Antibodies that specifically bind to ASM protein

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 Art

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

[0003] In the brains of patients with neurodegenerative diseases such as Alzheimer's disease, the activity of the ASM protein is significantly increased compared to that of healthy individuals. In relation to this, Korean Patent Registration No. 10-1521117 discloses that if the activity of the overexpressed ASM protein is inhibited or the expression of the ASM protein is suppressed, the accumulation of β-amyloid is suppressed, learning ability and memory are improved, and neurodegenerative diseases can be treated. Also, recently, it has been known that the activity of the ASM protein is increased in neurological diseases such as depression, and that suppressing the expression or activity of the above ASM protein has the effect of improving depression.

[0004] However, no substance has been developed that directly suppresses the expression or activity of the ASM protein; only a few inhibitors that indirectly suppress ASM protein expression have been identified. For example, there are tricyclic antidepressants used to treat depression, such as amitriptyline, desipramine, and mipramine. These tricyclic antidepressants were not developed as ASM protein inhibitors, but various studies have shown that they have an ASM protein inhibitory effect. The main pharmacological action of tricyclic antidepressants is to inhibit the reuptake of neurotransmitters in nerve cells and increase their activity, and it has been confirmed that their action as ASM inhibitors is an incidental effect. However, tricyclic antidepressants can act on the nervous system and nerve cells to induce side effects such as blurred vision, increased photosensitivity, and vomiting. [Overview of the project] [Problems that the invention aims to solve]

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

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

[0007] Another object of the present invention is to provide an application for detecting ASM proteins using the above-mentioned antibody or its antigen-binding fragment. [Means for solving the problem]

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

[0009] Furthermore, the present invention provides nucleic acids that encode the above-mentioned antibody or its antigen-binding fragment.

[0010] Furthermore, the present invention provides an expression vector containing the above-mentioned nucleic acid.

[0011] Furthermore, the present invention provides a host cell containing the above-mentioned nucleic acid or expression vector.

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

[0013] Furthermore, the present invention provides a composition and kit for detecting ASM proteins, comprising the above-mentioned antibody or its antigen-binding fragment.

[0014] Furthermore, the present invention provides an ASM protein detection method comprising the step of reacting the above-mentioned antibody or its antigen-binding fragment with a sample. [Effects of the Invention]

[0015] The antibody or antigen-binding fragment thereof according to the present invention binds specifically to the ASM protein with high binding affinity, and can therefore be used for detecting the ASM protein or diagnosing diseases caused by overexpression of the ASM protein. [Modes for carrying out 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 the ASM (acid sphingomyelinase) protein.

[0018] In this specification, the term "ASM (acid sphingomyelinase) protein" refers to an enzyme that is one of the SMase (sphingomyelinase) family, which regulates sphingolipid metabolism. The above-mentioned ASM protein catalyzes the process of breaking down sphingomyelin into ceramide and phosphorylcholine, and can be classified as alkaline, neutral, or acidic depending on the pH at which it exhibits optimal enzymatic activity.

[0019] The above ASM protein can include any type of ASM protein known in the ordinary art. Specifically, the above ASM protein is of mammalian origin, and more specifically, may be of human, monkey, rat, or mouse origin. Furthermore, the above ASM protein can include any amino acid sequence known in the ordinary art as an ASM protein. As an example, the above ASM protein can be a polypeptide consisting of the amino acid sequence described in Sequence ID No. 139 or a nucleic acid encoding it.

[0020] The above-mentioned ASM protein may have one or more amino acids added, deleted, or substituted in the amino acid sequence described in SEQ ID NO: 139, as 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 of the overall protein, i.e., its polarity or hydrophobicity. Furthermore, the above-mentioned ASM protein may have 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more homology to the amino acid sequence described in SEQ ID NO: 139.

[0021] In this specification, the term "antibody" refers to an immune protein that binds to an antigen to inhibit or remove its action. The antibodies described above can include any type of antibody known in the ordinary art. Specifically, the antibodies may include IgM, IgD, IgG, IgA, and IgE, which may each contain heavy chains made from μ, δ, γ, α, and ε, which are genes encoding heavy chain invariant regions. Typically, antibody technology primarily uses IgG, which is further composed of isotypes of IgG1, IgG2, IgG3, or IgG4, each of which may have different structural and functional properties. The antibodies described above can include humanized antibodies containing minimal sequences derived from non-human antibodies, human antibodies composed of human-derived sequences, or chimeric antibodies containing a mixture of sequences from different species.

[0022] The above-mentioned IgG can form a very stable Y-shaped structure (approximately 150 kDa) made up of two heavy-chain proteins of approximately 50 kDa and two light-chain proteins of approximately 25 kDa. The light and heavy chains constituting the antibody are divided into a variable region with a different amino acid sequence and a constant region with the same amino acid sequence. In this case, the heavy-chain constant region includes CH1, hinge (H), CH2, and CH3 domains, and each domain is composed of two β-sheets that can be linked by an intramolecular disulfide bond. At this time, the two variable regions of the heavy and light chains bind together to form an antigen-binding site, and one antigen-binding site can be present on each of the two arms of the Y-shape. In a full-length antibody, the portion that can bind to an antigen is called the Fab (antibody binding fragment), and the portion that cannot bind to an antigen is called the Fc (crystallizable fragment). The Fab and Fc can be linked together by a hinge.

[0023] In one embodiment of the present invention, the IgG is a human-derived IgG, specifically, it can include a human IgG heavy chain constant region composed of the amino acid sequence set forth in SEQ ID NO: 137 and a human light chain λ constant region composed of the amino acid sequence set forth in SEQ ID NO: 138. Further, the human-derived IgG can include a human IgG heavy chain constant region composed of the nucleotide sequence set forth in SEQ ID NO: 140 and a human light chain λ constant region composed of the nucleotide sequence set forth in SEQ ID NO: 141.

[0024] The antibody according to the present invention can include not only the full-length antibody but also all of its antigen-binding fragments. Specifically, the antigen-binding fragment can mean a part excluding the Fc that functions to transmit the binding stimulation to the antigen to cells, complement, etc. As an example, the antigen-binding fragments of the antibody can include all of Fab, scFv, F(ab)2, and Fv, and can also include third-generation antibody fragments such as single domain antibodies and minibodies.

[0025] As an example, the antibody or its antigen-binding fragment can include a heavy chain CDR1 composed of the amino acid sequence set forth in SEQ ID NO: 89, a heavy chain CDR2 in which five or fewer amino acids are substituted in the polypeptide composed of the amino acid sequence set forth in SEQ ID NO: 90, a heavy chain variable region composed of the amino acid sequence set forth in SEQ ID NO: 91, and a polypeptide composed of the amino acid sequence set forth in SEQ ID NO: 92 or a light chain CDR2 in which five or fewer amino acids are substituted in the polypeptide, and a polypeptide composed of the amino acid sequence set forth in SEQ ID NO: 94 or a light chain CDR3 in which two or fewer amino acids are substituted in the polypeptide, and a light chain variable region composed thereof can be included.

[0026] The term "CDR (complementarity determining region)" refers to a highly variable region within the heavy and light chain variable regions of an antibody, which has a different amino acid sequence for each antibody. It is the site that actually binds to the 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 it can exist beneath the loop. The heavy chain and light chain each have three ring structures, and these six ring structures combine to make direct contact with the antigen. For convenience, the antigen-binding sites CDRs with the above six ring structures will be referred to as heavy chain CDR1, heavy chain CD2, heavy chain CDR3, light chain CDR1, light chain CDR2, or light chain CDR3, respectively.

[0027] In the antibody or antigen-binding fragment thereof according to the present invention, the heavy chain CDR2 in which five or fewer amino acids are substituted in a polypeptide composed of the amino acid sequence described in SEQ ID NO: 90 may be a heavy chain CDR2 in which five or fewer amino acids selected from the group consisting of the 2nd, 4th to 10th, 12th, 13th, and 17th amino acids from the N-terminus of the polypeptide are substituted.

[0028] As an example, the 2nd, 4th, 6th, 7th, 9th, 12th, or 13th amino acid from the N-terminus of the polypeptide composed of the amino acid sequence described in Sequence 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 isoleucine, which is the second amino acid from the N-terminus, may be substituted with leucine, valine, or threonine; the tyrosine, which is the fourth amino acid, may be substituted with phenylalanine or tryptophan; the serine, which is the sixth amino acid, may be substituted with glycine or asparagine; the glycine, which is the seventh amino acid, may be substituted with valine; the isoleucine, which is the ninth amino acid, may be substituted with proline, alanine, or valine; the alanine, which is the twelfth amino acid, may be substituted with serine; and the aspartic acid, which is the thirteenth amino acid, may be substituted with asparagine or alanine. Furthermore, the tenth amino acid from the N-terminus of the polypeptide composed of the amino acid sequence described in Sequence ID No. 90 may be substituted with a basic amino acid, and specifically, the basic amino acid may include arginine, histidine, and lycine. More specifically, the tyrosine, which is the tenth amino acid from the N-terminus, may be substituted with arginine.Furthermore, the 17th amino acid from the N-terminus of the polypeptide composed of the amino acid sequence described in Sequence ID No. 90 is substituted with an acidic amino acid, specifically, the acidic amino acid may include aspartic acid and glutamic acid. More specifically, the glycine, which is the 17th amino acid from the N-terminus, may be substituted with aspartic acid or glutamic acid. Also, the 5th amino acid from the N-terminus of the polypeptide composed of the amino acid sequence described in Sequence ID No. 90 is a neutral or acidic amino acid, and the neutral or acidic amino acid is as described above. More specifically, the glycine, which is the 5th amino acid from the N-terminus, may be substituted with alanine, aspartic acid, serine, or proline. Furthermore, the 8th amino acid from the N-terminus of the polypeptide composed of the amino acid sequence described in Sequence 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 eighth amino acid from the N-terminus, asparagine, 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 are substituted in the polypeptide composed of the amino acid sequence described in SEQ ID NO: 90 may be a polypeptide composed of the amino acid sequences described 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 are substituted in a polypeptide composed of the amino acid sequence described in SEQ ID NO: 93 may be a light chain in which four or fewer amino acids selected from the group consisting of the 3rd to 7th amino acids from the N-terminus of the polypeptide are substituted.

[0031] In one embodiment of the present invention, the light chain CDR2 in which five or fewer amino acids are substituted in the polypeptide composed of the amino acid sequence described in SEQ ID NO: 93 may be a polypeptide composed of the amino acid sequences described 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 are substituted in a polypeptide composed of the amino acid sequence described in SEQ ID NO: 94 may be a light chain CDR2 in which two or fewer amino acids selected from the group consisting of the 6th and 8th amino acids from the N-terminus of the polypeptide are substituted.

[0033] As an example, the sixth and eighth amino acids from the N-terminus of the polypeptide composed of the amino acid sequence described in Sequence ID No. 94 are neutral amino acids, and these neutral amino acids are as described above. More specifically, the serine amino acids that are the sixth and eighth from the N-terminus may be replaced with tryptophan and glycine, respectively.

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

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

[0036] The above-mentioned antibody or its antigen-binding fragment can be produced by methods for producing monoclonal antibodies that are well known in the ordinary art, and these production methods can be appropriately modified by ordinary artisans. For example, the above-mentioned antibody can be obtained by producing hybridomas using B lymphocytes obtained from animals immunized with the antigen, or by producing it using phage display technology.

[0037] Furthermore, the present invention provides nucleic acids that encode the above-mentioned antibody or its antigen-binding fragment.

[0038] The nucleic acid-encoded antibody or antigen-binding fragment thereof according to the present invention may have the characteristics described above. As long as the amino acid sequence constituting the antibody or antigen-binding fragment thereof according to the present invention is known, the nucleic acid sequence encoding it is also obvious to a person skilled in the art. Furthermore, the above 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 therefrom is maintained.

[0039] Furthermore, the present invention provides an expression vector containing the above-mentioned nucleic acid.

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

[0041] In this specification, the term "expression vector" refers to a means for expressing a target gene in a host cell, and can include plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. The expression vector may contain elements necessary for generating peptides from the nucleic acids it contains. Specifically, the expression vector may include signal sequences, origins of replication, marker genes, promoters, transcription termination sequences, and the like. In this case, the nucleic acid encoding the antibody or its antigen-binding fragment according to the present invention can be operably linked to the promoter.

[0042] For example, an expression vector used in prokaryotic cells may include a promoter to initiate transcription, a ribosome binding site for initiating sequencing, and transcription and sequencing termination sequences. An expression vector used in eukaryotic cells may include a promoter and polyadenylation sequence derived from mammals or mammalian viruses.

[0043] Furthermore, any marker gene commonly known in the art can be used as the marker gene included in the expression vector, and specifically, it may be an antibiotic resistance gene. Specifically, the antibiotic resistance gene may be a gene that exhibits resistance to antibiotics including ampicillin, gentamicin, cabenicillin, chloramphenicol, streptomycin, kanamycin, neomycin, and tetracycline.

[0044] Furthermore, the present invention provides a host cell containing the above-mentioned nucleic acid or expression vector.

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

[0046] The host cells described above can be any type of cell known in the ordinary art to be usable for producing antibodies or their antigen-binding fragments. Specifically, the host cells may be prokaryotic cells, yeast, or eukaryotic cells. The prokaryotic cells may include Escherichia coli (E. coli), Bacillus strains, Streptomyces strains, Psudomonas strains, Stapirococcus strains, etc., and the yeast may include Saccharomyces cerevisiae, etc. The eukaryotic cells mentioned above 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, among others.

[0047] Furthermore, the host cells described above may have been transfused with the nucleic acids or expression vectors mentioned above by conventional methods in the field of normal technology. Specifically, the transfusion can be performed by methods such as transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroacupuncture, and gene gun. Moreover, the methods described above can be appropriately modified by conventional technicians.

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

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

[0050] The above culture can be carried out using a culture medium appropriate to the type of host cell used for production, and may include appropriate supplements as needed. Furthermore, the above culture can be carried out in an environment appropriate to the type of host cell.

[0051] The production method according to the present invention may further include a step of recovering antibodies or antigen-binding fragments thereof produced in host cells. The recovery can be carried out by conventional methods in the ordinary art, which can be appropriately modified by ordinary technicians as needed. For example, the recovery is carried out by removing impurities using centrifugation or ultrafiltration, and further purifying the obtained result by chromatography or the like. Examples of chromatography include affinity chromatography, cationic chromatography, and hydrophobic interaction chromatography.

[0052] Furthermore, the present invention provides a composition and kit for detecting ASM proteins, comprising the above-mentioned antibody or its antigen-binding fragment.

[0053] The antibody or antigen-binding fragment contained in the ASM protein detection composition and kit according to the present invention may have the characteristics described above.

[0054] Furthermore, the above composition may include ligands that can specifically bind to the antibody or its antigen-binding fragment according to the present invention. The ligands may be conjugates labeled with a detection agent such as a chromogenic enzyme, a fluorescent substance, a radioisotope, or a colloid, and ligands treated with streptavidin or avidin. In addition to the reagents described above, the detection composition of the present invention may further include distilled water or a buffer solution to stably maintain these structures.

[0055] Furthermore, the above kit may be bound to a solid substrate to facilitate subsequent steps such as washing the antibodies or antigen-binding fragments contained therein or separating the complexes. In this case, the solid substrate can be a synthetic resin, nitrocellulose, a glass substrate, a metal substrate, glass fibers, microspheres, or microbeads. Examples of synthetic resins include polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, or nylon.

[0056] Furthermore, the above kit can be manufactured by conventional manufacturing methods well known to those skilled in the art, and may further contain buffers, stabilizers, inactive proteins, and the like.

[0057] Furthermore, the present invention provides an ASM protein detection method comprising the step of reacting the above-mentioned antibody or its antigen-binding fragment with a sample.

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

[0059] The above-mentioned samples can include any type of sample as long as it is suitable for detecting ASM proteins. Furthermore, methods for detecting target proteins using antibodies or their antigen-binding fragments are well known in the ordinary art and can be carried out by ordinary technicians with appropriate modifications as needed. [Examples]

[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. Any device having substantially the same configuration and the same effects as the technical concept described in the claims of the present invention is included within the technical scope of the present invention.

[0061] [Example 1. Production of an antibody that specifically binds to the ASM (acid sphingomyelinase) protein] We created a variant of antibody #9104, which specifically binds to the human ASM protein (SEQ ID NO: 139).

[0062] First, the VH and VL genes of the #9104 antibody were amplified using a standard method with random primers to induce random mutations in the heavy and light chain CDR sequences of the #9104 antibody. The amplified VH and VL genes were ligated to the mouse heavy chain invariant region 1 (CH1, SEQ ID NO: 142) and light chain invariant region (CL, SEQ ID NO: 143), respectively, and inserted into a pComb3xss phagemide vector in scFab form. A #9104 random mutagenesis library was prepared using this library, and scFabs that specifically bind to the human ASM protein were selected by a standard screening method. Expression vectors were prepared so that the selected scFabs are expressed in a form in which the human heavy chain invariant region and human light chain λ invariant region, consisting of the nucleotide sequences described in SEQ ID NO: 137 or 138, respectively, are ligated to the carboxyl terminus of the heavy chain variable region and light chain variable region, respectively. As a result, the amino acid sequences and nucleic acid sequences of the heavy chain variable region of the selected scFabs are shown in Table 1 below, and the amino acid sequences and nucleic acid sequences of the light chain variable region are shown in Table 2 below.

[0063] [Table 1] JPEG0007899473000002.jpg236170JPEG0007899473000003.jpg237170JPEG000 7899473000004.jpg242170JPEG0007899473000005.jpg242170JPEG0007899473 000006.jpg238170JPEG0007899473000007.jpg238170JPEG0007899473000008. jpg238170JPEG0007899473000009.jpg238170JPEG0007899473000010.jpg34170

[0064] [Table 2] JPEG0007899473000012.jpg238170JPEG0007899473000013.jpg220170JPEG0007899473000014.jpg218170 JPEG0007899473000015.jpg219170JPEG0007899473000016.jpg219170JPEG0007899473000017.jpg219170 JPEG0007899473000018.jpg218170JPEG0007899473000019.jpg219170JPEG0007899473000020.jpg217170 JPEG0007899473000021.jpg220170JPEG0007899473000022.jpg218170JPEG0007899473000023.jpg218170

[0065] In this study, a mammalian expression vector was used. The resulting expression vector was used to transform the ExpiCHO cell line, and a full-length antibody that binds to the human ASM protein was produced.

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

[0067] [Table 3] JPEG0007899473000025.jpg243170JPEG0007899473000026.jpg119170

[0068] [Table 4] JPEG0007899473000028.jpg244170JPEG0007899473000029.jpg245170JPEG0007899473000030.jpg131170

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

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

[0071] First, an antibody that specifically binds to the ASM protein produced in Example 1 was captured using an anti-human IgG Fc capture (AHC) biosensor, and a recombinant human ASM protein solution of 1.25, 2.5, 5, 10, or 20 nM was added thereto. After adding the human ASM protein solution, the binding phase of the reaction product was observed for approximately 1,200 seconds, then 1× kinetics buffer (ForteBio) was added, and the separation phase of the reaction product was observed for approximately 1,500 seconds. The association constant (K) for each antibody was determined using Octet® analysis software (Pall Life Sciences). a ), separation constant:K d ) and equilibrium dissociation constant (K DThe following was determined. As a result, Table 5 shows the binding affinity of antibodies with mutations in the heavy chain CDR portion described in Table 1 to the human ASM protein, and Table 6 shows the binding affinity of antibodies with mutations in the light chain CDR portion described in Table 2 to the human ASM protein.

[0072] [Table 5]

[0073] [Table 6] JPEG0007899473000033.jpg68170

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

[0075] [Experimental Example 2: Combinations of heavy and light chain variable regions] Forty antibodies were created by combining the eight heavy chain variable regions and five light chain variable regions of the #9104 antibody selected above (Table 7).

[0076] [Table 7] JPEG0007899473000035.jpg214170

[0077] After expressing the antibodies on a small scale in the ExpiCHO cell line as described above, their binding affinity to human ASM protein was confirmed as described in Experimental Example 1. At this time, antibodies produced using the combination of the #9104v-H33 heavy chain variable region and the #9104v-L23 light chain variable region were not expressed and were therefore excluded from the analysis of their binding affinity to ASM protein. The results of the binding affinity of the above antibodies to human ASM protein are shown in Table 8.

[0078] [Table 8] JPEG0007899473000037.jpg213170

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

Claims

1. An antibody or an antigen-binding fragment thereof, It specifically binds to the ASM (acid sphingomyelinase) protein, The antibody or its antigen-binding fragment A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 97, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 116, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 98, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 116, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 99, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 116, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 101, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 116, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 103, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 116, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 108, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 116, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 110, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 116, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 111, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 116, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 97, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 117, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 98, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 117, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 99, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 117, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 101, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 117, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 103, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 117, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 108, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 117, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 110, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 117, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 111, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 117, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 97, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 118, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 98, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 118, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 99, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 118, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 101, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 118, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 103, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 118, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 108, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 118, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 110, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 118, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 97, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 134, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 98, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 134, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 99, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 134, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 101, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 134, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 103, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 134, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 108, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 134, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 110, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 134, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 111, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 134, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 97, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 135, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 98, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 135, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 99, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 135, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 101, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 135, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 103, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 135, and 94, respectively; A heavy chain variable region comprising heavy chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 89, 108, and 91, respectively, and a light chain variable region comprising light chains CDR1, CDR2, and CDR3 consisting of amino acid sequences shown in SEQ ID NOs. 92, 135, and 94, respectively; A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 110, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 135, and 94, respectively; or A heavy chain variable region comprising heavy chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 89, 111, and 91, respectively, and a light chain variable region comprising light chain CDR1, CDR2, and CDR3 consisting of the amino acid sequences shown in SEQ ID NOs. 92, 135, and 94, respectively. An antibody or its antigen-binding fragment, including the above.

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

3. A nucleic acid encoding an antibody or an antigen-binding fragment thereof as described in claim 1.

4. An expression vector comprising the nucleic acid described in claim 3.

5. A host cell comprising the nucleic acid described in claim 3 or the expression vector described in claim 4.

6. A method for producing an antibody or an antigen-binding fragment that specifically binds to an ASM protein, comprising the step of culturing the host cells described in claim 5 to produce an antibody or an antigen-binding fragment thereof.

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