Antibodies that specifically bind to ASM proteins

Abstract

The present invention relates to antibodies that specifically bind to ASM (acid sphingomyelinase) protein and uses thereof. Specifically, the antibodies or antigen-binding fragments thereof according to the present invention specifically bind to ASM protein with high binding affinity and can be used for detecting ASM protein and diagnosing diseases caused by overexpression of ASM protein.

Description

[Technical Field]

[0001] The present invention relates to an antibody that specifically binds to an ASM (acid sphingomyelinase) protein. [Background technology]

[0002] Sphingolipid metabolism regulates normal cell signaling, and abnormal changes in sphingolipid metabolism affect various neurodegenerative diseases, including Alzheimer's disease. Acid sphingomyelinase (ASM), an enzyme that regulates sphingolipid metabolism, is expressed in almost all types of cells and plays an important role in sphingolipid metabolism and cell membrane turnover.

[0003] In the brains of patients with neurodegenerative diseases such as Alzheimer's disease, ASM protein activity is significantly increased compared to healthy individuals. In this regard, Korean Patent Registration No. 10-1521117 discloses that inhibiting the activity of overexpressed ASM protein or suppressing its expression inhibits the accumulation of β-amyloid, improves learning ability and memory, and can treat neurodegenerative diseases. Furthermore, ASM protein activity has recently been found to be increased in neurological disorders such as depression, and suppressing the expression or activity of the ASM protein has been shown to be effective in alleviating depression.

[0004] However, no substance that directly inhibits ASM protein expression or activity has been developed, and only a few inhibitors that indirectly inhibit ASM protein expression have been identified. For example, tricyclic antidepressants, such as amitriptyline, desipramine, and mipramine, are used to treat depression. Although these tricyclic antidepressants were not developed specifically as ASM protein inhibitors, various studies have demonstrated their ASM protein inhibitory effects. The primary pharmacological effect of tricyclic antidepressants is to inhibit the reuptake of neurotransmitters in neurons and increase their activity, and their ASM inhibitory effect has been confirmed to be a secondary effect. However, tricyclic antidepressants can affect the nervous system and neurons, causing side effects such as blurred vision, increased light sensitivity, and vomiting. Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to an ASM protein.

[0006] Another object of the present invention is to provide a method for producing the above antibody or antigen-binding fragment thereof.

[0007] It is still another object of the present invention to provide a use of the above antibody or antigen-binding fragment thereof for detecting ASM proteins. [Means for solving the problem]

[0008] In order to achieve the above-mentioned objects, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to an ASM (acid sphingomyelinase) protein.

[0009] The present invention also provides a nucleic acid encoding the above antibody or antigen-binding fragment thereof.

[0010] The present invention also provides an expression vector comprising the nucleic acid.

[0011] The present invention also provides a host cell containing the above nucleic acid or expression vector.

[0012] The present invention also provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to an ASM protein, comprising the step of culturing the above-mentioned host cell to produce the antibody or antigen-binding fragment thereof.

[0013] The present invention also provides a composition and kit for detecting ASM protein, which comprises the above-mentioned antibody or an antigen-binding fragment thereof.

[0014] The present invention also provides a method for detecting an ASM protein, which comprises the step of reacting the above-mentioned antibody or antigen-binding fragment thereof with a sample. [Effects of the Invention]

[0015] The antibody or antigen-binding fragment thereof according to the present invention binds specifically to ASM protein with high binding strength and can therefore be used for detecting ASM protein and diagnosing diseases caused by overexpression of ASM protein. DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention will be described in detail below.

[0017] The present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to an ASM (acid sphingomyelinase) protein.

[0018] The term "ASM (acid sphingomyelinase) protein" as used herein refers to an enzyme that is a member of the SMase (sphingomyelinase) family, which regulates sphingolipid metabolism. ASM proteins catalyze the decomposition of sphingomyelin into ceramide and phosphorylcholine, and can be classified as alkaline, neutral, or acidic depending on the pH at which they exhibit optimal enzymatic activity.

[0019] The ASM protein can include any type of ASM protein known in the art. Specifically, the ASM protein can be derived from a mammal, more specifically, from a human, monkey, rat, or mouse. The ASM protein can also include any amino acid sequence known in the art as an ASM protein. For example, the ASM protein can be a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 139 or a nucleic acid encoding the polypeptide.

[0020] The ASM protein may have one or more amino acids added, deleted, or substituted in the amino acid sequence set forth in SEQ ID NO: 139, so long as it maintains the same or equivalent biological activity. In this case, the amino acid substitution may be a conservative substitution that does not affect or only slightly affects the charge, i.e., polarity or hydrophobicity, of the entire protein. In addition, the ASM protein may have 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more homology with the amino acid sequence set forth in SEQ ID NO: 139.

[0021] As used herein, the term "antibody" refers to an immune protein that binds to an antigen to inhibit or eliminate the antigen. The antibody may include any type of antibody known in the art. Specifically, the antibody may include IgM, IgD, IgG, IgA, and IgE, each of which may contain a heavy chain made from μ, δ, γ, α, and ε genes encoding heavy chain constant regions. Generally, IgG is primarily used in antibody technology, but it may be further divided into IgG1, IgG2, IgG3, and IgG4 isotypes, each of which may have different structural and functional properties. The antibody may include any of humanized antibodies containing minimal sequences derived from non-human antibodies, human antibodies composed of sequences derived from humans, and chimeric antibodies containing a mixture of sequences derived from different species.

[0022] IgG can form a highly stable Y-shaped structure (approximately 150 kDa) composed of two heavy chain proteins of approximately 50 kDa and two light chain proteins of approximately 25 kDa. The light and heavy chains that make up an antibody are divided into variable regions, whose amino acid sequences differ among antibodies, and constant regions, whose amino acid sequences are the same. The heavy chain constant region contains CH1, hinge (H), CH2, and CH3 domains, each of which is composed of two β-sheets and may be connected by intramolecular disulfide bonds. The heavy and light chain variable regions bind to form an antigen-binding site, which can be present in each of the two arms of the Y structure. The portion of a full-length antibody that can bind to an antigen is called Fab (antibody binding fragment), and the portion that cannot bind to an antigen is called Fc (crystallizable fragment), and Fab and Fc can be linked by a hinge.

[0023] In one embodiment of the present invention, the IgG is an IgG of human origin, and specifically may comprise a human IgG heavy chain constant region consisting of the amino acid sequence set forth in SEQ ID NO: 137 and a human light chain λ constant region consisting of the amino acid sequence set forth in SEQ ID NO: 138. Furthermore, the human-derived IgG may comprise a human IgG heavy chain constant region consisting of the nucleotide sequence set forth in SEQ ID NO: 140 and a human light chain λ constant region consisting of the nucleotide sequence set forth in SEQ ID NO: 141.

[0024] The antibodies of the present invention may include not only full-length antibodies but also all of their antigen-binding fragments. Specifically, the antigen-binding fragment may refer to a portion excluding Fc, which functions to transmit antigen-binding stimulation to cells, complement, etc. For example, the antigen-binding fragment of an antibody may include all of Fab, scFv, F(ab)2, and Fv, as well as third-generation antibody fragments such as single domain antibodies and minibodies.

[0025] For example, the antibody or antigen-binding fragment thereof may comprise a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 89, a heavy chain CDR2 consisting of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 90 in which five or fewer amino acids have been substituted, and a heavy chain variable region consisting of the amino acid sequence set forth in SEQ ID NO: 91, and a light chain variable region consisting of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 92 or the above polypeptide in which five or fewer amino acids have been substituted, and a light chain CDR3 consisting of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 94 or the above polypeptide in which two or fewer amino acids have been substituted.

[0026] The term "CDR (complementarity determining region)" refers to a hypervariable region in the heavy and light chain variable regions of an antibody, which has a different amino acid sequence for each antibody, and refers to the site that actually binds to an antigen. In the three-dimensional structure of an antibody, the CDR is loop-shaped and located on the surface of the antibody, and a framework region (FR) that structurally supports the CDR may be present below the loop. The heavy and light chains each have three ring structures, and these six ring structures combine to directly contact the antigen. For convenience, the CDRs, which are antigen-binding sites having the six ring structures, will be referred to as heavy chain CDR1, heavy chain CD2, heavy chain CDR3, light chain CDR1, light chain CDR2, and light chain CDR3, respectively.

[0027] In the antibody or antigen-binding fragment thereof according to the present invention, the heavy chain CDR2 having five or fewer amino acid substitutions in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 90 may have five or fewer amino acids substituted selected from the group consisting of the amino acids at positions 2, 4 to 10, 12, 13, and 17 from the N-terminus of the polypeptide.

[0028] For example, the second, fourth, sixth, seventh, ninth, twelfth, or thirteenth amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 90 may be substituted with a neutral amino acid. Specifically, the neutral amino acid may include alanine, glycine, leucine, isoleucine, proline, valine, phenylalanine, tryptophan, tyrosine, serine, threonine, cysteine, methionine, asparagine, and glutamine. More specifically, the second amino acid from the N-terminus, isoleucine, may be substituted with leucine, valine, or threonine; the fourth amino acid, tyrosine, may be substituted with phenylalanine or tryptophan; the sixth amino acid, serine, may be substituted with glycine or asparagine; the seventh amino acid, glycine, may be substituted with valine; the ninth amino acid, isoleucine, may be substituted with proline, alanine, or valine; the twelfth amino acid, alanine, may be substituted with serine; and the thirteenth amino acid, aspartic acid, may be substituted with asparagine or alanine. The tenth amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 90 may be substituted with a basic amino acid. Specific examples of the basic amino acid include arginine, histidine, and lysine. More specifically, the tenth amino acid from the N-terminus, tyrosine, may be substituted with arginine.Furthermore, the 17th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 90 is substituted with an acidic amino acid, and specifically, the acidic amino acid can include aspartic acid and glutamic acid. More specifically, the 17th amino acid from the N-terminus, glycine, may be substituted with aspartic acid or glutamic acid. Furthermore, the 5th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 90 is a neutral or acidic amino acid, and the neutral or acidic amino acid is as described above. More specifically, the 5th amino acid from the N-terminus, glycine, may be substituted with alanine, aspartic acid, serine, or proline. Furthermore, the 8th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 90 is a neutral, acidic, or basic amino acid, and the neutral, acidic, or basic amino acid is as described above. More specifically, the asparagine, which is the eighth amino acid from the N-terminus, may be substituted with aspartic acid, lysine, isoleucine, threonine, valine, glycine, or tyrosine.

[0029] In one embodiment of the present invention, the heavy chain CDR2 in which five or fewer amino acids have been substituted in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 90 may be a polypeptide consisting of the amino acid sequences set forth in SEQ ID NOs: 95 to 111, respectively.

[0030] In the antibody or antigen-binding fragment thereof according to the present invention, the light chain CDR2 in which five or fewer amino acids have been substituted in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 93 may be in which four or fewer amino acids selected from the group consisting of the third to seventh amino acids from the N-terminus of the above polypeptide have been substituted.

[0031] In one embodiment of the present invention, the light chain CDR2 in which five or fewer amino acids have been substituted in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 93 may be a polypeptide consisting of the amino acid sequences set forth in SEQ ID NOs: 112 to 135, respectively.

[0032] In the antibody or antigen-binding fragment thereof according to the present invention, the light chain CDR2 in which two or fewer amino acids have been substituted in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 94 may be in which two or fewer amino acids have been substituted selected from the group consisting of the sixth and eighth amino acids from the N-terminus of the above polypeptide.

[0033] For example, the sixth and eighth amino acids from the N-terminus of the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 94 are neutral amino acids, and the neutral amino acids are as described above. More specifically, the serine amino acids at the sixth and eighth positions from the N-terminus may be substituted with tryptophan and glycine, respectively.

[0034] In one embodiment of the present invention, the light chain CDR3 in which two or fewer amino acids have been substituted in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 94 may be a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 136.

[0035] Furthermore, the antibodies or antigen-binding fragments thereof according to the present invention may be modified as needed. Specifically, the antibodies or antigen-binding fragments thereof may be modified by conjugation, glycosylation, label attachment, or a combination thereof. Specifically, the antibodies or antigen-binding fragments thereof may be modified with horseradish peroxidase (HRP), alkaline phosphatase, hapten, biotin, streptavidin, fluorescent substances, radioactive substances, quantum dots, polyethylene glycol (PEG), histidine tags, etc. Furthermore, the antibodies or antigen-binding fragments thereof may be conjugated with other drugs as needed.

[0036] The antibody or antigen-binding fragment thereof can be produced by a method for producing a monoclonal antibody well known in the art, and the method can be modified as appropriate by a person skilled in the art. For example, the antibody can be obtained by producing a hybridoma using B lymphocytes obtained from an animal immunized with an antigen, or can be produced using phage display technology.

[0037] The present invention also provides a nucleic acid encoding the above antibody or antigen-binding fragment thereof.

[0038] The antibody or antigen-binding fragment thereof encoded by the nucleic acid of the present invention may have the characteristics described above. Since the amino acid sequence constituting the antibody or antigen-binding fragment thereof of the present invention is known, the nucleic acid sequence encoding it will be obvious to those skilled in the art. Furthermore, the nucleic acid sequence may have one or more bases added, deleted, or substituted, as long as the activity of the antibody or antigen-binding fragment thereof translated from the nucleic acid sequence is maintained.

[0039] The present invention also provides an expression vector comprising the nucleic acid.

[0040] The nucleic acid contained in the expression vector according to the present invention may encode an antibody or antigen-binding fragment thereof having the above-mentioned characteristics.

[0041] As used herein, the term "expression vector" refers to a means for expressing a gene of interest in a host cell, and may include a plasmid vector, a cosmid vector, a bacteriophage vector, a viral vector, and the like. The expression vector may contain elements necessary for producing a peptide from a nucleic acid contained therein. Specifically, the expression vector may contain a signal sequence, an origin of replication, a marker gene, a promoter, a transcription termination sequence, and the like. In this case, the nucleic acid encoding the antibody or antigen-binding fragment thereof according to the present invention may be operably linked to a promoter.

[0042] For example, an expression vector used in a prokaryotic cell can include a promoter to drive transcription, a ribosome binding site for the initiation of translation, and a transcription and translation termination sequence, whereas an expression vector used in a eukaryotic cell can include a promoter and polyadenylation sequence derived from a mammal or a mammalian virus.

[0043] The marker gene contained in the expression vector may be any gene known in the art, specifically, an antibiotic resistance gene, which may confer resistance to antibiotics such as ampicillin, gentamicin, cavenicillin, chloramphenicol, streptomycin, kanamycin, neomycin, and tetracycline.

[0044] The present invention also provides a host cell containing the above nucleic acid or expression vector.

[0045] The nucleic acid or expression vector contained in the host cell of the present invention can have the characteristics described above. For example, the nucleic acid can encode an antibody or antigen-binding fragment thereof that specifically binds to an ASM protein of the present invention, and the expression vector can include the nucleic acid described above.

[0046] The host cell can be any type of cell known in the art as being usable for producing antibodies or antigen-binding fragments thereof. Specifically, the host cell can be a prokaryotic cell, yeast, or eukaryotic cell. Prokaryotic cells include Escherichia coli (E. coli), Bacillus strains, Streptomyces strains, Pseudomonas strains, Staphylococcus strains, etc., and yeasts include Saccharomyces cerevisiae, etc. The above-mentioned eukaryotic cells 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, and HT1080.

[0047] The host cells may be transfected with the nucleic acid or expression vector described above by a conventional method in the art. Specifically, the transfection may be performed by transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroacupuncture, gene gun, etc. The above-described methods may be modified as appropriate by those skilled in the art.

[0048] The present invention also provides a method for producing an antibody or antigen-binding fragment thereof that specifically binds to an ASM protein, comprising the step of culturing the above-mentioned host cell to produce the antibody or antigen-binding fragment thereof.

[0049] The antibody or antigen-binding fragment thereof produced by the production method according to the present invention may have the characteristics described above.

[0050] The culture may be performed using a culture medium appropriate for the type of host cell used in the production, and may contain appropriate supplements as needed. The culture may also be performed in an environment appropriate for the type of host cell.

[0051] The production method according to the present invention may further comprise a step of recovering the antibody or antigen-binding fragment thereof produced in the host cell. The recovery may be carried out by a conventional method in the art, which may be modified as needed by a skilled artisan. For example, the recovery may be carried out by removing impurities using centrifugation or ultrafiltration, followed by further purification of the resulting product by chromatography or the like. Examples of such chromatography include affinity chromatography, cation chromatography, and hydrophobic interaction chromatography.

[0052] The present invention also provides a composition and kit for detecting ASM protein, which comprises the above-mentioned antibody or an antigen-binding fragment thereof.

[0053] The antibody or antigen-binding fragment thereof contained in the composition and kit for detecting ASM protein according to the present invention can have the above-mentioned characteristics.

[0054] The composition may also contain a ligand capable of specifically binding to the antibody or antigen-binding fragment thereof according to the present invention. The ligand may be a conjugate labeled with a detector such as a chromogenic enzyme, a fluorescent substance, a radioisotope, or a colloid, or a ligand treated with streptavidin or avidin. In addition to the above-mentioned reagents, the detection composition of the present invention may further contain distilled water or a buffer solution that stably maintains the structure of these reagents.

[0055] The kit may be bound to a solid substrate to facilitate subsequent steps such as washing the antibody or antigen-binding fragment thereof contained therein, separating the complex, etc. In this case, the solid substrate may be a synthetic resin, nitrocellulose, glass substrate, metal substrate, glass fiber, microspheres, or microbeads. Examples of synthetic resins include polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, and nylon.

[0056] The kit can be produced by conventional production methods well known to those skilled in the art, and can further include buffers, stabilizers, inactive proteins, and the like.

[0057] The present invention also provides a method for detecting an ASM protein, which comprises the step of reacting the above-mentioned antibody or antigen-binding fragment thereof with a sample.

[0058] The antibody or antigen-binding fragment thereof used in the method for detecting ASM protein according to the present invention can have the characteristics described above.

[0059] The sample may include any type of sample for detecting an ASM protein. Methods for detecting a target protein using an antibody or an antigen-binding fragment thereof are well known in the art and can be performed by a skilled artisan with appropriate modifications as needed. [Example]

[0060] The present invention will be described in detail below based on the following examples. However, the following examples are for illustrative purposes only and do not limit the present invention. Anything that has substantially the same configuration and the same effects as the technical idea described in the claims of the present invention is included in the technical scope of the present invention.

[0061] Example 1: Preparation of antibodies that specifically bind to ASM (acid sphingomyelinase) protein The mutants were produced from antibody #9104, which is an antibody that specifically binds to human ASM protein (SEQ ID NO: 139).

[0062] First, the VH and VL genes of antibody #9104 were amplified by standard methods using random primers to induce random mutations in the heavy and light chain CDR sequences of antibody #9104. The amplified VH and VL genes were ligated to mouse heavy chain constant region 1 (CH1, SEQ ID NO: 142) and light chain constant region (CL, SEQ ID NO: 143), respectively, and inserted into the pComb3xss phagemid vector in the form of scFab. A #9104 random mutagenesis library was then constructed using standard methods. The library was then screened to select scFabs that specifically bind to human ASM protein. Expression vectors were constructed to express the selected scFabs in the form of a human heavy chain constant region and a human light chain λ constant region, each consisting of the nucleotide sequences set forth in SEQ ID NO: 137 or 138, linked to the carboxy terminus of the heavy chain variable region and light chain variable region, respectively. The amino acid and nucleic acid sequences of the heavy chain variable regions of the selected scFabs are shown in Table 1 below, and the amino acid and nucleic acid sequences of the light chain variable regions are shown in Table 2 below.

[0063] [Table 1] JPEG2025538573000002.jpg236170JPEG2025538573000003.jpg237170JPEG202 5538573000004.jpg242170JPEG2025538573000005.jpg242170JPEG2025538573 000006.jpg238170JPEG2025538573000007.jpg238170JPEG2025538573000008. jpg238170JPEG2025538573000009.jpg238170JPEG2025538573000010.jpg34170

[0064] [Table 2] JPEG2025538573000012.jpg238170JPEG2025538573000013.jpg220170JPEG2025538573000014.jpg218170 JPEG2025538573000015.jpg219170JPEG2025538573000016.jpg219170JPEG2025538573000017.jpg219170 JPEG2025538573000018.jpg218170JPEG2025538573000019.jpg219170JPEG2025538573000020.jpg217170 JPEG2025538573000021.jpg220170JPEG2025538573000022.jpg218170JPEG2025538573000023.jpg218170

[0065] The expression vector used was a mammalian expression vector. The constructed expression vector was transformed into an ExpiCHO cell line to produce a full-length antibody that binds to human ASM protein.

[0066] Example 2: Determination of complementarity determining regions (CDRs) The complementarity determining regions of the scFv obtained above were confirmed by standard methods. As a result, the CDR sequences of the heavy chain variable region and the light chain variable region are shown in Table 3 and Table 4, respectively.

[0067] [Table 3] JPEG2025538573000025.jpg243170JPEG2025538573000026.jpg119170

[0068] [Table 4] JPEG2025538573000028.jpg244170JPEG2025538573000029.jpg245170JPEG2025538573000030.jpg131170

[0069] As shown in Tables 3 and 4, it was confirmed that CDR1 and CDR3 of the heavy chain variable region and CDR1 of the light chain variable region have the same sequences as those of antibody #9104, but CDR2 of the heavy chain variable region and CDR2 and CDR3 of the light chain variable region have sequences in which some of the amino acids constituting them have been substituted.

[0070] [Experimental Example 1: Confirmation of binding affinity with ASM protein] The binding affinity and interaction kinetics of the antibodies specifically binding to ASM protein obtained above were measured using the Octet® QK384 system (Pall Life Sciences).

[0071] First, an anti-human IgG Fc capture (AHC) biosensor was used to capture the antibody specifically binding to the ASM protein prepared in Example 1 above, and then 1.25, 2.5, 5, 10, or 20 nM recombinant human ASM protein solution was added. After the addition of the human ASM protein solution, the association phase of the reaction was observed for approximately 1,200 seconds. Then, 1x kinetics buffer (ForteBio) was added, and the separation phase of the reaction was observed for approximately 1,500 seconds. The adsorption rate constant (K) for each antibody was calculated using Octet® analysis software (Pall Life Sciences). a ), separation constant:K d ) and the equilibrium dissociation constant (K D) were determined. As a result, the binding ability of antibody #9104, in which the heavy chain CDR portion has been mutated as shown in Table 1, to human ASM protein, is shown in Table 5, and the binding ability of antibody #9104, in which the light chain CDR portion has been mutated as shown in Table 2, to human ASM protein, is shown in Table 6.

[0072] [Table 5]

[0073] [Table 6] JPEG2025538573000033.jpg68170

[0074] As shown in Tables 5 and 6, the antibody produced in Example 1 was 10 -10 ~10 -9 It binds to human ASM protein with M level binding affinity.

[0075] [Experimental Example 2. Combination of heavy and light chain variable regions] Eight types of heavy chain variable regions and five types of light chain variable regions of the #9104 antibody selected above were combined to produce 40 types of antibodies (Table 7).

[0076] [Table 7] JPEG2025538573000035.jpg214170

[0077] After small-scale expression in the ExpiCHO cell line as described above, the binding ability to human ASM protein was confirmed as described in Experimental Example 1. In this study, an antibody produced by combining the #9104v-H33 heavy chain variable region and the #9104v-L23 light chain variable region was excluded from the analysis of binding ability to ASM protein because it was not expressed. The resulting binding ability of the above antibodies to human ASM protein is shown in Table 8.

[0078] [Table 8] JPEG2025538573000037.jpg213170

[0079] As shown in Table 8, the antibodies produced were 10 -10 ~10 -9 It binds to human ASM protein with M level binding affinity.

Claims

1. An antibody or an antigen-binding fragment thereof that specifically binds to ASM (acid sphingomyelinase) protein.

2. The antibody or antigen-binding fragment thereof of claim 1 , wherein the ASM protein is derived from a mammal.

3. The antibody or antigen-binding fragment thereof is a heavy chain variable region consisting of a heavy chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 89, a heavy chain CDR2 consisting of the amino acid sequence set forth in SEQ ID NO: 90 in which five or fewer amino acids have been substituted, and a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 91; and The antibody or antigen-binding fragment thereof according to claim 1, comprising a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence set forth in SEQ ID NO: 92, a light chain CDR2 consisting of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 93, or the above polypeptide in which five or fewer amino acids have been substituted, and a light chain CDR3 consisting of a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 94, or the above polypeptide in which two or fewer amino acids have been substituted.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain CDR2 having five or fewer amino acid substitutions in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 90 has five or fewer amino acids substituted selected from the group consisting of the second, fourth to tenth, twelfth, thirteenth, and seventeenth amino acids from the N-terminus of the polypeptide.

5. The antibody or antigen-binding fragment thereof according to claim 3, wherein the heavy chain CDR2 in which five or fewer amino acids have been substituted in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 90 is a polypeptide consisting of the amino acid sequences set forth in SEQ ID NOs: 95 to 111, respectively.

6. The antibody or antigen-binding fragment thereof according to claim 3, wherein the light chain CDR2 in which five or fewer amino acids have been substituted in a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 93 has four or fewer amino acids selected from the group consisting of the third to seventh amino acids from the N-terminus of the polypeptide.

7. The antibody or antigen-binding fragment thereof according to claim 3, wherein the light chain CDR2 in which five or fewer amino acids have been substituted in the polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 93 is a polypeptide consisting of the amino acid sequences set forth in SEQ ID NOs: 112 to 135, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 3, wherein the light chain CDR3 having two or fewer amino acid substitutions in a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 94 has two or fewer amino acids selected from the group consisting of the sixth and eighth amino acids from the N-terminus of the polypeptide.

9. The antibody or antigen-binding fragment thereof of claim 3, wherein the light chain CDR3 in which two or fewer amino acids have been substituted in a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 94 is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO:

136.

10. A nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. An expression vector comprising the nucleic acid of claim 10.

12. A host cell comprising the nucleic acid of claim 10 or the expression vector of claim 11.

13. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to an ASM protein, comprising the step of culturing the host cell of claim 12 to produce the antibody or antigen-binding fragment thereof.

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