Cell-based botulinum neurotoxin detection method

Abstract

Serotype-specific cell-based detection methods for characterizing botulinum neurotoxins are described, as well as genetically modified cells for use in such detection methods. This detection method and genetically modified cell can distinguish between different serotypes of botulinum neurotoxin having the same SNARE protein substrate specificity despite lacking this differentiation in the parental cell line. This differentiation is accomplished without the use of serotype-specific neutralizing antibodies and without the use of competitive heavy chains from botulinum neurotoxins.

Description

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 333,282, filed on April 21, 2022. These and all other referenced external materials are incorporated herein by reference in their entirety. When the definition or use of a term in a reference incorporated by reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein is deemed to be authoritative. Field of the Invention

[0002] The field of the invention is cell-based detection methods for proteases, in particular botulinum neurotoxin. Background Art

[0003] The following description includes information that may be helpful in understanding the present invention. This is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is it an admission that any publication explicitly or implicitly referenced is prior art.

[0004] The background description includes information that may be helpful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is it an admission that any publication explicitly or implicitly referenced is prior art.

[0005] Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are among the most potent toxins known. These toxins are a well-recognized source of food poisoning, often resulting in severe injury or even death to the victim. There are seven structurally related botulinum neurotoxins or serotypes (BoNT / AG), each consisting of a heavy chain (≈100 KD) and a light chain (~50 KD). The heavy chain mediates entry of the toxin into target cells via receptor-mediated endocytosis. Once internalized, the light chain translocates from the lumen of endosomal vesicles into the cytoplasm and acts as a zinc-dependent protease to cleave proteins that mediate vesicle-target membrane fusion (“substrate proteins”).

[0006] These BoNT substrate proteins include the plasma membrane protein syntaxin, the peripheral membrane protein SNAP-25, and the vesicle membrane protein synaptobrevin (Syb). Collectively, these proteins are referred to as SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins. Cleavage of SNARE proteins blocks vesicle fusion with the plasma membrane and eliminates neurotransmitter release at the neuromuscular junction. Among the SNARE proteins, syntaxin and SNAP-25 are usually located on the target membrane and are therefore referred to as t-SNAREs, while synaptobrevin is only present in synaptic vesicles within synapses and is referred to as v-SNAREs. Together, these three proteins form a complex that is considered to be the minimal mechanism mediating fusion between the vesicle membrane and the plasma membrane. BoNT / A, E, and C1 cleave SNAP-25, and BoNT / B, D, F, and G cleave synaptobrevin (Syb) at single but distinct sites. In addition to SNAP-25, BoNT / C also cleaves syntaxin.

[0007] Due to their threat as a source of food poisoning and as a weapon of bioterrorism, there is a need for accurate and sensitive detection of BoNTs. Currently, the "gold standard" method for detecting toxins is to perform toxicity assays in mice. This method requires large numbers of mice and is very time-consuming, but the use of this assay raises ethical issues. A number of amplified immunoassay systems based on the use of anti-toxin antibodies have also been developed. However, because the activity of BoNTs depends on proteolytic activity and proper translocation across the cell membrane and subsequent processing, conventional immunoassays targeting the neurotoxins themselves are insufficient.

[0008] HPLC separation and immunoassays can be used to detect specific peptide fragments generated by the proteolytic activity of these toxins, but these methods are time consuming and complex. Therefore, new and improved methods and compositions are needed for the detection of BoNTs.

[0009] Based on the expression of cells carrying reporter peptides Cell-based assays based on resonance energy transfer (FRET) measurements have been used to detect BoNTs, with the reporter peptides incorporating a FRET pair of peptide-separated fluorophores corresponding to cleavage moieties of SNARE proteins. In this FRET assay, two fluorescent amino acid derivatives or peptides are separated by a short synthetic peptide (usually 12-35 amino acids) that contains the cleavable moiety of the SNARE protein substrate of the BoNT. In the intact reporter peptide, the fluorescence signal of one of these fluorescent moieties (i.e., the donor fluorophore) is quenched by the other fluorescent moiety (the acceptor). At the same time, upon excitation of the donor fluorophore, an increase in fluorescence from the acceptor fluorophore is observed. Cleavage of the peptide separates the fluorophores, allowing a decrease in FRET to be detected (i.e., an increase in fluorescence from the donor fluorophore and a decrease in fluorescence from the acceptor fluorophore). Fluorescence quenching assays utilize a very similar approach, with the transfer of FRET energy to the acceptor moiety producing no fluorescence. In this quenching assay, the loss of FRET observed upon separation of the donor and acceptor moieties can be characterized by an increase in fluorescence observed from the donor fluorophore.

[0010] FRET detection has been successfully used to detect BoNTs. (See, e.g., U.S. Patent Application No. 2004 / 0191887 to Chapman, filed October 28, 2003, U.S. Patent Application No. 2006 / 0134722 to Chapman, filed December 20, 2004, U.S. Patent Application No. 7,208,285 to Steward (April 2007), U.S. Patent Application No. 7,183,066 to Fernandez-Salas (February 2007), and US2011 / 0033866 (published February 2010), and has been adapted for use in living cells. All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. When a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0011] Although some success has been achieved in applying FRET detection to BoNT detection, its sensitivity and specificity are still not ideal for many purposes. Alternatively, cell-based detection methods have also been used that utilize fluorophore pairs that are arranged so that no significant FRET occurs. (See, e.g., U.S. Pat. No. 9,274,121 to Atapattu and Tucker).

[0012] An obvious limitation of this cell-based assay is that natural botulinum neurotoxin substrates or reporter constructs containing such proteins are easily cleaved by more than one botulinum neurotoxin serotype. For example, serotypes A, C, and E are able to cleave SNAP25 and reporter constructs containing SNAP25. Similarly, serotypes B, D, F, and G are able to cleave synaptic vesicle-associated membrane proteins and reporter constructs containing synaptic vesicle-associated membrane proteins.

[0013] Therefore, there is still a need for cell-based assays for BoNT that can distinguish between different BoNT serotypes. Summary of the invention

[0014] The present subject matter provides compositions and methods for characterizing botulinum neurotoxins (BoNTs). Surprisingly, the inventors have discovered that neuroblastoma cell lines genetically modified to produce reporter peptides or reporter constructs sensitive to botulinum neurotoxins can exhibit serotype specificity that the parental cell line does not exhibit.

[0015] Embodiments of the present inventive concepts include cells produced by genetic modification of a parental neuronal cell line, the cells comprising a reporter construct, wherein the reporter construct comprises a peptide that is a substrate for BoNT / A and BoNT / E. Such cells are more than 100-fold more selective for intoxication by serotype A botulinum neurotoxin (BoNT / A) than by serotype E botulinum neurotoxin (BoNT / E), whereas the parental cell line is susceptible to BoNT / A and BoNT / E. The parental neuronal cell line is a human neuronal cell line or a human neuroblastoma cell line, such as LA-N-2, SH-SY5Y, N2a, SiMa, NS-20Y, NIE-115, NG108-C15, NSC-34, NSC-19, M4b, CNh, G4b, HT-22, or PC12. In some embodiments, the cells remain viable after exposure to a concentration of 0.017% w / v polysorbate 20 or polysorbate 80 for up to 72 hours.

[0016] Embodiments of the present inventive concepts include a method of selectively quantifying BoNT / A by obtaining a first emission measurement from a genetically modified cell as described above at a first wavelength, obtaining a second emission measurement from the cell at a second wavelength, and dividing the first measurement by the second measurement to obtain a first emission ratio. The cell is then contacted with a solution containing a botulinum neurotoxin. After a period of time (e.g., 24 hours, 48 ​​hours, 72 hours, 96 hours, or an interval between these values), a third measurement is obtained from the cell at the first wavelength and a fourth measurement is obtained from the cell at the second wavelength. The third measurement is divided by the fourth measurement to obtain a second emission ratio. A decrease in the second emission ratio relative to the first emission ratio indicates the presence of a botulinum neurotoxin in the solution that renders the cell susceptible. This method can provide a detection limit of 50 fM or less for BoNT / A. In some embodiments, when the botulinum neurotoxin is BoNT / E at a concentration of 0.5 nM, the first emission ratio is indistinguishable from the second emission ratio. In some embodiments, the solution further includes one or more pharmaceutical excipients, such as surfactants (eg, polysorbate 20, polysorbate 80), that are not removed prior to contacting with the cells.

[0017] Embodiments of the present inventive concept may include improving a cell-based botulinum neurotoxin (BoNT) detection method by transfecting human neuroblastoma cells that are intoxicated by botulinum neurotoxin to produce genetically modified cells that express a reporter construct. A portion of the reporter construct can be cleaved by the protease activity of the BoNT. The genetically modified cells express multiple SV2 subtypes or GD1a and GT1b gangliosides. The method also includes contacting the genetically modified cells with the BoNT. In some embodiments, the genetically modified cells express SV2A, SV2b and / or SV2c, or two of these subtypes. Similarly, in some embodiments, the genetically modified cells express multiple SV2 subtypes, GD1a and GT1b. The reporter construct may include a membrane binding portion; a linking portion including a cleavage site that is a substrate for the BoNT protease activity; and a reporter portion including an unstable reporter molecule (such as a first fluorophore). This unstable reporter molecule can be a fluorescent peptide. The reporter portion is coupled to the membrane-binding portion via a linker portion such that cleavage of the cleavage site results in the release of an unstable reporter molecule into the cytoplasm of the cell, wherein the unstable reporter molecule undergoes proteolysis in the cytoplasm of the cell during the time course of the cell-based assay. In some embodiments, wherein the reporter construct further comprises a reference reporter molecule (e.g., a second fluorophore) coupled to the membrane-binding portion, wherein the reference reporter molecule remains coupled to the cleaved membrane-binding portion of the cleavage site and is not degraded upon cleavage of the cleavage site. In some embodiments, the improvement is relative to an EC based assay using culture medium and temperature optimized for cell-based assays using mouse cells transformed to express the reporter construct. 50 , EC for cell-based assays using culture medium and temperature optimization using genetically modified cells 50 Reduced by 50%.

[0018] Various objects, features, aspects and advantages of the present subject matter will become more apparent from the following detailed description of preferred embodiments taken in conjunction with the accompanying drawings, in which like numerals represent like components. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : Figure 1 A graphic representation depicting typical growth of the 5E9 and 8F4 genetically modified cell lines is provided.

[0020] Figure 2 : Figure 2 Histograms depicting typical viability of 5E9 and 8F4 genetically modified cell lines are provided.

[0021] Figure 3 : Figure 3 A graphic representation depicting the typical growth of a 2C2 transfected cell line of a 2C2 genetically modified cell line is provided.

[0022] Figure 4 : Figure 4 A histogram depicting typical viability of the 6h42C2 genetically modified cell line is provided.

[0023] Figure 5 : Figure 5 A graphical representation depicting typical comparative growth curves of 5E9, 8F4, and 2C2 transfected cell lines is provided.

[0024] Figure 6 : Figure 6 A graphical representation is provided depicting the typical response of a genetically unmodified human neuroblastoma cell line to BoNT / A and BoNT / E, as shown by prior art immunoassays directed against serotype-specific fragments of SNAP25.

[0025] Figure 7 : Figure 7 A graphical representation depicting the typical response of the genetically modified cell line 2C2 to BoNT / A and BoNT / E is provided, shown as a YFP / CFP normalized ratio.

[0026] Figure 8 : Figure 8 Provides a description Graphical representation of a typical comparison of the sensitivity of 2C2 and 2C2 transformed cell lines to BoNT / E responses.

[0027] Fig. 9 : Fig. 9 Provides a description Graphical representation of a typical comparison of the relative sensitivity of 2C2, 5E9, and BF4 transformed cell lines to BoNT / A.

[0028] Fig.10 : Fig.10 Provides a graph depicting the effects of typical culture time, culture temperature, and cell density on EC for cell-based BoNT / A assays using genetically modified human neuroblastoma cells. 50 Graph of the impact of .

[0029] Fig.11 : Fig.11 Representative photomicrographs depicting the expression of SV2A, SV2B, and SV2C in the 8F4 human neuroblastoma genetically modified cell line are provided.

[0030] Fig.12 : Fig.12 Representative photomicrographs depicting the expression of SV2A, SV2B, and SV2C in the 5E9 human neuroblastoma genetically modified cell line are provided.

[0031] Fig.13 : Fig.13Representative photomicrographs depicting the expression of SV2A, SV2B, and SV2C in the 8F4 human neuroblastoma genetically modified cell line are provided.

[0032] Figure 14: Figure 14 provides representative micrographs depicting the expression of SV2A, SV2B and SV2C in the 9C5 human neuroblastoma genetically modified cell line.

[0033] Fig.15 : Fig.15 Provides a description of SV2A, SV2B and SV2C in Representative micrographs of expression in cell lines. Cell line expression and Figure 11 to 1 4. The same reporter construct was expressed in human neuroblastoma-transformed cells.

[0034] Fig.16 : Fig.16 Representative micrographs depicting the expression of SV2 isoforms in 2C2-transformed human neuroblastoma cells using polyclonal antibodies are provided.

[0035] Fig.17 : Fig.17 Provided are human neuroblastoma cells, human neuroblastoma cells genetically modified to express a BoNT / A sensitive reporter construct, and human neuroblastoma cells expressing the same reporter construct. Representative micrographs of the ganglioside GD1a and GT1b content of cells.

[0036] Fig.18 : Fig.18 Shows Cells and genetically modified human neuroblastoma cells expressing the same reporter construct were exposed to different concentrations of polysorbate 20 (P20, Fig.18 The results for the reporter constructs were shown in Figure 19 (upper panel) and polysorbate 80 (P80, lower panel). Cell viability was characterized by measuring the fluorescence emission of the fluorescent portion of the reporter construct. Fig.18 The left side shows the fluorescence emission of the portion of the reporter construct that is degraded when cells are exposed to BoNT / A. Fig.18 The right side shows the fluorescence emission of the cells normalized as described above. DETAILED DESCRIPTION

[0037] The following description includes information that may be helpful in understanding the present invention. This is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, nor is it an admission that any publication explicitly or implicitly referenced is prior art.

[0038] The present invention provides compositions and methods for selective cell-based detection of botulinum neurotoxins (BoNTs). These include and contain cells that have been genetically modified to express a reporter construct, wherein a portion of the reporter construct can serve as a substrate for the BoNT proteolytic activity to be characterized. Surprisingly, the inventors have discovered that such genetically modified cells can exhibit a previously unobserved high degree of selectivity between different BoNT serotypes that utilize the same SNARE proteins as substrates, and that this selectivity is a result of the characteristics and function of the cells, rather than characteristics of the reporter construct.

[0039] In some embodiments, the genetically modified cells are derived from LA-N-2 cells (i.e., LA-N-2 cells are parental cell lines). In some embodiments, the genetically modified cells are derived from SH-SY5Y cells (i.e., SH-SY5Y cells are parental cell lines). In some embodiments, the genetically modified cells are derived from N2a cells (i.e., N2a cells are parental cell lines). In some embodiments, the genetically modified cells are derived from SiMa cells (i.e., SiMa cells are parental cell lines). In some embodiments, the genetically modified cells are derived from NS-20Y cells (i.e., NS-20Y cells are parental cell lines). In some embodiments, the genetically modified cells are derived from NIE-115 cells (i.e., NIE-115 cells are parental cell lines). In some embodiments, the genetically modified cells are derived from NG108-C15 cells (i.e., NG108-C115 cells are parental cell lines). In some embodiments, the genetically modified cells are derived from NSC-34 cells (i.e., NSC-34 cells are parental cell lines). In some embodiments, the genetically modified cells are derived from NSC-19 cells (i.e., NSC-19 cells are the parent cell line). In some embodiments, the genetically modified cells are derived from M4b cells (i.e., M4b cells are the parent cell line). In some embodiments, the genetically modified cells are derived from CNh cells (i.e., CNh cells are the parent cell line). In some embodiments, the genetically modified cells are derived from G4b cells (i.e., G4b cells are the parent cell line). In some embodiments, the genetically modified cells are derived from CNh cells (i.e., CNh cells are the parent cell line). In some embodiments, the genetically modified cells are derived from G HT-22 cells (i.e., HT-22 cells are the parent cell line). In some embodiments, the genetically modified cells are derived from G PC12 cells (i.e., PC12 cells are the parent cell line). PC12

[0040] Surprisingly, the degree of selectivity can differ between the genetically modified cell line and the parental cell line from which the genetically modified cell line is derived, thereby allowing the generation of a BoNT that can provide a high degree of discrimination between BoNT serotypes (e.g., serotype A and serotype E) with the same substrate protein specificity (e.g., EC 50 Different from cell lines with 100-fold or greater differences.

[0041] The following discussion provides many example embodiments of the subject matter of the present invention. Although each embodiment represents a single combination of inventive elements, the subject matter of the present invention is considered to include all possible combinations of disclosed elements. Therefore, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the subject matter of the present invention is also considered to include other remaining combinations of A, B, C, or D, even if not clearly disclosed.

[0042] As used herein, unless the context otherwise dictates, the term "coupled to" is intended to include direct coupling (where two elements coupled to each other are in contact with each other) and indirect direct coupling (where at least one additional element is located between two elements). Therefore, the terms "coupled to" and "coupled with ... are synonymous. In some embodiments, the numerical values ​​of the properties of the amount, properties (such as concentration), reaction conditions, etc., of the expression components used to describe and claim certain embodiments of the present invention should be understood to be modified by the term "about" in some cases. Therefore, in some embodiments, the numerical parameters set forth in the written description and the attached claims are approximate values ​​and can vary according to the desired properties sought to be obtained in a particular embodiment. In some embodiments, the numerical parameters should be interpreted according to the number of reported significant figures and by applying ordinary rounding techniques. Although the numerical ranges and parameters of the wide range of some embodiments of the present invention are approximate values, the numerical values ​​set forth in the specific examples are reported as accurately as possible. The numerical values ​​presented in some embodiments of the present invention may contain certain errors, which are necessarily caused by the standard deviations found in their respective test measurements.

[0043] As used in the detailed description of the present invention and in the claims that follow, the meanings of "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Additionally, as used in the description herein, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise.

[0044] The enumeration of numerical ranges herein is only intended as a simple method, for referring to each individual value falling within the range respectively. Unless otherwise specified herein, each individual value is incorporated into the specification, just as quoted separately in this article. Unless otherwise specified herein or obviously contradictory with context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (for example, "such as (such as)") provided for some embodiments herein is only intended to better illustrate the present invention, rather than to limit the scope of the present invention. Any language in the specification should not be interpreted as representing any unclaimed element that is crucial to the practice of the present invention.

[0045] The grouping of the alternative elements or embodiments of the present invention disclosed herein should not be construed as limiting. Each group member can be quoted and claimed individually, or quoted and claimed in any combination with other members of the group or other elements found herein. For convenience and / or patentability reasons, one or more members of a group can be included in the group or deleted from the group. When any such inclusion or deletion occurs, this specification is deemed to include the modified group at this time, thereby satisfying the written description of all Markush groups used in the appended claims.

[0046] In some embodiments, a reporter construct expressed in such cells can include (a) a reporter-containing portion coupled to (b) a linking moiety that includes a cleavage site, which in turn can serve as a substrate for the proteolytic activity of a characterized BoNT, and (c) a membrane-binding moiety coupled to the reporter-containing portion via the linking moiety. Interaction with the characterized BoNT cleaves the reporter-containing portion from the remainder of the reporter construct and releases it into the cytoplasm of the transformed cell. Once released, the reporter-containing portion is rapidly degraded by proteolytic activity in the cytoplasm.

[0047] In preferred embodiments, the reporter construct can include a reference reporter molecule that is retained with the membrane-bound moiety and does not undergo proteolysis after exposure of transformed cells to the characterized BoNT. In these embodiments, the reporter-containing moiety includes a fluorescent peptide (e.g., GFP or a GFP mutant-derived peptide), and the reporter construct can be arranged such that no useful interaction occurs between the fluorescent peptide and the fluorophore of the reference reporter molecule (e.g., a second, different fluorescent peptide). Resonance Energy Transfer (FRET) (e.g., less than 5% energy transfer). This advantageously allows measurements from a reference reporter molecule to be used to normalize measurements of reporter moieties from different test wells. For example, the emission of a "reporting" fluorophore that is proteolytically degraded upon BoNT activity release can be divided by the emission of a "reference" fluorophore that is not subject to such degradation to correct for differences in cell number, expression level, etc. between different wells of a test plate used in such a cell-based assay.

[0048] Such transformed cells can be used in methods for qualitatively and / or quantitatively characterizing BoNTs. An example of such a method can include (i) providing a composition comprising cells transformed to express a reporter construct as described above;

[0049] (ii) obtaining a baseline signal from the reporter molecule-containing portion of the reporter construct;

[0050] (iii) exposing the cells to the BoNT to be characterized;

[0051] (iv) obtaining a further signal from the reporter-containing portion of the reporter construct after sufficient time for the BoNT to cleave the reporter construct and degrade the released reporter portion in the cytoplasm of the cell; and (v) comparing the first emission measurement of step (ii) to the further measurement of step (iv).

[0052] Typically, these assays are performed at a temperature of 35°C to 39°C (preferably 37°C) with assay intervals of 24 hours to 72 hours. Temperature, time and media conditions can be optimized to suit specific assay needs.

[0053] The cleaved reporter-containing moiety is destroyed or otherwise degraded by the local environment, and the presence and / or quantity of the BoNT to be characterized is then evidenced by a decrease in the reporter signal. In the context of the present application, it is expected that degradation of the released reporter protein will generally occur intracellularly through at least one of two pathways, either by targeting the protein to the ubiquitin-dependent process of the proteasome, or through the autophagy-lysosomal pathway. In one pathway, the proteasome is the enzyme. In the lysosomal pathway, the enzyme of interest is expected to be a hydrolase, particularly including a family of proteases known as cathepsins.

[0054] BoNTs suitable for characterization include BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, BoNT / G, and BoNT / H. A linking portion of a suitable reporter construct may include a cleavage site having a sequence selected to correspond to the substrate specificity of the BoNT to be characterized. Contemplated cleavage site sequences may advantageously include SNARE proteins, motifs, or muteins. A "mutein" of a protein is herein construed as having at least 30% identity to the corresponding native protein, including, for example, compositions having at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% identity to the native protein. Changes in identity may include any one or more of additions, deletions, and substitutions. Contemplated muteins include fragments, truncated proteins, and fusion proteins.

[0055] As described above, the reporter molecule portion of the reporter construct can include a fluorescent protein or peptide, such as a green fluorescent protein (GFP) mutation, such as a yellow fluorescent protein (YFP), lemon yellow, Venus and YPet proteins. The sequences of such fluorescent proteins mentioned herein can be modified to include specific features (e.g., spectra) or truncated to a desired size.

[0056] As described above, the portion of the reporter construct that remains associated with the cell membrane after cleavage of the characterized BoNT can include a second fluorescent protein, such as cyan fluorescent protein (CFP), mCherry, or mStrawberry. The emission spectrum of this second fluorophore can be selected to minimize overlap with the emission spectrum of the fluorophore comprising the reporter molecule portion in order to simplify data normalization.

[0057] Any suitable reporter construct can be used in the cells and methods of the present invention. SEQ ID NO. 1 (provided in the accompanying sequence listing) is an example of a suitable reporter construct that does not provide useful FRET when intact. The reporter construct includes a portion corresponding to SNAP25, a flanking spacer peptide, and a YFP portion that is released by the proteolytic activity of the BoNT / A and BoNT / E light chains and rapidly degrades after release. The reporter construct also includes a CFP portion that is not degraded after BoNT / A or BoNT / E proteolysis, and its emission can be used to normalize the emission measurement of YFP during cell-based detection.

[0058] This reporter construct can be introduced into the cell by any suitable means, including introduction of a plasmid or viral genome encoding the reporter construct. This plasmid or viral genome can incorporate a transcriptional promoter that is positioned to affect transcription of the portion of the plasmid or viral genome encoding the reporter construct. This plasmid or viral genome can contain additional coding regions that allow screening of transformed cells, such as regions encoding antibiotic resistance.

[0059] In a preferred cell-based assay for characterizing a particular BoNT, cells containing a reporter construct will exhibit a baseline signal from the reporter moiety and then show a diminished signal from the reporter moiety after exposure. In reporter constructs that incorporate YFP into the reporter moiety and use CFP for normalization, both YFP emission (top, Ex500, Em526) and CFP emission (middle, Ex434, Em470) from individual excitations are collected. These emissions are then background subtracted and the YFP emission is divided by the CFP emission to control for cell density and reporter gene expression in individual cells. The emission ratio (YFP / CFP, bottom) is how the reporter is analyzed.

[0060] After exposure to the BoNT, the reporter-containing moiety is destroyed or otherwise degraded much faster than before exposure. In preferred embodiments, it is contemplated that the destruction or other degradation of the reporter-containing moiety occurs at least twice as fast after exposure as before exposure, but more preferably, the rate after exposure is at least 5 times, at least 10 times, at least 100 times, relative to the rate before exposure.

[0061] Cells used in the methods contemplated by the present invention are selected to be susceptible to intoxication by the BoNT to be characterized. Typically, these cells are of neuronal origin. Suitable cell lines include, but are not limited to, LA-N-2, SH-SY5Y, N2a, SiMa, NS-20Y, NIE-115, NG108-C15, NSC-34, NSC-19, M4b, CNh, G4b, HT-22, and PC12. Surprisingly, the inventors have discovered that there are unexpected differences in performance characteristics between different transformants modified to express the same reporter construct. For example, differences in sensitivity, BoNT serotype selectivity, and sensitivity to surfactants have been found that cannot be explained by the characteristics of the modified parental cell line.

[0062] In some embodiments of the present inventive concepts, a cell line susceptible to intoxication by BoNT (e.g., a neuroblastoma cell line) is genetically modified to express a reporter construct (e.g., a reporter construct represented by SEQ ID NO. 1) that is susceptible to cleavage by the light chain proteolytic activity of the BoNT of interest. As described above, such a reporter construct can include a fluorescent moiety that has utility in cell-based detection (e.g., by degradation after cleavage of the reporter construct or providing a normalized signal from the reporter construct). Such a reporter construct can be introduced by transforming the cell with an expression vector, plasmid, or virus that includes genetic information encoding such a reporter construct, which is prepared and administered by methods known in the art. Such transformation can be performed by any suitable method, including electroporation, infection by a viral vector (e.g., lentivirus), direct injection, and the like.

[0063] Genetically modified cells derived from cell lines can be identified by any suitable method. In some embodiments, such cells can be identified by selecting a selection marker in an expression vector, plasmid or viral genome that is incorporated into the encoding construct (e.g., by encoding antibiotic resistance). Alternatively, or in addition thereto, the genetically modified cells expressing the reporter construct can be identified and separated by the emission of the reporter construct fluorescent moiety expressed by fluorescence activated cell sorting (FACS). For example, droplets containing cells showing fluorescence associated with the reporter construct can be separated and amplified in culture.

[0064] As described above, the inventors surprisingly identified that cells exhibit a high degree of selectivity for a particular BoNT serotype compared to different BoNT serotypes with the same SNARE protein specificity (e.g., if EC 50, then exhibit a 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold or greater difference) where such selectivity is not observed in the corresponding cell line prior to transformation. Such cells can be identified by comparing the results of exposure to a range of concentrations of different BoNT serotypes upon cleavage of the reporter construct (e.g., by monitoring the loss of fluorescence in the fluorescent moiety so released). In some embodiments, this can be performed by culturing the genetically modified cells in the presence of BoNT in a series of wells of a microplate and then measuring fluorescence of the wells. Alternatively, such cells can be separated using FACS to isolate cells that respond to the desired BoNT serotype, for example, by contacting the genetically modified cells with a high concentration of BoNT and measuring a low fluorescence ratio (e.g., 0.1, 0.05, 0.05, 0.01 or less) between the fluorescent moiety that responds to BoNT and the fluorescent moiety that does not respond to BoNT. Cells in the population that are relatively unresponsive to the undesired BoNT can be identified from the population, for example, by contacting the population with a high concentration of the undesired BoNT and identifying cells that maintain a high ratio of fluorescence between the various fluorophores (e.g., greater than 50% of the fluorescence observed for untreated cells).

[0065] In these studies, relative concentrations of BoNTs may be initially determined using traditional mouse lethality assays or alternative methods that incorporate cellular internalization and processing steps and have been calibrated to provide results comparable to mouse lethality assays, thus providing directly comparable results between BoNT serotypes.

[0066] Example

[0067] Cells of the human neuroblastoma (human neuroblastoma) cell line were transformed with an expression vector encoding a reporter construct (SEQ ID NO. 1), the construct comprising:

[0068] · Reporter molecule containing YFP peptide

[0069] A linker containing a SNAP-25 derived cleavage site

[0070] The membrane-binding moiety is coupled to the reporter-containing moiety via a linker moiety

[0071] - A control moiety containing the CFP peptide coupled to the membrane binding moiety such that the coupling remains after cleavage of the linking moiety.

[0072] The same reporter construct was expressed in mouse A / E cell lines, the data are provided for comparison purposes.

[0073] like Figures 1 to 5As shown, the growth characteristics of several transformed cell lines derived from genetically modified human neuroblastoma (human neuroblastoma) cells and expressing reporter constructs were characterized. Figure 1 (cell lines 5E9 and 8F4) and Figure 3 As shown in (cell line 2C2), genetically modified human neuroblastoma cells expressing the reporter constructs described above showed vigorous growth in culture. Figure 2 (cell lines 5E9 and 8F4) and Figure 4 As shown in Figure 2 (cell line 2C2), the survival rate of the genetically modified cell line was maintained at 95% or higher through at least 168 hours of culture. Figure 5 Comparative results of genetically modified human neuroblastoma cell lines 5E9, 8F4, and 2C2 are provided, showing similar growth rates over time. These genetically modified cell lines were found to provide EC values ​​as low as about 0.1 pM to 1.1 pM for BoNT / A in an optimized cell-based assay. 50 s, the detection being performed by directly measuring fluorescence from the cells as described above. In the context of this application, EC 50 It is understood to be the concentration corresponding to the midpoint of a sigmoidal dose-response curve. Similarly, in the context of the present application, the lowest detectable dose (LDD) is understood to be the concentration corresponding to a response on a dose-response curve that is different from a negative control (e.g., a test condition that does not contain the compound for which the response is being tested, such as a BoNT or surfactant). For example, such a difference can be determined as a response that deviates from the negative control condition by at least 2 standard deviations, or provides clear evidence of the onset of a trend in the data.

[0074] Notably, a human neuroblastoma cell line, which serves as the parental cell line for the 5E9, 8F4, and 2C2 genetically modified cell lines, has been shown to be equally responsive to BoNT / A and BoNT / E in terms of cleavage of SNAP25, as determined by immunodetection characterization of the contents of the intoxicated cells. Cells of this human neuroblastoma cell line were treated with an equivalent concentration range of BoNT / A or BoNT / E (determined by mouse lethality assays) for 48 hours, lysed, and their contents characterized using an enzyme-linked immunosorbent assay. Briefly, the contents of the lysed cells were added to test wells coated with antibodies against SNAP25 epitopes exposed by BoNT / A or BoNT / E cleavage. Subsequently, the captured SNAP25 fragments were visualized using a polyclonal antibody against the SNAP25 sites present in the fragments generated by BoNT / A and BoNT / E cleavage. The results are shown in Figure 2. Figure 6 As shown, human neuroblastoma cell lines genetically modified to generate 5E9, 8F4, and 2C2 cell lines are unable to distinguish between BoNT / A and BoNT / E.

[0075] The reporter construct expressed by a genetically modified human neuronal cell line incorporates BoNT / A and BoNT / E cleavage sites and a recognition site derived from SNAP25 and can be cleaved by the proteolytic activity of BoNT / A or BoNT / E. Cells from the 2C2 genetically modified human neuroblastoma cell line were exposed to various concentrations of BoNT / A and BoNT / E, and emission from YFP and CFP was measured using independent excitation measurements. As described above, in this cell-based assay, YFP is proteolytically released by BoNT / A or BoNT / E and rapidly degraded in the cytoplasm, while CFP remains on the cell membrane and is protected from proteolysis. The normalized emission (i.e., YFP emission divided by CFP emission) emission measurements of these cells are shown in Figure 2. Figure 7 As shown. The genetically modified human neuronal cell line showed excellent sensitivity to BoNT / A, with normalized emission measurements made directly from the cells showing the lowest detectable dose (LDD, characterized by providing a significant reduction compared to medium without BoNT) using BoNT / A as low as 0.05 pM. Surprisingly, the same measurements made after exposure to BoNT / E showed almost no response in the range tested (up to 50 nM). It should be understood that this high sensitivity to BoNT / A and the high degree of discrimination between BoNT / A and BoNT / E were achieved by directly measuring intact cells in culture and in the absence of serotype-specific neutralizing antibodies.

[0076] As mentioned above, The cells are a mouse cell line expressing the same reporter construct as expressed in the 2C2 cell line. Figure 8 Figure 2 shows the results of cell-based assays performed under the same assay conditions (time, temperature, etc.). Comparison of BoNT / E dose response curves in 2C2 and 2C2 cell lines. As shown in the figure, compared with 2C2 cells, Cells of the cell line showed high sensitivity to BoNT / E.

[0077] Previously, cell-based assays were performed at 37°C. Studies of the sensitivity of BoNT / A and BoNT / E showed that the LDDs of BoNT / A and BoNT / E were similar, with values ​​within 30% of each other. Since different cell lines transformed to express the same reporter construct showed different degrees of serotype discrimination, this unexpected finding could not be attributed to the reporter construct itself. It is clear that the response of neuronal cell lines to different botulinum neurotoxin serotypes with the same substrate specificity can be altered during the genetic modification process to introduce a reporter construct that is sensitive to BoNT.

[0078] The differentiation between different botulinum serotypes against the same substrate protein within such a genetically modified cell is sufficient to render the genetically modified cell substantially non-responsive to one or more selected serotypes. Figure 7 and Figure 8 In the studies shown, despite the use of high nM concentrations of the neurotoxin, the 2C2 cell line did not respond to BoNT / E, so that EC could not be calculated. 50 In the context of the present application, a genetically modified cell that is responsive to a target BoNT is substantially non-responsive to a non-target BoNT serotype when a 1,000-fold, 200-fold, 3,000-fold, 5,000-fold, 8,000-fold, 10,000-fold or greater excess of the non-target BoNT is required to produce a response (e.g., LDD) from the cell that is equivalent to the response produced by the target BoNT. For example, Figure 7 As shown, when 2C2 cells were exposed to BoNT / A or BoNT / E, approximately 0.5 nM of BoNT / E was required to produce a response that corresponded to the response provided by 0.05 pM of BoNT / A under the same test conditions (i.e., an approximately 10,000-fold difference). It should be understood that this degree of discrimination was observed in the absence of antibodies or antibody fragments specific for the BoNT serotype that required desensitization. Similarly, this degree of discrimination was observed in the absence of a competing BoNT heavy chain or analog corresponding to the BoNT serotype that required desensitization.

[0079] Further studies have provided that in iBAM2+18mM NaCl medium, Comparison of sensitivity of transformed cells and 2C2 transformed cells expressing the same reporter construct to BoNT / E. When exposed to BoNT / E, Comparison results with the genetically modified 2C2 cell line are shown in Fig. 9 As shown in the figure, Sensitivity to BoNT / E was retained, whereas the genetically modified 2C2 cell line tested under the same conditions did not. Therefore, the difference in sensitivity to BoNT / E was not due to the characteristics of the reporter construct. Therefore, the difference in sensitivity to BoNT / E was not due to the characteristics of the reporter construct.

[0080] Also compared The sensitivity of the cell lines transformed with 2C2, 5E9 and BF4 to BoNT / A responses was studied. Fig. 9 As shown, human neuroblastoma-derived transformed cell lines 2C2, 5E9, and BF4 showed similar responses to increasing concentrations of BoNT / A and were significantly higher than those transformed with the same reporter construct. These cell lines exhibited an increased response (ie, improved sensitivity) to low concentrations of BoNT / A.

[0081] Cell-based assays based on genetically modified human neuroblastoma cell lines were shown to be affected by the initial seeding density of cells, the number of days in culture, and the temperature at which the cells were cultured. Fig.10 EC of BoNT / A for human neuroblastoma transformed cell lines expressing BoNT / A sensitive reporter constructs is shown. 50 Comparison of concentrations (ie, the concentration corresponding to the normalized emission from the reporter region at the midpoint of the dose / response curve).

[0082] Typically, the EC values ​​observed in BoNT / A assays using genetically modified human neuroblastoma cells are 50 Values ​​can be lower than those of mice engineered to express the same reporter construct. EC observed in cell lines 50 The inventors believe that at least some of the properties of genetically modified human neuroblastoma cells transformed to express a BoNT sensitivity reporter construct (described above) may be attributed to the expression of SV2 in these cells. Figures 10 to 13 Figure 3 shows the expression of human neuroblastoma cell lines ( ) expressing the same BoNT / A sensitivity reporter constructs when stained with antibodies specific for SV2A (anti-SV2A mouse mAb 119011), SV2B (anti-SV2B mouse mAb 119111), and SV2C (anti-SV2C rabbit pAb 119203). Figures 10 to 13 )and Cell lines (see Fig.15 ). Cells can be visualized by YFP fluorescence produced by the reporter construct; specific antibodies are labeled with Texas Red. As shown, genetically modified human neuroblastoma cells show expression of SV2A, SV2B, and SV2C at roughly similar levels to SV2A and SV2C. In contrast, mice expressing the same reporter construct Other studies using polyclonal antibodies against SV2 isoforms (SV2a, SV2b, SV2c) have shown that genetically modified human neuroblastoma cells can also show high expression of SV2b, such as Fig.16 shown.

[0083] Similar studies were performed to characterize human genetically modified neuroblastoma cells engineered to express a BoNT / A sensitivity reporter construct and human glioma cells expressing the same reporter construct. Ganglioside content of cell lines. Gangliosides were observed using anti-GD1a (mouse mAb, Millipore MAB5606Z) or anti-GT1b (mouse mAb, Millipore MAB5608). Fig.17As shown, genetically modified human neuroblastoma cells stain brightly for GD1a and GT1b gangliosides, while Cells stained predominantly for GD1a.

[0084] Without wishing to be bound by theory, the inventors believe that in a cell-based BoNT assay, such as a cell-based BoNT assay in which the cells express a BoNT sensitivity reporter construct, selecting a genetically modified human cell line or a genetically modified cell line that expresses a broader range of SV2 proteins and / or GD1a and GDTb gangliosides can provide improved assay performance relative to a mouse cell line. For example, in a cell-based BoNT assay in which the culture medium and culture conditions are optimized, the EC values ​​of an assay performed using a genetically modified human cell line expressing a BoNT sensitivity reporter construct (or a genetically modified cell line that expresses a broader range of SV2 proteins and / or GD1a and GDTb gangliosides) are as follows: 50 The EC may be 90%, 80%, 70%, 60%, 50%, 40%, 30%, 10%, 5%, 3%, 1%, 0.5%, 0.3%, 0.1% or less than the EC measured in a cell-based assay using transformed mouse cells expressing the same reporter construct with optimized culture medium and culture conditions. 50 .

[0085] As described above, cell-based assays utilizing genetically modified cells of the present invention can provide BoNT determination assays with excellent sensitivity and serotype specificity. Such cell-based assays can be applied to any type of sample suspected of the presence of BoNT or in which characterization of BoNT content is desired. Suitable sample types include foods, beverages, human or animal samples (e.g., tissue samples, blood, plasma, serum, lymph, tears, cerebrospinal fluid, saliva, urine, feces, etc.), and BoNT-containing pharmaceutical products. Such human or animal samples can be freshly obtained or preserved (e.g., by freezing or storage at reduced temperatures). In some embodiments, such human or animal samples can be treated prior to testing to reduce degradation of BoNT content, such as by the addition of protease inhibitors.

[0086] Commercial BoNT formulations (e.g., injectable pharmaceutical formulations) often include excipients that are generally considered incompatible with cell-based assays, such as surfactants, detergents, and peptides. Therefore, when cell-based assays are sensitive to such excipients, it may be necessary to remove them prior to testing such pharmaceutical formulations for BoNT content. These removal steps may include dialysis, diafiltration, size exclusion chromatography, etc., and in the case of surfactants, may be only partially successful due to the formation of micelles. Therefore, many pharmaceutical formulations may require extensive processing before being characterized using cell-based assays, which in turn affects the accuracy and precision of such assays. Surprisingly, the inventors found that genetically modified human neuroblastoma cells expressing the above-described BoNT sensitivity reporter constructs were relatively tolerant to surfactants that are commonly used as excipients for BoNT-containing pharmaceuticals.

[0087] Fig.18 Shows Cells and genetically modified human neuroblastoma cells expressing the same reporter construct were exposed to different concentrations of polysorbate 20 (P20, Fig.18 upper) and polysorbate 80 (P80, Fig.18 Cell viability was characterized by measuring the fluorescence emission of the fluorescent part of the reporter construct. Fig.18 The left side shows the fluorescence emission of the portion of the reporter construct that is degraded when cells are exposed to BoNT / A. Fig.18 The right side of the graph shows the fluorescence emission of the cells normalized as described above. As shown, genetically modified human neuroblastoma cells exhibit unexpected tolerance to both surfactants, which, combined with their high sensitivity to BoNT / A, allows for simple dilution of the BoNT / A-containing drug formulation prior to testing.

[0088] It will be apparent to those skilled in the art that, without departing from the inventive concept, further modifications may be made in addition to those described. Therefore, the subject matter of the present invention is not limited except in the spirit of the appended claims. Furthermore, in interpreting the specification and claims, all terms should be interpreted in as broad a manner as possible consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components or steps in a non-exclusive manner, indicating that the referenced elements, components or steps may exist, be used or be combined with other elements, components or steps not explicitly referenced. Where the specification claims involve at least one selected from A, B, C... and N, the text should be interpreted as requiring only one element of the group, rather than A plus N or B plus N, and so on.

Claims

1. A cell produced by genetically modifying a parental neuronal cell line, comprising a reporter construct, wherein the cell is more than 100-fold more selective for intoxication by serotype A botulinum neurotoxin (BoNT / A) than by serotype E botulinum neurotoxin (BoNT / E), wherein the parental cell line is susceptible to BoNT / A and BoNT / E, and wherein the reporter construct comprises a peptide that is a substrate for BoNT / A and BoNT / E.

2. The cell according to claim 1, wherein the parental neuronal cell line is a human neuronal cell line or a human neuroblastoma cell line.

3. The cell of claim 1, wherein the parental neuronal cell line is selected from the group consisting of LA-N-2, SH-SY5Y, N2a, SiMa, NS-20Y, NIE-115, NG108-C15, NSC-34, NSC-19, M4b, CNh, G4b, HT-22, and PC12.

4. The cell according to any one of claims 1 to 3, wherein the cell remains viable after exposure to polysorbate 20 or polysorbate 80 at a concentration of 0.017% w / v for 72 hours.

5. A method for selectively quantifying BoNT / A, comprising: obtaining a first emission measurement at a first wavelength from the cell of any one of claims 1 to 4; obtaining a second emission measurement from the cell at a second wavelength; dividing the first measurement value by the second measurement value to obtain a first emission ratio; contacting the cells with a solution comprising a botulinum neurotoxin; After a period of time, obtaining a third measurement from the cell at the first wavelength and obtaining a fourth measurement from the cell at the second wavelength; as well as dividing the third measured value by the fourth measured value to obtain a second emission ratio, wherein a decrease in the second emission ratio relative to the first emission ratio indicates the presence of a botulinum neurotoxin in the solution that renders the cell susceptible.

6. The method of claim 5, wherein the method has a limit of detection for BoNT / A of 50 fM or less.

7. The method of claim 5 or 6, wherein when the botulinum neurotoxin is BoNT / E at a concentration of 0.5 nM, the first emission ratio is indistinguishable from the second emission ratio.

8. The method of any one of claims 5 to 7, wherein the solution further comprises one or more pharmaceutical excipients that are not removed prior to contacting with the cells.

9. The method of claim 8, wherein the one or more pharmaceutical excipients comprises a surfactant.

10. The method according to claim 9, wherein the surfactant is polysorbate 20 or polysorbate 80.

11. The method of any one of claims 5 to 10, wherein the period of time is 72 hours.

12. The method according to any one of claims 5 to 11, wherein the solution is a human or animal sample.

13. The method of claim 12, wherein the human or animal sample is selected from the group consisting of blood, serum and plasma.

14. A method for improving cell-based detection of botulinum neurotoxin (BoNT), comprising: transfecting human neuroblastoma cells to generate genetically modified cells expressing a reporter construct, wherein the genetically modified cells express multiple SV2 isoforms or GD1a and GT1b gangliosides, wherein a portion of the reporter construct is cleavable by the protease activity of a BoNT; and The genetically modified cells are contacted with a BoNT.

15. The method of claim 14, wherein the genetically modified cells express SV2A, SV2b and SV2c.

16. The method of claim 14 or 15, wherein the genetically modified cells express multiple SV2 subtypes, GD1a and GT1b.

17. The method according to any one of claims 14 to 16, wherein the reporter construct comprises: membrane-bound portion; a linking moiety comprising a cleavage site, wherein the cleavage site is a substrate for BoNT protease activity; and A reporter portion comprising an unstable reporter molecule, wherein the reporter portion is coupled to the membrane binding portion via the linker portion such that cleavage of the cleavage site results in release of the unstable reporter molecule into the cytoplasm of the genetically modified cell, wherein the unstable reporter molecule is proteolytically cleaved in the cytoplasm of the genetically modified cell over the time course of the cell-based assay. The method of claim 17 , wherein the labile reporter molecule is a first fluorophore. The method of claim 18 , wherein the first fluorophore is a fluorescent peptide.

20. The method of any one of claims 14 to 19, wherein the reporter construct further comprises a reference reporter molecule coupled to the membrane-bound portion, wherein the reference reporter molecule remains coupled to the cleaved membrane-bound portion of the cleavage site.

21. The method of claim 20, wherein the reference reporter molecule is a second fluorophore.

22. The method of claim 20 or 21, wherein the reference reporter molecule is not degraded upon cleavage at the cleavage site.

23. The method of any one of claims 14 to 22, wherein the improvement comprises a second EC optimized for cell-based assays with respect to medium and temperature using murine cells transformed to express the reporter construct. 50 The first EC of a cell-based assay using culture medium and temperature optimization using the genetically modified cells 50 Reduced by 50%.

Images