HK1208706B - Cellular test systems for the determination of the biological activities of neurotoxin polypeptides - Google Patents

Description

Cell-based test system for determining the biological activity of neurotoxin peptides

The present invention relates to a method for generating neurotoxin-sensitive, neuronally differentiated cells, comprising the steps of: a) culturing tumor cells capable of differentiating into neuronal cells in a culture medium under conditions and for a time period that induce neuronal differentiation of the tumor cells; and b) culturing the tumor cells induced for neuronal differentiation in step a) for at least three days in a differentiation medium having an osmotic pressure of 100 to 270 mOsm/kg and comprising (i) B27 supplement and/or (ii) N2 supplement, thereby obtaining neurotoxin-sensitive, neuronally differentiated cells. The present invention also relates to neurotoxin-sensitive, neuronally differentiated cells obtainable by the method of the present invention. Furthermore, the present invention encompasses a method for determining the activity of a neurotoxin polypeptide, comprising the steps of: a) contacting the neurotoxin-sensitive, neuronally differentiated cells obtainable by the method of the present invention with a neurotoxin polypeptide; b) culturing the neurotoxin-sensitive, neuronally differentiated cells of step a) for 3 to 74 hours or 72 hours under conditions that allow the neurotoxin polypeptide to exert its biological activity; and c) determining the activity of the neurotoxin polypeptide in the cells after culturing according to step b). Finally, the present invention provides a culture medium comprising OptiMEM, FBS, B27 supplement, and N2 supplement.

Clostridium botulinum and Clostridium tetani produce potent neurotoxins, namely, botulinum toxin (BoNT) and tetanus toxin (TeNT), respectively. These clostridial neurotoxins (CNTs) specifically bind to neuronal cells and disrupt neurotransmitter release. Each toxin is synthesized as an inactive, unprocessed, single-chain protein of approximately 150 kDa. Post-translational processing involves the formation of disulfide bridges and limited proteolysis (cleavage) by bacterial proteases. The active neurotoxin consists of two chains, an N-terminal light chain of approximately 50 kDa and a heavy chain of approximately 100 kDa, which are connected by a disulfide bond. CNTs are structurally and functionally composed of three domains: a catalytic light chain, a heavy chain encompassing the translocation domain (N-terminal half), and a receptor binding domain (C-terminal half); see, e.g., Krieglstein 1990, Eur J Biochem 188, 39; Krieglstein 1991, Eur J Biochem 202, 41; Krieglstein 1994, J Protein Chem 13, 49. Clostridium botulinum neurotoxin is synthesized as a molecular complex comprising a 150 kDa neurotoxin protein and associated non-toxic proteins. The size of the complex varies depending on the Clostridium strain and the different neurotoxin serotypes, ranging from 300 kDa, to over 500 kDa, and 900 kDa. The non-toxic proteins in these complexes stabilize the neurotoxin and protect it from degradation; see Silberstein 2004, Pain Practice 4, S19–S26.

Clostridium botulinum secretes seven antigenically distinct serotypes, designated botulinum neurotoxins (BoNTs), A to G. All serotypes, along with the related tetanus neurotoxin (TeNT) secreted by Clostridium tetani, are Zn2 ⁺ -endoproteases that block synaptic exocytosis by cleaving SNARE proteins; see Couesnon, 2006, Microbiology, 152, 759. CNTs cause the flaccid muscle paralysis seen in botulism and tetanus; see Fischer, 2007, PNAS 104, 10447.

Regardless of its toxic effects, botulinum toxin complexes have been used as therapeutic agents in a variety of diseases. Clostridium botulinum toxin A serotype was approved for human use in the United States in 1989 for the treatment of strabismus, blepharospasm and other conditions. It is commercially available as a botulinum toxin A (BoNT/A) protein preparation, for example under the trade name BOTOX (Allergan Inc) or the trade name DYSPORT/RELOXIN (Ipsen Ltd). An improved, complex-free botulinum toxin A preparation is commercially available under the trade name XEOMIN (Merz Pharmaceuticals GmbH). For therapeutic applications, the preparation is injected directly into the muscle to be treated. At physiological pH, the toxin is released from the protein complex and produces the desired pharmacological effect. The effects of botulinum toxin are only temporary, which is why repeated administration of botulinum toxin is required to maintain a therapeutic effect.

Clostridial neurotoxins weaken active muscle strength and are effective treatments for strabismus, focal dystonias (including cervical dystonia, and benign essential blepharospasm). They have also been shown to relieve hemifacial spasms and focal spasticity and are effective for a wide range of other indications, such as gastrointestinal disorders, hyperhidrosis, and cosmetic wrinkle correction; see Jost 2007, Drugs 67, 669.

In the process of producing clostridial neurotoxins, the qualitative and quantitative determination of the neurotoxins and the quality control of biologically active neurotoxin polypeptides are particularly important. In addition, government agencies only accept simple, reliable and validated assays for the activity of Clostridium botulinum toxin. Currently, the mouse _LD50 bioassay, a lethality test, remains the "gold standard" used by drug manufacturers to analyze the efficacy of their preparations; see Arnon et al. (2001), JAMA 285, 1059-1070. However, in recent years, considerable efforts have been made to find alternative methods to alleviate the need for animal testing and all the disadvantages, costs and ethical concerns associated with this type of animal-based assay. In addition, regulatory agencies require pharmaceutical companies to apply the 3 "R" principle to the efficacy testing of Clostridium botulinum neurotoxins: "Reduce, Refine, Replace"; see Straughan, Altern. Lab. Anim. (2006), 34, 305-313. Therefore, cell-based test systems have been developed to provide a reasonable alternative to methods using living animals. However, to date, only three cell-based test systems that have been shown to be sufficiently sensitive to neurotoxin polypeptides are available for measuring neurotoxin biological activity. These cell-based test systems include the use of primary neurons (Pellett et al. (2011), Biochem.Biophys.Res.Commun.404,388-392) isolated from rodent embryos, induced pluripotent stem cells (Whitemarsh et al. (2012), Toxicol.Sci.126,426-35) of neuronal differentiation, and subclones of the SiMa cell line (WO 2010/105234A1).

However, isolating primary neurons requires sacrificing animals and is laborious and time-consuming. Furthermore, test systems using different primary neurons show significant variability. Similarly, generating neuronally differentiated induced pluripotent stem cells is difficult and time-consuming. Furthermore, storage of such cells is very cumbersome. Assays using tumor cell lines are often insufficiently sensitive to BoNTs. Furthermore, these tumor cell lines require complex differentiation protocols, which results in significant variability and/or high failure rates in assays using these cell lines.

In view of the foregoing, additional test systems for determining neurotoxin polypeptide activity that are acceptable to governmental agencies and that provide an alternative to animal-based test systems are highly desired.

Therefore, the technical problem of the present invention can be seen as providing a tool and a method that meets the aforementioned needs. This technical problem is solved by the embodiments characterized in the claims and below.

In a first aspect, the present invention relates to a method for generating neurotoxin-sensitive, neuronally differentiated cells, said method comprising the steps of:

a) culturing tumor cells capable of differentiating into neuronal cells in a culture medium under conditions and for a time period that induce neuronal differentiation of the tumor cells; and

b) culturing the tumor cells initiated for neuronal differentiation of a) in a differentiation medium having an osmotic pressure of 100 to 270 mOsm/kg and comprising (i) B27 supplement and/or (ii) N2 supplement for at least 3 days, thereby obtaining neurotoxin-sensitive, neuronally differentiated cells.

In the method of the present invention, tumor cells capable of differentiating into neuronal cells are first grown in a cell culture medium under conditions and time that induce neuronal differentiation of the tumor cells. Thereafter, the tumor cells thus induced to differentiate into neurons are transferred to a differentiation medium having a weight molar osmotic pressure concentration of 100 to 270 mOsm/kg. In addition, this differentiation medium contains at least (i) a B27 supplement and/or (ii) an N2 supplement. Alternatively, the differentiation medium may contain an NS21 supplement instead of the B27 supplement and/or the N2 supplement. Cultivation in the differentiation medium is carried out for at least 3 days. Thus, neurotoxin-sensitive, neuronally differentiated cells are obtained. Preferably, the differentiation medium comprises neurobasal medium.

The result of this novel differentiation method or protocol of the present invention is the acquisition of neurotoxin-sensitive, neuronally differentiated cells that exhibit significantly improved sensitivity to neurotoxin polypeptides, as shown in detail in the following examples.

Clostridium botulinum neurotoxins are characterized by their specific inhibition of neurotransmitter secretion from presynaptic nerve terminals. Selectivity for peripheral neurons is mediated by recognition of two distinct receptors, SV2 and GT1b. The physiological effects of the neurotoxins are based on cleavage of proteins of the so-called SNARE complex, following receptor binding and translocation of the neurotoxin's light chain. Determination of BoNT biological activity is an important aspect of the characterization of these neurotoxin proteins and is required, in particular, by regulatory agencies for the licensing of BoNT-containing products. Therefore, reliable tests for measuring BoNT biological activity are fundamental to the research, development, and marketing of BoNT-containing products. Furthermore, for ethical reasons, cell-based test systems should replace animal testing, which has been the predominant method to date. To establish such cell-based test systems, neuronal cells or cell lines with sufficiently high sensitivity to the botulinum neurotoxins are necessary. However, achieving such high sensitivity has previously required laborious differentiation methods of neuronal cell lines. As a result, only a few cell-based test systems are available. To determine the biological activity of botulinum toxin in a drug product, neuronal cells or cell lines should possess the following properties: First, the cells should be of human, neuronal origin to closely resemble the target, i.e., the human patient. Second, the cell system should be robust to excipients in the final product and, preferably, also to impurities in intermediate steps of the production process (process control). Third, the cell-based assay system should exhibit a dynamic measurement range that allows accurate determination of the biological activity of BoNT in a small tube (e.g., 50 U BoNT/A). Taking into account technical factors such as excipient solubility and cell culture medium volume, accurate measurement of BoNT concentrations below 1 pM is essential. To the best of the present inventors' knowledge, only three cell-based assay systems are currently available that demonstrate sufficiently high sensitivity for BoNTs. These include primary neurons from rodent embryos, neuronally differentiated induced pluripotent stem cells, and subclones of the SiMa cell line, as described elsewhere herein. However, these cell lines have been reported to exhibit sufficiently high sensitivity only after complex and laborious differentiation protocols, which are often associated with significant variability. In contrast, the present invention provides a simple, reliable and robust cell-based test system for measuring the biological activity of Clostridium botulinum neurotoxin (BoNT) that meets the above requirements and further improves upon the cell-based test systems described in the art.

More specifically, SiMa (human neuroblastoma) and P19 (murine embryonal carcinoma) tumor cells are first cultured as described in the art, then seeded onto multiwell plates and initiated for neuronal differentiation, as described elsewhere herein and in the Examples below. In a subsequent, new differentiation step, the cell culture medium is replaced with a differentiation medium comprising a medium having a low osmolality, such as 100 to 270 mOsm/kg. An example of such a medium having a low osmolality is neurobasal medium. After a period of differentiation (e.g., 3 to 7 days) during which the medium is replaced with fresh medium, if applicable, a neurotoxin polypeptide is added to the cell culture medium. In addition, GT1b is used in the methods of the present invention as a BoNT sensitivity enhancer. For example, 50 μM GT1b is added to the neurobasal medium during and/or prior to intoxication of the cells with the neurotoxin polypeptide. After an additional 72 hours of incubation with the neurotoxin polypeptide, the cells are terminated and analyzed for the biological activity of the neurotoxin polypeptide. As a result, it was found that the cells exhibited a significant increase in BoNT sensitivity.

The inventors have surprisingly discovered that the low osmolality of the differentiation medium is responsible for the increased sensitivity of neurotoxin-sensitive, neuron-differentiated cells to BoNTs produced by the methods of the present invention, as demonstrated in the following examples. This discovery was no trivial undertaking, as evidenced by the experimental history that culminated in the methods of the present invention: Initially, the inventors used a differentiation medium described in the art, comprising MEM + N2 supplement + B27 supplement; see Differentiation Medium 2, Examples. The sensitivity of neuron-differentiated cells to BoNTs achievable with this differentiation medium was approximately 1 to 2 pM. In a subsequent step, it was discovered that the addition of retinoic acid resulted in only a small increase in the sensitivity of neuron-differentiated cells to BoNTs. In contrast, a significant increase in sensitivity, approximately 7- to 10-fold, was achievable by using neurobasal medium. For example, the EC50 for SiMa cells was found to be 0.22 pmolar (2.18 x ^10-13 mol/l) when a differentiation medium comprising neurobasal medium + 2% B27 supplement + 1% GlutaMAX + 3 micromolar retinoic acid (RA) was used in the methods of the present invention. Furthermore, it was demonstrated that retinoic acid in the differentiation medium is not necessary for the improved sensitivity of neuronally differentiated cells, as long as neurobasal medium is included. Surprisingly, the inventors were able to demonstrate that improved sensitivity can be achieved even by using the differentiation medium described in the art (comprising MEM + N2 supplement + B27 supplement) that they used at the beginning of the experimental series, simply by diluting to an osmolality of 225 mOsm/kg. In contrast, undiluted differentiation medium containing the aforementioned components had an osmolality of 300 mOsm/kg. Thus, the increased sensitivity of neurotoxin-sensitive, neuronally differentiated cells to BoNTs produced by the methods of the present invention is a result of the low osmolality of the differentiation medium. In addition to improved sensitivity, the novel differentiation protocol of the present invention shows greater accuracy and robustness for testing the biological activity of Neurotoxin polypeptides compared to methods known in the art.

As used herein, the singular forms "a," "an," and "the" refer to both singular and plural references unless the context clearly dictates. By way of example, "a cell" refers to one or more than one cell.

As used herein, the term "about" when quantitatively describing a stated item, number, percentage, or term value refers to a range of plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the stated item, number, percentage, or term value. Preferably, the range is plus or minus 10%.

The term "comprising" as used herein is a synonym for "including" or "containing" and is inclusive or open-ended and does not exclude additional, uncited members, elements or method steps. It is obvious that the term "comprising" encompasses the term "consisting of. More specifically, the term "comprising" as used herein means that the claim covers all listed elements or method steps, but may also include additional, unnamed elements or method steps. For example, a method comprising steps a), b) and c) in its narrowest sense encompasses a method consisting of steps a), b) and c). The phrase "consisting of means that the composition (or device, or method) has the recited elements (or steps) and nothing else. In contrast, the term "comprising" may also encompass a method that includes other steps in addition to steps a), b) and c), such as steps d) and e).

Where numerical ranges are used herein such as "retinoic acid at a concentration between 0.1 and 0.5 micromolar," the range includes not only 0.1 and 0.5 micromolar, but also any value between 0.1 and 0.5 micromolar, for example, 0.2, 0.3, and 0.4 micromolar.

The term "in vitro" as used herein means outside of, or external to, an animal or human body. The term "in vivo" as used herein should be understood to include "ex vivo." The term "ex vivo" generally refers to tissues or cells that are removed from an animal or human body and cultured or propagated outside the body, such as in a culture vessel. The term "in vivo" as used herein means within, or internal to, an animal or human body.

The term "neurotoxin-susceptible cell" as used herein means a cell that is susceptible to a neurotoxin polypeptide that exhibits biological properties characteristic of the neurotoxin polypeptide, i.e., (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytosol, and/or (d) endoproteolytic cleavage of proteins involved in synaptic vesicle fusion. Thus, a "neurotoxin-susceptible cell" as referred to herein is susceptible to neurotoxin intoxication. More specifically, "susceptible to neurotoxin intoxication" as referred to herein means a cell that is capable of undergoing the full cellular mechanism by which a neurotoxin polypeptide (e.g., BoNT/A) cleaves a neurotoxin substrate (e.g., BoNT/A substrate SNAP-25) and encompasses binding of a neurotoxin to its corresponding receptor (e.g., BoNT/A binding to a BoNT/A receptor), internalization of the neurotoxin/receptor complex, translocation of the neurotoxin light chain from intracellular vesicles into the cytoplasm, and proteolytic cleavage of a neurotoxin substrate. Assays for determining the biological activity of neurotoxin polypeptides are well known in the art and are also described elsewhere herein (see, for example, Pellett et al., Withemarsh et al. Toxicological Sciences 126(2), 426-435 (2012), WO 2010/105234 A1). As will be appreciated by those skilled in the art, neurotoxin-sensitive cells are preferably able to first take up the neurotoxin and then undergo the entire cellular mechanism listed above. As used herein, a neurotoxin-sensitive cell can take up, for example, about 100 nanomoles (nM), about 10 nM, about 1 nM, about 500 picomoles (pM), about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 90 pM, about 80 pM, about 70 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 20 pM, about 10 pM, about 9 pM, about 8 pM, about 7 pM, about 6 pM, about 5 pM, about 4 pM, about 3 pM, about 2 pM, about 1 pM, about 0.5 pM, or about 0.1 pM of a neurotoxin polypeptide, or even one of the values below. EC50 values above 100 pM have been reported in the literature. By definition, a cell susceptible to neurotoxin intoxication must express, or be engineered to express, at least one neurotoxin receptor and at least one neurotoxin substrate. Receptors and substrates for neurotoxins are described in the art. Thus, the cell is preferably susceptible to a biologically active or mature neurotoxin polypeptide as defined herein. The term "neurotoxin-sensitive cell" as used herein comprises a cell or cell line, e.g., an isolated primary cell or a cell line thereof or a cell of an established cell line, preferably a neuroblastoma cell or a neuroblastoma cell line as defined herein. Preferably, a "neurotoxin-sensitive cell" as used herein is a cell that is sensitive to a biologically active or mature neurotoxin polypeptide as defined herein at a concentration of, e.g., about 1 nM or less, 500 pM or less, about 400 pM or less, about 300 pM or less, about 200 pM or less, about 100 pM or less, about 90 pM or less, about 80 pM or less, about 70 pM or less, about 60 pM or less, about 50 pM or less, about 40 pM or less, about 30 pM or less, about 20 pM or less, about 10 pM or less, about 9 pM or less. [0014] In some embodiments, the present invention provides a method for differentiating a drug or antibody from a subject to at least about 1 pM of a subject, wherein the subject is susceptible to neurotoxin intoxication. For example, by using the novel differentiation method of the present invention, very low EC50 values of less than 0.1 pM have been achieved for SiMa cells and EC50 values of less than 1 pM have been achieved for P19 cells. As is known in the art, "half-maximal effective concentration (EC50)" refers to the concentration of a drug, antibody, or toxic agent that induces a response that is halfway between baseline and maximum after a specified exposure time. It is often used as a measure of the efficacy of a drug. The EC50 of a graded dose response curve therefore represents the concentration of compound at which 50% of its maximal effect is observed.The EC50 of a quantal dose response curve represents the concentration of compound at which 50% of the population exhibits a response after a particular exposure time.

Methods for identifying cells or cell lines that are susceptible to neurotoxin poisoning and/or have the ability to take up neurotoxins, i.e., cells that are sensitive to neurotoxins as defined herein, are known in the art; see, for example, US 2012/0122128 A1. In one aspect, the biological activity of the neurotoxin polypeptide is derived from all of the above-mentioned biological properties. To date, only a few cell-based assays with sufficiently high sensitivity to neurotoxins that can be used to determine the biological activity of neurotoxins have been described in the art, as suggested elsewhere herein. In vivo assays for evaluating the biological activity of neurotoxins include, for example, the already mentioned mouse _LD50 assay and the ex vivo mouse hemidiaphragm assay, as described by Pearce et al. and Dressier et al.; see Pearce 1994, Toxicol.Appl.Pharmacol.128:69-77 and Dressier 2005, Mov.Disord.20:1617-1619. The method of the present invention provides a simple, reliable and robust cell-based test system with increased sensitivity to neurotoxin polypeptides compared to cell test systems described in the art that require complex differentiation protocols. Therefore, the method of the present invention provides an improved alternative to cell test systems or animal tests used in the art to determine the biological activity of neurotoxins. As known to those skilled in the art, the biological activity of neurotoxins is usually expressed in mouse units (MU). 1 MU is the amount of neurotoxic component that kills 50% of a specified mouse population after intraperitoneal injection, i.e., the mouse ip LD50.

The term "differentiation", "differentiated" as used herein refers to the process by which non-specialized or relatively less specialized cells become relatively more specialized. In the context of cell ontogeny, the adjective "differentiated" is a relative term. Therefore, a "differentiated cell" is a cell that has further developed downward in a certain developmental pathway compared to the cell to which it is compared. A differentiated cell can be, for example, a terminally differentiated cell, i.e., a fully specialized cell that is engaged in specialized functions in multiple tissues and organs of a organism and can, but does not need to be, post-mitotic. In another example, a differentiated cell can also be a progenitor cell within a differentiation lineage that is capable of further proliferation and/or differentiation. Similarly, a cell is "relatively more specialized" if it has further developed downward in a certain developmental pathway compared to the cell to which it is compared, and the cell to which it is compared is considered to be "non-specialized" or "relatively less specialized". A relatively more specialized cell may differ from an unspecialized or relatively less specialized cell in one or more indicative phenotypic characteristics, such as, for example, the presence, absence or expression level of a particular cellular component or product, e.g., RNA, protein, specific cellular marker or other substance, activity of certain biochemical pathways, morphological appearance, proliferative capacity and/or kinetics, differentiation potential and/or response to differentiation signals, etc., where such characteristics indicate that the relatively more specialized cell is further along the developmental pathway.

The term "neuronally differentiated cells" as used herein means cells that have reached the final neuronal differentiation state. For example, the mouse embryonic carcinoma cell P19 cell used in the method of the present invention first differentiates into neural progenitor cells, which are then further differentiated into neurons. The neural differentiation process can be tracked, for example, phenotypically (by phase contrast microscopy) and/or by the expression of neuronal differentiation markers; see, for example, Babuska et al. (2010), Prague Medical Report 111, 289-299 or Migliore and Shepherd, Nature Reviews Neuroscience 6, 810-818 (2005). Assays that can be used to measure the expression of the neuronal differentiation markers include, for example, PCR, RT-PCR, Northern blots, Western blots or Dot blots, immunoprecipitation assays, enzyme-linked immunosorbent assays (ELISA) or FACS analysis known in the art; see, for example, Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 3rd edition, 2001. Assays for testing other characteristics of neuronally differentiated cells are also known in the art.

The term "tumor cell capable of differentiating into a neuronal cell" as used herein means, for example, a neuroblastoma cell, an embryonal carcinoma cell, a teratocarcinoma cell, a neural hybrid cell (e.g., a neuron x glioblastoma cell), or a fibroblastoma cell. The method of the present invention has been exemplified using mouse and human tumor cells, namely P19 (murine embryonal carcinoma) and SiMa (human neuroblastoma) cells, respectively.

The term "neuroblastoma" as used herein means a cancer that develops from nerve cells found in several areas of the body. Neuroblastomas most commonly occur in or around the adrenal glands, which have a similar origin to nerve cells and are located on top of the kidneys. However, neuroblastomas can also develop in other areas of the abdomen where groups of nerve cells are present and in the chest, neck, and pelvis. The term "neuroblastoma cell" as used herein includes one or more neuroblastoma cells that are sensitive to neurotoxins and can differentiate into neuronal cells. Neuroblastoma cells can be primary neuroblastoma cells or primary neuroblastoma cell lines. The term also encompasses established neuroblastoma cells or cell lines. Neuroblastoma cells can be mammalian neuroblastoma cells, for example, rodent neuroblastoma cells such as rat or mouse neuroblastoma cells, or monkey neuroblastoma cells, such as rhesus monkey, macaque or cynomolgus monkey neuroblastoma cells or primate neuroblastoma cells such as chimpanzee neuroblastoma cells or, preferably, human neuroblastoma cells. Examples of established neuroblastoma cell lines include, for example, Neuro-2a (mouse neuroblastoma), Kelly (human neuroblastoma), SH-SY5Y (human neuroblastoma), or SiMa (human neuroblastoma). Human neuroblastoma cells are preferably used in the methods of the present invention to generate neurotoxin-sensitive, neuronally differentiated cells. More preferably, the neuroblastoma cells described herein are SiMa cells or SiMa cell lines. This is because SiMa cells are easily metastatic and in a BoNT-sensitive form. Furthermore, they have high sensitivity to BoNTs. Furthermore, the differentiation protocol for SiMa cells is simple and considerably shorter than for other neuroblastoma cells or cell lines. SiMa cells used in the methods of the present invention can be parental SiMa cells or (sub)clones derived therefrom.

In step a) of the aforementioned method of the present invention, "culturing" means culturing tumor cells capable of differentiating into neuronal cells as defined herein in a cell culture medium under conditions and for a time period that induce neuronal differentiation of the tumor cells. The term "priming" as used in cell differentiation processes is known in the art; see, for example, Khoo et al., PLOS ONE 6(5):e19025, (2011); Steindler, ILAR Journal 48(4), 323-338 (2007); Vazey and Connor, Stem Cell Research & Therapy 1:41 (2010). As used herein, "priming tumor cells to differentiate into neurons" means inducing neuronal differentiation of tumor cells as defined herein by the indicated cell culture conditions, time period, and cell culture medium. The priming of tumor cells to differentiate into neurons can be performed, for example, by culturing in a suitable cell culture medium, and optionally, by reducing the serum in the cell culture medium and/or adding retinoic acid. As known to those skilled in the art, the differentiation state of the tumor cells in which the tumor cells are "induced to differentiate into neurons" or "triggered to differentiate into neurons" is characterized by, for example, the expression of neuronal induction markers as set forth elsewhere herein. As will be apparent to those skilled in the art, the "triggered" or "induced" differentiation state of the tumor cells does not, however, yet represent the final neuronal differentiation state of the tumor cells as defined by the characteristics set forth elsewhere herein, but rather represents a previous differentiation step in the neuronal differentiation lineage. Only after performing step b) of the differentiation method of the invention are the tumor cells terminally differentiated, i.e., neuronally differentiated cells as specified herein. Culturing the tumor cells as defined herein in a culture medium under conditions and for a time that induce neuronal differentiation of the tumor cells means culturing the tumor cells in an appropriate cell culture medium at 37°C for 12 hours to 7 days, preferably 24 hours to 6 days, more preferably 36 hours to 5 days and is known in the art. Suitable cell culture media that can be used to induce neuronal differentiation of the tumor cells (also referred to herein as "priming medium") include, for example, OptiMEM, MEMα, RPMI-1640, minimal essential medium (MEM), Ham's F12 medium, Dulbeccos modified Eagle's Medium (DMEM) or DMEM:F12 (1:1). The priming medium may contain other components known in the art, such as serum (e.g., FBS), NEAA, retinoic acid, growth factors (such as NGF or FGF), vitamins, fatty acids, hormones and/or antibiotics. Suitable cell culture media for inducing neuronal differentiation of SiMa cells include, for example, 80 to 98.6% OptiMEM, 1 to 7.5% FBS, 0.2 to 5% B27 supplement, 0.2 to 5% N2 supplement, and, optionally, 0.1 to 2.5% non-essential amino acids (NEAA). Advantageously, the present inventors have found that a cell culture medium comprising 92.5% OPTI-α, 5% FBS, 1% non-essential amino acids, 1% B-27 supplement, and 0.5% N-2 supplement is particularly suitable for priming SiMa cells, as shown in the examples below. To induce neuronal differentiation of SiMa cells, the preferred culture time is 12 hours to 5 days. Preferably, priming of SiMa cells is performed under the cell culture conditions shown in the examples below. To prime P19 cells, for example, 90 to 99.8% MEM-α, 0.2 to 10% FBS, and 0.001 to 10 micromolar (μM) retinoic acid (RA), preferably 0.1 μM RA, can be used. In this case, the culture time is preferably 3 to 7 days.

The term "differentiation medium" as used in step b) of the method for producing neurotoxin-sensitive, neuron-differentiated cells of the present invention is a cell culture medium having an osmolality of 100 to 270 mOsm/kg. Preferably, the osmolality of the differentiation medium is between 120 and 250 mOsm/kg, more preferably between 150 and 240 mOsm/kg, even more preferably between 180 and 230 mOsm/kg, most preferably between 200 and 225 mOsm/kg, and most preferably about 225 mOsm/kg. As shown elsewhere herein and in the examples below, the inventors have surprisingly discovered that the low osmolality of the differentiation medium is responsible for the increased sensitivity of the neurotoxin-sensitive, neuron-differentiated cells produced by the method of the present invention to BoNTs. For example, neurobasal medium, MEM, DMEM:F12 or any basal medium known in the art can be used as a differentiation medium if adjusted to an appropriate weight molar osmotic pressure concentration, for example, by reducing the NaCl concentration in the medium formulation or by diluting the medium with water. In addition, the differentiation medium comprises at least one supplement. The supplement can be (i) a B27 supplement, (ii) an N2 supplement, (iii) an NS21 supplement, or (iv) a combination thereof. For example, a B27 supplement can be used in combination with an N2 supplement. B27 supplements are known in the art and are generally used for the growth and maintenance of neurons. In addition, B27 supplements are commercially available, for example, from Life Technologies. Typically, in the methods of the present invention, B27 supplements can be used at a concentration of 0.2% to 5%. N2 supplements (which can be obtained from, for example, Life Technologies) are chemically determined, serum-free supplements based on Bottenstein's N-1 formulation. N2 supplement is recommended for growth and expression of neuroblastoma and postmitotic neurons in primary cultures from the peripheral nervous system (PNS) and central nervous system (CNS). In the method of the present invention, N2 supplement can be used at a concentration of 0.2% to 5%. NS21 supplement is a redefined and modified B27 supplement, in which 21 different ingredients have been used for neuronal cultures, as described in, for example, Chen et al. (2008), Journal of Neuroscience Methods 171,239-247. Typically, NS21 supplement is used at a concentration of 0.2% to 5% in the method of the present invention. Other components used in the above differentiation medium include glutamine or GlutaMax, one or more antimicrobial agents, NEAA, FBS (0.1 to 20%), GT1b (0.1 to 300 μM), retinoic acid (RA) (1 nanomolar (nM) to 300 μM), growth factors (such as 100 ng/ml NGF, 40 ng/ml BDNF, 40 ng/ml CNTF, 5 ng/ml LIF, 5 ng/ml GDNF, TGF or FGF) or other differentiation factors known in the art (1% DMSO, 1 mM dibutyryl cAMP, 1 mM butyrate, 5 μg/ml celecoxib, Y-27632, SB431542, sonic hedgehog (SHH), etc.). The cells in step b) of this method of the present invention are preferably cultured in the differentiation medium for at least 3 days to obtain neurotoxin-sensitive, neuron-differentiated cells. The culture may also be longer, for example, at least 3.5 days, 4 days, 4.5 days, 5 days, 6 days, 7 days (1 week), 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days (2 weeks), 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days (3 weeks) or even longer. Preferably, P19 tumor cells are cultured for 2 to 3 weeks, and SiMa cells are cultured for 3 to 7 days. Preferred differentiation media and cell culture conditions that allow neuronal differentiation are shown in the examples below. Preferably, the neuronally differentiated cells represent terminally differentiated neuronal cells as defined elsewhere herein.

The term "weight molar osmotic pressure concentration" as used herein means the measurement of solute concentration, which can be defined as the number of osmoles (Osm) of solute per kilogram (kg) of solution (osmol/kg or Osm/kg). The weight molar osmotic pressure concentration of a solution is usually expressed in Osm/kg. Weight molar osmotic pressure concentration measures the number of osmoles of solute particles per unit mass of a solution. Molar osmotic pressure concentration is the measurement of the osmoles of solute per liter of solvent (osmol/l or Osm/l). Weight molar osmotic pressure concentration and molar osmotic pressure concentration can be measured on an osmometer.

In another aspect, the aforementioned method of the present invention may comprise additional steps. For example, the additional steps may encompass steps for determining the biological activity of a neurotoxin polypeptide as defined herein. To this end, neurotoxin-sensitive, neuron-differentiated cells obtained or obtainable by the method of the present invention are first contacted with a neurotoxin polypeptide. The term "contacting" as used in accordance with the method of the present invention refers to bringing the aforementioned cells into physical proximity with the neurotoxin to allow physical and/or chemical and/or biological interaction. The neurotoxin may be contained in a sample, preferably a biological sample such as cells, cell lysates, blood, plasma, serum or lymph. Suitable conditions that allow specific interactions are well known to those skilled in the art. The conditions will depend on the cells and neurotoxin to be applied in the method of the present invention and can be easily adjusted by a technician. In addition, a technician can also determine the time sufficient to allow interaction by routine experiments. For example, a specific amount of an isolated or recombinant neurotoxin polypeptide as defined herein or a variant thereof or a sample comprising a neurotoxin polypeptide can be added to neurotoxin-sensitive, neuron-differentiated cells. Thereafter, the cells are incubated with the neurotoxin polypeptide for at least 24 hours, preferably 48 hours, and more preferably 72 hours, under conditions that allow the neurotoxin polypeptide to exert its biological activity. As used herein, "conditions allowing a Neurotoxin polypeptide to exert its biological activity" are well known in the art. Subsequently, the cells are stopped, for example by adding a lysis buffer, and the biological activity of the Neurotoxin polypeptide is determined, for example as shown in the following examples.

The terms "neurotoxin," "neurotoxin polypeptide," or "neurotoxin protein," as used herein, refer to the seven different serotypes of Clostridium botulinum neurotoxin, namely, BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, as well as tetanus neurotoxin (TeNT), and variants thereof, as defined herein. The corresponding nucleic acid and amino acid sequences are known in the art; see, for example, the Uniprot or TREMBL sequence databases. Preferably, BoNT/A is used in the methods of the present invention (WO2009/114748). The corresponding receptors and substrates for the neurotoxins have been identified elsewhere herein. The neurotoxin polypeptide may be a naturally occurring neurotoxin or a non-naturally occurring neurotoxin. Naturally occurring neurotoxin polypeptides are produced by naturally occurring methods, including, for example, isoforms and subtypes produced from post-translational modifications, alternatively spliced transcripts, or spontaneous mutations. For example, a BoNT/A subtype is a BoNT/A1 subtype, a BoNT/A2 subtype, a BoNT/A3 subtype, a BoNT/A4 subtype, or a BoNT/A5 subtype. Naturally occurring neurotoxin polypeptides include the above sequences wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more amino acid residues have been added, substituted, or deleted. Commercially available pharmaceutical compositions comprising naturally occurring BoNT/As have been mentioned in the introduction. A non-naturally occurring neurotoxin polypeptide is any neurotoxin polypeptide whose structure has been modified with the aid of human manipulation, including, for example, neurotoxin polypeptides with altered amino acid sequences produced by random mutagenesis or rationally designed genetic engineering, and neurotoxin polypeptides produced by chemical synthesis. Such non-naturally occurring neurotoxin polypeptides have been described in the prior art.

In another aspect of the invention, the neurotoxin polypeptide, as defined herein, has an amino acid sequence that is at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, or a tetanus neurotoxin. As used herein, identity refers to sequence identity of amino acid sequences where the sequences are aligned so as to obtain the highest order match. This can be achieved using published techniques or computer program encoding methods such as, for example, BLASTP, BLASTN, FASTA, Altschul 1990, J Mol Biol 215, 403. In one aspect, the percent identity value is calculated over the entire amino acid sequence. A range of programs based on a variety of algorithms are available to the skilled artisan for comparing different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. For sequence alignment, the program PileUp (1987, J Mol Evolution 25, 351; Higgins 1989 CABIOS 5, 151) or the programs Gap and BestFit (Needleman and Wunsch 1970, J Mol Biol 48; 443; Smith and Waterman 1981, Adv Appl Math 2, 482) are used, which are part of the GCG software package (Genetics Computer Group 1991, 575 Science Drive, Madison, Wisconsin, USA 53711). In one aspect of the present invention, the sequence identity values indicated above in percentages (%) were determined over the entire sequence region using the program GAP with the following settings: gap weight: 50, length weight: 3, average matches: 10.000, and average mismatches: 0.000, which should always be used as standard settings for sequence alignments unless otherwise stated. It will be understood that the aforementioned variants will, in one aspect of the present invention, retain at least one of the biological properties of the neurotoxin, and in another aspect, will retain all of the biological properties of the cited neurotoxin polypeptides. In other aspects, the variants can be neurotoxins with improved or altered biological properties, for example, they may contain an improved cleavage site for enzyme recognition, or may be improved for receptor binding or any of the other properties described above.

In principle, the neurotoxins referred to herein comprise an N-terminal light chain and a C-terminal heavy chain. Neurotoxins are produced as single-chain precursor molecules, referred to as "unprocessed neurotoxin polypeptides". Due to subsequent processing, "processed neurotoxin polypeptides" are obtained. The processed neurotoxin polypeptides exhibit biological properties characteristic of neurotoxins, namely (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytosol, and/or (d) endoproteolytic cleavage of proteins involved in synaptic vesicle fusion. Therefore, processed neurotoxin polypeptides are referred to as biologically active or mature neurotoxin polypeptides. The biological activity of neurotoxin polypeptides, on the one hand, is due to all of the aforementioned biological properties, as shown elsewhere herein.

In another aspect, the neurotoxin polypeptide according to the method of the present invention may be a chimeric molecule. Said chimeric molecule, in one aspect, may have a single domain substitution. Thus, in another aspect, a portion of the neurotoxin heavy chain is replaced with a portion of the Fc domain of an antibody.

The term "amount" as used herein encompasses, for example, the absolute amount of a Neurotoxin polypeptide, the relative amount or concentration of said polypeptide as well as any value or parameter related thereto or derivable therefrom.

The term "determining an amount," e.g., determining the amount of a neurotoxin polypeptide, refers to measuring, for example, the absolute amount, relative amount, or concentration of a neurotoxin polypeptide in a quantitative or semi-quantitative manner. Suitable methods for detection are well known to those skilled in the art. It will be understood that the determination of the amount of a neurotoxin polypeptide also requires, in one aspect, calibration of the method by administering a standard solution having a predefined amount of the neurotoxin polypeptide. How to perform such calibration is well known to those skilled in the art.

As used herein, the term "determining the biological activity of a neurotoxin polypeptide" means measuring the biological activity of a neurotoxin protein, i.e., (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytosol, and/or (d) endoproteolytic cleavage of proteins involved in synaptic vesicle fusion. More specifically, the overall cellular mechanism by which a neurotoxin (e.g., BoNT/A) cleaves a neurotoxin substrate (e.g., SNAP-25) encompasses binding of the neurotoxin to its corresponding receptor (e.g., binding of BoNT/A to a BoNT/A receptor), internalization of the neurotoxin/receptor complex, translocation of the neurotoxin light chain from intracellular vesicles into the cytoplasm, and proteolytic cleavage of the neurotoxin substrate. In vitro and in vivo assays for determining the biological activity of neurotoxin polypeptides are well known in the art and have been mentioned elsewhere herein (see, for example, Pellett et al., Withemarsh et al. Toxicol. Sciences 126(2), 426-435 (2012), WO 2010/105234 A1).

As used herein, the term "sensitivity to neurotoxin (polypeptide) activity" refers to the lowest dose that an assay can consistently measure above the signal detected by a non-treated control or background signal.

In one aspect of the method of the present invention, the differentiation medium used in step b) of the aforementioned method of the present invention further comprises retinoic acid. Preferably, the retinoic acid is present in the neurobasal medium at a concentration between 0.01 micromolar (μM) and 300 μM. More preferably, for P19 tumor cells, retinoic acid is present at a concentration between 0.1 and 0.5 μM retinoic acid, i.e., at a concentration of 0.1, 0.2, 0.3, 0.4 or 0.5 μM retinoic acid; and for SiMa cells, at a concentration between 1 and 5 μM, i.e., at a concentration of 1, 2, 3, 4, or 5 μM, most preferably at a concentration of 3 μM. Retinoic acid is a known neural inducer that can induce differentiation of neuronal cells, including neuroblastoma cells. It has been found that adding low concentrations of retinoic acid to the differentiation medium used in step b) of the method of the present invention advantageously enhances the sensitivity of tumor cells to neurotoxin polypeptides.

In another aspect of the method of the present invention, the differentiation medium in step b) further comprises an antimicrobial agent and/or a cytostatic agent that inhibits the growth of non-neuronal cells. For example, penicillin-streptomycin can be used as an antimicrobial agent. For example, cytosine-1-β-D-arabinofuranoside (AraC) can be used as a cytostatic agent. The addition of the antimicrobial agent prevents bacterial growth in the cell culture medium, while the cytostatic agent inhibits the cell growth and proliferation of non-tumor/non-neuronal cells that might otherwise outgrow the tumor cells in the differentiation method of the present invention.

In still another aspect of the methods of the present invention, the differentiation medium in step b) further comprises GT1b. GT1b is a ganglioside that binds neurotoxins and potentially mediates their selectivity for neurons. Thus, GT1b can be used as an enhancer for BoNT uptake into tumor cells in the methods of the present invention. Preferably, GT1b is present at a concentration between 25 and 75 μM, i.e., 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, 50 μM, 55 μM, 60 μM, 65 μM, 70 μM, or 75 μM, more preferably 50 μM.

In another aspect of the method of the present invention, step a) of the method of the present invention comprises cell culture conditions comprising reducing serum and/or adding retinoic acid from the (cell) culture medium. As used herein, "reducing serum from the culture medium" means, for example, gradually reducing serum from 10% to 5% to 2.5% to 1% to 0% at intervals of 2 to 5 days. Alternatively, a serum-free cell culture medium can be used to replace the cell culture medium containing serum in one step. Removing the growth factors contained in the serum allows the differentiation of tumor cells as defined herein. Additionally or alternatively, retinoic acid can be added to the culture medium at low concentrations, as shown elsewhere herein.

In some aspects of the methods of the present invention, the tumor cells capable of differentiating into neuronal cells are SiMa cells, which are available from the DSMZ (German Collection of Microorganisms and Cell Cultures) under the accession number ACC 164. The SiMa cell line DSMZ ACC 164 is also referred to as the parental SiMa cell line. The SiMa cells used in the methods of the present invention may be parental SiMa cells or (sub)clones thereof. Such subclones are also known in the art; see, for example, WO 2010/105234, US 8,476,068 B2 or Ester Fernández-Salas, Joanne Wang, Yanira Molina, Jeremy B. Nelson, Birgitte P.S. Jacky, K. Roger Aoki—PLoS One; 20127(11)e49516.

SiMa cells can be cultured according to the DMSZ protocol. In one aspect, in step a) of the method of the present invention, culturing the SiMa cells in a cell culture medium under conditions and for a time that induce neuronal differentiation of the SiMa cells comprises culturing the SiMa cells at 37° C. in a cell culture (or growth) medium comprising at least OptiMEM, FBS, B27 supplement, N2 supplement, and optionally, NEAA for at least 12 hours (h), at least 24 hours, or preferably at least 36 hours.

In one aspect of the method of the present invention, the culture or priming medium for SiMa cells in step a) comprises 80% to 98.8% OptiMEM, 1 to 10% FBS, 0.2 to 5% B27 supplement, and/or 0.2 to 5% N2 supplement. Optionally, the culture medium for SiMa cells further comprises non-essential amino acids and/or antibiotics and/or retinoic acid.

Preferably, the culture or priming medium of the SiMa cells in step a) comprises 92.5% OptiMEM, 5% FBS, 1% B27 supplement, 0.5% N2 supplement and 1% NEAA.

In one aspect of the method of the present invention, the differentiation medium in step b) comprises a medium having an osmolality of 100 to 270 mOsm/kg. Preferably, the medium is neurobasal medium used at a concentration of 86 to 98.8%. The differentiation medium may further comprise one or more of the following ingredients: 0.2 to 5% B27 supplement and/or 0.2 to 5% N2 supplement; 0.5 to 2% non-essential amino acids (NEAA); 1 to 5 μM retinoic acid; 0.5 to 2% GlutaMAX and/or 25 to 75 μM GT1b.

Preferably, the differentiation medium in step b) comprises 97% neurobasal medium, 2% B27 supplement, 1% GlutaMAX, and 3 μM retinoic acid.

In one aspect of the method of the invention, the SiMa cells are cultured in step a) for at least 36 hours.

In one aspect of the method of the present invention, in step a), SiMa cells are cultured on a tissue culture dish coated with at least one compound selected from the group consisting of poly-L-lysine, poly-D-lysine, collagen, laminin, and gelatin. Methods for coating cell culture dishes are well known to those skilled in the art. Preferably, the tissue culture dish is coated with poly-L-lysine.

The present invention provides a novel differentiation protocol that significantly increases the sensitivity of SiMa cells to neurotoxin peptides. Furthermore, the assay system is more accurate and robust than prior art assays for the biological activity of neurotoxin peptides. In the first step of initiating neuronal differentiation of SiMa cells, SiMa cells were cultured in a novel priming medium comprising 92.5% Opti-, 5% FBS, 1% B27 supplement, 0.5% N2 supplement, and 1% NEAA. If applicable, one or more antimicrobial agents may also be added to the cell culture medium. Cells were then seeded onto coated multiwell plates. Optimal results were achieved using cell culture dishes coated with poly-L-lysine. Alternatively, collagen, poly-D-lysine, laminin, gelatin, or a combination thereof may be used to coat the cell culture dishes. After the seeded cells had been grown in the aforementioned growth medium or priming medium for at least 36 hours, the medium was replaced with six different differentiation media. After 3 to 7 days of differentiation, in which the differentiation medium was replaced with fresh differentiation medium, a neurotoxin polypeptide (exemplified by BoNT/A) was added to the differentiation medium, if applicable. After an additional 75 hours of incubation, the cells were terminated and the biological activity of the added neurotoxin polypeptide was analyzed. In contrast, differentiation medium with a low osmolality gave the best results: the use of a differentiation medium comprising neurobasal medium containing 2% B27 supplement, 1% GlutaMAX, and 3 μM retinoic acid provided an EC50 value of 0.22 pM. The differentiation medium had an osmolality of approximately 225 mOsm/kg. Similar results were observed with an EC50 value of 0.24 pM for a differentiation medium comprising MEM, 2% B27 supplement, 1% N2 supplement, and 3 μM retinoic acid, diluted to approximately 225 mOsm/kg. The relatively simple differentiation protocol of the present invention allows for high reproducibility and high sensitivity (EC50 below 0.3 pM; LLOD below 0.1 pM), which allows for highly diluted samples to be analyzed. Thus, the sensitivity of SiMa cells generated by the differentiation method of the present invention is preferably below 10 pM, below 5 pM, below 2 pM, below 1 pM, below 0.5 pM, or even below 0.3 pM.

In other aspects of the method of the present invention, the tumor cell capable of differentiating into a neuronal cell is a P19 cell, which can be obtained from DSMZ, for example, with ACC deposit number: 316, from ATCC with CRL-1825, or from ECACC with 95102107. P19 cells can be cultured according to the protocol of DSMZ. In one aspect, in step a) of the method of the present invention, under conditions and time that induce neuronal differentiation of the P19 cells, culturing P19 cells in a cell culture medium includes culturing the P19 tumor cells on a bacterial culture dish in a cell culture (or growth) medium comprising at least MEM-α and FBS for at least 3 days, 4 days, or preferably 5 days to form cell aggregates. In one aspect, the cell culture medium of P19 cells comprises retinoic acid, preferably at a concentration between 75 and 125 nM, more preferably at a concentration of 100 nM. In one aspect, the cell culture medium comprises 95% MEM-α, 5% FBS, and 0.1 μM retinoic acid. Preferably, P19 cell aggregates are harvested and isolated cells that have been initiated to differentiate into neurons are generated from the aggregates by protease treatment, such as trypsin treatment. In one aspect of the method of the invention, the P19 cells in step b) of the method of the invention are cultured in a differentiation medium having a low weight molar osmotic pressure concentration as defined herein, preferably in neurobasal medium having a weight molar osmotic pressure concentration of about 225 mOsm/kg for 2 to 3 weeks.

The present invention provides a novel differentiation protocol for P19 cells that significantly increases the sensitivity of cells to BoNT, as demonstrated in the following examples. In the differentiation protocol of the present invention, P19 cells were cultured as described in the prior art. To induce neuronal differentiation of P19 cells, the cells were plated on a bacterial culture dish (i.e., a cell-free culture dish) and 100 nM retinoic acid was added. After 5 days, the resulting cell aggregates were isolated and trypsinized. Thereafter, the cells were plated on multiwell plates and further cultured. The present inventors surprisingly discovered that the use of neurobasal medium (comprising 2% B27 supplement, 1% N2 supplement, and 1% GlutaMAX) and the addition of a low concentration of retinoic acid (100 nM) further increased the sensitivity of cells to BoNT. The growth of non-neuronal cells can be inhibited by the addition of a cytostatic drug (e.g., 5 μM RaC). Furthermore, it was discovered that when GT1b was added to the culture medium upon intoxication of cells with BoNT, the addition of GT1b enhanced BoNT uptake by the cells. For this purpose, for example, 50 μM GT1b was used. After 2 to 3 weeks of differentiation, in which case the culture medium had been replaced if necessary, BoNT was added to the cell culture medium. After 72 h of incubation with BoNT, the cells were terminated and analyzed by the methods shown in the following examples.

As illustrated for two different cell lines, the present invention provides a new cell-based test system for determining the biological activity of neurotoxins. The cell-based test system of the present invention is extremely sensitive and robust and provides an alternative to conventional animal testing. In addition, the cell-based test system of the present invention is a simple differentiation protocol compared to the cell-based test systems described in the art and allows high reproducibility and high sensitivity (EC50 for SiMa cells is less than 0.1 pM and for P19 cells is less than 1 pM), which allows for a high dilution of the sample containing the neurotoxin to be analyzed. In order to apply the potentially interfering substances at the lowest possible concentration, such a high dilution of the sample is extremely important for impurities contained in the excipient or sample to be analyzed. Finally, the cell-based test system of the present invention is more flexible and economical than the systems described in the art.

It should be understood that the definitions and explanations of the terms made above apply mutatis mutandis to all aspects described hereinafter in this specification unless otherwise stated.

The present invention also relates to neurotoxin-sensitive, neuronally differentiated cells obtainable or obtained by the methods of the present invention. Preferably, in the case of SiMa cells, the cells have an EC50 value of less than 10 pM, less than 5 pM, less than 2 pM, less than 1 pM, less than 0.5 pM, or even less than 0.3 pM. It is also preferred that the neuronally differentiated SiMa cells generated by the differentiation method of the present invention have a sensitivity to Clostridium botulinum neurotoxin that is increased by at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or even at least 10-fold compared to SiMa cells differentiated in a differentiation medium having an osmolality of about 290 to 350 mOsm/kg, typically used in neuronal differentiation methods described in the art. Similar results can be achieved using P19 cells; see the Examples.

Furthermore, the present invention provides a method for determining the activity of a neurotoxin polypeptide, comprising the steps of:

a) contacting neurotoxin-sensitive, neuronally differentiated cells, preferably neurotoxin-sensitive, neuronally differentiated cells obtainable or obtained by the method of the present invention, with a neurotoxin polypeptide;

b) culturing the neurotoxin-sensitive, neuronally differentiated cells of step a) for 3 to 74 hours or preferably 72 hours under conditions that allow the neurotoxin polypeptide to exert its biological activity; and

c) determining the biological activity of the Neurotoxin polypeptide in said cells after cultivation according to step b).

Specifically, the method for determining the activity of a neurotoxin polypeptide comprises the steps of:

a) culturing tumor cells capable of differentiating into neuronal cells in a culture medium under conditions and for a time period that induce neuronal differentiation of the tumor cells;

b) culturing the tumor cells initiated for neuronal differentiation of step a) in a differentiation medium having an osmolality of 100 to 270 mOsm/kg and comprising (i) B27 supplement and/or (ii) N2 supplement for at least 3 days, thereby obtaining neurotoxin-sensitive, neuronally differentiated cells;

c) contacting the neurotoxin-sensitive, neuronally differentiated cells of step b) with a neurotoxin polypeptide;

d) culturing the neurotoxin-sensitive, neuronally differentiated cells of step c) for 3 to 74 hours, or preferably for 72 hours, under conditions that allow the neurotoxin polypeptide to exert its biological activity; and

e) determining the biological activity of the Neurotoxin polypeptide in said cells after cultivation according to step d).

The biological activity of the neurotoxin polypeptide can be determined by methods described in the art (see, for example, Pellett et al., Withemarsh et al. Toxicological Sciences 126(2), 426-435 (2012), WO 2010/105234A1, WO 2009/114748, WO 2012/123370, WO 2013/131991). Preferably, the tumor cell capable of differentiating into a neuronal cell is a SiMa cell, more preferably a parental SiMa cell (DSMZ ACC 164). The neurotoxin polypeptide is preferably a BoNT/A.

The present invention also relates to the use of a cell culture medium and/or differentiation medium as specified in the method of the present invention for the ex vivo generation of neurotoxin-sensitive, neuronally differentiated cells from neuronally differentiated cells, preferably neuroblastoma cells.

In another aspect, the present invention provides a cell culture or priming medium comprising 80 to 98.8% OptiMEM, 1 to 10% FBS, 0.2 to 5% B27 supplement and/or 0.2 to 5% N2 supplement. Alternatively, 0.2 to 5% NS21 supplement may be used. Preferably, the medium comprises 93.5% OptiMEM, 5% FBS, 1% B27 supplement and 0.5% N2 supplement. The medium is particularly useful for priming SiMa cells. Opti-Reduced Serum Medium (Life Technologies) is a modification of Eagle's Minimum Essential Medium, buffered with HEPES and sodium bicarbonate and supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements and growth factors. In one aspect, the medium may further comprise an antimicrobial agent and/or non-essential amino acids (NEAA) as defined herein. The medium of the present invention is particularly useful in step a) of the method of generating neurotoxin-sensitive, neuronally differentiated cells of the present invention using SiMa cells.

Finally, the present invention relates to a kit suitable for performing the aforementioned method, the kit comprising a cell culture medium containing 80 to 98.8% OptiMEM, 1 to 10% FBS, 0.2 to 5% B27 supplement and/or 0.2 to 5% N2 supplement and, optionally, non-essential amino acids and/or an antimicrobial agent, and SiMa cells. Preferably, the cell culture medium comprises 93.5% OptiMEM, 5% FBS, 1% B27 supplement and 0.5% N2 supplement, and SiMa cells. The kit may further comprise a differentiation medium as defined herein having an osmolality of 100 to 270 mOsm/kg, preferably neurobasal medium, and at least one supplement as indicated herein, i.e., B27, N2 or NS21 supplement. Preferably, the differentiation medium comprises 78 to 98.3% culture medium, 1 to 10% FBS, 0.5 to 2% GlutaMAX, 0.2 to 5% B27 supplement, and/or 0.2 to 5% N2 supplement. Other preferred culture media that can be used in the kits of the present invention are shown in the following examples.

It should be understood that the kit of the present invention is for practicing the methods mentioned above herein. In one aspect, it is envisioned that all components are provided in a ready-to-use manner for practicing the methods mentioned above. In other aspects, the kit contains instructions for performing the methods. The instructions can be provided by a user manual in paper or electronic form. For example, the manual can include instructions for explaining the results obtained when using the kit of the present invention to perform the aforementioned methods.

All references cited in this specification are incorporated by reference with their entire disclosure content and the disclosure content specifically mentioned in this specification.

Abbreviations used herein:

OptiMEM: Opti-Reduced Serum Medium (Life Technologies) is a modification of Eagle's Minimum Essential Medium, buffered with HEPES and sodium bicarbonate and supplemented with hypoxanthine, thymidine, sodium pyruvate, L-glutamine, trace elements, and growth factors. Most cells grown in serum-supplemented medium can be transferred to Opti-Reduced Serum Medium with 50% reduced serum.

FBS: Fetal bovine serum is the most widely used serum supplement for in vitro cell culture of eukaryotic cells. FBS is commercially available from many manufacturers, such as Sigma-Aldrich, Invitrogen, Life Technologies, and others.

B27 supplement: B27 supplement is commonly used to grow and maintain neurons. B27 supplement is commercially available as a 50x or 100x stock solution, for example from Life Technologies.

N2 Supplement: Available commercially as 50x or 100x stock solutions, for example, from Life Technologies. N2 Supplement is recommended for the growth and expression of neuroblastoma cells and postmitotic neurons in primary cultures from the peripheral and central nervous systems. N2 Supplement can be used as an alternative to Bottenstein's N1 formulation. N2 Supplement can be used with neurobasal medium supplemented with growth factors such as bFGF or EGF, or with DMEM.

NS21 supplement: NS21 supplement is a redefined and modified B27 supplement, of which 21 different components have been used for neuronal cultures, as described, for example, in the publication by Chen et al. (2008), Journal of Neuroscience Methods 171, 239-247.

NEAA: Non-essential amino acid cell culture supplements commercially available from many manufacturers such as Invitrogen or Cyagen are typically provided as stock solutions containing several non-essential amino acids such as glycine, L-alanine, L-asparagine, L-glutamic acid, L-aspartic acid, L-serine, or L-proline. They are used as supplements to cell culture media for optimized cell growth.

P19 cells: P19 cells are embryonic carcinoma cell lines derived from teratocarcinomas derived from embryos in mice. This cell line is pluripotent and can differentiate into all three germ layer cell types. It is also the most characterized embryonic carcinoma (EC) cell line, which can be specifically induced into cardiomyocytes and neurons by different specific treatments. Exposing aggregated P19 cells to dimethyl sulfoxide can lead to differentiation into cardiac muscle and skeletal muscle. In addition, exposing P19 cells to retinoic acid (RA) can differentiate them into neurons (McBurney & Rogers (1982), Dev. Biol 89 (2): 503-508; Rudnicki, et al. (1990) Dev. Biol 138 (2): 348-358).

SiMa cells: SiMa cells correspond to a neuroblastoma (Nb) cell line that carries the major recurrent chromosomal alterations associated with poor prognosis in Nb, including amplification of N-MYC by formation of double minute bodies (dmin), der(1)t(1;17)(p35;q12) and der(22)t(17;22)(q22;p13), and loss of chromosome 11, documented at both early and late passages. In contrast to these cytogenetic features of poor prognosis, analysis of catecholamine synthesis measured by high-pressure liquid chromatography (HPLC) revealed a high degree of adrenergic differentiation with high rates of 3,4-dihydroxyphenylalanine (DOPA), norepinephrine, homovanillic acid (HVA), and vanillylmandelic acid (VMA) production. The contrastingly high degree of differentiation and poor prognostic combination of genetic markers make SiM a unique tool for studying the pathology and therapy of Nb (Marini et al., Cancer Genet. Cytogenet. (1999), 112: 161-4).

MEM-α: Minimum Essential Medium α (Invitrogen, Life Technologies)

Neurobasal Medium: This medium (Invitrogen, Life Technologies) is a basal medium that, when used with supplements, meets the specialized cell culture requirements of prenatal and embryonic neuronal cells. The medium can be used to grow neuronal cells from the hippocampus, cortex, and other regions of the brain. It allows for the long- and short-term maintenance of homogenous neuronal cell populations without the need for astrocyte feeder layers.

GlutaMax: GlutaMAX ^™ medium is a cell culture medium containing L-alanyl-L-glutamine, a stabilized dipeptide derived from L-glutamine, which prevents degradation and ammonia accumulation even during long-term culture. L-alanyl-L-glutamine dipeptide is extremely stable in aqueous solution and does not degrade into ammonia during storage or incubation like L-glutamine.

The present invention will now be illustrated by the following examples, which however are not to be construed as limiting the scope of the invention.

Example: Effects of Differences Between Neurobasal and MEM Media on Susceptibility to Clostridium botulinum neurotoxin (BoNT)

i) SiMa parental cells were cultured according to the DSMZ (German Collection of Microorganisms and Cell Cultures) protocol. One day before plating the cells in 96-well plates, the medium was changed to 92.5% OPTI- (Gibco, Life Technologies ^™ #51985) + 5% FBS (PAA#A15-152) + 1% non-essential amino acids (NEAA; Gibco, Life Technologies ^™ #11140-038) + 1% B-27 supplement (Gibco, Life Technologies ^™ #17504-044) + 0.5% N-2 supplement (Gibco, Life Technologies ^™ #17502-048) as "priming medium." Cells (30,000 cells/cm ² ) were plated in 96-well plates (TPP #92096). Five days later, Clostridium botulinum neurotoxin (BoNT) was added to the priming medium. 24 h after plating the cells, change the medium to one of the following differentiation media:

1. 97% Neurobasal (Gibco, Life Technologies ^™ #21103) + 2% B-27 supplement (Gibco, Life Technologies ^™ #17504-044) + 1% GlutaMAX ^™ (Gibco, Life Technologies ^™ #35050-038) + 3 μM retinoic acid (RA; Sigma-Aldrich #R2625)

or

2. 96% MEM (Gibco, Life Technologies ^™ #42360) + 2% B-27 supplement (see above) + 1% N-2 supplement (see above) + 1% NEAA (see above) + 3 μM RA (see above)

or

3. 96% MEM (see above) + 2% B-27 supplement (see above) + 1% N-2 supplement (see above) + 1% NEAA (see above) + 3 μM RA (see above) + Vitamin B12 + Fe + Zn (low)

or

4. 96% MEM (see above) + 2% B-27 supplement (see above) + 1% N-2 supplement (see above) + 1% NEAA (see above) + 3 μM RA (see above) + Vitamin B12 + Fe + Zn (high)

or

5. 96% MEM (see above) + 2% B-27 supplement (see above) + 1% N-2 supplement (see above) + 1% NEAA (see above) + 3 μM RA (see above); diluted to 225 mOsm/kg

or

6. 97% Neurobasal (see above) + 2% B-27 supplement (see above) + 1% GlutaMAX ^™ (see above) + 3 μM retinoic acid (see above) + Zn (high)

Four days after switching to differentiation medium, half of the medium in each well was replaced with fresh medium containing varying concentrations of BoNT/A (serial 1:2 dilutions starting at 10 pM in 11 steps plus a negative control without BoNT/A). 72 hours after adding BoNT, the medium was aspirated and lysis buffer containing nuclease (benzonase) was added. The plates were incubated at room temperature (RT) and then Roti-load 1 (Roth #K929.1) was added. Samples prepared as described above can be stored at temperatures below -70°C. Low temperature is critical because SNAP-25 appears to degrade specifically at higher storage temperatures. Samples were then separated using SDS-PAGE and subsequently analyzed by Western blot. EC50 values were determined and compared in Table 1 (averages of four experiments are given).

Table 1:

EC50 values in bold = high sensitivity to BoNT

Surprisingly, simply replacing MEM with Neurobasal medium increased the sensitivity of the cells to BoNT/A by 10-fold (2 vs. 1). Supplementing MEM with components of Neurobasal medium that MEM lacks (iron, vitamin B12, and zinc) did not affect the susceptibility of the cells to BoNT/A (2 vs. 3 and 4). However, diluting MEM to 225 mOsm/kg with sterile deionized water produced the same surprising effect as using Neurobasal medium. Thus, reducing the osmolality from 300 mOsm/kg to 225 mOsm/kg significantly increased the sensitivity of SiMa cells from ~2 pM to ~0.2 pM.

Table 2 shows the osmolality values of differentiation media 1 to 6.

Table 2: Osmolality of the tested differentiation media

For example, MEM has an osmolality of approximately 300 mOsm/kg. Typically, cell culture media used in the prior art have an osmolality between 290 and 350 mOsm/kg. Other ingredients do not substantially affect the osmolality of the cell culture medium. For supplements, an osmolality within the desired range is selected.

ii) P19 cells were cultured according to the protocol of DSMZ (German Collection of Microorganisms and Cell Cultures). 5 days before the cells were plated on 96-well plates, the cells were transferred to bacterial grade culture dishes and the culture medium was changed to 95% MEM-α (Gibco, Life Technologies ^TM #32571) supplemented with 5% FBS (PAA #A15-152) and 0.1 μM retinoic acid (RA; Sigma-Aldrich #R2625) as "priming medium". During the 5-day growth process, the cells formed clumps or aggregates. They were trypsinized and the cells (30.000 cells/cm ² ) were plated on 96-well plates (TPP #92096). After 5 days, Clostridium botulinum neurotoxin (BoNT) was added to the above-mentioned priming medium. 24 h after the cells were plated, the culture medium was changed to one of the following differentiation media:

1. 97% Neurobasal (Gibco, Life Technologies ^TM #21103) + 2% B-27 Supplement (Gibco, Life Technologies ^TM #17504-044) + 1% GlutaMAX ^TM (Gibco, Life Technologies ^TM #35050-038) + 3 μM retinoic acid (RA; Sigma-Aldrich #R2625); 225 mOsm/kg.

2. 95% MEM-α (Gibco, Life Technologies ^TM #32571) + 5% FBS (PAA #A15-152) + 0.1 μM retinoic acid (RA; Sigma-Aldrich #R2625); 300 mOsm/kg.

3. 90% MEM-α (Gibco, Life Technologies ^TM #32571) + 10% FBS (PAA #A15-152); 300 mOsm/kg.

4. 97% MEM-α (Gibco, Life Technologies ^™ #32571) + 2% B-27 supplement (see above) + 1% N-2 supplement (see above) + 1% NEAA (see above) + 1 μM retinoic acid (RA; Sigma-Aldrich #R2625); 300 mOsm/kg.

Four days after switching to differentiation medium, half of the medium in each well was replaced with fresh medium containing varying concentrations of BoNT/A (serial 1:2 dilutions starting at 10 pM in 11 steps plus a negative control without BoNT/A). 72 hours after adding BoNT, the medium was aspirated and lysis buffer containing nuclease (benzonase) was added. The plates were incubated at room temperature (RT) and then Roti-load 1 (Roth #K929.1) was added. Samples prepared as described above can be stored at temperatures below -70°C. Low temperature is critical because SNAP-25 appears to degrade specifically at higher storage temperatures. Samples were then separated using SDS-PAGE and subsequently analyzed by Western blot. EC50 values were determined and compared in Table 3 (averages of four experiments are given).

Table 3:

Differentiation medium EC50[pM] EC50[mol/l] 1. Neurobasal+B-27+GlutaMax+RA 1.10 2.α-MEM+5% FBS+RA 8.84 3.α-MEM + 10% FBS 17.7 4.α-MEM+B-27+NEAA+N-2+RA 12.5

Bold EC50 values = high sensitivity to BoNT.

Claims (18)

1. A method for generating neurotoxin-sensitive, neuronal-differentiated cells, the method comprising the steps of: a) Under conditions and time that induce neuronal differentiation of the tumor cells, tumor cells capable of differentiating into neurons are cultured in a culture medium, wherein the tumor cells are SiMa cells; and b) Culture the tumor cells of a) that have been induced to differentiate into neurons for at least 3 days in a differentiation medium having a weight molar osmolality of 120 to 250 mOsm/kg and containing (i) B27 supplement and/or (ii) N2 supplement, thereby obtaining neurotoxin-sensitive, neuron-differentiated cells with increased sensitivity to the clostridial neurotoxin BoNT compared to SiMa cells cultured in a differentiation medium having a weight molar osmolality of 290 to 350 mOsm/kg. 2. The method of claim 1, wherein the differentiation culture medium further comprises retinoic acid. 3. The method of claim 2, wherein the retinoic acid is present at a concentration between 0.01 μM and 300 μM. 4. The method of claim 1, wherein the differentiation medium further comprises an antibacterial agent and/or a cell inhibitor that inhibits the growth of non-neuronal cells. 5. The method of claim 1, wherein the differentiation medium further comprises GT1b. 6. The method of claim 5, wherein the GT1b is present at a concentration between 25 and 75 μM. 7. The method of claim 5, wherein the GT1b is present at a concentration of 50 μM. 8. The method of claim 1, wherein the conditions and timing for inducing neuronal differentiation of the cells in claim 1a) include reducing serum and/or adding retinoic acid from the culture medium. 9. The method of claim 1, wherein the tumor cells are SiMa cells (DSMZ ACC accession number: 164). 10. The method of claim 1 or 9, wherein the culture medium in step a) comprises 80 to 98.8% OptiMEM, 1 to 10% FBS, 0.2 to 5% B27 supplement and/or 0.2 to 5% N2 supplement and, optionally, non-essential amino acids and/or antibiotics. 11. The method of claim 1 or 9, wherein the differentiation medium in step b) has a weight molar osmotic concentration of 200 to 225 mOsm/kg. 12. The method of claim 11, wherein the differentiation medium in step b) comprises 78 to 98.3% neurobasal medium, 1 to 10% FBS, 0.5 to 2% GlutaMAX, 0.2 to 5% B27 supplement, and/or 0.2 to 5% N2 supplement. 13. The method of claim 1 or 9, wherein SiMa cells are cultured for at least 36 hours in step a). 14. The method of claim 1 or 9, wherein the culture medium in step a) further comprises an antimicrobial agent and/or non-essential amino acids (NEAA). 15. The method of claim 1 or 9, wherein in step a) SiMa cells are cultured on a tissue culture dish coated with at least one compound selected from the group consisting of: poly-L-lysine, poly-D-lysine, collagen, laminin and gelatin. 16. A method for determining the activity of neurotoxin peptides, the method comprising the steps of: a) Obtaining neurotoxin-sensitive, neuronal-differentiated cells by any one of claims 1 to 15, and contacting the cells with a neurotoxin polypeptide; b) Culture the neurotoxin-sensitive, neuronal-differentiated cells from step a) for 3 to 74 hours under conditions that allow the neurotoxin peptides to exert their biological activity; and c) After culturing according to step b), the biological activity of the neurotoxin peptides in the cells is determined. 17. The method of claim 16, wherein in step b), the neurotoxin-sensitive, neuronal-differentiated cells of step a) are cultured for 72 hours under conditions that allow the neurotoxin polypeptide to exert its biological activity. 18. The method of claim 1, wherein the culture medium in step a) comprises 93.5% OptiMEM, 5% FBS, 1% B27 supplement and 0.5% N2 supplement.