Anti-TLR2 antibodies and their applications

Abstract

This invention relates to novel applications of anti-TLR2 antibodies, and more particularly to the preventive or therapeutic applications of anti-TLR2 antibodies for multiple system atrophy. The anti-TLR2 antibodies according to this invention exhibit remarkable effects in reducing disease marker expression, decreasing inflammasomes, and restoring nerve bundles at the cellular and animal levels in multiple system atrophy, and can therefore be utilized in a variety of ways in the fields of prevention, improvement, and treatment of multiple system atrophy.

Description

Technical Field

[0001] The present invention relates to a novel use of an anti-TLR2 antibody, and more particularly, to a prophylactic or therapeutic use of an anti-TLR2 antibody for multiple system atrophy.

Background Art

[0002] As human lifespan increases, the incidence of neurodegenerative diseases including Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, Huntington's disease, Alzheimer's disease, multiple system atrophy, multi-infarct dementia, and age-related macular degeneration is also increasing. Currently, it is estimated that there are 24 million patients with neurodegenerative diseases worldwide. In addition, diseases induced by stroke or other forms of trauma or injury, which are other forms of neurodegenerative diseases, tend to increase every year.

[0003] According to the statistical data of the Health Insurance Review and Assessment Service, the total number of patients who received hospital treatment for multiple system atrophy in 2020 was 1,951. This is more than twice the number of 926 in 2016, an increase over a five-year period.

[0004] Among these, in particular, multiple system atrophy (MSA) is a rare progressive neurodegenerative disease characterized by the combined appearance of various symptoms. Patients with this disease have symptoms similar to Parkinson's disease, such as cerebellar ataxia and autonomic failure of the autonomic nervous system functions such as heartbeat, blood pressure, sweating, defecation, and bladder regulation. However, the exact cause of multiple system atrophy has not yet been clarified. Currently, no therapeutic agent has been developed, and drugs such as the combination of levodopa / carbidopa known as Sinemet, which is used for the treatment of Parkinson's disease patients and targets only symptom regulation, may be prescribed.

Summary of the Invention

Problems to be Solved by the Invention

[0005] Therefore, while conducting research to develop a therapeutic agent for multiple system atrophy, the present inventors confirmed the excellent therapeutic effect of anti-TLR2 antibodies on the symptoms of multiple system atrophy, which led to the completion of the present invention.

[0006] Therefore, an object of the present invention is to provide a composition for the prevention or treatment of multiple system atrophy, comprising an antibody or antigen-binding fragment thereof that specifically binds to TLR2 (Toll-Like Receptor 2).

[0007] Another object of the present invention is to provide a method for treating multiple system atrophy, comprising administering an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to TLR2 to an individual. [Means for solving the problem]

[0008] To achieve the above objective, the present invention provides a pharmaceutical composition for the prevention or treatment of multiple system atrophy, comprising an antibody or antigen-binding fragment thereof that specifically binds to a TLR2 comprising a heavy chain variable region including a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region including a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0009] The present invention also provides a veterinary composition for the prevention or treatment of multiple system atrophy, comprising an antibody or antigen-binding fragment thereof that specifically binds to a TLR2 comprising a heavy chain variable region including a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region including a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6.

[0010] Furthermore, the present invention provides a method for treating multiple system atrophy, comprising administering to an individual an effective amount of an antibody or antigen-binding fragment that specifically binds to a TLR2 comprising a heavy chain variable region including a heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, and a light chain variable region including a light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6. [Effects of the Invention]

[0011] The anti-TLR2 antibody according to the present invention exhibits remarkable effects in reducing disease marker expression, decreasing inflammasomes, and restoring nerve bundles at the cellular and animal levels in patients with multiple system atrophy, and can therefore be utilized in a variety of ways in the fields of prevention, improvement, and treatment of multiple system atrophy. [Brief explanation of the drawing]

[0012] [Figure 1] This figure shows the results of confirming the characteristics of an in vitro multiple system atrophy cell model using human neural stem cells. [Figure 2] This figure shows the results of confirming the DNA damage phenomenon in an in vitro multiple system atrophy cell model using human neural stem cells. [Figure 3] This figure shows the results of confirming the cellular senescence phenomenon in an in vitro multiple system atrophy cell model using human neural stem cells. [Figure 4] This figure shows the results of confirming the effect of NM-103 treatment on reducing GCI in an in vitro multiple system atrophy cell model using human neural stem cells. [Figure 5] This figure shows the results of evaluating the effect of NM-103 treatment on reducing DNA damage in an in vitro multiple system atrophy cell model using human neural stem cells. [Figure 6]This figure shows the results of confirming the effect of NM-103 treatment on reducing cellular senescence in an in vitro multiple system atrophy cell model using human neural stem cells. [Figure 7] This figure shows the results of an analysis of the survival rate of individuals treated with NM-103 in an in vivo model of multiple system atrophy. [Figure 8] This figure shows the results of confirming the effect of NM-103 treatment on improving motor function in an in vivo model of multiple system atrophy. [Figure 9] This figure shows the results of confirming the effect of NM-103 treatment on reducing GCI in an in vivo model of multiple system atrophy. [Figure 10A] This figure shows the results of confirming the neuroinflammatory reduction effect of NM-103 treatment in the cerebral cortex of an in vivo model of multiple system atrophy. [Figure 10B] This figure shows the results of confirming the neuroinflammatory reduction effect of NM-103 treatment in the cerebral white matter of an in vivo multiple system atrophy model. [Figure 10C] This figure shows the results of confirming the effect of NM-103 treatment on reducing neuroinflammation in the hippocampus of an in vivo model of multiple system atrophy. [Figure 10D] This figure shows the results of confirming the effect of NM-103 treatment on reducing neuroinflammation in the striatum of an in vivo model of multiple system atrophy. [Figure 10E] This figure shows the results of confirming the neuroinflammatory reduction effect of NM-103 treatment in the substantia nigra of the midbrain in an in vivo model of multiple system atrophy. [Figure 11] This figure shows the effect of NM-103 treatment on reducing inflammasomes in an in vivo model of multiple system atrophy. [Figure 12] This figure shows the effect of NM-103 treatment on nerve bundle recovery in an in vivo model of multiple system atrophy. [Modes for carrying out the invention]

[0013] The present invention will be described in detail below.

[0014] According to an aspect of the present invention, the present invention provides a composition for preventing or treating multiple system atrophy, comprising an antibody or an antigen-binding fragment thereof that specifically binds to TLR2, the antibody or antigen-binding fragment thereof comprising a heavy-chain variable region comprising a heavy-chain CDR1 having the amino acid sequence of SEQ ID NO: 1, a heavy-chain CDR2 having the amino acid sequence of SEQ ID NO: 2, and a heavy-chain CDR3 having the amino acid sequence of SEQ ID NO: 3, and a light-chain variable region comprising a light-chain CDR1 having the amino acid sequence of SEQ ID NO: 4, a light-chain CDR2 having the amino acid sequence of SEQ ID NO: 5, and a light-chain CDR3 having the amino acid sequence of SEQ ID NO: 6. The composition for preventing or treating multiple system atrophy according to the present invention may be a pharmaceutical composition or a veterinary composition.

[0015] As used herein, the term "antibody" means an anti-TLR2 antibody that specifically binds to TLR2. The scope of the present invention includes not only the complete antibody form that specifically binds to TLR2, but also antigen-binding fragments of the antibody molecule.

[0016] A complete antibody has a structure with two full-length light chains and two full-length heavy chains, and each light chain is linked to the heavy chain by a disulfide bond.

[0017] As used herein, the term "heavy chain" means a full-length heavy chain and fragments thereof, including a variable region domain VH containing an amino acid sequence having a sufficient variable region sequence for conferring specificity to an antigen, and three constant region domains CH1, CH2, and CH3. Also, as used herein, the term "light chain" means a full-length light chain and fragments thereof, including a variable region domain VL containing an amino acid sequence having a sufficient variable region sequence for conferring specificity to an antigen, and a constant region domain CL.

[0018] The aforementioned antibodies include subtypes of IgA, IgD, IgE, IgM, and IgG, and in particular, IgG includes IgG1, IgG2, IgG3, and IgG4. The heavy chain invariant regions have gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and have subclasses gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain invariant regions have kappa (κ) and lambda (λ) types.

[0019] Antibody antigen-binding fragments or antibody fragments refer to fragments that possess antigen-binding function, and include Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure that has variable regions of the light chain and heavy chain, an invariant region of the light chain and a first invariant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 is produced when the cysteine ​​residues in the hinge region of Fab' form disulfide bonds.

[0020] Fv represents the smallest antibody fragment, possessing only a heavy chain variable region and a light chain variable region. In double-chain Fv, the heavy chain variable region and the light chain variable region are linked by non-covalent bonds, while in single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked by covalent bonds via a peptide linker, or directly linked at the C-terminus, and can form dimer-like structures similar to double-chain Fv. Such antibody fragments can be produced using proteolytic enzymes (for example, Fab can be obtained by restrictive cleavage of a complete antibody with papain, and F(ab')2 can be obtained by cleavage with pepsin), or by using genetic engineering techniques.

[0021] The "Fv" fragment is an antibody fragment containing a complete antibody recognition and binding site. Such a region is a dimer in which one heavy chain variable domain and one light chain variable domain are linked.

[0022] The "Fab" fragment contains variable and invariant domains in the light chain and a variable and first invariant domain (CH1) in the heavy chain. The F(ab')2 antibody fragment generally contains a pair of Fab' fragments covalently linked by a cysteine ​​in the hinge region located at the C-terminus of the Fab' fragment.

[0023] A "single-stranded Fv (scFv)" antibody fragment is a structure consisting of a single polypeptide chain containing the VH and VL domains of the antibody. A polypeptide linker may be further included between the VH and VL domains to enable the scFv to form the desired structure for antigen binding.

[0024] In one example, the antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, scFv, Fab fragments, F(ab')2 fragments, disulfide-bound Fvs(sdFv), and anti-idiotype (anti-Id) antibodies or epitope-bound fragments of said antibodies.

[0025] The heavy chain invariant region may be selected from any of the isotypes gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε). For example, the invariant region may be gamma 1 (IgG1), gamma 2 (IgG2), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain invariant region may be kappa or lambda type.

[0026] The aforementioned monoclonal antibody refers to an antibody obtained from a substantially homogeneous antibody population; that is, an antibody in which the individual antibodies comprising the population are identical except for any spontaneous mutations that may exist in trace amounts. Because monoclonal antibodies are highly specific, they are induced against a single antigenic site. Typically, in contrast to ordinary (polyclonal) antibodies, which contain different antibodies against different determinants (epitopes), each monoclonal antibody points to a single determinant on the antigen.

[0027] An "epitope" refers to a protein determining site to which an antibody can specifically bind. Epitopes are typically composed of chemically active surface molecules, such as amino acids or their side chains, and generally possess not only specific three-dimensional structural features but also specific charge properties. Stereomorphic and non-stereomorphic epitopes are distinguished by the fact that binding to the former is lost in the presence of a denaturing solvent, while binding to the latter is not.

[0028] The aforementioned "humanized" non-human (e.g., mouse) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibodies) in which residues from the recipient's hypervariable region are replaced with residues from the hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, that possess the desired specificity, affinity, and capability.

[0029] The term "human antibody" refers to a molecule derived from human immunoglobulin, meaning that the entire amino acid sequence constituting the antibody, including the complementarity-determining region and structural region, is composed of human immunoglobulin.

[0030] The heavy chain and / or part of the light chain are identical or homologous to a corresponding sequence in an antibody belonging to a particular species or a particular antibody class or subclass, while the remaining chains are also identical or homologous to a "chimeric" antibody (immunoglobulin) that is also derived from another species or is identical or homologous to a corresponding sequence in an antibody belonging to another antibody class or subclass, as well as fragments of the antibody exhibiting the desired biological activity.

[0031] In the present invention, the "variable region" of the antibody refers to the light chain and heavy chain portions of the antibody molecule, including the amino acid sequences of the complementarity-determining region (CDR; i.e., CDR1, CDR2, and CDR3) and the skeletal region (FR). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0032] The "Complement Determining Region (CDR)" refers to the amino acid residues in the antibody's variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3.

[0033] The "skeletal region (FR)" consists of variable domain residues other than CDR residues. Each variable domain typically has four FRs: FR1, FR2, FR3, and FR4.

[0034] In a specific example of the present invention, the antibody that specifically binds to the TLR2 preferably includes a heavy chain variable region represented by the amino acid sequence of SEQ ID NO: 7.

[0035] In a specific example of the present invention, the antibody that specifically binds to the TLR2 preferably includes a light chain variable region represented by the amino acid sequence of SEQ ID NO: 8.

[0036] In a specific example of the present invention, the antibody that specifically binds to the TLR2 preferably includes a heavy chain represented by the amino acid sequence of SEQ ID NO: 9 and a light chain represented by the amino acid sequence of SEQ ID NO: 10.

[0037] The antibodies or antibody fragments of the present invention may include not only the sequences of the anti-TLR2 antibodies of the present invention described herein, but also their biological equivalents, within the range of being able to specifically recognize TLR2. For example, further modifications can be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are based on the relative similarity of amino acid side-chain substitutions, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, morphology, and type of amino acid side-chain substitutions reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar morphologies. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0038] Considering the variants with biologically equivalent activity described above, the antibodies of the present invention are analyzed to include sequences that exhibit substantial identity with the sequence described in the sequence number. Substantial identity means sequences that, when aligned to the sequence of the present invention and any other sequence to the greatest extent possible and analyzed using algorithms commonly used in the industry, exhibit a minimum of 90% homology, most preferably a minimum of 95% homology, 96% or higher, 97% or higher, 98% or higher, or 99% or higher homology. Alignment methods for sequence comparison are publicly known in the industry. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI and other sources and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx over the internet. BLAST is accessible at www.ncbi.nlm.nih.gov / BLAST / . The sequence homology comparison method using this program can be found at www.ncbi.nlm.nih.gov / BLAST / blast_help.html.

[0039] Based on this, the antibodies or antigen-binding fragments of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher homology with the explicitly described sequence or whole as described in the specification. Such homology may be determined by sequence comparison and / or alignment by methods known in the art. For example, the percentage sequence homology of the nucleic acids or proteins of the present invention may be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment, or macroscopic inspection.

[0040] In the present invention, "prevention" means any action that suppresses or delays the progression of clinical symptoms of a disease by administering the composition according to the present invention, and "treatment" means suppression of the progression of clinical symptoms of a disease, reduction or elimination of clinical symptoms of a disease.

[0041] If the composition of the present invention is a pharmaceutical composition, it may further contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers contained in the composition of the present invention are those commonly used in formulation and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the composition of the present invention may further contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like.

[0042] The pharmaceutical composition of the present invention may be administered orally or parenterally. When administered parenterally, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial injection, local injection, intranasal injection, intrapulmonary injection, or rectal injection.

[0043] When administered orally, proteins or peptides are digested; therefore, oral compositions need to be formulated to coat the active agent or protect it from digestion in the stomach. Furthermore, pharmaceutical compositions may be administered by any device that allows the active substance to reach target cells.

[0044] The appropriate dosage of the composition according to the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, condition, diet, administration time, route of administration, excretion rate, and response sensitivity, and a skilled, ordinary physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. For example, the daily dose of the pharmaceutical composition of the present invention is 0.0001 to 100 mg / kg. In this specification, the term "pharmaceutical effective dose" means an amount sufficient to prevent or treat cancer or infectious disease.

[0045] The pharmaceutical compositions of the present invention may be manufactured in unit volume form or contained in multi-volume containers by formulation using pharmaceutically acceptable carriers and / or excipients by a method readily available to a person with ordinary skill in the art to which the invention pertains. The dosage form may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may further contain a dispersant or stabilizer.

[0046] If the composition of the present invention is a veterinary composition, the composition of the present invention may further contain appropriate excipients and diluents commonly used in the manufacture of veterinary compositions.

[0047] Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil.

[0048] The veterinary compositions of the present invention may further contain fillers, anti-agglutinants, lubricants, wetting agents, spices, emulsifiers, preservatives, and the like. The veterinary compositions of the present invention may be formulated using methods well known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to an animal. The formulation may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, suppositories, sterile injection solutions, sterile topical preparations, and the like. The veterinary compositions of the present invention thus formulated may be administered via various routes, including orally, transdermally, subcutaneously, intravenously, dura materally, or intramuscularly.

[0049] The veterinary composition of the present invention may vary depending on the age, sex, and weight of the animal, but may be administered in amounts of 0.1 to 100 mg / kg once or several times a day. Furthermore, the dosage of β-NGF may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, etc. The aforementioned dosage does not limit the scope of the present invention in any respect.

[0050] In another aspect of the present invention, the present invention provides a method for treating multiple system atrophy, comprising administering to an individual an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to a TLR2 comprising a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6.

[0051] In specific examples of the present invention, the individual may be, but is not limited to, an individual that is expected to develop multiple system atrophy; an individual that has developed the disease; or an individual that has been determined to be cured.

[0052] Repetitive content has been omitted in consideration of the complexity of this specification, and terms not otherwise defined herein have the meanings commonly used in the art to which the present invention pertains. [Examples]

[0053] The present invention will be described in more detail below through examples. It will be obvious to those with ordinary skill in the art that these examples are merely illustrative and that the scope of the present invention is not limited by these examples.

[0054] [Example of experiment] Experimental Example 1: Method for differentiating human neural stem cells (hNSCs) into oligodendrocytes. To form oligospheres, hNSCs were cultured in oligodendrocyte progenitor cell (OPC) medium for 7 days. After dissociation into single OPCs, they were cultured in OPC medium for 5 days. After culturing, the medium was replaced with oligodendrocyte differentiation medium, and the cells were cultured for a further 14 days to obtain mature oligodendrocytes.

[0055] Experimental Example 2: Treatment of differentiating oligodendrocytes with extracellular α-synuclein. a-Syn conditioned medium (SCM) was treated at a concentration of 1000 ng / mL for 72 hours, and LacZ CM (LZCM), used as a control, was treated with the same volume as aSCM. aSCM was treated on oligodendrocyte differentiation day 11, and the experiment was conducted to coincide with the completion of oligodendrocyte differentiation and the completion of 72 hours of CM treatment.

[0056] Experimental Example 3. Treatment of differentiating oligodendrocytes with NM-103. NM-103 was used at concentrations of 10 μg / mL and 50 μg / mL, and pretreatment was performed 30 minutes before CM treatment.

[0057] Experimental Example 4. Immunofluorescence Staining Method Cells were fixed on coverslips with 4% PFA (in PBS). The fixed cells were treated with 0.1% Triton X-100 and then washed with PBS. The washed cells were treated with a blocking solution (5% bovine serum albumin and 3% goat serum in PBS) and then washed again. The cells were treated with a primary antibody diluted in the blocking solution for 16 hours. The cells treated with the primary antibody were washed. The washed cells were then treated with a secondary antibody conjugated with the fluorescent dyes Alexa488, Cy2, Rhodamine red-X, or Alexa647 for 2 hours. The cells treated with the secondary antibody were washed with PBS. The nuclei were further stained with Hoechst 33342. Slides were prepared using mounting solution on the coverslips. Subsequently, the slides are observed and analyzed using a Zeiss LSM 900 with Airyscan 2 (Zeiss, Oberkochen, Germany).

[0058] Experimental Example 5. Transgender Mouse Animal Experiment The aSyn A53T Tg mouse (line G2-3) was used as a disease model for MSA, in which the human aSyn A53T gene is expressed under the PrP promoter. NM-103 was administered to 6-month-old diseased mice, and in vivo efficacy evaluation was conducted.

[0059] Experimental Example 6. Immunofluorescence staining method using brain tissue sections Sectional fragments were obtained from mouse brain tissue fixed with 4% PFA using Cryotome equipment. These sectional fragments were infiltrated with a 0.2% Triton X-100 and 0.5% NGS (Normal Goat Serum) solution. After washing the sectional fragments with PBS, they were treated with a primary antibody for 16 hours. The primary antibody-treated sectional fragments were washed with PBS and then treated with a secondary antibody for 2 hours under light-shielding conditions, followed by immunofluorescence staining. The stained brain tissue was mounted using coverslips and slides and observed using a Zeiss LSM 900 with Airyscan 2 (Zeiss, Oberkochen, Germany).

[0060] Experimental Example 7. Myelin Sheath Analysis Using TEM Brain tissue was fixed with 4% formaldehyde and 1% glutaraldehyde, and stirred at 4°C for 16 hours. After stirring, it was fixed with 1% osmium tetroxide for 30 minutes. Brain tissue samples were treated with epoxy resin and then dehydrated with ethanol solutions (70%, 80%, 85%, 90%, 95%, 100%). Brain tissue sections were obtained using Ultramicotome, and then sample fragments were obtained on Cu grid and stained with uranyl acetate and lead citrate. The prepared tissues were imaged by Cryo-TEM to observe the myelin sheath.

[0061] [Examples] Example 1. Construction of an in vitro MSA disease cell model using human neural stem cells. Human neural stem cells (hNSCs) were differentiated to obtain oligodendrocytes (OLs). The differentiated OLs were treated for 72 hours with a cell culture medium containing a large amount of the disease-associated toxic protein α-synuclein (aSyn) (aSyn Conditioned Media; aSCM) to construct an in vitro multiple system atrophy (MSA) cell model. The results of the analysis of the constructed in vitro MSA cell model are shown in Figure 1.

[0062] As shown in Figure 1, when OLs differentiated from hNSCs were treated with aSCM, isotopic detection of TPPP / p25a and ubiquitin with aSyn was confirmed. The aforementioned TPPP / p25a ubiquitin is a characteristic feature of glial cytoplasmic inclusions (GCIs) found in MSA patients.

[0063] Example 2. Confirmation of DNA damage and cellular senescence in an in vitro MSA disease cell model. Immunohistochemical staining was used to confirm DNA damage and cellular senescence in the in vitro MSA disease cell model constructed in Example 1. The results of confirming DNA damage in the in vitro MSA disease cell model are shown in Figure 2, and the results of confirming cellular senescence are shown in Figure 3.

[0064] As shown in Figure 2, DNA damage associated with degenerative brain disease was confirmed in an in vitro MSA cell model induced by aSCM treatment.

[0065] As shown in Figure 3, we confirmed cellular senescence associated with degenerative brain disease in an in vitro MSA cell model induced by aSCM treatment.

[0066] Example 3. Construction of an in vitro MSA disease cell model treated with NM-103. The in vitro MSA disease cell model constructed in Example 1 was treated with NM-103 at a concentration of 50 μg / mL 30 minutes before being treated with aSCM for 72 hours. NM-103 is an anti-TLR2 (toll-like receptor 2) antibody, also known as tomaralimab. The specific sequence is shown in Table 1.

[0067] [Table 1]

[0068] Example 4. Evaluation of the efficacy of NM-103 in an in vitro MSA disease cell model. 4-4. GCI reduction, a disease phenomenon of MSA caused by NM-103 The GCI-reducing efficacy of NM-103 was evaluated in an in vitro MSA disease cell model, and the results are shown in Figure 4.

[0069] As shown in Figure 4, we confirmed that treating an in vitro MSA disease cell model with the anti-TLR2 antibody NM-103 reduced the GCI disease marker, specifically serine 129-phosphorylated α-synuclein (pS129-aSyn).

[0070] 4-2. Reduction of DNA damage, a disease phenomenon of MSA, caused by NM-103. The effect of NM-103 on reducing DNA damage was evaluated in an in vitro MSA disease cell model, and the results are shown in Figure 5.

[0071] As shown in Figure 5, we confirmed that treating an in vitro MSA disease cell model with the anti-TLR2 antibody NM-103 reduced the DNA damage marker 53BP1 (p53-binding protein 1).

[0072] 4-3. Reduction of cellular senescence symptoms, a disease phenomenon associated with MSA, by NM-103. The effect of NM-103 on reducing cellular senescence was evaluated in an in vitro MSA disease cell model, and the results are shown in Figure 6.

[0073] As shown in Figure 6, we confirmed that treating an in vitro MSA disease cell model with the anti-TLR2 antibody NM-103 reduced the cellular senescence marker H3K9m3.

[0074] Example 5. Therapeutic efficacy experiment of NM-103 in an MSA in vivo model using α-synuclein disease mutation overexpressing mice 5-1. Survival rate after NM-103 treatment In an in vivo model of MSA, the survival rate of individuals treated with NM-103 was analyzed. NM-103 was administered by tail vein intravenous (IV) injection. The results of the survival rate analysis are shown in Figure 7.

[0075] As shown in Figure 7, the MSA in vivo model (TG mouse) showed a gradual increase in mortality over time, with a survival rate of 50%. In contrast, the NM-103 administration groups (TG mouse + NM-103 3 mg / kg, TG mouse + NM-103 10 mg / kg) showed a survival rate of 100%.

[0076] 5-2. Effects of NM-103 treatment on improving athletic performance Through GST and Pole Test behavioral experiments, the effect of NM-103 treatment on improving motor performance in an MSA in vivo model was confirmed. The results of confirming the improvement in motor performance are shown in Figure 8.

[0077] As shown in Figure 8, the NM-103 administration groups (TG mouse + NM-103 3 mg / kg, TG mouse + NM-103 10 mg / kg) showed improved motor function.

[0078] 5-3. Decreased MSA pathology marker GCI due to NM-103 We confirmed the expression of the MSA marker GCI in an MSA in vivo model by NM-103 treatment through fluorescence staining analysis. The results of confirming GCI expression are shown in Figure 9.

[0079] As shown in Figure 9, the NM-103 administration groups (TG mouse + NM-103 3mpk, TG mouse + NM-103 10mpk) showed a decrease in GCI, a disease-specific marker for MSA, within oligodendrocytes.

[0080] 5-4. Reduction of neuroinflammation in MSA mouse models using NM-103. Through fluorescence staining analysis, the neuroinflammatory reduction effect of NM-103 treatment was confirmed in an in vivo model of MSA. The results of the neuroinflammatory reduction effects of NM-103 in the cerebral cortex, white matter, hippocampus, striatum, and substantia nigra are shown in Figures 10A-E, respectively.

[0081] As shown in Figures 10A-E, the NM-103 administration groups (TG mouse + NM-103 3mpk, TG mouse + NM-103 10mpk) showed reduced levels of glial cell-induced neuroinflammation (GFAP, Iba-I) markers in multiple brain regions (cerebral cortex, cerebral white matter, hippocampus, striatum, and substantia nigra).

[0082] 5-6. Reduction of inflammasomes in MSA mouse models by NM-103 The inflammasome reduction effect of NM-103 treatment was confirmed in an in vivo model of MSA using the FLICA assay. The results of the confirmed inflammasome reduction effect are shown in Figure 11.

[0083] As shown in Figure 11, the MSA model (TG) showed an increase in inflammasomes in oligodendrocytes. In contrast, the NM-103 administration groups (TG mouse + NM-103 3mpk, TG mouse + NM-103 10mpk) showed a decrease in inflammasomes.

[0084] 5-7. Restoration of nerve bundles in an MSA mouse model using NM-103 In an in vivo model of MSA, the effect of NM-103 treatment on nerve bundle restoration was confirmed. The results of confirming the nerve bundle restoration effect are shown in Figure 12.

[0085] As shown in Figure 12, the MSA model (TG) showed increased nerve myelin sheath damage and demyelination. In contrast, the NM-103 administration groups (TG mouse + NM-103 3mpk, TG mouse + NM-103 10mpk) showed nerve myelin protection and suppressed demyelination (i.e., nerve bundle recovery).

[0086] Having described in detail certain aspects of the present invention, it is clear to those with ordinary skill in the art that such specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, and A heavy chain variable region containing heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and A pharmaceutical composition for the prevention or treatment of multiple system atrophy, comprising a light chain variable region containing a light chain CDR3 having the amino acid sequence of SEQ ID NO: 6, and an antibody or antigen-binding fragment thereof that specifically binds to TLR2 (toll-like receptor 2).

2. The composition according to claim 1, wherein the antibody that specifically binds to the TLR2 includes a heavy chain variable region represented by the amino acid sequence of SEQ ID NO:

7.

3. The composition according to claim 1, wherein the antibody specifically binds to the TLR2 includes a light chain variable region represented by the amino acid sequence of SEQ ID NO:

8.

4. The antibody that specifically binds to the aforementioned TLR2 is The heavy chain represented by the amino acid sequence of Sequence ID No. 9, The composition according to claim 1, comprising a light chain represented by the amino acid sequence of Sequence ID No.

10.

5. Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, and A heavy chain variable region containing heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and A veterinary composition for the prevention or treatment of multiple system atrophy, comprising a light chain variable region containing a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6, and an antibody or antigen-binding fragment thereof that specifically binds to TLR2 (toll-like receptor 2).

6. Heavy chain CDR1 containing the amino acid sequence of SEQ ID NO: 1, Heavy chain CDR2 containing the amino acid sequence of SEQ ID NO: 2, and A heavy chain variable region containing heavy chain CDR3 containing the amino acid sequence of SEQ ID NO: 3, Light chain CDR1 containing the amino acid sequence of SEQ ID NO: 4, Light chain CDR2 containing the amino acid sequence of SEQ ID NO: 5, and A method for treating multiple system atrophy, comprising administering to an individual an effective amount of an antibody or its antigen-binding fragment that specifically binds to a TLR2 (Toll-Like Receptor 2) containing a light chain variable region including a light chain CDR3 containing the amino acid sequence of SEQ ID NO: 6.

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