IN VITRO METHODS OF TESTING A CLOSTRIDIUM BOTULINUM (NTB) NEUROTOXIN FOR THE ACTIVITY AND USE OF AN

专利摘要:
Summary Compositions and Methods for Toxicity Testing The present invention relates to compositions and methods for testing agents (e.g. detection and analysis of clostridium botulinum (ntb) neurotoxin). in particular, the present invention relates to the use of cells derived from induced human pluripotent stem cells (hips) for agent detection and analysis.
公开号:BR112014007717B1
申请号:R112014007717-7
申请日:2012-09-28
公开日:2021-07-20
发明作者:Eric Arthur Johnson；Sabine Pellett；Regina Clare Meyer Whitemarsh；William Howard Tepp
申请人:Cellsnap, Llc；
IPC主号:

专利说明:

[0001] This application claims priority to US Provisional Application No. 61/540,693, filed September 29, 2011, which is incorporated herein by reference in its entirety. FIELD OF THE INVENTION
[0002] The present invention relates to compositions and methods for the test agents (for example, detection and analysis of Clostridium botulinum (NTB) neurotoxin). In particular, the present invention relates to the use of cells derived from induced human pluripotent stem cells (hiPS) for agent detection and analysis. FUNDAMENTALS OF THE INVENTION
[0003] Botulinum neurotoxins (NTBs), synthesized by the soil-dwelling bacterium Clostridium botulinum Gram-positive, are the most toxic substances known to mankind and are the causative agents of the neuroparalytic disease botulism (Johnson E (2005) in Topley and Wilson's microbiology and microbial infections (Topley and Wilson microbiological and microbial infections, ed. SP Borriello, PR Murray, and G. Funke (Hodder Arnold, London, UK), pages 1035-1088.) Seven immunologically distinct serotypes of designated NTBs A to G have been described (Gimenez DF & Gimenez JA (1995) Int. J. Food Microbiol 27: 1-9.) NTBs are initially synthesized as a single chain polypeptide of ~150kDa, but post-translational proteolytic cleavage produces distinct heavy and light chains (CP and CL) of ~100kDa and ~50kDa linked by a disulfide bond.The CP is further functionally divided into CPC and CPN subdomains.The CPC domain is responsible for recognition oe binding to specific neuronal cell surface receptors that lead to endocytosis, while the CPN domain is responsible for channeling formation in the endocytic vesicle membrane and translocation and internalization of the CL across the endosomal membrane (Montecucco et al., (2004) Trends Microbiol 12: 442-446; Fischer A & Montal M (2007) J Biol Chem 282:29604-29611; Fisher A, et al. (2009) Proc Natl Acad Sci USA 106: 1330-1335). During translocation, the disulfide bond is cleaved, and CL is released into the cell cytosol and refolded to the active enzyme component as a zinc-dependent endopeptidase (Fischer et al., supra; Fischer A & M Montal (2007) ) Proc Natl Acad Sci USA 104: 10447-10452). CL then specifically targets and cleaves an intracellular SNARE protein in presynaptic vesicles, which leads to inhibition of neurotransmitter release. Each NTB serotype has a different cleavage target, with NTB/A and E cleaving SNAP-25 at different sites, NTB/B, D, F, and G cleaving VAMP/sinaptobrevin at different sites, and NTB/C both cleaving SNAP -25 and syntaxin (reviewed in Montecucco C & Schiavo, G (1994) Mol Microbiol 13:1-8).
[0004] Naturally occurring botulism is a rare but serious disease, with ~110 cases occurring per year in the United States and a fatality rate of ~5-10% (Johnson EA & Montecucco C (2008) in the Handbook of Clinical Neurology ( Manual of Clinical Neurology), ed. Andrew G. Engel (Elsevier, pages 333-368) Due to its extreme potency (estimated human lethal dose of 1 ng/kg body weight for NTB/A (Bossi P, et al ( 2006) Cell Mol Vida Sci 63:2196-2212)), the severity of the botulism disease and the high cost involved in treating cases, especially on a large scale, NTBs have been classified as a Selection Agent category A and present a serious threat as a weapon of bioterrorism (Arnon SS, et al. (2001) JAMA 285:1059-1070).
[0005] NTB/A, and a much smaller NTB/B range, are also being used as unique and important drugs for the treatment of a variety of neuromuscular and cosmetic disorders. Conditions for which the Food and Drug Administration has approved the use of NTBs include cosmetic treatments and to temporarily alleviate a variety of muscular spasticity, hyperhidrosis, and migraine disorders (Chaddock JA & Acharya K (2011) FEBS J 278:899-904). The cosmetic and clinical applications of NTBs are increasing and new formulations of NTBs for pharmaceutical purposes are being developed requiring clinical trials, determination of exact potency, and research into neutralizing antibodies. For example, NTBs are pharmaceutically administered for the treatment of pain disorders, voluntary muscle strength, focal dystonia, including cervical, cranial dystonia, and benign essential blepharospasm, hemifacial spasm, and focal spasticity, gastrointestinal disorders, hyperhidrosis, and correction of cosmetic wrinkles, blepharospasm, oromandibular dystonia, jaw opening type, jaw closure type, bruxism, Meige syndrome, lingual dystonia, eyelid apraxia, cervical opening dystonia, antecollis, retrocollis, laterocollis, torticollis, pharyngeal dystonia, laryngeal dystonia, spasmodic/adductor dysphonia, spasmodic/abductor dysphonia, spasmodic dyspnea, limb dystonia, arm dystonia, task-specific dystonia, writer's cramp, colic, golfer's cramp, leg dystonia, adduction of thigh, knee flexion of thigh abduction, knee extension, ankle flexion, ankle extension, equinus var o, foot deformity dystonia, striated toe, toe flexion, toe extension, axial dystonia, pisa syndrome, belly dancer dystonia, segmental dystonia, hemidystonia, generalized dystonia, lubag dystonia, dystonia in corticobasal degeneration, lubag dystonia, tardive dystonia, dystonia in spinocerebellar ataxia, dystonia in Parkinson's disease, dystonia in Huntington's disease, dystonia in Hallervorden-Spatz disease, dopa-induced dyskinesia/dopa-induced dystonia, tardive dyskinesias /tarddy dystonia, paroxysmal dyskinesias/dystonias, palatal myoclonus induced by non-kinesiogenic kinesiogenic action, myoclonus myochemia, rigidity, benign muscle cramps, hereditary chin tremor, paradoxical jaw muscle activity, hemimastigatory spasms, hyperetrophic branchial myopathy, hypertrophy anterior, nystagmus, supranuclear gaze palsy oscillopsy, continuous partial epilepsy, planning tion of surgery for spasmodic torticollis, abductor vocal cord paralysis, recalcitrant mutational dysphonia, upper esophageal sphincter dysfunction, vocal fold granuloma, Gilles de la Tourette stuttering syndrome, middle ear myoclonus, protective laryngeal closure, post laryngectomy, speech insufficiency, protective ptosis, entropion, sphincter of Odii dysfunction, pseudoachalasia, nonachalasia, esophageal motor diseases, vaginismus, postoperative immobilization tremor, bladder dysfunction, detrusor-sphincter dyssynergia, bladder sphincter spasm, hemifacial spasm, reinnervation dyskinesias, cosmetic crow's feet, facial asymmetries when frowning, mentalis undulation, rigid person syndrome, tetanus, prostate hyperplasia, adiposity, treatment of infantile cerebral palsy strabismus, concomitant mixed paralytic, after surgery of retinal detachment, after cataract surgery, in myositic strabismus of aphakia , myopathic strabismus, dissociated vertical deviation, as an adjunct to strabismus surgery, esotropia, exotropia, achalasia, anal fissures, hyperactivity of exocrine glands, Frey's syndrome, Crocodile's tear syndrome, hyperhidrosis, plantar rhinorrhea palmar axillary, relative hypersalivation cerebrovascular disease, Parkinson's disease, amyotrophic lateral sclerosis, spastic conditions, autoimmune processes of encephalitis and myelitis, multiple sclerosis, transverse myelitis, Devic syndrome, viral infections, bacterial infections, parasitic infections, fungal infections, post-apoplectic syndrome of hereditary spastic paraparesis, hemispheric infarction, brainstem infarction, myelon infarction, in central nervous system trauma, hemispherical injuries, brainstem injuries, myelon injury, in central nervous system hemorrhage, intracerebral hemorrhage, subarachnoid hemorrhage, subarachnoid hemorrhage, subdural, intraspinal hemorrhage, in neoplasia as, hemispheric tumors, brainstem tumors and myelon tumor. Thus, the quantitative and reliable detection of NTB activity in the environment, in foods, in pharmaceutical preparations, for the detection of antibodies, and in research applications is critical in the prevention of botulism, in the fight against terrorism, as well as the development of new drugs and patient safety and quality control and product assurance testing.
[0006] Many NTB detection methods have been published, and can be divided into four general categories (reviewed in Cai et al., (2007) Crit Rev Microbiol 33: 109-125): 1. in vitro assays that immunologically detect the presence of holotoxin but cannot distinguish between active and inactive states (ELISA), 2. endopeptidase assays that detect the enzymatic activity of CL toxin, but do not distinguish between biologically active holotoxin and CL alone, 3. in vivo assays (bioassay in rats), and lastly 4. in vivo simulation tests such as the diaphragm dome assay, local injection assays and cell-based assays using primary or immortalized cells. In order to detect fully active NTBs, a detection assay must measure all steps of the intoxication process (eg, CP binding to cell surface receptors, endocytosis, vesicle channel formation, disulfide bond cleavage, transduction of CL within the cell cytosol, and finally proteolytic cleavage of SNARE proteins). Only the rat bioassay and the in vivo simulation assays measured all these steps. The rat bioassay involves injecting rats intravenously or intraperitoneally with different dilutions of NTB, then observing rats with symptoms of botulism intoxication (limb paralysis, difficulty breathing, ruffled skin, etc.) (Hatheway CL (1988) in Laboratory diagnosis of infectious diseases: principles and practice (Laboratory diagnosis of infectious diseases: principles and practices), eds. BALOWS A, Hausler WH, Ohashi M & Turano MA (Springer-Verlag, New York), pages 111 -133; Schantz EJaK, DA (1978) Journal of the Association of Official Analytical Chemists (Journal of the Association of Official Analytical Chemists) 61:96-99) and finally death. Although the MBA is quantitative and can monitor all steps of poisoning, it has a large error rate, is not standardized across or within laboratories, requires a large number of animals and corresponding facilities and trained personnel. On-site diaphragm dome and injection tests reduce animal suffering and some are sensitive enough, but still require large numbers of animals and qualified personnel.
[0007] These clearly identified deficiencies of these trials have prompted a recommendation from regulatory agencies, including the FDA and USDA to develop a cell-based model that provides a specific, sensitive and quantitative alternative to the MBA (National Institute of Environmental Health Sciences, 2008). Several continuous cell lines, including neuro-2a and PC-12, have been used for toxicity assays, but they are not sensitive enough to compete with the MBA. Primary neurons derived from rat, or chicken, and neurons derived from embryonic stem cells are significantly more sensitive (Hall YH, et al. (2004) J Immunol Methods 288:55-60; Keller JE, Cai F & Neale EA ( 2004) Biochemistry 43:526-532; Lalli L, et al.(1999) J. Cell Sci. 112 (Pt 16): 2715-2724; Neale et al., (1999), J. Cell Biol 147:1249- 1260; Stahl AM, et al. (2007) J Biomol Screen 12: 370-377). The most sensitive cell type for toxicity testing and antibody detection described is the primary rat spinal cord cell (RSC) assay (Pellett et al., (2007) FEBS Lett 581:4803-4808), which is more sensitive than the MBA, reproducible, and correlates well with the rat bioassay (Pellett et al., (2010) J Pharmacol Toxicol Methods). In addition, embryonic stem cell-derived neurons have also been shown to be highly sensitive (McNutt et al., (2011) Biochem Biophys Res Commun 405:85-90; Pellett S, et al. (2011) Biochem Biophys Res Commun 404 :388-392; Kiris E, et al. (2011) Stem Cell Res). However, the CSR assay still requires the use of some animals and specialized personnel for cell preparation, and is not easily adaptable for standardization tests due to the need to continually prepare new batches of cells. SUMMARY OF THE INVENTION
[0008] The present invention relates to compositions and methods for the test agents (for example, detection and analysis of the neurotoxin of Clostridium botulinum (NTB)). In particular, the present invention relates to the use of cells derived from induced human pluripotent stem cells (hiPS) for agent detection and analysis.
[0009] Embodiments of the present invention provide neuronal cells (e.g., human (e.g., derived from iPS)) for use in research, screening, clinical and therapeutic applications. In some embodiments, the methods are used for the detection and analysis of NTB and neutralizing antibodies to NTB. Examples of embodiments are described here and below. Additional embodiments are described herein and are within the knowledge of one skilled in the art.
[0010] For example, in some embodiments, the present invention provides a method of assaying a clostrial species (e.g., Clostridium botulinum neurotoxin (NTB)) for activity, comprising: a) contacting a cell-derived neuronal cell induced human pluripotent stem (hiPS) with a composition comprising a NT; and b) testing NT for biological activity. In some embodiments, the clostrial species is Clostridium botulinum, Clostridium butyricum, or Clostridium baratii. In some embodiments, NTB encompasses all seven known serotypes, including serotypes A, B, C, D, E, F and G and the known subtypes of each serotype. In some embodiments, NT is recombinant, mutant, or chimeric NT. In some embodiments, the biological activity is cleavage of SNAP-25, VAMP or syntaxin. In some embodiments, the assay is qualitative, while in others it is quantitative. In some embodiments, NT is purified, while in others, it is in a complex, a solution, or a matrix. In some embodiments, NT is recombinant. In some embodiments, NT is conjugated to another molecule selected from therapeutic modalities, markers, imaging agents, enzymes, receptors, antibodies, or bioactive compounds. In some embodiments, the method further comprises the step of contacting NT with a test compound prior to contacting the hPS-derived neuronal cells. In some embodiments, the test compound is an antibody (e.g., a neutralizing antibody) or small molecular inhibitors of NT. In some embodiments, the neutralizing antibody is a purified sample or in a serum or antitoxin.
[0011] The present invention further provides a method of testing a closdrial species neurotoxin (e.g. NTB) for activity, comprising: a) contacting induced human pluripotent stem cell (hiPS) derived neuronal cells with a composition comprising a) an NT, and b) a neutralizing antibody; and b) testing NT for biological activity.
[0012] The present invention also relates to a method for determining the amount of biologically active NTB in a preparation comprising biologically active NTB and preferably a pharmaceutical preparation comprising biologically active NTB. In some embodiments, the method comprises the steps of: (a) contacting a neuronal cell derived from hiPS cells with a sample of a preparation comprising biologically active NTB; and (b) determining the amount of biologically active NTB present in the preparation by testing the sample for the biological activity of NTB.
[0013] In other embodiments, the present invention provides the use of induced human pluripotent stem cell (hiPS) derived neuronal cells to test NTB for activity.
[0014] Additional embodiments are described herein. DESCRIPTION OF THE FIGURES
[0015] Figure 1 shows NTB receptor expression in iPS neurons A. iPS cells were matured for 4, 7, 10, 14 and 21 days and analyzed by Western blot for NTB receptor expression levels. B. iPS cells were matured for 5, 10, 15 and 20 days and adult human brain cells were used to perform the quantitative PCR assays.
[0016] Figure 2 shows a comparison of the NTB/A1 sensitivity of iPS neurons seeded on seven different substrates (indicated on the right side).
[0017] Figure 3 shows the sensitivity of NTB/A1 of iPS neurons and RSC cells.
[0018] Figure 4 shows the time dependence on detecting NTB/A1 activities in iPS neurons.
[0019] Figure 5 shows the NTB/A1 uptake rate of iPS neurons compared to RSC cells.
[0020] Figure 6 shows the activity-dependent uptake of NTB/A1 by neurons. A. iPS neurons and RSC cells were exposed to 55 or 275 U of NTB/A1 in cell stimulation medium for 1, 5, 10 and 15 minutes, followed by removal of toxins and 24 h of incubation. B. iPS neurons were exposed to 55 U of NTB/A1 in both neuronal and cellular stimulation medium for 1, 5 and 10 min. C. Neurons were exposed to 1.7-55 U of NTB/A1 for 5 min in cell stimulation medium, washed twice with neuronal medium, and incubated for 24 h.
[0021] Figure 7 shows Western blot and densitometry data from antibody protection assay in iPS neurons. A. iPS neurons were exposed to 1.5 U of toxin and antibody for 24 h. B. iPS neurons were exposed to 55 U of toxin-antibody mixture in cell stimulation medium for 5 minutes, the mixture was removed, cells were washed twice and incubated for 24 h.
[0022] Figure 8 shows the sensitivity of iPS neurons to NTB/A complex and purified NTB/A A. SDS-PAGE on gel comparing purified NTB/A1 and NTB/A1 complex (Invitrogen ladder: Pre-stained pattern SeeBlue® Plus2). B. Sensitivity of iPS neurons to purified NTB/A1 toxin and NTB/A1 complex 48 h after toxin exposure.
[0023] Figure 9 shows the detection of NTB/B, C and E activity in iPS neurons. iPS neurons matured for 7 days and RSC cells were exposed to serial dilutions of NTB/B (A), /C (B) and /E (C) for 48 h in parallel. DEFINITIONS
[0024] To facilitate an understanding of the present invention, a series of terms and phrases are defined below:
As used herein, the term "host cell" refers to any eukaryotic or prokaryotic cell (e.g. mammalian cells, avian cells, amphibian cells, plant cells, fish cells and insect cells) , both located in vitro and in vivo.
As used herein, the term "cell culture" refers to any in vitro cell culture. Included within the scope of this term are continuous cell lines (eg, with an immortal phenotype), primary cell cultures, finite cell lines (eg, untransformed cells), and any other population of cells maintained in vitro, including oocytes and embryos.
[0027] As used herein, the term "toxic" refers to any harmful or detrimental effects on a cell or tissue as compared to the same cell or tissue prior to administration of the toxic agent.
As used herein, the term "pharmaceutical composition" refers to the combination of an active agent with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
As used herein, the term "pharmaceutically acceptable carrier" refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil/water emulsion or water/oil), and various types of wetting agents. The compositions can also include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants. (See, for example, Martin, Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Science), 15thEd., Mack Publ. Co., Easton, PA [1975]).
As used herein, the terms "detecting", "detecting" or "detecting" can describe either the general act of discovery or discernment or the specific observation of a detectably labeled composition.
[0031] As used herein, the term "purified" or "purify" refers to the removal of components (eg, contaminants) from a sample. For example, antibodies are purified by removing contaminating non-immunoglobulin proteins; they are also purified by removing immunoglobulin, which does not bind to the target molecule. Removal of non-immunoglobulin proteins and/or removal of immunoglobulins that do not bind to the target molecule results in an increase in the percentage of reactive target immunoglobulins in the sample. In another example, recombinant polypeptides are expressed in bacterial host cells and the polypeptides are purified by removing proteins from the host cells; the percentage of recombinant polypeptides is thus increased in the sample.
As used herein, the term "sample" is used in its broadest sense. In a sense, it is intended to include a sample or culture obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from animals (including humans), and include fluids, solids, tissues, and gases. Biological samples include cells, tissues, blood products such as plasma, serum and the like. These examples are not, however, to be interpreted as limiting the types of samples applicable to the present invention. In some embodiments, the sample may also be a sample of a preparation comprising biologically active NTB, such as an NTB preparation to be applied for pharmaceutical or cosmetic purposes. In addition, in one aspect of disclosure, the sample may also be an environmental sample or a food sample suspected of containing NTBs. DETAILED DESCRIPTION OF THE INVENTION
[0033] The present invention relates to compositions and methods for test agents (e.g. detection and analysis of the neurotoxin of Clostridium botulinum (NTB)). In particular, the present invention relates to the use of cells derived from induced human pluripotent stem cells (hiPS) for agent detection and analysis.
[0034] Embodiments of the present invention provide systems and methods for detecting and analyzing NTB. In some embodiments, the systems and assays utilize human iPS-derived neurons as a highly sensitive and reproducible platform for detecting botulinum neurotoxin (NTB). In some embodiments, the neurons are a 98% pure panneuronal population of GABAergic, dopaminergic, and glutamatergic neurons and are produced and cryopreserved as differentiated cells. In another aspect, neurons are an essentially pure panneuronal population of GABAergic, dopaminergic, and glutamatergic neurons and are produced and cryopreserved as differentiated cells, where the cells are at least 70%, at least 80%, at least 90%, at least 95% or at least 96% pure with respect to neuronal cells. Experiments performed during the course of developing embodiments of the present invention demonstrated that cells express all receptors and substrates necessary for NTB intoxication by all NTB serotypes. NTB detection assays have demonstrated that iPS-derived neurons are highly sensitive to quantitative detection of NTB/A, B, C and E and neutralizing antibodies.
[0035] In November 2007, two independent groups showed for the first time that human fibroblast cells can be reprogrammed into pluripotent stem cells simply by activating a small set of silenced genes (Takahashi K, et al (2007) Cell 131 :861-872; Yu J, et al (2007) Science 318:1917-1920). These cells have been called induced pluripotent stem cells and can be maintained and cryopreserved similar to cell lines. This discovery opens the opportunity for the development of a large number of human iPS-derived cell models that fully functionally resemble differentiated human somatic cells and do not require any use of animals.
[0036] In selecting iPS cells for use in the systems and methods described herein, it is preferred that the cells can be reliably and reproducibly produced and generate pure populations of differentiated cells in sufficient quantities for such studies. In some embodiments, these cells are available from Cellular Dynamics Inc. (Madison, WI).
[0037] Experiments conducted during the course of development of embodiments of the present invention demonstrated that human iPS-derived neurons are highly sensitive, selective, and species-specific cell model for the detection of botulinum neurotoxins, neutralizing antibodies, and inhibitors , and for NTB cell entry and trafficking studies. These neurons are suitable for replacing MBA power determination with NTB, as well as for antibody detection, inhibitor scanning, and research applications. I. Cells
As described herein, embodiments of the present invention provide pluripotent stem cell derivatives for use in detecting and analyzing agents such as NTB. In some embodiments, the cells are human (e.g., neuronal cells derived from induced human pluripotent stem cells (hiPS) or derived from human embryonic stem cells). Methods of generating iPS cells are described, for example, in Yu et al., Science. May 8, 2009; 324 (5928):797-801. Epub 2009, WO2011056971 and WO2011025852, each of which is incorporated herein by reference in its entirety. In some embodiments, iPS cells are differentiated into neurons using suitable methods (for example, those described in US Patent Applications US2010/0279403 and US2010/0216181, each of which is incorporated herein by reference in its entirety).
[0039] In some embodiments, neurons are a 98% pure panneuronal population of GABAergic, dopaminergic, and glutamatergic neurons and are produced and cryopreserved as differentiated cells. In some embodiments, commercially available neuronally derived iPS cells (for example, those available from Cellular Dynamics Inc. (Madison, WI) or GlobalStem, (Rockville, MD)) are used, although other sources may be used. In some embodiments, the cells are neuronal hiPS cells.
[0040] In another aspect, cells are neurons are an essentially pure pan-neuronal population of GABAergic, dopaminergic, and glutamatergic neurons and are produced and cryopreserved as differentiated cells, in which the cells are at least 70%, at least 80%, at least 90%, at least 95% or at least 96% pure with respect to neuronal cells.
[0041] The present invention is not limited to the cells described herein. Additional cell lines and primary cell cultures can be used. For example, in some embodiments, cholinergic neurons are used.
In some embodiments, suitable cell lines express receptors and substrates necessary or sufficient for NTB intoxication.
[0043] In some embodiments, the present invention provides systems and kits that include the cell lines described herein, along with the necessary, sufficient, or useful components to effect NTB detection and analysis. For example, in some embodiments, kits comprise cells and cell culture reagents (e.g., plates, buffers, etc.), assay reagents, controls (NTB positive and negative and/or inhibitor controls) and instructions to perform and analyze the tests.
[0044] In one aspect of the invention, the hiPS cell-derived neuronal cell is obtained or has been obtained by differentiation and/or maturation of a hiPS cell generated by a process as noted above for a neuronal cell. This differentiation of neuronal cells, in one aspect, can be achieved by culturing the hiPS cells at about 37°C and under about 5% CO2. In one aspect, the medium for the culture can be Neurobasal medium supplemented with B27 and glutamax (Invitrogen, Inc., USA). In yet another aspect, the cells are cultured on poly-lysine coated plates and, in yet another aspect, the plates are further coated with matrigel (BD Bioscience, USA). In one aspect, a 96-well plate is used for cultivation where cells are grown at a density of about 40,000 cells per well. In one aspect, cells can seed for about 24 hours. Later, the cells are allowed in one aspect to mature for about 2 to about 28 days, in one aspect to about 4 to about 14, or in one aspect to about 4 to about 7 days.
In one aspect, said neuronal cells derived from hiPS cells are obtained by a process of differentiation and maturation essentially as described in the appended examples, below.
[0046] Embodiments of the present invention also relate to a neuronal cell derived from a hiPS cell obtained by the above-mentioned differentiation and maturation process.
[0047] In one aspect, such neuronal cells derived from hiPS cells is characterized by the presence of one or more and, in one aspect of all, of the following markers: β3-tubulin, NeuN, vGAT, vGLUT2, NSE-specific neuronal enolase) or Tbrl (T domain transcription factor 1). For β3-tubulin or NeuN the number of positive cells in a neuronal cell culture derived from hiPS cells of the invention is predicted to be about 99%. For vGAT or vGLUT2 it is predicted, in one aspect, that at least a part of the cultured cells are positive for said markers.
[0048] In one aspect, such a neuronal cell derived from hiPS cells is characterized by the absence of one or more and, in one aspect of all, of the following markers: GFAP, TH, NNE (non-neuronal enolase), Tbr2 ( T domain transcription factor 2), or NoGo a,C. For GFAP, the number of positive cells in a culture of neuronal cells derived from hiPS cells is predicted to be significantly low and, in one aspect, below about 5% or even below about 1% for the remaining markers. it is predicted, in one respect, that they are, at most, present below a detectable amount.
[0049] The presence or absence or amount of the aforementioned markers can, in one aspect, be determined by conventional immunological techniques. For example, markers can be determined by immunohistological staining techniques, Western blot analysis of lysed cells or, as long as β3-tubulin or NeuN are concerned, via FACS analysis. Other detection techniques are described (see, for example, US2012/0178083, US2008/0280301, Englund, 2005, J Neurosci 25:247-251; Dupuis 2002, Neurobiology of Diseases 10:358-365, each of which is herein incorporated by reference).
[0050] In another aspect, the neuronal cell derived from hiPS cells is characterized by at least one and, in one aspect more, of the following electrophysiological properties: inhibition of Na2+ channels by tertrodotoxin (TTX), inhibition of channels of K+ by tetraethylammonium, inhibition of L-type Ca2+ channels by nifedipine, inhibition of P/Q-type Ca2+ channels by agatoxin IVA or inhibition of N-type Ca2+ channels by w-conotoxin GVIA. Such electrophysiological properties can be tested by means of standard electrophysiological measurements including, for example, patch-clamp measurements, before and after treating the cells with the respective inhibitors.
[0051] In another aspect, the neuronal cell derived from hiPS cells is characterized by sensitivity to NTB and, in one aspect, to NTB/A. Furthermore, cells, in one aspect, are also sensitive to other neurotoxic compounds in a dose-dependent manner and, in particular, to at least one or all of the following compounds: staurosporine, ATP competitive kinase inhibitor, chlorpromazoin or phenothiazine . Sensitivity towards the aforementioned compounds can be determined in, for example, cell viability assays.
[0052] In one aspect neuronal cells derived from hiPS cells are also sensitive with respect to neurite proliferation for at least one and in one aspect all of the following compounds: antimycin A, mitomycin C, MK571, PD98092 or staurosporine .
[0053] Furthermore, in one aspect, neuronal cells derived from hiPS cells are sensitive to potential loss of mitochondrial membrane for at least one or, in one aspect, all of the following compounds: antimycin A or valinomycin. II. Tests and Uses
[0054] Embodiments of the present invention provide compositions and methods for assaying NTB. Assays can be used in research, clinical, diagnostic and therapeutic applications.
In some embodiments, assays use pluripotent cells (e.g., hiPS-derived neuronal cells). The use of human cells offers the advantage of a species-specific model. Furthermore, neurons derived from pluripotent cells are representative of normal, healthy neurons, as opposed to neurons derived from cancer cell lines or modified cell lines, which cannot be the reflex of somatic neurons.
In some embodiments, assays use neuronal cells, e.g., hiPS-derived neuronal cells to control NTB potency. In other embodiments, NTB toxicity control assays. In yet other embodiments, control assays to detect the presence of or properties of neutralizing antibodies to NTB or other biopharmaceuticals for activity. In some embodiments, assays are quantitative, while in others they are qualitative.
[0057] In some embodiments, cells are first cultured in a suitable matrix. In some embodiments, cells are cultured in order to achieve maturation of neurons. Pluripotent cells used in embodiments of the present invention provide the advantage of faster maturation than cells used in existing assays. In some embodiments, cells are then exposed to toxin (e.g., NTB) for an appropriate period of time. After exposure to a toxin, the desired parameters (eg EC50) are calculated using appropriate methods. The assays described herein are suitable for detecting both purified NTB and NTB in complexes (eg, a complex with other proteins, as found in native configurations and some pharmaceutical preparations). The assays described herein are suitable for detecting any number of NTB serotypes (e.g., NTB/A, B, C, D, E, F and G) or variants or chimeras thereof. In some embodiments, NTBs are expressed recombinantly. In other embodiments, they are purified from bacterial cells.
[0058] In some embodiments, antibody protection assays are performed to test for neutralizing antibodies. Although NTBs are used effectively in treating a large number of patients with different conditions (reviewed in Dhaked et al., Indian J. Med. Res. 132:489-503), some will develop neutralizing antibodies, which will prevent the success of other treatments. For example, it is estimated that in the treatment of cervical dystonias that ~5% of treated patients will develop neutralizing NTB antibodies that prevent further treatment (Kessler et al., (1999) J Neurol 246:265-274). Currently, patients are not monitored throughout their treatments for the development of neutralizing antibodies because a highly sensitive and quantitative assay is not commercially available (Sesardic et al., (2004), Mov Disord 19 Suppl 8:S85-91) . The testing platform presented here using iPS neurons provides sensitive and quantitative detection of neutralizing NTB antibodies in the serum of patients who have received repeated therapeutic or cosmetic injections of NTB. Neutralizing antibodies can be detected in any number of sample types (eg purified antibodies, serum, antitoxins, etc.).
[0059] In some embodiments, small molecule inhibitors of NTBs are tested (e.g., for drug research or control). For example, in some embodiments, cells are either exposed to NTB first, then inhibitor, co-exposed to both, or cells are exposed to inhibitor first, then NTB.
[0060] Any number of suitable endpoint measurements can be used to test NTB activity. Examples include, but are not limited to, Western blot, neurotransmitter release, ELISA (Nuss JE, et al. (2010) J Biomol Screen 15:42-51) or intracellularly expressed reporters such as, for example, FRET sensors ( Dong et al. (2004) Proc. Natl. Acad. Sci. USA 101:14701-14706).
[0061] In some embodiments, the assays described herein find use in quality control assays during the production of NTBs or their derivatives as pharmaceuticals or for research purposes, in diagnostic evaluation, optimization, and treatment dosing clinical and in the selection of the therapeutic modality.
[0062] Additional applications of the assays described herein include, but are not limited to, inhibitor detection and diagnostic, clinical, control and research uses.
[0063] In some embodiments, the present invention provides a method for assaying a Clostridium botulinum (NTB) neurotoxin for activity, comprising: a) contacting a hiPS-derived neuronal cell with a composition comprising an NTB substrate ; and b) testing the NTB for biological activity.
[0064] In one aspect of said method, the test may comprise determining the presence or absence of biological activity of NTB. Such a test may sometimes also be referred to herein as a qualitative test. It should be understood that, based on the presence or absence of biological activity of NTB, one can conclude on the presence or absence of biologically active NTB in a composition comprising, or suspected of containing, such biologically active NTB. Furthermore, in yet another aspect, the assay may involve determining the amount of biologically active NTB in a composition comprising biologically active NTB. It should be understood that the amount of biologically active NTB can be derived from the amount of biological activity tested for said NTB in the composition. Such an assay may sometimes also be referred to as a quantitative assay herein.
[0065] In one aspect of the method of the present invention, NTB is a neurotoxin selected from different serotype groups of Clostridium neurotoxins, for example, is selected from, for example, NTB/A, NTB/B , NTB/C1, NTB/D, NTB/E, NTB/F, or NTB/G. Furthermore, in one aspect, tetanus toxin (TeNT) can be used as a neurotoxin in the methods according to the present invention.
[0066] The bacteria Clostridium botulinum and Clostridium tetani naturally produce these highly potent neurotoxins, for example, botulinum toxins (NTBs) and tetanus toxin (TeNT), respectively. These neurotoxins specifically bind to neuronal cells and disrupt neurotransmitter release. Each toxin is synthesized as an inactive, single-chain protein of approximately 150 kDa. Post-translational processing involves the formation of disulfide bridges and limited proteolysis (nicking) by the bacterial protease(s). Active double-chain neurotoxin consists of two chains, an N-terminal light chain of approximately 50 kDa and a heavy chain of approximately 100 kDa, linked by a disulfide bridge. Neurotoxins structurally comprise three domains, for example the catalytic light chain, the heavy chain comprising the translocation domain (N-terminal half) and the receptor binding domain (C-terminal half), see for example Krieglstein 1990, Eur J Biochem 188, 39; Krieglstein 1991, Eur J Biochem 202, 41; Krieglstein 1994, J. Protein Chem 13, 49. The structures of NTB polypeptides and TeNT polypeptides have been described in the aforementioned references.
[0067] The seven antigenically distinct serotypes of NTBs and TeNT are Zn2+ endoproteases that block synaptic exocytosis by cleaving SNARE proteins. Neurotoxins cause flaccid muscle paralysis seen in botulism and tetanus disorders, see Fischer 2007, Proc Natl. Academic Sci. USA 104, 10447.
[0068] In yet another aspect, the activity of a modified NTB or TeNT can be tested in the method of the invention. Such modified NTB can be derived from said NTB/A, NTB/B, NTB/C1, NTB/D, NTB/E, NTB/F, or NTB/G of TeNT by introducing at least one substitution, addition and /or deletion in the NTB or TeNT amino acid sequence. Such modification of NTB or TeNT, then, can have an amino acid sequence being 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 any of the NTBs or TeNT referred to above. The term "identical" as used herein refers to sequence identity, characterized by determining the number of identical amino acids between two amino acid sequences, wherein the sequences are aligned so that the highest order combination is obtained. It can be calculated using published techniques or methods encoded in computer programs such as, for example, BLASTP and FASTA (Altschul 1990, J Mol Biol 215, 403). Percent identity values are, in one aspect, calculated over the complete amino acid sequence. A number of programs based on a variety of algorithms are available for the skilled worker to compare different sequences. In this context, the algorithms of Needleman and Wunsch or Smith and Waterman give particularly reliable results. To perform sequence alignments, the PileUp program (Higgins 1989, CABIOS 5, 151) or the Gap and BestFit programs (Needleman 1970, J Mol Biol 48; 443; Smith 1981, Adv. Appl Math 2, 482), which do part of the GCG software package (Genetics Computer Group 1991, 575 Science Drive, Madison, Wisconsin, USA 53711) may be used. The sequence identity values recited above in percent (%) are determined, in another aspect of the invention, using the GAP program over the entire sequence region with the following definitions: Gap Weight: 50, Length Weight: 3, Combination Average: 10,000 and Average Mismatch: 0.000, which, unless otherwise noted, will always be used as default settings for sequence alignments.
[0069] In one aspect, each of the aforementioned modified NTB or TeNT polypeptides retain one or more and, in another aspect, all the biological properties of the respective unmodified polypeptide, i.e. the NTB/A, NTB/B, NTB /C1, NTB/D, NTB/E, NTB/F, NTB/G or TeNT. Those skilled in the art will appreciate that full biological activity is maintained after proteolytic activation, although it is conceivable that the unprocessed precursor may exert some biological functions or be partially active. "Biological properties" as used herein refers to: (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane into the cytosol, and/or (d) endoproteolytic cleavage of involved proteins in synaptic vesicle membrane fusion. In vivo assays to assess biological activity include the rat LD50 assay and the ex vivo rat hemidiaphragm assay, as described by, for example, Dressler et al. (Dressler 2005 Mov Disord 20:1617-1619, Keller 2006 Neuroscience 139:629-637). Biological activity is usually expressed in rat units (MU). As used herein, 1 MU is the amount of neurotoxic component, which kills 50% of a specific mouse population after intraperitoneal injection, i.e., i.p. LD50 of mouse. In another aspect, the modified polypeptides may have improved or modified biological properties, for example, they may comprise cleavage sites that are improved for enzyme recognition or may be improved for binding to the receptor or any other property specified above.
[0070] In one aspect, modified NTBs or TeNT can be tested for one or more and, in one aspect, all of the above biological activities by the method described herein.
[0071] In one aspect of the invention, a modified NTB or TeNT is selected from, for example, hybrid NTB or NTB/TeNT, redirected NTBs, redirected TeNT, and chimeric NTBs or TeNT. Modified NTBs and TeNT are described.
[0072] In one aspect of the method of the invention, contacting comprises bringing at least two different components into physical proximity, such as to allow physical and/or chemical interaction of said components. In the above-mentioned method, the hiPS-derived neuronal cell is contacted with a composition that comprises or is suspected of containing biologically active NTB. The contact is carried out for a time and under conditions sufficient to allow the biologically active NTB comprised in the composition to exert its biological activity on the neuronal cell derived from hiPS cells. In one aspect, therefore, contact should allow for (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane to the cytosol, and/or (d) endoproteolytic cleavage of proteins involved in membrane fusion of the synaptic vesicle or substrates mimicking said process in the neuronal cell derived from hiPS cells. The person skilled in the art is well aware that the conditions must be applied for a particular culture of neuronal cells derived from hiPS cells. Contacting can, in one aspect, be carried out in a cell culture system in which neuronal cell derived from hiPS cells are cultured in well plates in a suitable culture medium and under suitable culture conditions, adding to the culture medium from a sample of the composition to be tested for NTB activity by the methods described herein.
[0073] In one aspect, suitable growing conditions include growing at about 37°C and under about 5% CO2. In one aspect, the Neurobasal culture medium is supplemented with B27 and glutamax (Invitrogen, Inc., USA). In yet another aspect, neuronal cells derived from hiPS cells are cultured on poly-lysine coated plates and in yet another aspect plates are used which are further coated with matrigel (BD Bioscience, USA). In one aspect, a 96-well plate is used for cultivation in which cells are grown at a density of about 10,000 to about 100,000 cells, in an additional aspect about 20,000 to about 60,000 cells, and still in one aspect about 40,000 cells per well. In one aspect, cells can propagate for about 24 hours. In one aspect, below, the cells are allowed to mature for about 2 to about 28 days, in one aspect, to about 4 to about 14, or, in one aspect, to about 4 to about 7 days before of the contact being made.
[0074] In yet one aspect, said contact is performed as described in the attached Examples, below.
[0075] In one aspect of the method of the present invention, the composition comprising biologically active NTB is a composition known to comprise biologically active NTB or a composition suspected of comprising biologically active NTB. The composition may include other ingredients in addition to said biologically active NTB, such as, for example, a solvent and/or agents such as proteins and suitable stabilizers, in one aspect, the complexing proteins of NTBs (HA70, HA17, HA33 , or NTNH (NBP)), or other protein stabilizers. The composition may also further comprise proteins that facilitate the biological activity of the NTB, for example, by enhancing any of the biological activities of the NTBs referred to elsewhere herein. In yet one aspect, the composition may comprise more than one NTB.
[0076] In one aspect, the composition is a lysate of botulinum cells or other bacterial cells or non-bacterial cells comprising biologically active NTB. In one aspect, such a composition is also an NTB preparation obtained from such a cell lysate by partial purification, for example a crude extract, or NTB purification, for example, a purified NTB preparation. In another aspect, the composition is an artificial composition comprising the mixed components. In yet one aspect, the composition is a preparation to be used as a pharmaceutical composition as defined elsewhere herein.
[0077] In one aspect of the method of the present invention, the NTB assay for biological activity is performed by determining the endoproteolytic cleavage of proteins involved in synaptic vesicle membrane fusion or other substrates being cleaved by biologically active NTB, if present , in the neuronal cell derived from hiPS cells.
In some embodiments, proteins involved in synaptic vesicle membrane fusion or other substrates have a cleavage site recognized by the neurotoxin NTB or TeNT being tested. A neurotoxin cleavage site, as used herein, refers to the cleavage site that is recognized and cleaved by the endogenous protease of a neurotoxin polypeptide. Cleavage sites that are recognized by neurotoxin proteases are described (see, for example, EP 1 926 744 B1; incorporated herein by reference in its entirety). In principle, a neurotoxin cleavage site can be a cleavage site that occurs naturally on a substrate or that is an artificially designed cleavage site recognized and cleaved by the neurotoxin polypeptide protease.
A neurotoxin cleavage site recognized and cleaved by the NTB/A protease, in one aspect of the invention, is derived from a protein that is sensitive to cleavage by NTB/A. In one aspect, such a protein is human SNAP25A or B, or a paralogous homologue, or ortholog thereof from rat, mouse, bovine, Danio, Carassius, Xenopus, Torpedo, Strongylocentrotus, Loligo, Lymnaea or Aplysia. Suitable cleavage sites derived from said proteins are disclosed in EP 1 926 744 B1.
A neurotoxin cleavage site recognized and cleaved by the NTB/B protease, in one aspect of the invention, is derived from a protein that is sensitive to cleavage by NTB/B. In one aspect, such a protein is human or rat VAMP-1, VAMP-2 and VAMP-3/cellubrevin, bovine VAMP-2, rat VAMP-2 or VAMP-3, VAMP-1, VAMP-2 or Chicken VAMP-3, Torpedo VAMP-1, Strongylocentrotus VAMP, sybA, synB, synC, synD, or Drosophila syn, Hirudo VAMP, Xenopus VAMP-2 or VAMP-3, VAMP-1 or VAMP-2 from Danio, VAMP from Loligo, VAMP from Lymnaea, VAMP from Aplysia or Caenorhabditis of the SNB1 type or any ortholog, paralog or homologue thereof. Suitable cleavage sites derived from said proteins are disclosed, for example, in EP 1926744 B1.
A neurotoxin cleavage site recognized and cleaved by the NTB/C1 protease in one aspect of the invention is derived from a protein that is sensitive to cleavage by NTB/C1. In one aspect, such a protein is Syntaxin 1A, Syntaxin 1B1, Syntaxin 2-1, Syntaxin 2-2, Syntaxin 2-3, Syntaxin 3A or Syntaxin 1B2 from human and rat, Syntaxin 1A, Syntaxin 1B1 or Syntaxin 1B2 from bovine or rat , Rat Syntaxin 2 or Rat Syntaxin 3, Syntaxin 1A, Syntaxin 1B1, Syntaxin1B2, Syntaxin 2, Syntaxin 3A, Syntaxin 3B or 3C Rat Syntaxin 1A or Chicken Syntaxin 2; Syntaxin 1A or Syntaxin 1B from Xenopus, Syntaxin 1A, Syntaxin 1B or Syntaxin 3 from Danio, Syntaxin 1A or Syntaxin 1B from Torpedo, Syntaxin 1A or Syntaxin 1B from Strongylocentrotus, Syntaxin 1A or Syntaxin 1B from Drosophila, Syntaxin 1B or Hirudo, Syntaxin 1A or Syntaxin 1B from Loligo, Syntaxin 1A or Syntaxin 1B from Lymnaea or any ortholog, homologue paralog thereof. Suitable protein-derived cleavage sites are disclosed, for example, in EP 1926744 B1.
A neurotoxin cleavage site recognized and cleaved by the NTB/D protease, in one aspect of the invention, is derived from a protein that is sensitive to cleavage by NTB/D. In one aspect, such a protein is human or rat VAMP-1, VAMP-2 and VAMP-3/cellubrevin, bovine VAMP-2, rat VAMP-2 or VAMP-3, VAMP-1, VAMP-2 or Chicken VAMP-3, Torpedo VAMP-1, Strongylocentrotus VAMP, synA, synB, synC, synD, or Drosophila syn, Hirudo VAMP, Xenopus VAMP-2 or VAMP-3, VAMP-1 or VAMP-2 from Danio, VAMP from Loligo, VAMP from Lymnaea, VAMP from Aplysia or Caenorhabditis of the SNB1 type or any ortholog, paralog or homologue thereof. Suitable cleavage sites derived from the proteins are disclosed, for example, in EP 1 926 744 B1.
A neurotoxin cleavage site recognized and cleaved by the NTB/E protease, in one aspect of the invention, is derived from a protein that is sensitive to cleavage by NTB/E. In one aspect, such a protein is, such a protein is human SNAP-25A or B, or a rat, mouse, bovine, Danio, Carassius, Xenopus, Torpedo, Strongylocentrotus, Loligo, Lymnaea, or a homologue, ortholog or paralog thereof, or Aplysia. Suitable cleavage sites derived from the proteins are disclosed, for example, in EP 1 926 744 B1.
A neurotoxin cleavage site recognized and cleaved by the NTB/F protease, in one aspect of the invention, is derived from a protein that is sensitive to cleavage by NTB/F. In one aspect, such a protein is, such a protein is human or rat VAMP-1, VAMP-2 and VAMP-3/cellubrevin, bovine VAMP-2, rat VAMP-2 or VAMP-3, VAMP- 1, Chicken VAMP-2 or VAMP-3, Torpedo VAMP-1, Strongylocentrotus VAMP, synA, synB, synC, synD, or Drosophila syn, Hirudo VAMP, Xenopus VAMP-2 or VAMP-3, VAMP -1 or Danio VAMP-2, Loligo VAMP, Lymnaea VAMP, Aplysia or Caenorhabditis VAMP of the SNB1 type or any ortholog, paralog or homologue thereof. Suitable cleavage sites derived from the proteins are disclosed, for example, in EP 1 926 744 B1.
A neurotoxin cleavage site recognized and cleaved by the NTB/G protease, in one aspect of the invention, is derived from a protein that is sensitive to cleavage by NTB/G. In one aspect, such a protein is, such a protein is human or rat VAMP-1, VAMP-2 and VAMP-3/cellubrevin, bovine VAMP-2, rat VAMP-2 or VAMP-3, VAMP-1 , chicken VAMP-2 or VAMP-3, Torpedo VAMP-1, Strongylocentrotus VAMP, synA, synB, synC, synD, or Drosophila syn, Hirudo VAMP, Xenopus VAMP-2 or VAMP-3, VAMP- 1 or Danio VAMP-2, Loligo VAMP, Lymnaea VAMP, Aplysia or Caenorhabditis VAMP of the SNB1 type or any ortholog, paralog or a homolog thereof. Suitable cleavage sites derived from the disclosed proteins are, for example, in EP 1 926 744 B1.
A neurotoxin cleavage site recognized and cleaved by the TeNT protease, in one aspect of the invention, is derived from a protein that is sensitive to cleavage by TeNT. In one aspect, such a protein is human or rat VAMP-1, VAMP-2 and VAMP-3/cellubrevin, bovine VAMP-2, rat VAMP-2 or VAMP-3, VAMP-1, VAMP-2 or Chicken VAMP-3, Torpedo VAMP-1, Strongylocentrotus VAMP, synA, synB, synC, synD, or Drosophila syn, Hirudo VAMP, Xenopus VAMP-2 or VAMP-3, VAMP-1 or VAMP-2 from Danio, VAMP from Loligo, VAMP from Lymnaea, VAMP from Aplysia or Caenorhabditis of the SNB1 type or any ortholog, paralog or homologue thereof. Suitable cleavage sites derived from the proteins are disclosed, for example, in EP 1 926 744 B1.
A neurotoxin cleavage site recognized and cleaved by NTB proteases, in another aspect of the invention, is obtained from the autocatalytic cleavage sites found in NTB proteins. In aspects, a neurotoxin cleavage site to be used in accordance with the present invention and which is derived from the autocatalytic cleavage site of a given NTB or TeNT comprises at least 6, at least 8, at least 10 or, at least 15 consecutive residues including NTB/D residues 250Tyr-251Tyr, NTB/B residues 256Phe-257Phe, NTB/C1 residues 257Phe-258Tyr, NTB/D residues 257Phe-258Phe, residues of NTB/E 239Pro-240Leu, NTB/F residues 254Pro-255Leu, NTB/G residues 256Phe-257Phe, TeNT residues 259Ile-260Tyr, NTB/A residues Phe266-Gly267, NTB/ residues B Phe272-Gly273, NTB/C1 residues Phe273-Gly274, NTB/D residues Phe273-Gly274, NTB/E residues Phe255-Gly256, NTB/F residues Phe270-Gly271, NTB/ residues G Phe272-Gly273 or TeNT residues Phe275-Gly276. Suitable cleavage sites derived from the proteins are disclosed, for example, in EP 1 926 744 B1.
[0088] In one aspect of the present invention, cleavage of the cleavage sites for the aforementioned neurotoxin for NTBs and TeNT can be tested by determining one or more cleavage products obtained by cleavage of the aforementioned proteins. Protein-derived products can be determined by means of antibodies that specifically bind to said cleaved products, but not to the uncleaved proteins. The binding of such antibodies which specifically bind to the products can be determined by techniques described herein or elsewhere. For example, antibodies that specifically bind can be covalently or non-covalently bound to a detectable label. Such detectable label may be a detectable moiety covalently linked to the antibody that specifically binds, or may be a detecting agent, such as a detecting antibody or aptamere that specifically binds to the binding antibody specifically and allows detection, for example, through a detectable moiety covalently attached thereto. Various types of such immunoassays can be used in this way to determine cleaved products and thus to test the biological activity of an NTB or TeNT.
In one aspect, the cleavage of proteins having a neurotoxin cleavage site as defined herein can be determined by Western B1ot. In another aspect, said cleavage may be determined by means of ELISA, RIA or other immunological assay formats, including those mentioned elsewhere herein.
[0090] In another aspect, an artificial substrate can be used, which comprises a neurotoxin cleavage site as specified above and which, after cleavage at said site is changed in at least one physical and/or chemical property . For example, substrates envisioned in such an aspect may comprise a first and a second portion capable of physically and/or chemically interacting with each other and separated by a linker having the neurotoxin cleavage site. As a result of the cleavage of the cleavage site, the aforementioned interaction between the two halves will change. Suitable radicals are, for example, donor and acceptor fluorophores which exhibit resonant energy transfer, in the uncleaved state, said resonant energy transfer being interrupted after cleavage. Alternatively, a fluorophore and a quencher can be applied, wherein the quenching effect of the quencher is reversed after cleavage. Substrates of said type are described, for example, in any one of EP 1 438 586 B1, EP 2 208 067 A1, EP 1 543 329 A2, EP 1 869 459 B1, EP 2 293 064 B1, EP 1 920 248 B1, EP 2 264 458 A1, EP 2 332 959 A2, WO 2011/47241, EP 1 901 069 B1, EP 2 293 064 B1, EP 1 807 698 B1 or EP 2 107 112 B1, each of which is incorporated herein by reference in your totality.
[0091] In yet a specific aspect of the method of the invention, the test is performed by determining the amount of cleaved SNAP-25 present in the neuronal cell derived from hiPS cells by determining the amount of cleaved SNAP-25 using a first antibody and , in one aspect, a monoclonal antibody that specifically binds to said cleaved SNAP-25. Furthermore, the total SNAP-25 present in the cells, e.g., cleaved and uncleaved SNAP-25, is determined by a second antibody and, in one aspect, a polyclonal antibody that binds to said total SNAP-25. In one aspect, the bound amount of the first antibody and the bound amount of the second antibody can be determined by a detection agent, in one aspect, by one or more detection antibodies that allow to distinguish between the bound amount of the first and the bound amount. of the second antibody. For example, a first detection antibody coupled to a first label and binding to the first antibody and a second detection antibody coupled to a second label and binding to the second bound antibody may be used. The amount of antibody first bound and second bound, and therefore the amount of SNAP-25 cleaved and total, can thereafter be derived from the amount of first and second markers determined. In one aspect, a first label provided herein may be an enzyme such as horseradish peroxidase. In another aspect, a second label provided herein may be an enzyme such as alkaline phosphatase. Labels can be used to catalyze the conversion of non-fluorescent detectable substrates to fluorescent products.
[0092] In yet another aspect of the method of the invention, the assay is performed by determining the release of neurotransmitters, for example, in the culture medium. The amount of neurotransmitter released or not released can be determined by techniques described here or any other.
[0093] Advantageously, the methods contemplated by the present invention are based on non-animal resources, such as, for example, neuronal cells derived from hiPS cells, and therefore avoids testing in animals. Neuronal cells derived from hiPS cells, however, allow testing of all the necessary biological activities of NTBs, eg, (a) receptor binding, (b) internalization, (c) translocation across the endosomal membrane to the cytosol, and/or (d) endoproteolytic cleavage of proteins involved in synaptic vesicle membrane fusion. Thus, the methods can be used for security or quality control measures, as well as for the development of NTBs with modified biological properties that generally require, for example, large-scale control approaches.
[0094] The present invention also relates to a method for determining the amount of biologically active NTB in a preparation comprising biologically active NTB, comprising the steps of: (a) contacting a neuronal cell derived from hiPS cells with a sample of said preparation ; and (b) determining the amount of biologically active NTB present in the preparation by assaying the sample for the biological activity of NTB.
[0095] The invention also relates to the use of neuronal cell derived from hiPS cells to test NTB activity in a composition as specified herein.
[0096] In one aspect, the assay encompasses the determination of the presence or absence of NTB and/or biologically active NTB activity. Qualitative testing for biologically active NTB can be used, for example, in applications for the purpose of risk assessment, in order to avoid any damage caused by NTBs, for example, as a safety control measure during the manufacturing process or during the pharmaceutical or cosmetic applications of NTBs or for the prevention of criminal conduct based on NTBs, such as bioterrorism.
[0097] In another aspect, the test encompasses the determination of the amount of NTB activity and/or biologically active NTB. The quantitative assay for biologically active NTB can be used in-process for NTB manufacturing or to set an appropriate dose of biologically active NTB for cosmetic or pharmaceutical applications. Accordingly, such an assay may also be useful as a means for quality control or for formulating correct pharmaceuticals or cosmetics.
[0098] The invention also relates to the use of neuronal cell derived from hiPS cells to determine the amount of biologically active NTB in a preparation comprising said biologically active NTB as specified herein.
The references cited in this specification above are hereby incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification. EXPERIMENTAL
[0100] The following examples are provided to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting its scope. Example 1 A. Methods
[0101] Neuronal cells: Human iPS-derived neurons were supplied frozen by Cellular Dynamics Inc. (Madison, WI). Neurons were thawed according to Cellular Dynamics instructions, and live cells were counted by Trypan Blue exclusion assay. Cells were seeded at a density of 40,000 cells per well in 96-well plates (TPP, MidSci) coated with 0.01% poly-L-ornithine (Sigma) and 8.3 μg/cm2 matrigel (BD Biosciences), unless otherwise noted, and were incubated in neuronal media (Neurobasal supplemented with B27 and glutamax, all from Invitrogen and supplied by CDI) at 37°C, 5% CO2 for the indicated maturation times. At the end of 24 hours after seeding, the medium was changed completely, and half of the medium was replaced every 2-3 days thereafter.
[0102] Primary rat spinal cord cells (RSC) were prepared as previously described (Pellett et al., 2007 and 2010), and seeded in matrigel-coated 96-well plates at a density of 75,000 cells per well.
[0103] Botulinum neurotoxin: Pure botulinum neurotoxin (NTB) A, B, C and E (150 kDa) and NTB/A complex were prepared from C. botulinum strains Hall A hyper, Okra B, Brazil C, and Beluga E as previously described ( Malizio et al., (2000) Methods Mol Biol 145:27-39; Prabakaran et al., (2001), Toxicon 39: 1515-1531 ), whereby purification of NTB/C was performed by the method of Malizio et al. (2000) with the additional step of adding 0.2 mg/ml yeast RNA (Sigma) to the culture prior to ammonium sulfate precipitation. Toxins were dissolved in phosphate buffered saline, pH 7.4 and 40% glycerol and stored at -20°C until use. The activity of NTB/A, /B, /C, /E and NTB/A complex preparations was determined by the rat bioassay (Hatheway CL (1988), supra; EJ Schantz and Kautter DA (1978) J. Assoc Off Anal Chem 61:96-99), and specific toxicity was 7 x 107LD 50 rat units/mg (NTB/A1), 7.7 x 107LD 50 Units/mg (NTB/A1 complex), 1 x 108LD 50 Units/mg (NTB/B), 1.1 x 107LD 50 Units/mg (NTB/C), and 7.6 x 107LD 50 Units/mg (NTB/E).
[0104] Neuronal toxicity assays: For all neuronal toxicity assays, iPS neurons were exposed to a series of dilutions of NTB in 50 μL of neuronal medium, as indicated, unless otherwise indicated. Primary rat spinal cord cells were used as control cells in some assays, as indicated in the results. All samples were tested a minimum of three times, and a toxin-free negative control was always included. After the specific exposure time, the protein solution was removed and cells were lysed in 50 µL of 1 x LDS sample buffer (Invitrogen). Cell lysates were analyzed by Western blot for SNAP-25 or VAMP2 cleavage as described previously (Pellett et al., (2007), supra; Pellett et al., (2010), supra). Cleaved and non-cleaved bands were quantified by densitometry using a Foto/Analyst FX system and TotalLab Quant software (Fotodyne). Data plots were prepared and EC50s derived using PRISM software.
[0105] Matrix Selection: To select the ideal surface matrix, neurons were seeded in seven different matrices. The matrices consisted of poly-D-lysine coated plates (Biosciences BD) coated with either 1.0 μg/cm2 laminin (PDL laminin) or 8.3 mg/cm2 matrigel (PDL matrigel), plates coated with 0.01 % poly-L-ornithine followed by coating with either 1.0 μg/cm2 laminin (OLP laminin) or 8.3 mg/cm2 matrigel (OLP matrigel) PLO-laminin coated plates purchased from BD Biosciences (OLP-laminin ( BD)), PDL coated plates from BD Biosciences (PDL (BD)) or 0.01% OLP coated plates (OLP (CDI)). Neurons were allowed to mature for 14 days, and sensitivity to NTB/D was determined by exposing neurons to a series of toxin dilutions for 48 h. Some of the neurons were kept for 6 weeks and retested as above.
[0106] Analysis of receptor expression: For the analysis of receptor expression, iPS neurons were placed in 24-well plates at a density of 210,000 cells/well in a volume of 0.75 mL. Cells from three wells, respectively, were harvested at 4, 7, 10, 14, and 21 days after plating in 75 µL of 1 x LDS sample buffer (Invitrogen). Cell lysates were analyzed by Western blot for the expression of SV2A, B and C isoforms, synaptotagmin I and II, SNAP-25, VAMP, using an antibody that recognizes VAMP 2 or an antibody that recognizes VAMP 1, 2, and isoforms 3, and syntaxin. Beta-actin was used as a loading control, and primary rat spinal cord cells were used as a positive control. The SV2C antibody (Janz R & Sudhof TC (1999) Neuroscience 94: 1279-1290) was generously provided by Roger Janz. All other antibodies were from Synaptic Systems (Gottingen, Germany). All antibodies recognize human proteins.
[0107] For real-time qPCR mRNA analysis, three independent cultures of iPS neurons were maintained for the indicated times, respectively. Cells were lysed directly on the plate and RNA was purified using the RNeasy Mini Kit and the RNase-free DNase Kit (Qiagen, Valencia, CA). For a positive control, adult human brain total RNA was purchased (Agilent Technologies, Santa Clara, CA). For all samples, cDNA was generated using the SuperScript VILO cDNA Synthesis Kit (Life Technologies, Carlsbad, CA). Real-time qPCR amplification was performed in LightCycler® 480 II (Roche, Basel, Switzerland) using the TaqMan® Gene Expression Master Mix and the following TaqMan Human Gene expression assays: SNAP25 (Hs00938962_ml); STX1A (Hs00270282_ml); STX1B (Hs01041315_ml); VAMP1 (Hs00249911_ml); VAMP2 (Hs00360269_ml); VAMP3 (Hs00922166_ml); SV2A (Hs00372069_ml); SV2B (Hs00208178_ml); SV2C (Hs00392676_ml); SYT1 (Hs00194572_ml); SYT2 (Hs00980604_ml) and the human endogenous GAPDH Primary Control Set (all from Life Technologies). Derivative analyzes of Quant/2a Abs were performed on all samples and Cp values were converted to fold shift versus Cp normalization pathway for endogenous GAPDH expression. Mean and standard deviation were calculated for each gene in the three biological replicas. Technical quadruplicate PCR reactions were performed for each gene.
[0108] Activity-dependent NTB/A1 uptake assays: NTB/A1 was diluted to a concentration of 55 or 275 U by 50 μL of cell stimulation medium (Custom Neurobasal Invitrogen medium containing 2.2 mM CaCl and 56 mM of KCl, supplemented with B27 and glutamax) or neuronal and added to 4-day matured iPS CDI neurons and RSC cells. Cells were incubated with toxin for 1, 5, 10 and 15 min, respectively. For the negative control, the respective toxin-free culture medium was added to the cells. The toxin was removed and cells were immediately washed twice with 200 µL of neuronal medium followed by incubation in 200 µL of fresh neuronal medium for 24 h. Samples were collected in replicates of 4.
[0109] To determine the minimum concentration required for NTB/A1 activity-dependent toxin absorption within 5 min, the toxin was diluted to concentrations between 1.72 and 55 U by 50 μL of cell stimulation medium. Four-day-matured iPS neurons were exposed to toxin dilutions for 5 min, followed by removal of toxins and two washes with neuronal medium and incubation in neuronal medium for 24 h. All dilutions were tested in replicates of 4.
[0110] Antibody protection analysis: NTB/A1 specific antibodies were prepared according to Johnson et al., 1993. Inhibition of NTB/A1 activity in iPS neurons by neutralizing antibodies was analyzed by two different methods. For the first assay, the 55 U of NTB/A1 was combined with the antibody serially diluted in cell stimulation medium and incubated for 1h at 37°C to allow for antibody-toxin interaction. Four-day-matured iPS neurons were exposed to the toxin-antibody mixture for 5 minutes, followed by removal of the toxin-antibody mixture and two steps of washing with neuronal medium and incubation in neuronal medium for 24 h. For the second test, 1.5 U of NTB/A was combined with serial dilutions of antibody in neuronal medium and incubated at 37°C for 1 h. iPS neurons were exposed to toxin-antibody mixtures for 24 h. The comparison with the bioassay in rats used serial dilution of the antibody pre-incubated with 5-10 U of NTB/A1 for 1.5 h at room temperature, in a volume of 167 μL. The volume was adjusted to 500 microliters and injected into four rats by dilution. B. Results
[0111] Expression receptors of iPS neurons important for NTB intoxication: In order to determine whether human iPS-derived neurons can be used to detect NTB activity, the expression of receptors and enzyme targets required for NTB cell entry and catalytic activity were analyzed by Western blot and quantitative PCR (qPCR), respectively (Figure 1). Western blot resulted in signals for SV2A, a weak band for SV2B, synaptotagmin 1, syntaxin, SNAP-25, VAMP2, and beta-actin, which did not change over a time period of 21 days after cell plating (Figure 1A). While VAMP2 was detected with a VAMP2 specific antibody, an antibody that recognizes all three VAMP isoforms, resulted in no signal, indicating that VAMP2 is the predominant VAMP isoform in iPS neurons. Rat spinal cord primary cell lysate was used as a positive control for antibody detection, and the different intensities of neuron bands against RSC cells could be due to differences in antibody recognition or different expression levels. Analysis of mRNA levels of the same proteins by qPCR indicated expression of all studied proteins (Figure 2B) including SV2B and C isoforms, synaptotagmin 2, and VAMP1 and 3, which were not detected by Western B1ot. However, the mRNA levels of these isoforms were at least 200 times lower than those of the isoforms detected by Western blot (SV2A, synaptotagmin 1 and VAMP2). Thus, qPCR data corroborate Western blot data and indicate that iPS neurons primarily express isoforms of these proteins SV2A, synaptotagmin 1 and VAMP2, which is consistent with neurons representing forebrain neurons (Janz R & Sudhof TC (1999) ) Neuroscience 94: 1279-1290). Expression levels of all proteins did not change over the study period, indicating that cells are fully matured for 4 days after plating and remain stable for at least 21 days.
[0112] Surface matrix does not influence the quality of cells for the NTB/A1 assay: To determine whether the plating matrix influenced the sensitivity to NTB, neurons plated in seven different matrices were tested for the sensitivity of NTB/A1 . Neurons have been linked to and matured in all matrices, forming an ever-increasing network of axons and dendrites. Significant morphological differences were observed between cells grown in plates with or without laminin or matrigel. Cells grown on PDL (BD Biosciences) laminin or matrigel plates or on PLO (Cellular Dynamics) laminin or matrigel plates grew mainly in a monolayer but formed some aggregates with long axons extending from them, mainly around the perimeter of the plates . In contrast, cells grown in PLO or PDL plates remained in a single cell monolayer with axons and dendrites that span between the cell networks. Cells grown in PLO laminin (BD Biosciences) resembled cells grown in OLP or PDL plates.
[0113] Neurons were exposed to a series of NTB/A dilutions after 14 days of maturation, and Western blot analysis of cell lysates indicated that cleavage and SNAP-25 was nearly identical for all substrates tested (Figure two). The detection limit was 0.05 rats LD50 units, and cleavage was complete between 1.75 and 3.5 U. EC50s ranged from 0.21 to 0.31.
[0114] These data indicate that cells can be placed on any surface matrices tested by this assay. All of the following experiments were performed on PLO-Matrigel coated plates. In order to reduce cell aggregation, TPP plates (MidSci) which have a smoother surface area were used. This completely eliminated aggregation around the perimeter of the well.
[0115] Cell maturation time of 4-14 days provides an excellent and sensitive NTB/A1 test platform: In order to determine whether NTB cell maturation time affects the sensitivity of iPS neurons, the cells were analyzed for the sensitivity of NTB/A1 at 4 and 7 days after plating in parallel using the same toxin dilutions. Primary rat spinal cord cells (RSC cells) were also tested in parallel to compare the sensitivity of iPS neurons to RSC cells, which are currently the most sensitive cells described for the detection of NTB (Pellet et al., 2007, supra ;Pellett et al., (2010) J Pharmacol Toxicol Methods). The resulting data consistently showed no statistically significant difference in the sensitivity of cells matured for 4 or 7 days, with EC50s of ~0.3 U (Figure 3). This is similar to the EC50 observed above for day 14 cells (Figure 2) and for RSC cells. The dose-response curve for iPS neurons was significantly steeper than for RSC cells, and 100% cleavage was achieved at 1.75 U, whereas 100% cleavage was not achieved in RSC cells at the concentrations of toxin used. This is likely due to the high purity of iPS neurons. Thus, cells matured for 4-14 days provide a highly sensitive, reproducible cell-based model for NTB/A1 detection and quantification. Furthermore, testing four different iPS cell lots did not indicate any major difference in NTB/A1 sensitivity.
[0116] Sensitivity of iPS neurons increases with longer exposure times: The time dependence of NTB detection in iPS neurons was examined by exposing the cells to NTB/A1 dilution series and sampling 6, 16, 24 , and 48 h after the addition of the toxin. The resulting data consistently indicated that the 48 hr exposure provided greater sensitivity, with a 3-fold increase compared to a 24-hour trial and a 6-fold increase compared to a 16 hr trial (Figure 4 ). The 6 h toxin exposure resulted in a ~130-fold decrease in sensitivity, with an EC50 of about 40 units.
[0117] iPS neurons have a faster NTB/A1 uptake rate than RSC cells: NTB/A1 uptake rate in iPS neurons compared to RSC cells was examined by exposing iPS neurons and RSC cells to 82 U of NTB/A1 in parallel and evaluating SNAP-25 cleavage at 2, 4, 6, 8, and 10 h. Two different means were used to differentiate between activity-dependent and activity-independent toxin uptake, as neuronal activity has been reported to result in rapid absorption of NTBs (Keller et al., (2004), supra). The first medium was the neuronal medium (NM), and the second was the cell stimulation medium (CSM), which is a modified version of the neuronal medium that contains 56 mM KCl and 2.2 mM CaCl2 to stimulate cell activity. neuronal cells chemically.
[0118] iPS neurons resulted in significantly earlier and more complete cleavage of SNAP-25 than RSC cells. In iPS neurons, 100% of SNAP-25 cleavage was performed in 8 h and « 50%) of SNAP-25 cleavage in « 4 h (Figure 5). RSC cells, in contrast, achieved only « 70-80% SNAP-25 cleavage after 10 h, and 50% SNAP-25 cleavage was observed within 6 h. No difference was observed between neuronal and cellular stimulation medium for any type of cell, which indicates that over the time period the neuronal activity tested does not affect NTB uptake in cells. These data indicate that iPS neurons are significantly more sensitive to NTB/D than RSC cells and take up toxin at a faster rate, although this test does not differentiate between faster toxin uptake and SNAP-25 cleavage plus fast.
[0119] iPS neurons capture NTB/A1 significantly faster than RSC cells in an activity-dependent assay: In order to analyze whether other iPS neurons capture NTB in an activity-dependent manner, iPS neurons and RSC cells matured for four days were exposed to 55 and 275 U of NTB/A1 in cell stimulation medium, respectively. Cells were exposed to toxin for 1, 5, 10 or 15 minutes, followed by complete removal of toxin and incubation in neuronal medium for 24 hours to allow SNAP-25 cleavage. Significant SNAP-25 cleavage was observed in iPS neurons as early as 1 min after exposure with 55 U of NTB/A1 (Figure 6A). After 5 minutes, about 75% of SNAP-25 was cleaved, and there was no significant change with longer exposures to the toxin, indicating complete absorption in less than 5 minutes. Exposure to 275 U resulted in complete cleavage of SNAP-25 at all exposure times tested (Figure 6A). In contrast, exposure of RSC cells to 55 Units of NTB/A1 did not result in significant SNAP-25 cleavage after exposure times of up to 15 minutes, and only about 30-40% SNAP-25 was cleaved after one exposure of at least 10 minutes at 275 U (Figure 6A). This indicates that iPS neurons take up NTB/A1 in an activity-dependent manner, and that this uptake occurs markedly more efficiently and faster than in RSC cells.
[0120] In order to confirm that the rapid uptake in iPS neurons is activity-dependent, the neurons were exposed to 55 U of NTB/A for 1, 5, or 10 minutes amidst cell stimulation or neuronal media. There was significantly more SNAP-25 cleavage in cells treated with cell stimulation medium, with 50% SNAP-25 cleavage observed from 1 min and 70% cleavage after 5 min (Figure 6B). In neuronal media, in contrast, only about 20% of SNAP-25 was cleaved after 10 minutes (Figure 6B). This indicates that the rapid uptake of NTB/A1 in iPS neurons is activity dependent.
[0121] In order to determine the concentration dependence of NTB/A1 uptake dependent activity by iPS neurons, cells were exposed to 1.7-55 U of NTB/A1 in cell stimulation medium for 5 min. After removal of toxins, cells were incubated for 24 hours to allow SNAP-25 cleavage to occur. An increase in concentration dependence was observed in SNAP-25 cleavage with increasing toxin concentration, with 50% of SNAP-25 cleavage occurring at about 30 U (Figure 6C).
[0122] NTB/A1 specific antibodies protect iPS neurons from SNAP-25 cleavage by NTB/A1: NTB iPS assay specificity was confirmed by an antibody protection assay with two different assay formats. In the first assay, cells were exposed to toxin antibody mixtures for 24 h, using the minimum amount of toxin necessary to achieve nearly complete SNAP-25 cleavage (1.5 units). In the second assay, cells were exposed to mixtures of 55 U NTB/D and serially diluted antibody for 5 minutes in cell stimulation medium, followed by removal of toxins and incubation for 24 h. The first assay produced significantly higher sensitivity in detecting antibodies. Neurons were fully protected against SNAP-25 cleavage with as little as 0.0025 µL of antibody. Significant partial protection was observed up to 0.000625 µL of antibody (Figure 7A). The same pattern of protection was observed previously when the antibody was tested on RSC cells using 0.5 U NTB/A1 and a 48 h exposure. Testing the same antibody dilutions per bioassay in mice indicated at least ~10-fold greater sensitivity of the cell-based assays compared to the mouse bioassay, as also the previous data indicated. The RSC assay has been shown to be more sensitive in detecting neutralizing antibodies than the mouse bioassay (Pellett, S. 2007, supra). The second assay (activity-dependent) was about 10-fold less sensitive, requiring 0.016 μL of antibody per 50 μL of full protection, and partial protection was observed at 0.004 μL (Figure 7B).
[0123] This confirms the specificity of this assay, and indicates that iPS neurons provide an excellent and highly sensitive assay for detecting neutralizing antibodies, and that a longer exposure with less toxin is more sensitive than an activity-dependent assay that requires more toxin but a shorter exposure time. Activity-dependent assay is useful for certain purposes, such as controlling compounds or antitoxins that may be cytotoxic or need to be dissolved in solvents that can damage neurons over time.
[0124] Detection of NTB/A1 toxin in its natural complex is more sensitive than detection of purified toxin: NTBs are expressed in clostridia as a complex with several other proteins (non-toxic nongemagglutinin protein (NTNH) and hemagglutinins (HA) in the NTB/A case (reviewed in Johnson EA & M Bradshaw (2001) Toxicon 39: 1703-1722) The non-toxic complex proteins are believed to protect the toxin from pH degradation of the gastrointestinal tract (Oguma et al., (2000) Microbial Foodborne Diseases: Mechanism of Pathogenesis and Toxin Synthesis 273-293) The most widely used medical preparations of NTB/A (BOTOX® and Dysport® preparations) consist of all the toxin complex, although newer formulations containing only purified NTB (Xeomin® preparation) have already been approved by the FDA, in order to determine whether NTB/A1 in its natural complex is detected with equal sensitivity as NTB/A 1 pure in iPS neurons, cells were exposed to equal amounts of NTB/A1 or purified NTB/A1 complex in parallel. The complex is made up of about 24% NTB/A1 and 76% other non-toxic associated proteins as determined by densitometry (Fig. 8A). The preparation of purified NTB/A1 and NTB/A1 complex had similar specific activities (7 x 107U/mg and 7.3 x 107 U/mg). In a direct comparison, significantly less of the toxin component of the complex was needed to achieve total SNAP-25 cleavage (Figure 8B). This finding indicates that the non-toxic proteins in the complex increase NTB/1A activity in this assay, possibly due to a protective effect in the neuronal milieu.
[0125] iPS neurons are a highly sensitive cell model for the detection of NTB serotypes B, C and E: NTB receptor analysis indicated that iPS neurons express the SNARE proteins and receptors necessary for entry into cells from all NTB serotypes (Figure 1). NTB/A and /E cleave SNAP-25, NTB/B cleave VAMP and NTB/C cleave SNAP-25 and syntaxin ( Humeau et al., (2000) Biochimie 82: 427-446). To test the sensitivity of neurons of different serotypes, serial dilutions of NTB/B, C, or E were added to iPS neurons or RSC cells for 48 h in parallel, and cell lysates were analyzed by Western blot for cleavage of the respective neuronal substrate. iPS neurons consistently detected all NTB serotypes with equal or greater sensitivity than RSC cells (Figure 9). The EC50s values for iPS neurons and RSC cells were 15.71 U and 29.22 U for NTB/B (Figure 9A), 0.4 U and 0.36 U for NTB/C (Figure 9B) and 1, 79 U for NTB/E in iPS neurons (Figure 9C). An EC50 value of NTB/E in RSC cells cannot be determined with the PRISM software, but is estimated to be similar to that of iPS neurons (Figure 9C).
[0126] All publications, patents, patent applications and accession numbers mentioned in the above specification are hereby incorporated by reference in their entirety. While the invention has been described in connection with specific embodiments, it is to be understood that the invention, as claimed, should not be unduly limited to such specific embodiments. Indeed, various modifications and variations of the described compositions and methods of the invention will be apparent to those of ordinary skill in the art and are intended to be within the scope of the following claims.

权利要求:
Claims (16)
[0001]
1. In vitro method of testing a Clostridium botulinum (NTB) neurotoxin for activity, CHARACTERIZED by comprising: a) contacting an induced human pluripotent stem cell (hiPS) derived neuronal cell with a composition comprising an NTB; and b) testing said NTB for biological activity.
[0002]
2. In vitro method according to claim 1, CHARACTERIZED by the fact that it further comprises the step of contacting said NTB with a test compound before contacting said hPS-derived neuronal cells.
[0003]
3. In vitro method, according to claim 2, CHARACTERIZED by the fact that said test compound is an antibody.
[0004]
4. In vitro method according to claim 2, CHARACTERIZED by the fact that said test compound is an antibody.
[0005]
5. In vitro method, according to claim 3, CHARACTERIZED by the fact that said antibody is a neutralizing antibody.
[0006]
6. In vitro method according to claim 4, CHARACTERIZED by the fact that said neutralizing antibody is in a sample selected from the group consisting of purified antibodies, serum and antitoxins.
[0007]
7. In vitro method of testing a Clostridium botulinum (NTB) neurotoxin for activity, CHARACTERIZED by comprising: a) contacting an induced human pluripotent stem cell (hiPS)-derived neuronal cell with a composition comprising i) a NTB, and ii) a neutralizing antibody; and b) testing said NTB for biological activity.
[0008]
8. In vitro method according to claim 6, CHARACTERIZED by the fact that said NTB has a serotype selected from the group consisting of A, B, C, E and modified variants of said NTB.
[0009]
9. In vitro method, according to any one of claims 1 to 7, CHARACTERIZED by the fact that said biological activity is selected from the group consisting of SNAP-25 cleavage, VAMP2 cleavage and neurotransmitter release.
[0010]
10. In vitro method, according to any one of claims 1 to 8, CHARACTERIZED by the fact that said test is qualitative.
[0011]
11. In vitro method, according to any one of claims 1 to 9, CHARACTERIZED by the fact that said test is quantitative.
[0012]
12. In vitro method, according to any one of claims 1 to 10, CHARACTERIZED by the fact that said NTB is purified.
[0013]
13. In vitro method, according to any one of claims 1 to 10, CHARACTERIZED by the fact that said NTB is in a complex.
[0014]
14. In vitro method, according to any one of claims 6 to 12, CHARACTERIZED by the fact that said neutralizing antibody is in a sample selected from the group consisting of purified antibodies, serum and antitoxins.
[0015]
15. In vitro method for determining the amount of biologically active NTB in a preparation comprising biologically active NTB, CHARACTERIZED in that the method comprises the steps of: (a) contacting a neuronal cell derived from a hiPS cell with a sample of a preparation comprising Biologically active NTB, and (b) determining the amount of biologically active NTB present in the preparation by testing said sample for the biological activity of NTB.
[0016]
16. Use of an Induced Human Pluripotent Stem Cell (hiPS) Derived Neuronal Cell CHARACTERIZED by the fact that it is to test an NTB for in vitro activity.

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法律状态:
2018-01-23| B25G| Requested change of headquarter approved|Owner name: CELLSNAP, LLC (US) |

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-08-13| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-05-04| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-07-13| B350| Update of information on the portal [chapter 15.35 patent gazette]|

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2021-07-20| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 28/09/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161540693P| true| 2011-09-29|2011-09-29|

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US61/540,693|2011-09-29|

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PCT/US2012/057825|WO2013049508A1|2011-09-29|2012-09-28|Compositions and methods for toxigenicity testing|

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