomes with GT1 b and GST-Sytll, and PBS as negative control (upper part), fractions of undiluted liposomes separated by SEC (middle), and positive controls, i.e. 0.02 mM GT1 b and 7.2 mg/mL GST-Sytll (lower part).
  • the dotted upper line represents the negative control for OD254 (PBS), the dotted lower line (two lines coinciding) represent the negative controls for GST-Sytll and GT1 b, respectively.
  • Liposome were composed of (A) 20 mM SPC/Cholesterol (69.75:29.75), 0.2 mM GT1 b, and 0.72 mg/mL GST-Sytll and (B) 20 mM DOPC/Cholesterol (18: 1 ), 0.2 mM GT1 b, and 0.72 mg/mL GST-Sytll.
  • Figure 8 Sandwich dot blot for detection of dual-receptor integration into liposomal membranes.
  • Sample composition was (1 ) 20 mM DOPC/Cholesterol (18: 1 ), (2) DOPC/Cholesterol (18: 1 ), 0.5 mM GT1 b, and 0.72 mg/mL GST-Sytll, (4&6) 0.1 mg/mL GT1 b, and (5&6) 7.2 mg/mL GST-Sytll, respectively.
  • Figure 9 VAMPtide fluorescence and cleavage.
  • cleavage buffers without BoNT/B were used as negative controls.
  • D-F Modified cleavage buffer (20 mM HEPES pH 7.4, 0.05 mM ZnS0 4 , 2 mM TCEP) was used to detect cleavage of 10 ⁇ VAMPtide by different concentrations of (D) BoNT/B (filled symbols), (E) LH N B (filled symbols), and (F) scLH N B (filled symbols). Modified cleavage buffer without toxin (open circles) was used as negative control.
  • Figure 10 Sensitivity of VAMPtide immunoassay and encapsulation efficiency of VAMPtide into liposomes.
  • Figure 11 Test for non-specific membrane binding of VAMPtide with protease protection assay.
  • Figure 13 Cleavage of PL150 substrate by BoNT/A in the presence of liposomes.
  • Figure 14 Immunological detection of BoNT/A and BoNT/B cleavage substrates.
  • Figure 15 Sensitivity of List Biological Laboratories cleavage substrates for BoNT/A and BoNT/B.
  • Figure 16 Separation of A-liposomes by SEC.
  • A-liposomes analysed for liposomes via the count rate determined by dynamic light scattering (black bars) and for receptors, i.e. GST-SV2c (shaded bars) and GT1 b (dotted bars), respectively, via ELISA.
  • A-liposomes were composed of 40 mM DOPC/Cholesterol, 0.2 mM GT1 b, and 0.0125 mg/mL GST-SV2c.
  • Figure 17 Release of cleavage substrate upon lysis of A-FAL and B-FAL.
  • FAL were composed of 40 mM DOPC/Cholesterol, 0.2 mM GT1 b and either 0.008 mg/mL GST-SV2c and 200 ⁇ PL50 (for A-FAL) or 0.72 mg/mL GST-Sytll and 200 ⁇ PL150 (for B-FAL) before treatment with BioBeads SM-2 and dialysis.
  • the reactions was performed in 10 mM HEPES, supplemented with 75 ⁇ ZnS0 4 .
  • Figure 18 Cleavage substrate in B-FAL SEC-fractions.
  • B-FAL SEC fractions of B-FAL, analysed for liposomes via the count rate determined by dynamic light scattering (black bars) and for (A) PL150 and (B) VAMPtide 0 .
  • B-FAL were composed of 40 mM DOPC/Cholesterol, 0.2 mM GT1 b, 0.72 mg/mL GST-Sytll and 200 ⁇ BoNT/B cleavage substrate before treatment with BioBeads SM-2 and dialysis.
  • SEC fraction were diluted 1/2000 in 10 mM HEPES buffer containing 0.1 % Triton X-100 prior to coating on ELISA microplates at 4°C over night.
  • Example 1 illustrate the invention but are not to be construed as being limiting.
  • BoNT/B truncation mutants consisting of residues 1 to 863 and lacking the H c -fragment; either as single polypeptide chain (sc) or dichain hydrolysed into LC and HC after R441 ; both available from A. Rummel, Hannover Medical School (MHH), Institute for Toxicology).
  • Lyophilised Trisialoganglioside GT1 b from bovine brain with a total mass of 2180 Da was purchased from Merck KGaA (Darmstadt, Germany). The ganglioside was dissolved in a 2:1 mixture of Chloroform (CHCI 3 ) and methanol (CH 3 OH) and stored at -20°C.
  • CHCI 3 Chloroform
  • CH 3 OH methanol
  • the modified version used here is truncated, containing 61 amino acids of the N-terminus, with 30 amino acids representing the transmembrane domain.
  • the truncated Sytll also contains GST (Glutathione S-transferase) coupled to the N-terminus, without hindering binding of BoNT/B H c .
  • GST-Sytll is bound in Triton X-100 micelles due to the purification protocol used by Andreas Rummel's group (Rummel et al. [80]).
  • mouse anti-GT1 b antibodies (Millipore, Billerica, MA, USA) or rabbit polyclonal anti-GST antibody (Bethyl Laboratories Inc., Montgomery, TX, USA) at a concentration of 0.1 pg/mL, and peroxidase coupled goat anti-mouse, or goat anti-rabbit antibodies (Sigma-Aldrich, Buchs, Switzerland) at 1/1000 dilution were used.
  • TMB (3, 3', 5, 5'- tetramethylbenzidine; Sigma-Aldrich, Buchs, Switzerland) or 4CN (4-chloro-1 -naphthol; Bio- Rad, Laboratories Inc., Hercules, CA, USA) were used as substrates for ELISA or dot blot experiments, respectively.
  • VAMPtide (List Biological Laboratories, Inc., Campbell, CA, USA) was used as a peptide substrate for measuring proteolytic activity of BoNT/B LC.
  • VAMPtide is an oligopeptide with o- Abz (o-Aminobenzoic acid) as donor fluorophor coupled to the N-terminus, and DNP (2,4- Dinitrophenyl) as acceptor/quencher with an absorbance spectrum overlapping the emission spectrum of the donor fluorophor coupled to the C-terminus.
  • the peptide contains a recognition sequence specific for the cleavage by BoNT/B LC.
  • FRET Forster resonance energy transfer
  • VAMPtide assay For immunological detection of uncleaved VAMPtide molecules, a 1/1000 dilution of polyclonal rabbit antibody generated against aa 1 -81 of the cytoplasmic part of rat cellubrevin (anti-VAMP 1/2/3; Synaptic Systems GmbH, Gottingen, Germany) was used. Binding of the anti-VAMP antibody was detected via peroxidase coupled anti-rabbit antibody (Sigma-Aldrich, Buchs, Switzerland). VAMPtide assay
  • VAMPtide assay for the detection of BoNT cleavage activity was performed according to the manufacturer's instructions. In short, lyophilised VAMPtide was resuspended in DMSO resulting in a 5 mM stock solution and stored at -20°C. Prior to the experiments, stock solution was diluted in 20 mM HEPES to 250 ⁇ .
  • the reaction buffer for hydrolysis of VAMPtide by BoNT/B was 20 mM HEPES (pH 7.4), 0.05 mM ZnS04, 5 mM DTT and 0.2% Tween-20.
  • the hydrolysis buffer contained 50 mM HEPES (pH 6.3) and 0.05% Tween-20.
  • An alternative lipid mixture may be prepared as follows. Soybean asolectin, dioleoyl L-K- phoshatidylcholine, bovine brain phospholipids or alternatively a phosphatidic acid (PA) lipid mixture (consisting of dioleoyl L-alpha-phosphatidylcholine 70%, phosphatidic acid 20% and cholesterol 10%) are dissolved in chloroform, dried to a thin film under a gentle N 2 flow and vacuum pumped for at least 2 h to remove residual traces of organic solvent. Production of LUVs (large unilamellar vesicles)
  • PA phosphatidic acid
  • the required volume of the respective lipid mixture was dissolved in a 1 : 1 mixture of methanol (CH 3 OH) and dichloromethane (CH 2 CI 2 ), and dried at room temperature under vacuum at 1400 rpm in a Eppendorf concentrator 5301 with a F 45-48-1 1 Rotor (Eppendorf AG, Hamburg, Germany) until complete evaporation of the organic solvents (1 -2 h). Upon complete evaporation of solvent, the lipids formed a thin yellow film on the walls of the vials. For longer storage, vials were sealed under N 2 -atmosphere and stored at -20°C.
  • the lipid film was resuspended in appropriate volumes of 20 mM HEPES buffer (pH 7.4) as aqueous medium. The vial was shaken until complete solution of the lipids, giving a turbid white emulsion of multilamellar vesicles (MLV). Examination under Zeiss Dialux 20 EB light microscope using 100-fold object lens (100/1.25 oil immersion) plus 10 fold ocular (Periplan GF 10x/18) magnification allowed for visual control that the MLV had been formed.
  • MLV multilamellar vesicles
  • the vial with the emulsion was shaken with 1400 rpm in an Eppendorf Thermomixer comfort (Eppendorf AG, Hamburg, Germany) at 30°C for 30 minutes until the lipid film was completely dissolved in the aqueous medium.
  • LUVs were obtained by extrusion through polycarbonate (PC) membranes (Nucleopore track-etched polycarbonate membrane; Whatman pic, Maidstone, UK) with defined pore sizes (400, 200, and 100 nm) in a MiniExtruder (Avanti Polar Lipids Inc., Alabaster, AL, USA).
  • the equipment was assembled according to the manufacturer's instructions and, for production of LUVs, the MLV emulsion was passed through a 400 nm, and subsequently through 200 and 100 nm pore size PC-membrane. Homogeneity and size were controlled via DLS (Dynamic Light Scattering) with a Brookhaven Instruments BI-200SM research goniometer and particle sizer (Brookhaven Instruments, Holtsville, NY, USA) at an angle of 90° at 25°C sample temperature. Data was processed with Brookhaven Instruments Particle Sizing Software (Brookhaven Instruments, Holtsville, NY, USA). While LUVs are preferred, liposomes may also be prepared as follows. Production of DRVs ( dehydra tion-rehydra tion vesicles)
  • VAMPtide or other any other substrate according to the invention is diluted in the reaction buffer (preferably 20 mM HEPES with ZnS0 4 and TCEP at pH 7.2-7.4 as defined herein above; see "aqueous medium”) at concentrations of 100-200 ⁇ and added to a lipid film giving an emulsion of 40 mM DOPC/Cholesterol (13/1 ), 100-200 ⁇ substrate in the reaction buffer.
  • the emulsion is extruded eleven times each through polycarbonate membranes with pore diameters of 200 nm and 100 nm (and, if preferred, 50 nm) pore diameter. Then, lactose or trehalose is added to give a final concentration of 1 to 5%, preferably 3.5%.
  • the (clear) liposome solution is cooled to 4°C, to -20°C, and subsequently to -80°C.
  • the frozen emulsion is then subjected to freeze-drying over night until complete evaporation of the contained liquid.
  • Reconstitution is carried out first by adding 1/10 of the original liquid volume as 10 times concentrated buffer (preferably 10x HEPES).
  • concentrated buffer preferably 10x HEPES.
  • the lyophilizate plus added buffer is left for 30 minutes at room temperature, and then vigorously shaken, centrifuged, and again vigorously shaken. Subsequently, liquid (i.e. distilled H 2 0) is added till the original level of liquid volume is reached.
  • liquid i.e. distilled H 2 0
  • VAMPtide or other any other substrate is diluted in the reaction buffer (preferably 20 mM HEPES with ZnS0 4 and TCEP at pH 7.2-7.4 as defined herein above; see "aqueous medium”) at concentrations of 100-200 ⁇ and added to a lipid film giving an emulsion of 40 mM DOPC/Cholesterol (13/1 ), 100-200 ⁇ substrate in the reaction buffer.
  • the vessel containing the emulsion is placed into a 0°C waterbath.
  • the transducer tip of the sonicator such as Branson sonifier
  • Different durations and sonication times can be applied until the emulsion turns from milky to opalescent.
  • Metallic particles from the transducer tip can be removed by centrifugation.
  • liposome emulsions were subjected to SEC (size exclusion chromatography) columns with a gel bed volume of approximately 600 ⁇ . for separation from not-integrated and/or not-encapsulated compounds.
  • SEC columns allowed for easy and quick separation of 10- 00 ⁇ of sample via centrifugation in a tabletop centrifuge. Due to the small gel-volume the dilution effect was relatively low (1 -2 times dilution). The technique works as common gravitational SEC-column, just that the gravitational force is replaced by centrifugal force.
  • the gel in this case a 50%-slurry of Sepharose 4B (Sigma-Aldrich, Buchs, Switzerland) in 20 mM HEPES (pH 7.4), is filled into the column, i.e. a 0.5 ml. Eppendorf cup pierced on the bottom and stuffed with 1 mm glass fibres. For separation, 10-100 ⁇ _ of the sample are loaded slowly onto the gel bed, assuring that none of the sample passes along the sides of the SEC column. Centrifugation of the tube at 1200 RCF for 45 s elutes the first fraction of the sample, including the components with the biggest hydrodynamic volume.
  • GT1 b For production of receptor liposomes, GT1 b was added to the lipid mixture dissolved in organic solvent and either dried under vacuum or under constant laminar flow of gaseous N 2 . Due to the high transition phase of GT1 b gangliosides, the lipid solution cannot be dried out completely. Accordingly, instead of a lipid film the lipid mixture is present in a highly viscous gel-like state. For complete solvation of the gel, the whole suspension is pipetted up and down in aqueous medium and subsequently shaken as described above. GST-Sytll is added with the aqueous medium in the respective concentration. Likewise, for encapsulation experiments, VAMPtide is added with the aqueous medium in the respective concentration to dried lipid film.
  • the emulsion is incubated with pre-hydrated Bio-Beads SM-2 Adsorbents (Bio-Rad, Laboratories Inc., Hercules, CA, USA). Bio-Beads plus GST-Sytll liposomes are incubated under rotation at 4°C for 2.5 h. Thereafter, another portion of SM2 beads is added and incubated with the emulsion for another 2h. To separate Bio-Beads with bound Triton X-100 from the liposome, the mixture is kept still for 5 min to allow settling of the beads. After transferring the liposomes in the supernatant to a clean vial, the remainder is centrifuged at 5.000xg RCF for 30 seconds and the resulting supernatant pooled with the first one.
  • Bio-Beads SM-2 Adsorbents Bio-Rad, Laboratories Inc., Hercules, CA, USA. Bio-Beads plus GST-Sytll liposomes are
  • receptors, VAMPtide, receptor liposomes, or VAMPtide liposomes are coated on MaxiSorp microtiter plates (Nunc, Langenselbold, Germany) at 4°C overnight. After 60 min of blocking, sample cavities are washed and incubated for 60 min with the respective antibodies, followed by incubation with species-specific peroxidase coupled antibodies for 30 min.
  • the aqueous liposomes lumen provides a cell-like reaction compartment. Due to the multiple and complex reactions (binding of BoNT/B, translocation of BoNT/B LC and VAMPtide cleavage) that are supposed to take place in the assay, preferably LUV with a size of approximately 100-200 nm are used to provide for a sufficiently large reaction volume. For the reproducibility and functionality of the assay it is preferable that the liposome emulsion were consistent concerning their lamellarities and diameters. Accordingly, emulsions with mainly MLVs obtained by reconstitution of lipid films were subjected to an extrusion process.
  • Figure 3C exemplarily shows estimates of the distribution of liposome diameters in a liposome emulsion from 20 mM lipids (SPC/Cholesterol/Tocopherol). After eleven times extrusion through 400 nm and subsequently eleven times extrusion through 100 nm pore diameter PC membranes, the ratio of liposome diameters measured in the emulsion (intensity) resembled a Gaussian distribution.
  • lipids such as DOPC (1 ,2-Dioleoyl-sn- glycero-3-phosphocholine) or POPC (1 -Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), due to their low transition phase temperature (approximately 0°C) and their structural properties (Steven Burgess, director R&D, Avanti Polar Lipids, personal communication). Accordingly, not only SPC, but also DOPC, both in combination with Cholesterol, were tested. DLS measurements were performed for the liposomes derived from different lipid formulations (Table 2).
  • Characteristics of liposome emulsions from different lipid formulations Mean diameter and calculated lipid molecules per liposome and liposomes per mL, respectively.
  • Lipid formulation Lipid .
  • a head group area, calculated on the basis of the molar ratio of the lipid formulation and on 0.70 nm 2 , 0.28 nm 2 , and
  • the quantity of lipid molecules per liposome was calculated with formula (1 ), using the average diameter of the lipid head groups (a), the outer liposome diameter (d), and the inner liposome diameter, which is the outer diameter reduced by 5 nm (the average thickness of a lipid double membrane).
  • the total number of liposomes ( ⁇ //, ⁇ 0 ) in one millilitre of the respective emulsion was calculated using formula (2), with the concentration of the lipid mixture in Mol (c /( p d ), the Avogadro constant of 6.022 x 10 23 mol "1 (N A ) and division by 1000 for conversion from L to mL [40].
  • a pH-shift provides for enhanced translocation of BoNT LC via the BoNT H N transmembrane channel from the cis side (i.e. endosomal lumen or medium surrounding the liposomes) to the trans side (i.e. cytosol or liposomal lumen) of a membrane [44].
  • a pH-shift with DMG (3,3-Dimethylglutaric acid) would influence the binding affinity of BoNT/B to the respective receptors we performed the following experiment.
  • BoNT/B was coated on a microtiter plate and GST-Sytll used as antigen, which was detected by anti-GST and a respective peroxidase coupled anti-species antibody.
  • the pH was kept at pH 7.2 for 90 min, shifted to pH 5.2 after 60 min and incubated for another 30 min, or kept at pH 5.2 for the entire 90 min during incubation of GST-Sytll with the coated toxin, respectively.
  • Figure 5 shows, that if pH was shifted from pH 7.2 to 5.2 even more toxin bound to coated GST-Sytll than if incubated at pH 7.2 only. If binding occurred solely at pH 5.2, instead of pH 7.2, the effect was even more pronounced, and binding increased by almost 100%. This is especially interesting, as it has been proposed, that a conformational change in BoNT HC structure takes place upon an acidic pH shift [45].
  • Figure 6A shows that dot blot analysis was able to detect both receptors in dialysed and undialysed receptor liposome samples and that liposomes without any receptors yielded no unspecific signal.
  • ELISA analysis of liposomes after dialysis with 100 kDa molecular weight cut-off ( WCO) against buffer showed that if receptors were integrated into liposome membranes they could only be detected at much higher concentrations (Figure 6B). It was found, that similar signal intensities as, e.g. for isolated GST-Sytll, required 200 to 400 times higher receptor concentrations.
  • GT1 b inserted in liposome membranes yielded even lower signal, if compared to isolated GT1 b.
  • Figure 8B shows a distinct signal when liposomes with both receptors integrated were blotted. Yet, controls with isolated receptors only also showed colouration, although the signal intensity was much lower and the spot much more diffuse. This was only observed, when isolated receptor was present that in theory can be detected by the detection antibody. As the employed antibody pairings showed neither cross reactions in this (negative control with PBS) nor in other experiments, a possible explanation might be, that due to the porous structure of the membrane, the isolated receptors did not only bind to the coated anti-receptor antibodies, but also to the membrane structure itself - in spite of blocking with PBS supplemented with 1 % BSA.
  • VAMPtide fluorescence without the necessity of the actual cleavage reaction
  • VAMPtide uq VAMPtide; List Biological Laboratories, Inc., Campbell, CA, USA
  • This peptide contains the complete VAMPtide peptide sequence with a coupled o-Abz fluorophor, but without any quencher molecule.
  • emission at 418 nm (after excitation at 321 nm) displayed the highest signal intensity, although the S/N-ratio was a little below the maximum found at 410 nm.
  • 418 nm also represents the emission readout wavelength recommended by the manufacturer, these parameters were used for further analysis.
  • the cleavage buffer As the liposome lumen presents the reaction compartment for the cleavage reaction, the cleavage buffer has to be confined to the liposome interior.
  • the cleavage buffer recommended by the manufacturer is comprised of HEPES, ZnS0 4 , DTT as reducing agent and Tween-20 to increase toxin's solubilisation. Due to its size and steric hindrance, tris(2- carboyethyl)phosphine (TCEP) cannot freely diffuse through membranes. Accordingly, it has been tested as a substitute for DTT as reducing agent [45,59,60,6,61 ,62].
  • the detergent Tween-20 may present a problem when used within liposomes, as it may compromise the integrity of cell membranes, possibly facilitating cells lysis [63]. Also, Tween-20 may intercalate between the membrane bilayers and form mixed micelles with isolated phospholipids and membrane proteins. Accordingly, if Tween-20 was used, there might be a certain risk, that a fraction of the liposomes could be transformed from LUV into mixed micelles with lipid and detergent molecules in the membrane. Hence, attempts were made to replace DTT in the cleavage buffer by TCEP and to elucidate how the absence of Tween-20 affects VAMPtide cleavage.
  • the cleavage activity of 20 nM BoNT/B was tested with different TCEP concentrations, ranging from 0.75 to 5 mM, and compared with the cleavage activity in buffer supplemented with 5 mM DTT.
  • the cleavage reaction was performed at 37°C and fluorescence was measured with excitation and emission wavelengths of 321 nm and 418 nm, respectively.
  • 2 mM TCEP was found to be the concentration, which yielded the highest cleavage activity (Figure 9B). Omitting Tween-20 in the cleavage buffer did not reduce the cleavage activity. Instead, cleavage activity increased considerably ( Figure 9C).
  • replacing 5 mM DTT by 2 mM TCEP and omitting Tween-20 in the cleavage buffer did not affect VAMPtide cleavage considerably.
  • Tween-20 may help to disperse non-specifically bound enzyme or substrate from the walls of the microtiter well, so that more enzyme and substrate are available for interaction [66]. So, apparently specificity of VAMPtide cleavage by BoNT/B LC should not be impaired by the absence of Tween-20 in the cleavage buffer. As our results confirm good cleavage activity even without Tween-20, the described buffer composition without Tween-20 and with 2 mM TCEP in 20 mM HEPES (pH 7.4) supplemented with 0.05 mM ZnS0 4 is preferred.
  • BoNT/B Apart from BoNT/B, we tested the cleavage activity of isolated BoNT/B LC, TeNT, LH N B, and scLH N B in the modified cleavage buffer. Early experiments showed that isolated BoNT/B LC did not exhibit any cleavage activity under these conditions. Instead, supplementation of 0.2% Tween-20 restored most of the cleavage activity of isolated BoNT/B LC, while DTT supplementation restored the function only partly (data not shown). As expected, the cleavage reaction did neither work for TeNT, as VAMPtide did not contain the specific recognition site for TeNT LC. In contrast to isolated BoNT/B LC and TeNT, BoNT/B and BoNT/B complex displayed typical cleavage activity, with a limit of detection of 2 and 5 nM, respectively ( Figure 9D & Table 3).
  • Toxin Buffer type 1 M SD 2 nM SD 5 nM SD 10 nM SD 20 nM SD derivate
  • LH N Band scLH N B are BoNT/B truncation mutants devoid of the receptor-binding domain. Also, scLH N B is still connected to the BoNT/B LC not only via disulphide, but also via a peptide bond. As the LC in these toxin derivatives is still active, VAMPtide could be successfully cleaved under the modified buffer conditions, both with detection limits of 1 nM.
  • VAMPtide molecules are incorporated into liposomes by adding them together with the aqueous medium for lipid film reconstitution. Upon forming of MLV, VAMPtide molecules become enclosed by liposomal membranes. With subsequent extrusion through PC membranes, a distinct portion of the molecules gets encapsulated, while another part stays outside the liposomes. To test for the actual encapsulation rate of VAMPtide molecules into the liposomes, without the need for any cleavage reaction, we used a VAMP antibody and corresponding peroxidase coupled anti-species antibody for detection.
  • sensitivity is shown for VAMPtide coated in concentrations ranging from 5 to 100 ng/mL.
  • the assay showed excellent sensitivity with a detection limit as low as 5 ng/mL.
  • VAMP antibody detected VAMPtide at concentrations as low as 2.5 ⁇ , while not detecting unquenched VAMPtide calibration peptide (100 ⁇ ), unquenched SUQ- FITC (100 ⁇ ), GT1 b (50 pg/mL) or empty liposomes (data not shown).
  • anti- VAMP antibody recognized GST-Sytll at a concentration of 7.2 mg/mL.
  • SD Standard deviation +/- 23.14 +/- 16.19
  • EE[%] values were very high, with approximately 73 and 67% of all VAMPtide molecules encapsulated into liposomes, i.e. 146.14 +/- 46.28 ⁇ (original 200 ⁇ ) or 66.61 +/- 16.19 ⁇ (original 100 ⁇ ) VAMPtide encapsulated.
  • High Standard Deviations (SD) were due to the narrow range of linear detection in the ELISA immunoassay ( Figure 10C&D). It is evident, that if a lower original VAMPtide concentration was used, the encapsulation efficiency is also negatively affected.
  • the percentage of encapsulated VAMPtide corresponds to 14.6 +/- 4.6 times and 6.7 +/- 1.6 times of the concentration recommended by the manufacturer (10 ⁇ ) for sensitive detection of BoNT/B cleavage activity.
  • concentration recommended by the manufacturer (10 ⁇ ) for sensitive detection of BoNT/B cleavage activity.
  • fluorescence readout of unquenched calibration peptide of VAMPtide encapsulated into liposomes was slightly affected by high liposome concentration (data not shown). Accordingly, high encapsulation efficiencies would later allow for dilution of the liposomes, thus reducing the interference with the fluorescence measurement.
  • simple extrusion encapsulates a high percentage of VAMPtide molecules into liposomes.
  • DNP 2,4-dinitrophenol
  • o-Abz o-Aminobenzoic acid
  • FRET acceptor and donor fluorophor respectively.
  • DNP exhibits hydrophobic properties [71] and o-Abz may also dissolve in nonpolar solvents [72].
  • VAMPtide is only loosely associated to the liposomes and/or integrated only by part into the liposome membranes [73]. This could influence the calculated encapsulation efficiency, as BoNT/B could cleave the associated VAMPtide molecules without any binding or translocation needed.
  • VAMPtide molecules which are only loosely associated to the liposomes, and VAMPtide molecules that are partly integrated into the membrane, but whose peptide chains extend into the liposome exterior, would be cleaved by Proteinase K (Sigma-Aldrich, Buchs, Switzerland).
  • Proteinase K is a broadspectrum serine protease, which predominantly cleaves the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups [74].
  • VAMPtide liposomes from the previous experiment with approximately 146.14 +/- 46.28 ⁇ VAMPtide encapsulated (original 200 ⁇ ) were subjected to digestion for 60 min at 37°C with 50 and 100 Mg/mL of Proteinase K, respectively. After digestion, VAMPtide liposomes were fractioned by SEC, and measured with VAMP antibody in an ELISA assay ( Figure 1 1 B).
  • Triton X-100 Prior to coating on microtiter plates for ELISA analysis, Triton X-100 was added at a concentration of 3% to lyse liposomes and to guarantee detection of encapsulated and not-encapsulated VAMPtide. As coating took place at 4°C, no more VAMPtide cleavage took place at this point, even if Proteinase K was present. The immunoassay results showed that signal intensities in the liposome fractions two and three in samples digested with Proteinase K were only little lower than those, which were found in undigested samples.
  • VAMPtide molecules are either thoroughly encapsulated into the liposomes and/or that they may be integrated into the liposome membrane in whichever modality and cannot be cleaved by an endopeptidase such as Proteinase K, even at concentrations as high as 100 g/mL.
  • Cleavage assays were performed in black 96 half-well microplates (Greiner Bio-One GmbH, Frickenhausen, Germany) with a reaction volume 100 ⁇ _.
  • reaction buffer composition corresponding to the respective substrate; see below
  • only components HPES, ZnCI 2 , ZnS0 4 , TCEP (with an adjusted pH to 7.0), DTT, and ultrapure H 2 0
  • TCEP with an adjusted pH to 7.0
  • DTT dimethyl sulfoxide
  • ultrapure H 2 0 molecular biology grade reagents
  • the respective substrate is mixed with 10 ⁇ _ of the corresponding l Otimes concentrated reaction buffer (RB) and the toxin or respective sample. Finally, H 2 0 (ultrapure; 37°C) is added to give a final volume of 100 ⁇ !_ (in black 96 half-well microplates pre-warmed to 37°C). The measurement is then performed with a SpectraMax GeminiXS or any other spectro-fluorometry microplate reader at 37°C assay temperature. For signal readout the samples are excited at the excitation and emission wavelength combination of the respective substrate
  • PL50 (Pharmaleads, Paris, France) is a peptide, which is intramolecularly quenched by fluorescence resonance energy transfer (FRET). Pyrenylalanine (Pya) is the fluorophore Para-nitro-phenylalanine (Nop) the acceptor chromophore. PL50 contains a specific cleavage site for BoNT/A LC [83,84].
  • Lyophilised PL50 stock is dissolved in 80:20 ultrapure H 2 0 and DMF ( ⁇ , ⁇ -Dimethylformamide) and for experiments with PL50 the following reaction buffer (RBp L5 o) is used: 10 mM HEPES (pH 6.2), 75 ⁇ ZnS0 4 , 2.5 mM TCEP (pH 7.0).
  • PL50 is used at an assay concentration of 10 ⁇ .
  • sequence information of PL50 is as follows: [(Ac-156-203) SNAP-25](Nop197, Pya200, Nle202):
  • PL150 (Pharmaleads, Paris, France) is a peptide, which is intramolecularly quenched by fluorescence resonance energy transfer (FRET). Pyrenylalanin (Pya) is the fluorophore Para-nitro-phenylalanin (Nop) the acceptor chromophore. PL150 contains a specific cleavage site for BoNT/B LC [85,86,87]. Lyophilised PL150 stock is dissolved in ultrapure H 2 0 and for experiments with PL150 the following reaction buffer (RBpuso) is used: 10 mM HEPES (pH 7.2), 75 ⁇ ZnS0 4 , 2.5 mM TCEP (pH 7.0).
  • PL150 is used at an assay concentration of 10 ⁇ .
  • sequence information of PL150 is as follows: [(Ac-60-94)VAMP](Pya74,Nop77): 6 'V ⁇ cLSELDDRADALQAG-Pya-SQ-Nop-ESSAAKLKRKYWWKNLKWH ? 9i Results
  • SNAPtidei A F (SNAPtide (DABCYL/5-IAF); List Biological Laboratories Inc., Campbell, CA, USA) is a peptide, which is labelled using 5-lodoacetamido- fluorescein to obtain an S-fluoresceinyl cysteine fluorophore on the C-terminal.
  • the acceptor chromophore is DABCYL [88].
  • Lyophilised SNAPtide !A F stock is dissolved in ultrapure H 2 0 and for experiments with SNAPtide, AF , the following reaction buffer ( BsNAPtide-iAF) is used: 20 mM HEPES (pH 7.5), 15 mM ZnCI 2 , 1 .25 mM DTT, 0.1 % Tween-20.
  • BsNAPtide-iAF the following reaction buffer
  • SNAPtidei AF is used at an assay concentration of 10 ⁇ .
  • SNAPtide F iTc SNAPtide (FITC/DABCYL); List Biological Laboratories Inc., Campbell, CA, USA) is a peptide, which is intramolecularly quenched by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the N-terminally-linked fluorophore is fluorescein-thiocarbamoyi (FITC) and the acceptor chromophore is DABCYL [89].
  • Lyophilised SNAPtide F rrc stock is dissolved in ultrapure H 2 0 and for experiments with SNAPtide F rrc, the following reaction buffer (RB SNA p tide -F!Tc) is used: 20 mM HEPES (pH 7.5), 15 mM ZnCI 2 , 1.25 mM DTT, 0.1 % Tween-20.
  • SNAPtide 0 _Abz SNAPtide (o-Abz/Dnp); List Biological Laboratories Inc., Campbell, CA, USA) is a peptide, which is intramolecularly quenched by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the N-terminally-linked fluorophore is o- aminobenzoic acid (o-Abz) and the acceptor chromophore is a 2,4-dinitrophenyl group (Dnp) [89,90,91].
  • VAMPtide 0 .Abz (VAMPtide (o-Abz/Dnp); List Biological Laboratories Inc., Campbell, CA, USA) is a peptide, which is intramolecularly quenched by fluorescence resonance energy transfer (FRET).
  • FRET fluorescence resonance energy transfer
  • the N-terminally-linked fluorophore is o- aminobenzoic acid (o-Abz) and the acceptor chromophore is a 2,4-dinitrophenyl group (Dnp).
  • Lyophilised VAMPtide o Abz stock is dissolved in ultrapure H 2 0 and for experiments with VAMPtide 0 .
  • reaction buffer (RB V AMPtide-o-Abz) is used: 10 mM HEPES (pH 7.2), 0.075 mM ZnS0 4 , 2.5 mM TCEP (pH 7.0).
  • Bound VAMPtide 0 -Abz e was detected with affinity purified rabbit@VAMP1 ,2,3, (SynapticSystems GmbH, Goettingen, Germany). The antibodyy was subsequently detected with goat@rabbit-HRP (BioFX Laboratories Inc., Owings Mills MD, USA) and TMB (Pierce Biotechnology Inc., Rockford IL, USA) as substrate. As can be seen in Fig. 14B, at least 50 nM of VAMPtide 0 . A bz could be detected using the described ELISA setup.
  • SV2c represents a suitable protein receptor [92,93]. Therefore, truncated and modified GST-SV2c (kindly provided by A. Rumme!, MH Hannover, Hannover, Germany) was used.
  • the SV2c used for GST-SV2c was derived from human and contains the complete luminal domain (aa454-579) plus a C-terminal transmembrane domain (aa580-603) and a N-terminally attached GST-tag.
  • GT1 b just as for BoNT/B, also serves as ganglioside receptor for BoNT/A (Matreya, pleasant Gap, PA, USA).
  • the lipid receptor (ganglioside GT1 b) was evaporated together with the used lipids (40 mM DOPC/Cholesterol; same as for B- liposomes) from organic solution to form a lipid film; GT1 b was used at the same concentrations described for B-liposomes.
  • the protein receptor for BoNT/A (GST- SV2c) was applied to the lipid film together with the future reaction buffer (RB PL5 o); GST-SV2c was used at 12.5 or 8 g/mL (final concentration in the liposome emulsion). Except for the receptors used, the production process for A-liposomes is the same as for B-liposomes, including removal of Triton X-100 by treatment with BioBeads SM-2. Results
  • the A-liposomes were analysed via dynamic light scattering and were found to have an average diameter of approximately 190 nm. After separation of the A-liposomes by size exclusion chromatography the derived fractions were analysed regarding the contained receptors. Detection of receptors (GST-SV2c & GT1 b) in A-Liposome fractions after SEC was performed on ELISA microplates. Samples were diluted 1/100 and coated at 4°C over night. As can be seen in Fig. 16, both receptors can be found in the same fractions as the liposomes. As can be seen in Fig. 16, the majority of receptor molecules could be found in the same fractions as the liposomes. Accordingly, we were able to produce A-liposomes with receptors specific for binding of BoNT/A.
  • the receptors i.e. protein receptor GST-Sytll for B-FAL and GST-SV2c for A-FAL, respectively, and lipid receptor GT1 b for both FAL, are used at the same concentration as for production of B- and A-liposomes, respectively.
  • GT1 b is evaporated from organic solvent (Methanol/Dichloromethane 1 :1 ) together with the lipids (40 mM DOPC/Cholesterol).
  • the resulting lipid film is hydrated in a mixture of GST-Sytll or GST-SV2c, the respective substrate, i.e.
  • Cleavage substrates are used at a concentration of 200 ⁇ .
  • the resulting emulsion consisting of multilamellar and multivesicular vesicles of different sizes, is subsequently extruded through with a MiniExtruder (Avanti Polar Lipids, Alabaster, AL, USA) through track-etched polycarbonate membranes with pore diameters ranging from 50-400 nm.
  • MiniExtruder Advanti Polar Lipids, Alabaster, AL, USA
  • A-FAL-PL50 and B-FALp L150 were tested with this method to elucidate whether the substrate molecules are really encapsulated into the liposomes and do not reside in the membrane or adhere to the liposome surface. Both FAL types were also diluted 1/20 and treated with either 100 pM BoNT/A or BoNT/B in the respective RB. If the substrate only resides in the liposome lumen, then addition of 0.1 % Triton X-100 should cause lysis of the liposomes and release of the substrate, which can subsequently be cleaved by BoNT/B in the surrounding medium. As can be seen by the sharp increase in fluorescence following the addition of Triton X-100 (Fig.
  • the liposome lysis causes release of the substrate molecules, which are then cleaved by the BoNT molecules in the surrounding medium. Accordingly, the majority of substrate molecules contained in A- and B-FAL are located in the liposome lumen. Cleavage substrates in B-FAL
  • Bottrill K (2003) Growing old disgracefully: the cosmetic use of botulinum toxin. AT LA 31 (4):381 -391 .
  • SV2 is the protein receptor for botulinum neurotoxin A. Science, 312: 592-596.
  • SV2 is the protein receptor for botulinum neurotoxin A. Science 312: 592-596.

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Abstract

Cette invention concerne un procédé pour déterminer la présence, la quantité et/ou l'activité d'une neurotoxine clostridiale dans un échantillon, ce procédé comprenant ou étant constitué par les étapes suivantes : (a) mettre en contact l'échantillon avec un liposome, ce liposome comprenant (aa) au moins un récepteur sur sa surface extérieure, ce récepteur étant capable de se lier à ladite neurotoxine et comprenant ou étant constitué par (i) un glycolipide et (ii) un peptide ou une protéine; et (ab) un substrat à l'intérieur du liposome, ce substrat (i) pouvant être clivé par la peptidase contenue dans la neurotoxine et (ii) générant un signal détectable lors du clivage, le signal détectable étant de préférence généré par (1) le donneur d'une paire FRET, ce donneur manifestant une fluorescence accrue lors du clivage par ladite peptidase, (2) un composé luminescent qui se forme lors du clivage, ou (3) une enzyme qui se forme lors du clivage; et (b) déterminer si un accroissement du signal se produit ou non par rapport à un échantillon dépourvu de neurotoxine, cet accroissement indiquant la présence de ladite neurotoxine et/ou le degré de cet accroissement indiquant la quantité et/ou l'activité de ladite neurotoxine dans cet échantillon.
PCT/EP2012/064068 2011-07-19 2012-07-18 Moyens et procédés pour déterminer la présence de neurotoxines clostridiales WO2013011055A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US14/233,740 US20140287433A1 (en) 2011-07-19 2012-07-18 Means and methods for determining clostridial neurotoxins
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