Pharmaceutical composition for treating brain diseases comprising antibody specifically binding to ASM protein as active ingredient

Abstract

The present invention relates to use of an antibody that specifically binds to an acid sphingomyelinase (ASM) protein. Particularly, the antibody or antigen-binding fragment thereof according to the present invention specifically binds to the ASM protein with high binding affinity, significantly inhibits the activity of the ASM protein present in the cell membrane, and, in an Alzheimer's disease animal model, does not exhibit toxicity and exhibits the effects of improving cognitive memory, inhibiting the activity of the ASM protein, inhibiting the accumulation of amyloid-β and tau protein, and inhibiting the occurrence of neuroinflammation, and thus can be effectively used in the treatment of brain diseases.

Description

All marker genes known in the art may be used as a marker gene included in the expression vector, and may be specifically an antibiotic resistant gene. Specifically, the antibiotic resistant gene may be a gene showing resistance to antibiotics including ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, neomycin, tetracycline, and the like. In still another aspect of the present disclosure, the pharmaceutical composition according to the present disclosure may include as an active ingredient a host cell including the nucleic acid or expression vector as described above. All types of cells known to be usable to produce antibodies or antigen-binding fragments thereof in the art may be used as a host cell. Specifically, the host cell may be a prokaryotic cell, yeast, or a eukaryotic cell. Examples of the prokaryotic cell may include E. coli , the genus Bacillus strains, the genus Streptomyces strain, the genus Pseudomonas strain, the genus Staphylococcus strain, and the like, and examples of the yeast may include Saccharomyces cerevisiae and the like. Example of the eukaryotic cell may include COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, HEK293, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PERC6, SP2 / 0, NS-0, U20S, HT1080, and the like. The host cell may be transduced with the nucleic acid or expression vector as described above according to a method known in the art. Specifically, the transduction may be performed by transient transfection, microinjection, transduction, cell infusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, a gene gun, or the like. The methods may also be appropriately modified by a person skilled in the art. The brain disease may include all types of brain diseases known in the art and, specifically, the brain disease may be a degenerative brain disease. As an example, the degenerative brain disease may be a disease in which the expression or aggregation level of amyloid-p is higher or at risk of being higher than normal. For example, the degenerative brain disease may be dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, mild cognitive impairment, cerebral amyloid angiopathy, Down syndrome, amyloid stroke, systemic amyloid disease, Dutch type amyloidosis, Niemann-Pick disease, senile dementia, amyotrophic lateral sclerosis, spinocerebellar atrophy, Tourette's syndrome, Friedrich's ataxia, Machado-Joseph's disease, Lewy body dementia, dystonia, progressive supranuclear palsy, or frontotemporal dementia. The pharmaceutical composition according to the present disclosure may include an antibody or antigen-binding fragment thereof, which is an active ingredient and specifically binds to an ASM protein, in 10 to 95 wt% relative to the total weight thereof. In addition, the pharmaceutical composition of the present disclosure may further include, in addition to the above active ingredient, at least one active ingredient that exhibits the same or similar function to the above ingredient. The pharmaceutical composition of the present disclosure may include a carrier, a diluent, an excipient, or a mixture thereof that is commonly used in biological preparations. Any pharmaceutically acceptable carrier may be used as long as it is suitable for in vivo delivery of the composition. Specifically, the carrier may be a compound, saline, sterile water, Ringer's solution, a dextrose solution, a maltodextrin solution, glycerol, ethanol, or a mixture thereof, described in Merck Index, 13th ed., Merck & Co. Inc. In addition, common additives, such as an antioxidant, a buffer, and a bacteriostatic agent, may be added as needed. When the composition is formulated into a preparation, a diluent or an excipient that is commonly used, such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant may be added. The composition of the present disclosure may be formulated into an oral preparation or a parenteral preparation. Examples of the oral preparation may include a solid preparation and a liquid preparation. The solid preparation may be a tablet, a pill, a powder, granules, a capsule, or a troche, and these solid preparations may be prepared by adding at least one excipient to the composition. The excipient may be starch, calcium carbonate, sucrose, lactose, gelatin, or a mixture thereof. The solid preparation may include a lubricant, and examples thereof may be magnesium stearate, talc, and the like. The liquid formulation may be a suspension, an oral liquid, an emulsion, or a syrup. The liquid formulation may include an excipient, such as a wetting agent, a sweetener, an aromatic agent, or a preservative, and the like. Examples of the parenteral preparation may include an injection, a suppository, a respiratory inhalation powder, a spray aerosol, a powder, a cream, and the like. The injection may include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, and the like. Examples of the non-aqueous solvent or suspension may use propylene glycol, polyethylene glycol, a vegetable oil such as olive oil, an injectable ester such as ethylolate, and the like. Furthermore, the present disclosure provides a health functional food for preventing or alleviating a brain disease, the health functional food including as an active ingredient an antibody or antigen-binding fragment thereof that specifically binds to an ASM protein. The antibody or antigen-binding fragment thereof that specifically binds to an ASM protein and included as an active ingredient in the health functional food according to the present disclosure may have the characteristics as described above. The antibody or antigen-binding fragment thereof that specifically binds to an ASM protein of the present disclosure may be added as it is to a food or may be used along with other foods or food ingredients. The content of the added active ingredient may be determined according to the purpose, and may be usually 0.01 to 90 parts by weight relative to the total weight of the food. The form and kind of health functional food are not particularly limited. Specifically, the health functional food may be in the forms of a tablet, a capsule, a powder, granules, a liquid, and a pill. The health functional food may include as additional ingredients, several flavoring agents, sweeteners, or natural carbohydrates. The sweetener may be a natural or synthetic sweetener, and examples of the natural sweetener include thaumatin, a stevia extract, and the like. Examples of the synthetic sweetener are saccharin, aspartame, and the like. In addition, the natural carbohydrates may be monosaccharides, disaccharides, polysaccharides, oligosaccharides, and sugar alcohols. The health functional food of the present disclosure may further include, in addition to the above-described additional ingredients, nutritional supplements, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and the like. These ingredients may be used either alone or in combination. The proportion of such an additive may be selected within a range of 0.01 to 0.1 parts by weight relative to 100 parts by weight of the composition of the present disclosure. In accordance with aspects of the present disclosure, the present disclosure provides a method for preventing, alleviating, or treating a brain disease, the method including a step of administering to a subject an antibody or antigen-binding fragment thereof that specifically binds to an ASM protein. The antibody or antigen-binding fragment thereof that specifically binds to an ASM protein administered for preventing, alleviating, or treating a brain disease according to the present disclosure may have the characteristics as described above. The subject may be a mammal and particularly a human. The administration may be conducted orally or parenterally according to the desired method. Examples of the parenteral administration may include an intraperitoneal, rectal, subcutaneous, intravenous, intramuscular, or intrathoracic injection. In addition, the active ingredient may be administered in a pharmaceutically effective amount. The pharmaceutically effective amount may vary depending on the type or severity of disease, the activity of a drug, the sensitivity of a patient to a drug, the time of administration, the route of administration, the duration of treatment, a drug to be used with the composition, and the like. However, for desirable effects, the amount of the active ingredient included in the pharmaceutical composition according to the present disclosure may be 0.0001 to 1,000 mg / kg, specifically 0.001 to 500 mg / kg. The administration may be performed once or several times a day. The administration may be alone or in combination with other medicines. In the combinative administration, the administration may be performed sequentially or simultaneously. In accordance with aspects of the present disclosure, the present disclosure provides an antibody or antigenbinding fragment thereof that specifically binds to an ASM protein for use in a method for preventing, alleviating, or treating a brain disease. The antibody or antigen-binding fragment thereof that specifically binds to an ASM protein for use in a method for preventing, alleviating, or treating a brain disease according to the present disclosure may have the characteristics as described above. MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present disclosure will be described in detail by the following examples. However, the following exemplary embodiments are merely for illustrating the present disclosure, and are not intended to limit the scope of the present disclosure. Any embodiment that has substantially the same constitution as the technical idea described in the claims of the disclosure and achieves the same effects of action are included in the technical scope of the present invention. Example 1: Production of Antibodies Specifically Binding to Acid Sphingomyelinase (ASM) Protein Antibodies that specifically bind to human ASM protein (SEQ ID NO: 66) and mouse ASM protein (SEQ ID NO: 67) were produced as follows. Specifically, phages with scFv specifically binding to the human ASM protein or mouse ASM protein were selected by performing panning through a typical method using a recombinant human and mouse ASM protein and the human synthetic scFv-phage library. Nucleic acid sequences encoding the selected scFv were analyzed, and amino acid sequences translated therefrom were identified. Expression vectors were produced such that a mouse heavy chain constant region and a mouse light chain X constant region consisting of the nucleotide sequences as set forth in SEQ ID NOS: 75 and 77, respectively, were linked to the carboxy termini of the heavy chain variable region and the light chain variable region of each of the identified scFv sequences. The amino acid sequences and nucleic acid sequences of the heavy chain variable regions constituting scFv contained in the produced expression vectors are shown in Table 1, and the amino acid sequences and nucleic acid sequences of the light chain 5 variable regions thereof are disclosed in Table 2. TABLE 1 Antibody # Sequence Sequence Number #9101 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAP GKGLEWVSGIYPNGGNKYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKNAYRFDYWGQGTLVTVS S SEQ ID NO: 1 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCC TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCA CCTTTAGCAATTATGCTATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGGGATCTATCCTAATGG TGGTAATAAATATTACGCTGATTCTGTAAAAGGTCGGTTCA CCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAA ATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTG TGCGAAAAATGCTTATCGTTTCGACTACTGGGGCCAGGGTA CACTGGTCACCGTGAGCTCA SEQ ID NO: 2 #9102 EVQLLE S GGGLVQPGGSLRL S CAASGFTFSGYYMSWVRQAP GKGLEWVSLISPGSGSIYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKSWHHFDYWGQGTLVTVSS SEQ ID NO: 3 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCC TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCA CCTTTAGCGGTTATTATATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCATTGATCTCTCCTGGTAG TGGTAGTATATATTACGCTGATTCTGTAAAAGGTCGGTTCA CCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAA ATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTG TGCGAAATCTTGGCATCATTTCGACTACTGGGGCCAGGGTA CACTGGTCACCGTGAGCTCA SEQ ID NO: 4 #9104 EVQLLE S GGGLVQPGGSLRL S CAASGFTFSNYYMSWVRQAP GKGLEWVSGIYYGSGNIYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARDTPGFDYWGQGTLVTVS S SEQ ID NO: 5 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCC SEQ ID TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCA CCTTTAGCAATTATTATATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGGGATCTATTATGGTAG TGGTAATATATATTACGCTGATTCTGTAAAAGGTCGGTTCA CCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAA ATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTG TGCGAGAGATACGCCTGGGTTCGACTACTGGGGCCAGGGTA CACTGGTCACCGTGAGCTCA NO: 6 #9108 EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAP GKGLEWVSAIYPGGGSIYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARDVLGLTPKPFDYWGQGTLVTVSS SEQ ID NO: 7 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCC TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCA CCTTTAGCAATTATGCTATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCGATCTATCCTGGTGG TGGTAGTATATATTACGCTGATTCTGTAAAAGGTCGGTTCA CCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAA ATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTG TGCGAGAGATGTTTTGGGTCTGACTCCTAAGCCGTTCGACT ACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA SEQ ID NO: 8 #9113 EVQLLE S GGGLVQPGGSLRL S CAASGFTFS SYYMSWVRQAP GKGLEWVS SIS PGGS SIYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCAKGASLFDYWGQGTLVTVSS SEQ ID NO: 9 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCC TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCA CCTTTAGCAGTTATTATATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCATCGATCTCTCCTGGTGG TAGTAGTATATATTACGCTGATTCTGTAAAAGGTCGGTTCA CCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAA ATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTG TGCGAAAGGGGCGTCTCTGTTCGACTACTGGGGCCAGGGTA CACTGGTCACCGTGAGCTCA SEQ ID NO: 10 #9123 EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAMSWVRQAP GKGLEWVSAISYGGGNIYYADSVKGRFTISRDNSKNTLYLQ MNSLRAEDTAVYYCARVGGMCTRRQCYYDYGMDVWGQGTLV TVSS SEQ ID NO: 11 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCC TGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCCGGATTCA CCTTTAGCGATTATGCTATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCGATCTCTTATGGTGG TGGTAATATATATTACGCTGATTCTGTAAAAGGTCGGTTCA CCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAA SEQ ID NO: 12 ATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTG TGCGAGAGTTGGTGGTATGTGTACTAGGCGTCAGTGTTATT ATGATTATGGTATGGACGTCTGGGGCCAGGGTACACTGGTC ACCGTGAGCTCA TABLE 2 Antibody # Sequence SEQ ID NO #9101 QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNYVSWYQQLPG TAPKLLIYADSKRPSGVPDRFSGSKSGTSASLAIGGLRSEDE ADYYCGSWDYSLNAYVFGGGTKLTVL SEQ ID NO: 13 CAGTCTGTGCTGACTCAGCCACCCTCAGCTAGCGGGACCCCC GGGCAGAGGGTCACCATCTCTTGTAGTGGCTCTTCATCCAAT ATTGGCAATAATTATGTCTCCTGGTACCAGCAGCTCCCAGGA ACGGCCCCCAAACTCCTCATCTATGCTGATAGTAAGCGGCCA AGCGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC TCAGCCTCGCTGGCCATCGGTGGGCTCCGGTCCGAGGATGAG GCTGATTATTACTGTGGTTCTTGGGATTATAGCCTGAATGCT TATGTCTTCGGCGGAGGCACCAAGCTTACGGTCCTA SEQ ID NO: 14 #9102 QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNPVYWYQQLPG TAPKLLIYANNQRPSGVPDRFSGSKSGTSASLAISGLRSEDE ADYYCAAWDS SLS GYVFGGGTKLTVL SEQ ID NO: 15 CAGTCTGTGCTGACTCAGCCACCCTCAGCTAGCGGGACCCCC GGGCAGAGGGTCACCATCTCTTGTAGTGGCTCTTCATCTAAT ATTGGCAATAATCCTGTCTACTGGTACCAGCAGCTCCCAGGA ACGGCCCCCAAACTCCTCATCTATGCTAATAATCAGCGGCCA AGCGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC TCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAG GCTGATTATTACTGTGCTGCTTGGGATTCTAGCCTGAGTGGT TATGTCTTCGGCGGAGGCACCAAGCTTACGGTCCTA SEQ ID NO: 16 #9104 QSVLTQPPSASGTPGQRVTISCTGSSSNIGNNAVNWYQQLPG TAPKLLIYYDSHRPSGVPDRFSGSKSGTSASLAISGLRSEDE ADYYCGAWDYSLSAYVFGGGTKLTVL SEQ ID NO: 17 CAGTCTGTGCTGACTCAGCCACCCTCAGCTAGCGGGACCCCC GGGCAGAGGGTCACCATCTCTTGTACTGGCTCTTCATCTAAT ATTGGCAATAATGCTGTCAACTGGTACCAGCAGCTCCCAGGA ACGGCCCCCAAACTCCTCATCTATTATGATAGTCATCGGCCA AGCGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC TCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAG GCTGATTATTACTGTGGTGCTTGGGATTATAGCCTGAGTGCT TATGTCTTCGGCGGAGGCACCAAGCTTACGGTCCTA SEQ ID NO: 18 #9108 QSVLTQPPSASGTPGQRVTLSCTGSSSNIGSNTVYWYQQLPG TAPKLLIYANSQRPSGVPDRFSGSKSGTSASLAISGLRSEDE ADYYCGSWDY SLS GYVFGGGTKLTVL SEQ ID NO: 19 CAGTCTGTGCTGACTCAGCCACCCTCAGCTAGCGGGACCCCC GGGCAGAGGGTCACCCTCTCTTGTACTGGCTCTTCATCTAAT ATTGGCAGTAATACTGTCTACTGGTACCAGCAGCTCCCAGGA ACGGCCCCCAAACTCCTCATCTATGCTAATAGTCAGCGGCCA AGCGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC TCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAG GCTGATTATTACTGTGGTTCTTGGGATTATAGCCTGAGTGGT TATGTCTTCGGCGGAGGCACCAAGCTTACGGTCCTA SEQ ID NO: 20 #9113 QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNAVSWYQQLPG TAPKLLIYSDNKRPSGVPDRFSGSKSGTSASLAISGLRSEDE ADYYCGTWDASLNAYVFGGGTKLTVL SEQ ID NO: 21 CAGTCTGTGCTGACTCAGCCACCCTCAGCTAGTGGGACCCCC GGGCAGAGGGTCACCATCTCTTGTAGTGGCTCTTCATCTAAT ATTGGCAATAATGCTGTCTCCTGGTACCAGCAGCTCCCAGGA ACGGCCCCCAAACTCCTCATCTATTCTGATAATAAGCGGCCA AGCGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC TCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAG GCTGATTATTACTGTGGTACTTGGGATGCTAGCCTGAATGCT TATGTCTTCGGCGGAGGCACCAAGCTTACGGTCCTA SEQ ID NO: 22 #9123 QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPG TAPKLLIYDNSKRPSGVPDRFSGSKSGTSASLAISGLRSEDE ADYYCGTWDDSL S GYVFGGGTKLTVL SEQ ID NO: 23 CAGTCTGTGCTGACTCAGCCACCCTCAGCTAGCGGGACCCCC GGGCAGAGGGTCACCATCTCTTGTAGTGGCTCTTCATCTAAT ATTGGCAGTAATACTGTCAACTGGTACCAGCAGCTCCCAGGA ACGGCCCCCAAACTCCTCATCTATGATAATAGTAAGCGGCCA AGCGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACC TCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAAGATGAG GCTGATTATTACTGTGGTACTTGGGATGATAGCCTGAGTGGT TATGTCTTCGGCGGAGGCACCAAGCTTACGGTCCTA SEQ ID NO: 24 The expression vector employed a mammalian expression vector. The produced expression vectors were transformed into the CHO or HEK293 cell line to produce full-length 5 antibodies binding to human ASM protein, or to both human and mouse ASM proteins. Example 2: Determination of Complementarity Determining Regions (CDRs) 5       The complementarity determining regions in the produced scFv fragments were identified by a conventional method, and as a result, the CDR sequences of the heavy chain variable regions are shown in Table 3 and the CDR sequences of the light chain variable regions are shown in Table 4. 10 TABLE 3 Antibody # CDR Sequence SEQ ID NO #9101 CDR1 NYAMS SEQ ID NO 25 CDR2 GIYPNGGNKYYADSVKG SEQ ID NO 26 CDR3 NAYRFDY SEQ ID NO 27 #9102 CDR1 GYYMS SEQ ID NO 28 CDR2 LISPGSGSIYYADSVKG SEQ ID NO 29 CDR3 SWHHFDY SEQ ID NO 30 #9104 CDR1 NYYMS SEQ ID NO 31 CDR2 GIYYGSGNIYYADSVKG SEQ ID NO 32 CDR3 DTPGFDY SEQ ID NO 33 #9108 CDR1 NYAMS SEQ ID NO 34 CDR2 AIYPGGGSIYYADSVKG SEQ ID NO 35 CDR3 DVLGLTPKPFDY SEQ ID NO 36 #9113 CDR1 SYYMS SEQ ID NO 37 CDR2 SISPGGSSIYYADSVKG SEQ ID NO 38 CDR3 GASLFDY SEQ ID NO 39 #9123 CDR1 DYAMS SEQ ID NO 40 CDR2 AISYGGGNIYYADSVKG SEQ ID NO 41 CDR3 VGGMCTRRQCYYDYGMDV SEQ ID NO 42 TABLE 4 Antibody # CDR Sequence SEQ ID NO #9101 CDR1 SGSSSNIGNNYVS SEQ ID NO 43 CDR2 ADSKRPS SEQ ID NO 44 CDR3 GSWDYSLNAYV SEQ ID NO 45 #9102 CDR1 SGSSSNIGNNPVY SEQ ID NO 46 CDR2 ANNQRPS SEQ ID NO 47 CDR3 AAWDSSLSGYV SEQ ID NO 48 #9104 CDR1 TGSSSNIGNNAVN SEQ ID NO 49 CDR2 YDSHRPS SEQ ID NO 50 CDR3 GAWDYSLSAYV SEQ ID NO 51 #9108 CDR1 TGSSSNIGSNTVY SEQ ID NO 52 CDR2 ANSQRPS SEQ ID NO 53 CDR3 GSWDYSLSGYV SEQ ID NO 54 #9113 CDR1 SGSSSNIGNNAVS SEQ ID NO 55 CDR2 SDNKRPS SEQ ID NO 56 CDR3 GTWDASLNAYV SEQ ID NO 57 #9123 CDR1 SGSSSNIGSNTVN SEQ ID NO 58 CDR2 DNSKRPS SEQ ID NO 59 CDR3 GTWDDSLSGYV SEQ ID NO 60 Experimental Example 1: Confirmation of Binding to ASM Protein The binding of the produced antibodies, specifically 5 binding to the ASM protein, to the ASM protein were analyzed by ELISA. First, 100 pl of 1 pg / mL recombinant human ASM protein (R&D systems) was added to a multi-array 96-well plate (Thermo scientific) and left at 4°C for 16 hours to coat the 10 plate. Then, 200 pl of PBS containing 5% BSA was added to the coated plate, followed by incubation at 37°C for about 2 hours, and then the plate was washed three times with PBS containing 0.05% Tween 20. The plate was treated with the anti-ASM antibody at 0.0001, 0.001, 0.01, 0.1, 1, 10, or 100 nM, followed by incubation at 37°C for about 1 hours. After the incubation, the plate was washed three times with PBS containing 0.05% Tween, and 100 pl of goat-anti-mouse IgG-HRP antibody (Jackson Immunoresearch) as a secondary antibody was added. After incubation again at 37°C for about 1 hour, the plate was washed three times with PBS containing 0.05% Tween. Then, 100 pl of a tetramethylbenzidine (TMB) substrate was added thereto, followed by incubation at room temperature for additional about 5 minutes, and then the reaction was terminated by 100 pl of a 2 N sulfuric acid solution, and the absorbance was measured at a wavelength of 450 nm. As a result, the measured absorbance was shown in FIG. 2 and the EC50 values of antibodies calculated from the absorbance values are shown in Table 5. TABLE 5 Antibody # EC50 (nM) #9101 0.0815 #9102 0.0954 #9104 1.948 #9108 0.3269 #9123 0.7578 As shown in FIG. 2, all of the five types of antibodies produced above were bound to the ASM protein in a concentration-dependent manner. In particular, as shown in Table 5, antibody #9101 showed the lowest EC50 value and thus had the highest binding ability to ASM protein. Experimental Example 2: Confirmation of Binding Affinity with ASM Protein The binding affinity of the produced antibodies, specifically binding to the ASM protein, with the ASM protein and the interactive dynamics therebetween were measured using an Octet® QK384 system (Pall Life Sciences). First, 20 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 40 mM N-hydroxysulfosuccinimide (sulfo-NHS) solutions were treated on an AR2G sensor (ForteBio) to activate carboxyl groups therein. Meanwhile, the 10 pg / mL recombinant human ASM protein or 2.5 pg / mL recombinant mouse ASM protein was prepared by dilution in a 10 mM sodium acetate solution (pH 5.0) (ForteBio). The solution having the diluted ASM protein was added to the AR2G sensor having activated carboxyl groups to obtain the AR2G sensor in which the recombinant human or mouse ASM protein was immobilized. A 1 M ethanol amine (ForteBio) was added to the sensor in which the obtained ASM protein was immobilized, to inactivate the remaining unreacted carboxyl groups. The antibodies produced in Example 1 were added at concentrations of 0.4, 2, or 10 nM, and the association phase of the reaction product was observed for up to about 900 seconds. Thereafter, a 1x kinetics buffer (ForteBio) was added, and the dissociation phase of the reaction product was observed for about 1,200 seconds. The association constant (Kon), dissociation 5 constant (Kdis), and equilibrium dissociation constant (KD) of each antibody were determined using Octet® analysis software (Pall Life® Sciences). As a result, the analysis results for the recombinant human ASM protein are shown in Table 6, and the analysis results for the recombinant mouse 10 ASM protein are shown in Table 7. TABLE 6 Antibody # KD (M) Kon(1 / Ms) Kdis(1 / s) RMax Full R2 #9101 1.16E-09 6.26E+05 7.28E-04 0.4527 0.9861 #9102 2.52E-10 4.16E+05 1.05E-04 0.8652 0.997 #9104 2.09E-10 3.92E+05 8.18E-05 0.3324 0.9759 #9108 1.19E-10 2.49E+05 2.96E-05 0.45 0.999 #9113 5.64E-10 1.16E+06 6.56E-04 0.2868 0.9635 TABLE 7 Antibody # KD (M) Kon(1 / Ms) Kdis(1 / s) RMax Full R2 #9101 4.25E-11 3.76E+06 1.59E-04 0.0185 0.5527 #9102 4.98E-10 4.89E+05 2.43E-04 0.4281 0.9944 #9104 6.38E-10 3.10E+05 1.98E-04 0.7027 0.9971 #9108 3.39E-09 4.59E+05 1.56E-03 0.3921 0.9865 #9113 <1.0E-12 1.00E+06 <1.0E-07 0 0 15 As shown in Table 6, five types of antibodies produced in Example 1 were bound to the human ASM protein with a binding affinity of 10-10 to 10-9 M. Meanwhile, as shown in Table 7, only the antibodies #9102, #9104, and #9108 showed a binding affinity of 10-10 to 10-9 M to the mouse ASM protein. Experimental Example 3: Identification of Antigenic Determinant of ASM Protein The sites of the ASM protein to which the present inventive antibodies were bound, that is, antigenic determinants were investigated by hydrogen deuterium exchange mass spectrometry (HDX-MS). First, the 1,000 pg / mL ASM protein, 1,000 pg / mL #9101 mIgG1, 1,250 pg / mL #9102 mIgG2, 1,000 pg / mL #9104 mIgG4, 1,000 pg / mL #9108 mIgG1, 3,300 pg / mL #9113 mIgG1, or 2,932 pg / mL #9123 mIgG1 antibodies were serially diluted 2-fold to prepare up to 128-fold dilutions. Then, 1 pl of each of the prepared dilutions was taken, and mixed with an equal amount of 10 mg / ml sinapinic acid matrix (K200 MALDI kit, CovalX), and 1 pl of the mixture was dispensed in SCOUT 384 MALDI plates, followed by crystallization at room temperature. Thereafter, the crystals were measured using an MALDI mass spectrometer. For the cross-link data for non-covalent interaction analysis, 1 pl of each of the 128-fold dilutions was mixed with an equal amount of a 2 mg / ml K200 stabilizer reagent (K200 MALDI kit, CovalX), followed by incubation at room temperature for 3 hours. After the reaction was completed, 1 pl of the mixed solution was subjected to the measurement using an MALDI mass spectrometer, and the measurement results were analyzed in a high-mass MALDI MS mode. As a result, the peptide sequences of the sites that 5 were identified to have significant difference of deuterium incorporation in the ASM protein are shown in Table 8, and human ASM protein structure models indicating the binding sites on ASM protein to each antibody are shown in FIG. 3. The deuterium exchange heat map of each antibody in the 10 saposin domain of the ASM protein is shown in FIG. 4. TABLE 8 Antibody # Peptide region Amino acid sequence #9101 135-159 PTVPKPPPKPPSPPAPGAPVSRILF #9102 135-159 PTVPKPPPKPPSPPAPGAPVSRILF 218-228 SGLGPAGPFDM #9104 53-72 TAINLGLKKEPNVARVGSVA 135-159 PTVPKPPPKPPSPPAPGAPVSRILF #9108 53-72 TAINLGLKKEPNVARVGSVA 135-155 PTVPKPPPKPPSPPAPGAPVS 259-269 VRKFLGPVPVY #9113 53-72 TAINLGLKKEPNVARVGSVA 101-123 VWRRSVLSPSEACGLLLGSTCGH 135-155 PTVPKPPPKPPSPPAPGAPVS 259-269 VRKFLGPVPVY #9123 259-269 VRKFLGPVPVY As shown in Table 8 and FIG. 3, the antibodies according 15 to the present disclosure were bound to the fragment of amino acids at positions 53 to 72 (SEQ ID NO: 68, TAINLGLKKEPNVARVGSVA), the fragment of amino acids at positions 101 to 123 (SEQ ID NO: 69, VWRRSVLSPSEACGLLLGSTCGH), the fragment of amino acids at positions 135 to 159 (SEQ ID NO: 70, PTVPKPPPKPPSPPAPGAPVSRILF), the fragment of amino acids at positions 135 to 155 (SEQ ID NO: 71, PTVPKPPPKPPSPPAPGAPVS), the fragment of amino acid at positions 218 to 228 (SEQ ID NO: 72, SGLGPAGPFDM), or the fragment of amino acids at positions 259 to 269 (SEQ ID NO: 73, VRKFLGPVPVY) from the N-terminus of the ASM protein. Especially, as shown in FIG. 4, the antibodies according to the present disclosure were mainly bound to a-helix 1 and a-helix 2 in the saposin domain. Experimental Example 4: Inhibition of ASM protein activity 4-1. Inhibition of ASM protein activity It was examined whether the produced antibodies, specifically binding to the ASM protein, inhibited the activity of the ASM protein located in the cellular membrane. First, fibroblasts (Coriell Institute) of Alzheimer's patients were treated with the antibodies produced in Example 1 at a concentration of 3 pg / ml, and incubated at 37°C for 24 hours. Thereafter, the cells were collected, and the cell membrane was separated using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermo) according to the manufacturer's protocol, followed by protein extraction. The extracted protein as a sample was placed into an insert for UPLC and subjected to UPLC analysis (Waters, 186004800) by a common method, and the results are shown in FIG. 5. The antibody #9105 confirmed not to bind to the ASM protein was used as a control group. As shown in FIG. 5, the antibody #9104 among the six types of antibodies produced in Example 1 significantly inhibited the activity of the ASM protein increased in the cellular membrane of Alzheimer's patients. 4-2. Inhibition of ASM protein activity according to treatment concentration of anti-ASM antibody The change in activity of the ASM protein located in the cellular membrane was examined by the same method as in Experimental Example 4-1 except for the treatment with antibody #9104 produced in Example 1 at a concentration of 0.03, 0.3, 3, 30, 300, or 3,000 ng / ml. As a result, the analyzed ASM protein activity inhibition (%) is shown in FIG. 6 and Table 9. Antibody #9105 was used as a control group. TABLE 9 Treatment concentration (ng / ml) ASM protein activity inhibition (%) #9104 #9105 0.03 0 0.3 7.9 3 16 30 21 300 28 3, 000 32 9 As shown in FIG. 6 and Table 9, antibody #9104 according to the present disclosure significantly inhibited the activity of the ASM protein located in the cellular membrane depending on the treatment concentration. However, antibody #9105 used as a control group slightly inhibited the activity of the ASM protein located in the cellular membrane only at a treatment concentration of 3,000 ng / ml or more. Experimental Example 5: Sphingolipid metabolism alteration The alteration of sphingolipid metabolism in the cellular membrane of the cells treated with antibody #9104 at 3 pg / ml as described in Experimental Example 4-1 was examined through changes in sphingomyelin and ceramide contents. The experiment was performed by a usual method, and the measured sphingomyelin and ceramide contents are shown in FIG. 7. As shown in FIG. 7, the content of ceramide present in the cellular membrane of fibroblasts of Alzheimer's patients was significantly reduced by the treatment with antibody #9104, but there was no significant change in content of sphingomyelin. The result indicated that the antibody according to the present disclosure inhibited the decomposition of sphingomyelin by ASM protein. Experimental Example 6: Cognitive memory improvement The change in cognitive memory of mice was examined by administering antibody #9104 to APP / PS1 mice, Alzheimer's disease animal models. 6-1. Administration of anti-ASM antibody APP / PS1 mice aged 7 months were intraperitoneally administered with 324 pl of antibody #9104 at a dose of 50 mg / kg for 8 weeks. The administration was performed twice a week (with an interval of 3 days), and antibody #9104 was administered a total of 16 times (FIG. 8). The descendant mice born from the crossbreeding of C57BL / 6 and APP / PS1 mice and not treated with any substance were used for a nontreatment control group, and APP / PS1 mice treated with 324 pl of PBS were used for a negative control group. 6-2. Cognitive memory improvement-(l) A behavioral test was conducted from the 41st day to the last day of administration of antibody #9104 to examine a change in cognitive memory by the administration of the antibody. First, the repulsion of each mouse against a tester was eliminated by repeatedly holding the tail of the mouse and placing the mouse on the arm and the back of the hand of the tester for 5 minutes from the 41st day of administration, and thereafter, the repulsion of the mouse against water was eliminated by acclimating the mouse to water for 30 minutes, before the test. A platform was installed while cues were removed from four sides of a water tank, and a small triangular water tank was installed based on the platform. The mouse was placed in the water tank and trained to swim and climb onto the platform 10 times. The mouse was allowed to stay for 10 seconds whenever the mouse climbed onto the platform 10 seconds after swim training, and after 10 seconds, the mouse was allowed to re-enter the water. This was repeated 10 times, during which the mouse was trained to climb onto the platform in various directions while the direction of approach to the platform was changed. A Morris water maze test was conducted from the next day, the 42nd day to the 51st day of administration. Specifically, cues were placed on the four sides of the water tank, and each mouse was tested four times (a time limit of 60 seconds for each time) at the same time every day, and this was repeated a total of 10 rounds to record the time required to climb onto the platform. On the 52nd day of administration, a probe test was conducted to record the existing location where the platform has been present in the water tank with the platform removed, and the path length and speed of swimming past other quadrants. As a result, the Morris water maze test results are shown in FIG. 9, and the probe test results are shown in FIGS. 10 to 12. As shown in FIG. 9, as the test was repeated, the escape latency time of the mice administered with antibody #9104 was significantly reduced. The mice of the negative control group showed an escape latency time of 30 seconds on average, indicating the impairment of cognitive function, and the mice of the non-treatment control group showed a reduction in escape latency time with the repetition of the test. Specifically, the group administered with antibody #9104 showed a 31.5% reduction in escape latency time in the 9th round and a 30.8% reduction in the 10th round, compared with the mice of the negative control group. As shown in FIGS. 10 and 11, the mice administered with antibody #9104 showed the time spent in the target quadrant significantly longer than the time spent in the non-target quadrants. This result was similar to that of the nontreatment control group, and was differentiated from that of the negative control group showing little difference between the time spent in the target quadrant and the time spent in the non-target quadrants. As shown in FIG. 12C, the frequency of crossing platform was significantly increased in the group administered with antibody #9104 but decreased in the negative control group. As shown in FIGS. 12A and 12B, all the mouse groups were similar in view of path length and swim speed, indicating that the administration of antibody #9104 did not affect exercise ability. 6-3. Cognitive memory improvement-(2) The change in cognitive memory of mice was examined by administering antibody #9104 at a dose of 50 mg / kg to APP / PS1 mice, Alzheimer's disease animal models. Therefore, antibody #9104 was administered by the same method as in Experimental Example 6-1 and a contextual and cued fear conditioning test was conducted by a common method, and then the results are shown in FIG. 13. As shown in FIG. 13, the mice administered with antibody #9104 showed a significant increase in freezing according to the contextual or tone condition, but the mice of the negative control group showed a reduction in freezing. The results indicated that the antibody of the present disclosure significantly improved the cognitive memory in Alzheimer's disease animal models. Experimental Example 7: ASM protein activity 7-1. ASM protein activity The activity of the ASM protein present in the blood and brain tissue of the animal models confirmed to have improved cognitive memory in Experimental Example 6 was examined. Specifically, proteins were obtained from the blood plasma of the mice by a common method, and the obtained proteins were used as a sample and subjected to UPLC by the same method as in Experimental Example 4-1. As a result, the results obtained by calculating the ASM activity (%) of the mice of the non-treatment control group and the mouse group administered with #9104 relative to the activity of the ASM protein of the mice of the negative control group are shown in FIG. 14. As shown in FIG, 14, the activity of the ASM protein present in the blood plasma was inhibited by about 64% by administration of #9104. 7-2. ASM protein level ELISA analysis was performed to examine whether the inhibition of ASM protein activity confirmed in Experimental Example 7-1 was due to a reduction in expression level of the protein. The blood plasma tissue obtained in Experimental Example 7-1 was used as a sample, and the analysis was performed using the mouse ASM ELISA kit (Mybiosource) according to the manufacturer's protocol. The results are shown in FIG. 15. As shown in FIG. 15, the ASM protein level was significantly increased in the mice administered with antibody #9104 rather than the non-treatment control group or the negative control group. The results indicated that the antibody according to the present disclosure inhibited the activity of the ASM protein without affecting the ASM protein level. Experimental Example 8: Inhibition of amyloid-p(A£) accumulation The accumulation of amyloid-p present in the brain tissue of the animal models confirmed to have improved cognitive memory in Experimental Example 6 was examined by thioflavin-S staining, western blotting using 6E10 antibody, and ELISA. 8-1. Inhibition of Ap accumulation-(1) The animal models of Experimental Example 6 were anesthetized with 2.5% avertin, and the chest cavity was opened to obtain the blood from the heart by using a 1-cc syringe. After the blood was obtained, perfusion was performed with 20 ml of PBS, and additional perfusion was performed with 20-30 ml of 4% paraformaldehyde (PFA). The mouse brain was excised, and left overnight while placed in a 4% PFA solution, and then the tissue was cut into a 30-pm thick slices by a vibratome. The obtained brain tissue slices were placed in a 12-well plate, and then 1 ml of a PBS solution was added, followed by stirring at 33 rpm for 5 minutes at room temperature. The PBS solution was removed, and Ap plaques were stained for 10 minutes by treatment with 500 ^ of a 0.5% thioflavin-S reagent diluted in ethanol. Thereafter, the slices were washed two times with 50% ethanol and washed once with 1 ml of a PBS solution. The slices were mounted by addition of mount media containing DAPI. For staining of all types of Ap proteins, the brain tissue slices were placed in a 12-well plate, and then 1 ml of a PBS solution was added, followed by stirring at 33 rpm for 5 minutes at room temperature, and then the PBS solution was removed. Thereafter, 1 ml of a PBS solution containing 0.2% Triton X-100 was added thereto, and stirred at 33 rpm for 1 hour. After stirring, the PBS solution containing Triton X-100 was removed, and the slices were pre-treated with a PBS solution containing 0.05% Triton X-100 for 1 hour. A mixture of 6E10 antibody (Biolegend) and a PBS solution containing 0.05% Triton X-100 at a volume ratio of 1:1 was added at an amount 500 pl of to the pre-treated brain tissue slices. The slices were incubated at 4°C overnight with stirring, and washed three times with a PBS solution containing Triton X-100. The slices were treated with a mouse-488 secondary antibody, incubated at room temperature for 2 hours, and then washed three times with a PBS solution containing Triton X-100. The slices were mounted by addition of mount media containing DAPI. The stained brain tissue slices were imaged at a magnification of 20 times by using a laser-scanning confocal microscope (FV3000, Olympus), and the stained area was analyzed by using MetaMorph software (Molecular Devices). The analysis results are shown in FIGS. 16 and 17. As shown in FIGS. 16 and 17, dense-core and diffuse amyloid-p plaques present in the cortex and hippocampus of the brain were significantly reduced by the treatment with antibody #9104 . 8-2. Inhibition of Ap accumulation-(2) The obtained brain tissue was separated into the cortex and the hippocampus, and RIPA buffer containing 100 mM PMSF and 1% protease inhibitor was added thereto, followed by tissue homogenization. The homogenized tissues were centrifuged for 10 minutes under conditions of 4°C and 13,000 rpm, to obtain supernatants. The obtained supernatants were further centrifuged for 30 minutes under the same conditions, to obtain supernatants again, and these supernatants were used as samples to measure the levels of Ap therein by using an amyloid-p40 ELISA kit (KHB3481, Invitrogen) and an amyloid-p42 ELISA kit (KHB3441, Invitrogen). The obtained results are shown FIG. 18. As shown in FIG. 18, Ap40 present in the cortex and hippocampus of the brain was reduced to 30.9% and 30.9%, respectively, by antibody #9104, and these reductions were significant compared with the negative control group. In addition, Ap42 was significantly reduced by the administration of antibody #9104 compared with the negative control group. Experimental Example 9: Tau protein accumulation The brain tissue slices obtained in Experimental Example 8 were used to examine the change in tau accumulation by staining the tau protein. Specifically, the experiment was conducted under the same conditions and method as in Experimental Example 8-1 except that anti-AT8 antibody (ThermoFisher Scientific, MN1020) was used as a primary antibody. The analysis results are shown in FIG. 19. As shown in FIG 19, the tau protein present in the cortex and hippocampus of the brain was significantly reduced by the treatment with antibody #9104 . Experimental Example 10: Alleviation of neuroinflammation The neuroinflammation alleviating effect by antibody #9104 in the brain tissue of the animal models confirmed to have improved cognitive memory in Experimental Example 6 was examined through activation of microglia and astrocytes. The experiment was conducted under the same conditions and method as in Experimental Example 8-1 except that anti-lba-1 antibody (Wako, 019-19941) or anti-GFAP antibody (Dako, N1506) was used as a primary antibody, and rabbit-488 secondary antibody was used as a secondary antibody. The analysis results are shown in FIGS. 20 and 21. As shown in FIGS. 20 and 21, the expressions of lba-1 and GFAP proteins present in the cortex and hippocampus of the brain were significantly reduced, and thus the activity of microglia and astrocytes in the brain tissue was inhibited by #9104 antibody. The results indicated that the antibody according to the present disclosure had an effect of alleviating brain neuroinflammation. Experimental Example 11: Toxicity The toxicity of antibody #9104 was examined by the following method. The experiment was conducted while antibody #9104 was administered in Experimental Example 6. Specifically, the 2022279866   22 Jun 2026 Response survival rate was assessed by visually observing the movement and abnormal signs of mice twice a week, and the mice were weighed on the same day and time once a week.     The mice after completion of the 5 administration were necropsied by a common method, and organs were observed to determine the presence or absence of organ toxicity due to #9104 antibody. As a result, the changes in survival rate and body weight of the mice are shown in FIG. 22, and the observation results of organ 10 toxicity are shown in FIG. 23. As shown in FIGS. 22 and 23, there were no changes in survival rate and body weight by the treatment with antibody #9104 antibody, and the organs were also confirmed to be normal. 15        The results indicated that the antibody according to the present disclosure can be significantly used in the treatment of a brain disease, such as Alzheimer's disease, even without toxicity. 20        In this specification, the terms “comprise”, “comprises”, “comprising” or similar terms are intended to mean a non-exclusive inclusion, such that a system, method or apparatus that comprises a list of elements does not include those elements solely, but may well include 25 other elements not listed. The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement or any form of suggestion that the prior art forms part of 30 the common general knowledge in Australia.

Claims

1. A pharmaceutical composition for preventing or treating a brain disease, wherein the pharmaceutical composition comprises as an 5     active ingredient an antibody or antigen-binding fragment thereofthat specifically binds to an acid sphingomyelinase (ASM) protein wherein the antibody or antigen-binding fragment thereof comprises:a heavy chain variable region comprising heavy chain CDR1,10     CDR2, and CDR3 consisting of the amino acid sequences set forthin SEQ ID NOS: 31, 32, and 33, respectively, and a light chainvariable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOS: 49, 50, and 51, respectively,15     wherein the brain disease is a degenerative brain disease,wherein the degenerative brain disease is Alzheimer's disease.

2. The pharmaceutical composition of claim 1, wherein the ASM protein is derived from a mammal.

203. The pharmaceutical composition of claim 1 or claim 2, wherein the ASM protein is a polypeptide consisting of an amino acid sequence set forth in SEQ ID NO: 66 or 67.25  4. A health functional food for preventing or alleviating a braindisease, the health functional food comprising as an active2022279866   22 Jun 2026ingredient an antibody or antigen-binding fragment thereof that specifically binds to an ASM protein;wherein the antibody or antigen-binding fragment thereof comprises:5     a heavy chain variable region comprising heavy chain CDR1,CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOS: 31, 32, and 33, respectively, and a light chainvariable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOS:10      49, 50, and 51, respectively,wherein the brain disease is a degenerative brain disease, wherein the degenerative brain disease is Alzheimer's disease.

5. A method for preventing, alleviating, or treating a brain 15     disease, the method including a step of administering to asubject an antibody or antigen-binding fragment thereof that specifically binds to an ASM proteinwherein the antibody or antigen-binding fragment thereof comprises:20     a heavy chain variable region comprising heavy chain CDR1,CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOS: 31, 32, and 33, respectively, and a light chainvariable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOS:25      49, 50, and 51, respectively,wherein the brain disease is a degenerative brain disease,2022279866   22 Jun 2026wherein the degenerative brain disease is Alzheimer's disease.

6. Use of an antibody or antigen-binding fragment thereof that specifically binds to an ASM protein in the manufacture of a 5     medicament for preventing, alleviating, or treating a braindiseasewherein the antibody or antigen-binding fragment thereof comprises:a heavy chain variable region comprising heavy chain CDR1,10     CDR2, and CDR3 consisting of the amino acid sequences set forthin SEQ ID NOS: 31, 32, and 33, respectively, and a light chainvariable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences set forth in SEQ ID NOS: 49, 50, and 51, respectively,15     wherein the brain disease is a degenerative brain disease,wherein the degenerative brain disease is Alzheimer's disease.

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