WO2014092227A1 - Composition pour le traitement de maladies associées à l'élimination de l'alpha-synucléine extracellulaire et procédé pour le criblage d'un agent thérapeutique pour lesdites maladies - Google Patents

Composition pour le traitement de maladies associées à l'élimination de l'alpha-synucléine extracellulaire et procédé pour le criblage d'un agent thérapeutique pour lesdites maladies Download PDF

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WO2014092227A1
WO2014092227A1 PCT/KR2012/011024 KR2012011024W WO2014092227A1 WO 2014092227 A1 WO2014092227 A1 WO 2014092227A1 KR 2012011024 W KR2012011024 W KR 2012011024W WO 2014092227 A1 WO2014092227 A1 WO 2014092227A1
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synuclein
antibodies
antibody
cells
microglia
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PCT/KR2012/011024
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Korean (ko)
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이승재
배은진
이혜진
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건국대학교 산학협력단
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the present invention relates to compositions for treating diseases associated with extracellular ⁇ -synuclein removal and methods for screening the same.
  • Parkinson's disease is a chronic progressive motor nervous system disorder. About 50,000 Americans are diagnosed with PD every year. The main symptoms of these neurodegenerative disorders are tremor, stiffness, motor slowness and balance disorder. Many PD patients also suffer from a variety of other symptoms, including emotional changes, memory loss, speech disorders, or difficulty sleeping.
  • PD is caused by the specificity and progressive neuronal loss of midbrain dopamine (DA) neurons.
  • DA midbrain dopamine
  • these neurons produce dopamine, a chemical transporter responsible for signal transduction between melanocytes and striatum, resulting in smooth and intentional muscle activity.
  • dopamine excites the nerve cells of the striatum so that they can be impaired, impairing their ability to direct and control their movements.
  • Korean Patent Publication No. 1020080106928 is administered by administering 7-chloro-4-aminoquinoline compounds such as amodiaquine or glafenine.
  • Methods and kits for treating Parkinson's disease or inhibiting Parkinson's disease are characterized.
  • ⁇ -synuclein is a protein present in the protoplasts located at presynaptic sites (Iwai A, Masliah E, Yoshimoto M, Ge N, Flanagan L, de Silva HA, Kittel A, Saitoh T (1995). Neuron 14: 467-475).
  • the present invention has been made in view of the above necessity, and an object of the present invention is to provide a novel Parkinson's disease (PD) and Lewy body dementia (DLB) therapeutic composition.
  • PD Parkinson's disease
  • DLB Lewy body dementia
  • Another object of the present invention is to screen candidate candidates for Parkinson's Disease (PD) and Lewy Body Dementia (DLB).
  • PD Parkinson's Disease
  • DLB Lewy Body Dementia
  • the present invention provides an antibody that specifically binds to the ⁇ -synuclein peptide set forth in SEQ ID NO: 1.
  • the present invention provides a composition for preventing or treating Parkinson's disease, comprising the antibody of the present invention as an active ingredient.
  • the present invention provides a composition for preventing or treating Lewy Body Dementia (DLB) comprising the antibody of the present invention as an active ingredient.
  • DLB Lewy Body Dementia
  • the present invention corresponds to a case where the candidate substance and the extracellular ⁇ -synuclein protein are contacted with the microglial cell under an environment suitable for the two components to interact, and the uptake and degradation rate of the ⁇ -synuclein protein are increased.
  • a method for identifying a candidate for Parkinson's disease or Lewy body dementia (DLB) treatment wherein the candidate is determined to be a candidate for Parkinson's disease or Lewy Body Dementia (DLB).
  • the candidate is preferably an antibody antisense oligonucleotide or a compound, but is not limited thereto.
  • epitope refers to a protein determinant capable of specifically binding to an antibody.
  • Epitopes usually consist of a group of chemically active surface molecules, such as amino acids or sugar side chains, and generally have specific three dimensional structural characteristics as well as specific charge characteristics. Three-dimensional epitopes and non-stereo epitopes are distinguished in that the binding to the former is lost but not to the latter in the presence of a denatured solvent.
  • the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of one or more antibodies of the invention in combination with a pharmaceutically acceptable diluent, carrier, solubilizer, emulsifier, preservative and / or adjuvant do.
  • the acceptable formulation material is nontoxic at the dosages and concentrations used for the recipient.
  • a pharmaceutical composition comprising a therapeutically effective amount of an antibody of the invention.
  • preferably acceptable formulation materials are nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition modifies, maintains or preserves, for example, the pH, osmolarity, viscosity, purity, color, isotonicity, flavor, sterilization, stability, dissolution or release rate, adsorption or permeation of the composition.
  • suitable formulation materials include, but are not limited to: amino acids (eg, glycine, glutamine, asparagine, arginine or lysine); Antimicrobial agents; Antioxidants (eg, ascorbic acid, sodium sulfate or sodium hydrogen sulfide); Buffer solutions (eg, borate, bicarbonate, tris-HCl, citrate, phosphate or other organic acids); Volume control agents (eg mannitol or glycine); Chelating agents (eg ethylenediamine tetraacetic acid (EDTA); complexing agents (eg caffeine, polyvinylpyrrolidone, ⁇ -cyclodextrin or hydroxypropyl- ⁇ -cyclodextrin); fillers; monosaccharides; Disaccharides; and other carbohydrates (e.g.
  • amino acids eg, glycine, glutamine, asparagine, arginine or lysine
  • Antimicrobial agents eg, ascorbic acid, sodium
  • glucose, mannose, or dextrins proteins
  • proteins e.g. serum albumin, gelatin or immunoglobulins
  • pigments e.g. anti-microbial and diluents
  • emulsifiers hydrophilic polymers (e.g. poly Vinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (e.g. sodium); preservatives (e.g. benzalkonium chloride, benzoic acid, salicylic acid, chimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine Sorbic acid or hydrogen peroxide); solvents (e.g.
  • glycerin propylene glycol or polyethylene glycol
  • sugar alcohols e.g. mannitol or sorbitol
  • suspending agents e.g., surfactants or Wetting agents (e.g., polysorbates such as pluronic, PEG, sorbitan esters, polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol and tiloxapal); stability enhancers ( For example sucrose or sorbitol); enteric enhancers (e.g. alkali metal halide compounds, preferably sodium chloride, potassium chloride, mannitol sorbitol); transport excipients; diluents; prostheses; and / or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES, 18th Edition, (AR Gennaro, ed.), 1990, obtained from Publishing Company.
  • the optimal pharmaceutical composition will be determined by one of ordinary skill in the art, for example, according to the intended route of administration, mode of transport and dosage required. See, for example, REMINGTON'S PHARMACEUTICAL SCIENCES above.
  • the composition affects the rate of in vivo clearance, rate of in vivo release, stability, and physical state of the antibodies of the invention.
  • the primary excipient or carrier in the pharmaceutical composition is aqueous or non-aqueous in nature.
  • a suitable excipient or carrier may be an injectable, physiological saline or artificial cerebrospinal fluid that is generally capable of supplementing other substances in the composition for parenteral administration.
  • Neutral buffered saline or physiological saline mixed with serum albumin is further typical excipients.
  • the pharmaceutical composition of the present invention comprises tris buffer of about pH 7.0-8.5 or acetate buffer of about pH 4.0-5.5, which further comprises sorbitol, sucrose, Tween-20 and / or Suitable substituents thereof.
  • the antibody compositions of the present invention may be formulated with selected compositions having the desired purity and optimal formulation formulations.
  • compositions of the invention can be selected for parenteral administration.
  • the composition may be selected for inhalation or transport through the digestive tract, such as oral. Preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • the formulation component is preferably present at an acceptable concentration at the site of administration.
  • buffers are used to maintain the composition at physiological or slightly lower pH, generally in the range of about pH 5 to about pH 8.
  • the formulations can also be administered orally.
  • Antibodies of the invention administered in this manner may be formulated with or without a carrier generally used in the preparation of solid pharmaceutical formulations such as tablets and capsules.
  • the capsule may be designed to release the effective portion of the formulation at a gastrointestinal tract location where the bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents may be included to facilitate uptake of the antibodies of the invention. Diluents, flavors, low melting waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents and binders may also be used.
  • compositions of the invention are preferably provided comprising an effective amount of one or multiple antibodies of the invention in a mixture with non-toxic excipients suitable for the manufacture of tablets.
  • excipients include, but are not limited to: inert diluents such as calcium carbonate, sodium carbonate or sodium bicarbonate, lactose or calcium phosphate; Or binders such as starch, gelatin or acacia; Or lubricants such as magnesium stearate, stearic acid or talc.
  • compositions used for administration in vivo are generally provided as sterile preparations. It can be sterilized by filtration through sterile filtration membranes. When the composition is lyophilized, it can be sterilized using the method before or after lyophilization and reconstitution.
  • the composition for parenteral administration can be stored in lyophilized form or as a solvent.
  • Parenteral compositions are generally stored in containers with sterile access ports, such as vials or intravenous solution bags with stoppers that can be inserted into subcutaneous needles.
  • the pharmaceutical composition Once the pharmaceutical composition has been formulated, it is stored in sterile vials in the form of solvents, suspensions, gels, emulsions, solids or dehydrogenated or lyophilized powders. Such formulations are stored in an easy-to-use form or in a form that requires reconstitution (eg, lyophilized) prior to administration.
  • an effective amount of a pharmaceutical composition containing an antibody of the invention to be used therapeutically depends, for example, on the therapeutic situation and the subject.
  • dosage levels suitable for treatment may be partially determined by the transport molecule, signs of the use of the antibody of the present invention, route and size of administration (weight, body surface area or organ size) and / or symptoms of the patient (age and overall health). Will vary depending on the condition.
  • the clinician may titrate the dosage and alter the route of administration to obtain the optimal therapeutic effect.
  • Typical dosages range from about 0.1 ⁇ g / kg up to about 30 mg / kg or more, depending on the factors mentioned above.
  • the dosage range is from 0.1 ⁇ g / kg to about 30 mg / kg; More preferably from 1 ⁇ g / kg to about 30 mg / kg; Even more preferably from 5 ⁇ g / kg to about 30 mg / kg.
  • compositions were administered by a single administration or by two or more administrations (with or without the same amount of the desired molecule) or by continuous infusion via an insertion device or catheter over time.
  • refining at a suitable dosage is an area of common work typically made by a person skilled in the art and done by a person skilled in the art. Appropriate doses were identified using appropriate dose-response data.
  • antibodies of the invention can be administered to a patient for an extended period of time. Routine administration of the antibodies of the present invention minimized immune adverse reactions or allergic reactions generally associated with antibodies to human antigens, such as non-complete human antibodies produced in non-human species, in non-human animals.
  • the route of administration of the pharmaceutical composition is according to known methods, including, for example: oral; Injection by intravenous, intraperitoneal, intracranial (brain parenchymal), intraventricular, intramuscular, intraocular, intraarterial, intraportal, intralesional routes; Sustained release system; Or injection using an insertion device.
  • the composition may be administered by injection or infusion or infusion device continuously.
  • composition can be administered topically through the insertion of a membrane, sponge or other suitable material in which the required molecules are absorbed or encapsulated.
  • a membrane, sponge or other suitable material in which the required molecules are absorbed or encapsulated.
  • the device when using an insertion device, the device is inserted into an appropriate tissue or organ and the required molecules are transported by diffusion, sustained-release concentrates or continuous administration.
  • fibrils and oligomers contain both SDS-resistant aggregates (multiple high molecular weights) and SDS soluble aggregates, showing 16 kDa molecular weight as monomers (FIG. 1H, I). ).
  • the inventors cultured rat primary microglia to determine the rate at which these microglia uptake the ⁇ -synuclein protein secreted from neuronal cells.
  • conditioned media obtained from differentiated SH-SY5Y neuroblastoma cells that overexpress human ⁇ -synuclein.
  • the conditioned medium contains both monomeric and SDS stable aggregates (FIG. 1I).
  • uptake of a-synuclein secreted from neuronal cells reached faster and higher levels in 274 antibody treated primary microglia (FIG. 3E, F), which antibody Suggests that it helps microglia in the removal of neuronal secreted extracellular a-synuclein.
  • Antibody-assisted clearance of ⁇ -synuclein aggregates is mediated by Fc ⁇ receptors resulting in rapid delivery to lysosomes
  • Fc ⁇ receptors were present with oligomer ⁇ -synuclein on the surface of BV-2 cells only in the presence of 274 antibodies, not in the presence of control IgG or Fab fragments of 274 antibodies (FIG. 5B), which was responsible for the uptake process of Fc ⁇ receptors. Prove your involvement. Fc ⁇ receptors were markedly expressed in BV-2 microglia and rat primary microglia but were not detected in astrocytic or differentiated neuroblastoma cells (FIG. 5C).
  • Type II and Type III (CD32 / CD16) Fc ⁇ receptors are low affinity receptors for IgG and require a multivalent antigen-antibody complex for high avidity (Ravetch JV, Bolland S (2001) Annu Rev Immunol 19: 275-290). It has a high affinity and cannot bind to these Fc ⁇ receptors.
  • control IgG or monoclonal antibody 274 was precultured with various amounts of ⁇ -synuclein fibrils and added to BV-2 cells. The level of absorbed antibody was directly proportional to the amount of foreign ⁇ -synuclein aggregates (FIG. 5D).
  • LDL low density lipoproteins
  • CTB cholera toxin B subunit
  • microglia To confirm the involvement of microglia in the blockade of cell delivery in the cells, we used the dual immunofluorescence staining to investigate the position of human ⁇ -synuclein and 274 antibodies in microglia.
  • mice injected with 274 antibodies The number of microglia containing human ⁇ -synuclein showed a significant increase ipsilaterally in mice injected with 274 antibodies (FIGS. 8A, B).
  • the injected antibody was also detected in microglia in ipsilaterally in tg mice injected with 274 antibodies (FIG. 8C, D), but not in mice injected with control IgG (FIG. 8D).
  • the co-location of ⁇ -synuclein and injected antibodies was consistently observed in tg mice injected with 274 antibodies (FIG. 8E), but not in mice injected with control IgG.
  • ⁇ -synuclein immunostaining was restricted to neuropil in non-tg mice with no differences observed between the control IgG and 274 antibody groups (FIG. 9E-G). ).
  • IgG-treated ⁇ -synuclein tg mice of astrocytes (FIG. 9F) and neuronal cells (FIG. 9G) in ⁇ -synuclein (FIG. 9E) in neocortex (FIG. 9H) and hippocampus (FIG. 9I). There was extensive accumulation.
  • ⁇ -synuclein tg mice treated with 274 antibodies FIG.
  • FIG. 9E were found to have astrocytes (FIG. 9F) and neurons ⁇ -synuclein (FIG. 9G) in neocortex (FIG. 9H) and hippocampus (FIG. 9I). There was a significant decrease in the accumulation of.
  • mice We also analyzed the striatum of passively immunized mice and found that the levels of ⁇ -synuclein were reduced to a lower extent compared to the cortex and hippocampus.
  • ⁇ -synuclein-containing microglia were observed mainly in neural networks and rarely around the blood vessels.
  • ⁇ -synuclein tg mice treated with IgG alone showed increased levels of pro-immune cytokines, TNF ⁇ (FIG. 10 I, J), and IL-6 (FIG. 10K, L), whereas 274 antibodies Low immunization reduced that level to baseline. This result is consistent with the result that 274 antibodies promote the removal of extracellular ⁇ -synuclein.
  • the antibody against ⁇ -synuclein specifically promotes the removal of extracellular ⁇ -synuclein aggregates by microglia and forms ⁇ -synuclein when complexed with specific antibodies.
  • Synuclein aggregates are absorbed through Fc ⁇ receptors on the surface of microglial cells and after absorption their immune complexes are transported along the transport pathway and cause more efficient delivery to lysosomes.
  • Removal of ⁇ -synuclein mediated by antibodies also inhibited the transfer of ⁇ -synuclein from cell to cell in the tg mouse model.
  • ⁇ -synuclein fibrils and oligomers show the properties of ⁇ -synuclein fibrils and oligomers.
  • AF EM image of fibrils ( A ), sonicated fibrils ( B ), and oligomers ( CF ).
  • Scale bar AC , 0.5 ⁇ m; DF , 30 nm.
  • G size exclusion chromatography of oligomers.
  • Asterisks (7 ml fractions) indicate oligomeric fractions. Monomers are present in 13-15 ml fractions.
  • Silver stained image of H SEC fractions.
  • I Western blot analysis of several forms of ⁇ -synuclein.
  • M monomers
  • F fibrils
  • O oligomers
  • CM conditioned medium of differentiated SH-SY5Y cells expressing ⁇ -synuclein.
  • Figure 2 shows the effect of monoclonal antibodies on the uptake of extracellular ⁇ -synuclein aggregates.
  • A uptake of ⁇ -synuclein fibrils in the presence of described monoclonal antibodies against ⁇ -synuclein or control IgG (-) in BV-2 microglia. 5 ⁇ g / ml ⁇ -synuclein fibrils (0.2 ⁇ M) Antibodies against control IgG or ⁇ -synuclein were preincubated for 5 minutes at room temperature and treated with BV-2 cells at 37 ° C. for 5 minutes. The amount of ⁇ -synuclein absorbed was analyzed by Western blot.
  • B the amount of ⁇ -synuclein absorbed was quantified and normalized to the level of beta-actin.
  • the number of y-axes indicates the amount of ⁇ -synuclein relative to the control.
  • n 4, * p ⁇ 0.05.
  • FIG. 3 shows the effect of 274 antibodies on uptake and degradation of extracellular ⁇ -synuclein in microglia.
  • Adsorbed ⁇ -synuclein aggregates were analyzed by western blotting at the time stated.
  • Graphs in A and C show the amount of ⁇ -synuclein compared to the maximum absorbed level with control IgG.
  • Graphs in B and D show the amount of ⁇ -synuclein remaining compared to that at time O. All values were normalized to beta-actin.
  • E , F show the effect of 274 antibodies on uptake of neuron secreted ⁇ -synuclein in primary microglia.
  • E primary rat microglia were treated with conditioned medium containing ⁇ -synuclein secreted from differentiated SH-SY5Y cells in the presence of control IgG or 274 antibodies.
  • a , C , E uptake rate.
  • the graph shows the amount of ⁇ -synuclein relative to the level of maximum absorption with control IgG. B , D , F , decomposition rate.
  • the graph shows the amount of ⁇ -synuclein remaining compared to that at time O. Pre-incubation time with ⁇ -synuclein fibrils was 12 hours ( B , D , F ). All ⁇ -synuclein data was normalized to beta-actin data. The curve of the graph is not significantly different.
  • FIG. 5 shows the role of Fc ⁇ receptors in the uptake of ⁇ -synuclein-antibody immune complexes.
  • FIG. 1 Shows indicate the co-existence of ⁇ -synuclein and Fc ⁇ receptors on the surface of the cells. Scale bar: 20 ⁇ m.
  • C Expression of Fc ⁇ receptors in several cell types. For BV-2, rat microglia, and rat astrocytes, anti-CD16 / CD32 antibodies specific to rodents were used, whereas for SH-SY5Y, anti-CD32 antibodies specific to human proteins were used. Expression of CD16 has not been reported in brain neurons in previous studies. Red, Fc ⁇ receptors; Blue, nuclear. Scale bar: 20 ⁇ m.
  • D uptake of antibody into BV-2 cells in the presence of several different amounts of ⁇ -synuclein fibrils or ⁇ -synuclein (0 -330 nM). Absorbed antibody was analyzed with anti-mouse IgG antibody. The bottom panel is beta-actin.
  • E Immunotype and microscopic results of ⁇ -synuclein fibrils and antibodies taken up in BV-2 cells. Scale bar: 20 ⁇ m. Quantification of fluorescence from antibodies absorbed at F , E. 150 cells were analyzed in three independent experiments (50 cells in each experiment). * p ⁇ 0.0001.
  • FIG. 6 shows altered intracellular transport and increased transport of lysosomes of uptake of ⁇ -synuclein aggregates.
  • A Altered intracellular transport of ⁇ -synuclein fibrils uptake in BV-2 cells. Fluorescently labeled tracers were treated with BV-2 microglia cells for 5 min at 37 ° C. with control IgG- ⁇ -synuclein mixture or ⁇ -synuclein-274 immune complex. Arrows indicate coexistence between ⁇ -synuclein and CTB in the presence of 274 antibody. Scale bar: 5 ⁇ m.
  • BE localization of ⁇ -synuclein aggregates absorbed in the endocytic compartment.
  • FIG. 7 shows astrocytic delivery in reduced neurons of ⁇ -synuclein in vivo following alone injection of 274 antibody.
  • a , B low-pan (20X) view of the cortex and hippocampus of PDGF- ⁇ -synuclein tg mice (line M) comparing the patterns of ⁇ -synuclein immunoreactivity. Scale bar: 200 ⁇ m.
  • FIG. 8 shows increased localization of ⁇ -synuclein and IgG in microglia cells in vivo after one injection of antibody.
  • PDGF- ⁇ -synuclein tg mice line M
  • images in the AE are obtained from the injection, ipsilateral region and dentate gyrus. Scale bar, 10 ⁇ m.
  • A double labeling with antibodies to ⁇ -synuclein (red) and microglia marker (Iba1, green).
  • B Image analysis for double label for measurement of proportion of Iba-1-positive cells co-located with ⁇ -synuclein.
  • N 8 per group, 9 months of age.
  • G percent of Iba-1-positive cells with ⁇ -synuclein after 1, 7, 14, and 28 days after injection.
  • I Image analysis for double labeling for proportional measurement of positive cells co-existing with ⁇ -synuclein.
  • N 8 per group, 9 months of age.
  • J double label with antibody against ⁇ -synuclein (red) and GFAP (green). Arrowheads indicate ⁇ -synuclein-positive neurons that do not coexist with GFAP; arrows indicate ⁇ -synuclein-positive cells coexist with GFAP.
  • FIG. 9 shows improved behavior and reduced ⁇ -synuclein accumulation in ⁇ -synuclein tg mice after passive immunization with 274 antibody.
  • A the total time traveled down the pole test.
  • B open field total spontaneous activity (number of crosses of photocell).
  • C Open field Total distance traveled 5 minutes after voluntary active testing.
  • D Open field Total number of roundings after 5 minutes of spontaneous active testing.
  • P ⁇ 0.05 by unilateral ANOVA with post hoc Dunnet's compared to AD , * non-tg and ⁇ -synuclein tg; # p ⁇ 0.05 by unilateral ANOVA with post hoc compared to IgG and 274 antibodies in ⁇ -synuclein tg.
  • E low magnification view (x20) of ⁇ -synuclein immunoreactivity in cortex and hippocampus. Squares show magnified sites in F and G. Scale bar, 250 ⁇ m.
  • F , G high magnification (x630) views ( F , astrocytes; G , neurons). Scale bar, 20 ⁇ m.
  • H- sh respectively neocortex, hippocampus, image analysis of glial and neuronal cells can be estimated (dissection method) representing the ⁇ - synuclein in the striatum. * Student's t by test p ⁇ 0.001.
  • K double labeled with antibody against ⁇ -synuclein (red) and neuronal marker (NeuN, green) in hippocampus. Arrow heads indicate coexistence.
  • L Image analysis for double labeling to assess the proportion of NeuN-positive cells co-existing with ⁇ -synuclein.
  • M double labeled with antibodies against ⁇ -synuclein (red) and GFAP (green) in the hippocampus. Arrowheads indicate ⁇ -synuclein-positive cells coexisting with astrocytes.
  • N Image analysis for double labeling for evaluation of the proportion of GFAP positive cells co-existing with antibodies to ⁇ -synuclein.
  • O double labeled with antibodies against ⁇ -synuclein (red) and Iba-1 (green) in hippocampus. Arrowheads indicate ⁇ -synuclein-positive cells coexist with microglia.
  • P Image analysis for double labeling for evaluation of the proportion of Iba-1-positive cells co-existing with antibodies to ⁇ -synuclein.
  • Q double labeled with antibodies against ⁇ -synuclein (red) and cathepsin D (green) in the hippocampus. Arrow heads indicate coexistence.
  • FIG. 10 shows reduced neurodegenerative and pre-inflammatory cytokines after passive immunization with 274 antibodies.
  • ⁇ -synuclein tg mice treated with IgG show neuronal loss in the hippocampus (arrow). Scale bar, 250 ⁇ m.
  • B Stereoscopic Analysis of NeuN in Hippocampus (Anatomy Method).
  • C high magnification views (x630), scale bars, 20 ⁇ m of sections immunostained with antibodies to synaptophysin in ⁇ -synuclein tg and non-tg mice treated with IgG or 274 antibodies, respectively.
  • D stereoscopic analysis of synaptophysin in the hippocampus (anatomist method).
  • ⁇ -synuclein tg mice treated with IgG show increased astrocytes in the neocortex and hippocampus. Scale bar, 250 ⁇ m.
  • F Image analysis of GFAP immune response expressed at absorbance. The measurement is performed on the entire hippocampus.
  • G high magnification view (x630), scale bar, 20 ⁇ m of hippocampal sections immunostained with antibodies against Iba-1 in ⁇ -synuclein tg and non-tg mice treated with IgG or 274 antibodies, respectively.
  • H Image analysis of the estimated number of microglia in the hippocampus. * p ⁇ 0.05 by unilateral ANOVA with post hoc Dunnet s compared to non-tg and ⁇ -synuclein tg; p ⁇ 0.05 by unilateral ANOVA with post hoc Fisher compared to IgG and 274 antibodies at # ⁇ -synuclein tg.
  • Antibodies used in the present invention include: ⁇ -synuclein monoclonal antibody (BD Biosciences, # 610787), ⁇ -synuclein polyclonal antibody (Cell Signaling Technology, # 2642), myc polyclonal antibody (Abcam, # ab9106), CD32 Polyclonal antibodies (United States Biological, # c2384-0B), and CD16 / CD32 monoclonal antibodies (Abcam, # ab25235), GM130 monoclonal antibodies (BD Biosciences, # G65120), cathepsin D monoclonal antibodies (Abcam, # ab6313), and caveolin-1 monoclonal antibody (BD Biosciences, # C13620).
  • ⁇ -synuclein monoclonal antibody BD Biosciences, # 610787
  • ⁇ -synuclein polyclonal antibody Cell Signaling Technology, # 2642
  • myc polyclonal antibody Abcam, # ab9106
  • CD32 Polyclonal antibodies
  • FITC-labeled cholera toxin B subunit was purchased from Sigma.
  • Bodipy-labeled GM1, Bodipy-FL LDL, and Alexa Fluor 568-attached Dextran were purchased from Invitrogen.
  • the antibodies used in the present invention do not distinguish between different forms.
  • 274 antibodies show immunoreactivity to both the extracellular and intraplasma forms of ⁇ -synuclein and to the monomer and aggregate forms.
  • Mixtures of control IgG, mouse IgG isotypes were prepared from normal mouse serum collected using protein A / G columns.
  • Example 2 Purification of ⁇ -synuclein and production of fibrils and oligomers
  • Wild-type human ⁇ -synuclein was purified as described by Lee HJ, Bae EJ, Jang A, Ho DH, Cho ED, Suk JE, Yun YM, Lee SJ (2011b) J Neurosci Methods 199: 249-257.
  • ⁇ -synuclein 3 mg / ml in PBS
  • ⁇ -synuclein was incubated at 37 ° C. for 2 weeks with continued stirring at 250 rpm.
  • ⁇ -synuclein was incubated for another week. After incubation, the protein was centrifuged at 100,000 ⁇ g for 1 hour and the pellet was resuspended in PBS.
  • ⁇ -synuclein oligomers were prepared by the methods described in Danzer KM, Haasen D, Karow AR, Moussaud S, Habeck M, Giese A, Kretzschmar H, Hengerer B, Kostka M (2007) J Neurosci 27: 9220-9232. Lyophilized ⁇ -synuclein was dissolved in 50 mM sodium phosphate, pH 7.0 with 20% ethanol to a final concentration of 0.1 mg / ml and stirred at 250 rpm for 4 hours at room temperature.
  • ⁇ -synuclein was lyophilized again and resuspended in 50 mM sodium phosphate, pH 7.0 with 10% ethanol of 1/2 starting volume. The protein was incubated at room temperature for 24 hours with the lid open for evaporation of residual ethanol.
  • SH-SY5Y cells were maintained and differentiated as described in Lee HJ, Khoshaghideh F, Patel S, Lee SJ (2004) J Neurosci 24: 1888-1896.
  • BV-2 microglia cell lines were maintained in DMEM with 5% fetal bovine serum (FBS) and penicillin and streptomycin.
  • Hybridoma cells were cultured in DMEM with 10% FBS and 100 ⁇ M hypoxanthine supplement (Invitrogen). Rat primary glial cultures were prepared as described in Lee HJ, Suk JE, Bae EJ, Lee SJ (2008) Biochem Biophys Res Commun 372: 423-428.
  • Differentiated SH-SY5Y cells were injected with adeno / ⁇ -syn. Two days after injection, cells were washed three times with DMEM and incubated in serum-free DMEM. After 18 hours of incubation at 37 ° C, the conditioned medium was collected and 4 ° C, 250x g in After centrifugation for 10 minutes, the supernatant was removed at 10,000 x 4 ° C to remove cellular debris. g in Centrifuge for 10 minutes. The supernatant was concentrated using an Amicon 10K MWCO filter (Millipore).
  • BV-2 microglia and astrocytes were divided into petri dishes the day before the experiment.
  • 0.2 ⁇ M ⁇ -synuclein fibrils or oligomers previously incubated with 5 ⁇ g / ml normal mouse IgG or ⁇ -synuclein antibodies at room temperature were added to the culture medium and cells. Monomer was added at 1 ⁇ M.
  • Conditioned medium (5 ⁇ concentrated) was preincubated with antibody for 5 minutes. Cells were then incubated at 37 ° C. and collected at the time indicated. To determine the rate of degradation, aggregates were taken up as above and cells were washed twice with cold PBS. Fresh culture medium was added and culture of cells was performed at 37 ° C. for the time stated.
  • BV-2 cells treated with 5 ⁇ g / ml of 274 antibody or control IgG were preincubated with various amounts of ⁇ -synuclein fibrils (0, 2.66, 13.3, 66.5, 133, and 330 nM).
  • ⁇ -synuclein fibrils (0, 2.66, 13.3, 66.5, 133, and 330 nM).
  • 5 ⁇ g / ml of 274 antibody preincubated with various amounts of ⁇ -synuclein monomer (0, 66.5, and 330 nM) was added to the cells.
  • Levels of absorbed antibodies were detected with anti-mouse IgG antibodies.
  • cells cultured on poly-L-lysine-coated coverslips were immobilized in 4% paraformaldehyde in PBS and taken up in 0.1% Triton X-100.
  • Cells were incubated in blocking solution (5% bovine serum albumin / 3% goat serum in PBS) prior to addition of the primary antibody diluted in blocking solution. After washing, the cells were incubated with fluorescent dye attached secondary antibody.
  • Nuclei were stained with TOPRO-3 iodide (Invitrogen) and coverslips mounted on slide glass using antifade reagent (Invitrogen). Olympus FV1000 confocal laser scanning microscope was used for the observation of the cells.
  • mice (line M) overexpressing ⁇ -synuclein derived from the platelet-derived growth factor-beta (PDGF-beta) promoter was used (Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M Takeda A, Sagara Y, Sisk A, Mucke L (2000) Science 287: 1265-1269).
  • This model was chosen because mice derived from these lines generate ⁇ -synuclein aggregates in both neuronal and glial cells distributed throughout the renal cortex, hippocampus, and striatum, similar to those described in Lewy body disease.
  • mice In these mice, ⁇ -synuclein accumulates in neural networks and neuronal cell bodies in the deep layers of the neocortex and hippocampus. ⁇ -synuclein begins to accumulate in neurons at 3 months of age and has maximum accumulation at 12 months of age. Thus, in this tg mouse model, ⁇ -synuclein pathology proceeds in a very constant and spatiotemporal order. All animal testing procedures were approved by the Experimental Animal Ethics Committee. All experiments were performed in female mice. Eight mice per group were used for each experiment. A total of 32 mice were used for this experiment; 16 mice were ⁇ -synuclein tg mice and the other 16 non-tg animal controls (9 months of age).
  • mice placed on Koft stereotactic devices and coordinates hippocampus: AP: 2.0 mm, outer 1.5 mm, depth 1.3 mm
  • mice placed in Franklin and Paxinos (Franklin KBJ, Paxinos G, (2008) The mouse brain in stereotaxic coordinates , Ed 3. Burlington, MA: Elsevier).
  • Hamilton syringes connected to a hydraulic system were used for antibody delivery for injection of the solution.
  • the needles were held for 5 minutes after completion of injection.
  • mice were unilaterally injected (right) for comparison with the contralateral side. Mice were survived 4 weeks after antibody injection. An additional group of twelve ⁇ -synuclein tg mice (3 months old) were unilaterally injected into the hippocampus with antibodies against ⁇ -synuclein (clone 274; 1 mg / ml) and at 1, 7, 14, and 28 days after injection Sacrificed for analysis. Mice were anesthetized with chloral hydrate and perfused to the heart with 0.9% saline according to the National Institutes of Health guidelines for human treatment of animals. The brain was removed and fixed for 48 hours at 4 °C in phosphate-buffer 4% paraformaldehyde, pH 7.4 for neuropathology analysis.
  • mice A total of 32 mice were used in this experiment, of which 16 were ⁇ -synuclein tg mice (line M, 10 months old) and the remaining 16 were non-tg animal controls (10 months old).
  • mice were survived for 1 month after the first injection and as described in Masliah E, Rockenstein E, Veinbergs I, Mallory M, Hashimoto M, Takeda A, Sagara Y, Sisk A, Mucke L (2000) Science 287: 1265-1269.
  • pole test a test for motor function and a spontaneous activity in the open field were performed. Behavioral tests were performed to perform mouse neuropathology analysis.
  • ⁇ -synuclein accumulation was performed on a series of sections, pre-floating, derived from non-tg and ⁇ -synuclein tg mice treated with antibodies to IgG or ⁇ -synuclein (clone 274). Sections were overnight incubated with anti- ⁇ -synuclein antibody (1: 500, affinity purified rabbit polyclonal; Millipore Bioscience Research Reagents) at 4 ° C., followed by biotinylated goat anti-earth IgG (1: 100; Vector Laboratories) After treatment with Avidin D-horseradish peroxidase (1: 200, ABC Elite; Vector Laboratories), it was detected with diaminobenzidine (DAB).
  • anti- ⁇ -synuclein antibody 1: 500, affinity purified rabbit polyclonal; Millipore Bioscience Research Reagents
  • biotinylated goat anti-earth IgG 1: 100; Vector Laboratories
  • Sections were imaged with an Olympus brightfield digital microscope. For each case, three sections were analyzed by dissection analysis using the Stereo-Investigator System (MBF Bioscience) and the results were averaged and expressed in number / mm 3 . To analyze the effect of antibodies on neurodegenerative and neuroinflammatory responses, sections were analyzed for NeuN (Millipore), glial fibrillary acidic protein (GFAP) (Millipore), Iba1 (Wako), IL6 (Novus Biologicals), and tumor necrosis factor Mouse monoclonal antibody against Abcam) and detected with DAB.
  • NeuN Millipore
  • GFAP glial fibrillary acidic protein
  • Iba1 Wako
  • IL6 Novus Biologicals
  • tumor necrosis factor Mouse monoclonal antibody against Abcam tumor necrosis factor Mouse monoclonal antibody against Abcam
  • Sections immunostained with antibody GFAP and Iba-1 were imaged with an Olympus brightfield digital microscope and absorbance and cell counts were analyzed with an Image Pro-Plus system. Sections immunostained with antibodies to NeuN were analyzed by dissection analysis using Stereo-Investigator System (MBF Bioscience) and the results were averaged and expressed in number / mm 3 .
  • ⁇ -synuclein (Millipore Bioscience Research Reagents, affinity purified polyclonal, 1: 500), mouse IgG (to detect 274 antibody distribution), and Double labeling with a combination of antibodies to Iba-1 (microglia marker).
  • ⁇ -synuclein immunoreactive structures were detected by the Tyramide Signal Amplification-Direct (Red) system (1: 100; NEN Life Sciences) while mouse IgG or Iba-1 were fluorescein isothiocyanate (FITC) -attached horse anti-mouse antibodies (1:75; Vector Laboratories).
  • FITC fluorescein isothiocyanate
  • ⁇ -synuclein was detected by the Tyramide Signal Amplification-Direct (Red) system (1: 100; NEN Life Sciences), while NeuN or GFAP was detected by FITC.

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Abstract

La présente invention concerne une composition pour le traitement de maladies associées à l'élimination de l'alpha-synucléine extracellulaire et un procédé pour le criblage d'un agent thérapeutique pour lesdites maladies.
PCT/KR2012/011024 2012-12-14 2012-12-17 Composition pour le traitement de maladies associées à l'élimination de l'alpha-synucléine extracellulaire et procédé pour le criblage d'un agent thérapeutique pour lesdites maladies WO2014092227A1 (fr)

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US20070232556A1 (en) * 2006-03-31 2007-10-04 Montine Thomas J Methods and compositions for the treatment of neurological diseases and disorders
KR20120090672A (ko) * 2011-02-08 2012-08-17 건국대학교 산학협력단 신규 항-α-시누클레인 단일클론항체 및 이를 이용한 ＥＬＩＳＡ 시스템

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WO2006020581A2 (fr) * 2004-08-09 2006-02-23 Elan Pharmaceuticals, Inc. Prevention et traitement de maladies synucleinopathique et amyloidogenique
US20070232556A1 (en) * 2006-03-31 2007-10-04 Montine Thomas J Methods and compositions for the treatment of neurological diseases and disorders
KR20120090672A (ko) * 2011-02-08 2012-08-17 건국대학교 산학협력단 신규 항-α-시누클레인 단일클론항체 및 이를 이용한 ＥＬＩＳＡ 시스템

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LEE, HE-JIN ET AL.: "Enzyme-linked immunosorbent assays for a-synuclein with species and multimeric state specificities", J. NEUROSCI. METHODS, vol. 199, no. 2, 31 May 2011 (2011-05-31), pages 249 - 257, XP028238899, DOI: doi:10.1016/j.jneumeth.2011.05.020 *

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