Antibody specifically binding to acidic sphingomyelinase protein

Abstract

The present invention relates to an antibody capable of specifically binding to an acid sphingomyelinase (ASM) protein, and a use thereof, and more particularly, to an antibody capable of specifically binding to an acid sphingomyelinase (ASM) protein. Specifically, the antibody or the antigen-binding fragment thereof according to the present invention binds to acidic sphingomyelin protein with a specifically high binding force, and thus can be used for detecting acidic sphingomyelin protein or diagnosing diseases caused by overexpression of acidic sphingomyelin protein.

Description

Technical Field

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

[0002] Sphingolipid metabolism regulates normal cell signal transduction, and abnormal changes in sphingolipid metabolism affect various neurodegenerative diseases including Alzheimer's disease. Acid sphingomyelinase (ASM) protein, as 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 cell membrane turnover.

[0003] Compared with normal people, the activity of acid sphingomyelinase protein is significantly increased in the brains of patients with neurodegenerative diseases such as Alzheimer's disease. In this regard, Korean Patent No. 10-1521117 discloses that when the activity of overexpressed acid sphingomyelinase protein is inhibited or the expression of acid sphingomyelinase protein is inhibited, the accumulation of amyloid-β protein can be inhibited and learning ability and memory can be improved, thereby treating neurodegenerative diseases. And recently, it is known that the activity of acid sphingomyelinase protein is also increased in neurological diseases such as depression, so inhibiting the expression or activity of the acid sphingomyelinase protein has the effect of improving depression.

[0004] However, substances that directly inhibit the expression or activity of acid sphingomyelinase protein have not been developed yet, but several inhibitors that indirectly inhibit the expression of acid sphingomyelinase protein have been identified. For example, there are tricyclic antidepressants for treating depression, which include amitriptyline, desipramine, mipramine, etc. Although these tricyclic antidepressants are not developed as acid sphingomyelinase protein inhibitors, they have been shown to exhibit acid sphingomyelinase protein inhibitory effects through various research results. The main pharmacological mechanism of tricyclic antidepressants is to increase the activity of neurotransmitters in nerve cells by inhibiting the reuptake of neurotransmitters, and it is confirmed that their role as acid sphingomyelinase inhibitors is a side effect. However, since tricyclic antidepressants act on the nervous system and nerve cells, they may cause side effects such as blurred vision, increased light sensitivity, and vomiting. Summary of the Invention

[0005] Technical Problem

[0006] An object of the present invention is to provide an antibody or an antigen-binding fragment thereof that specifically binds to acid sphingomyelinase protein.

[0007] Another object of the present invention is to provide a method for preparing the antibody or its antigen-binding fragment.

[0008] Still another object of the present invention is to provide the use of the antibody or its antigen-binding fragment for detecting acid sphingomyelinase protein.

[0009] Technical solutions

[0010] To achieve the above object, the present invention provides an antibody or its antigen-binding fragment that specifically binds to acid sphingomyelinase protein.

[0011] Moreover, the present invention provides a nucleic acid encoding the antibody or its antigen-binding fragment.

[0012] Furthermore, the present invention provides an expression vector containing the nucleic acid.

[0013] In addition, the present invention provides a host cell containing the nucleic acid or the expression vector.

[0014] Also, the present invention provides a method for producing an antibody or its antigen-binding fragment that specifically binds to acid sphingomyelinase protein, the method comprising the step of culturing the host cell to produce the antibody or its antigen-binding fragment.

[0015] And, the present invention provides a composition and a kit for detecting acid sphingomyelinase protein, which contain the antibody or its antigen-binding fragment.

[0016] Furthermore, the present invention provides a method for detecting acid sphingomyelinase protein, the method comprising the step of reacting the antibody or its antigen-binding fragment with a sample.

[0017] Effects of the invention

[0018] The antibody or its antigen-binding fragment of the present invention binds to acid sphingomyelinase protein with a specifically high binding affinity, and thus can be used for detecting acid sphingomyelinase protein or diagnosing diseases caused by overexpression of acid sphingomyelinase protein. Detailed implementation manners

[0019] Hereinafter, the present invention will be described in detail.

[0020] The present invention provides an antibody or its antigen-binding fragment that specifically binds to Acid sphingomyelinase (ASM) protein.

[0021] As used herein, the term "Acid sphingomyelinase (ASM) protein" refers to an enzyme that is one of the sphingomyelinase (SMase) family that regulates sphingolipid metabolism. The acid sphingomyelinase protein promotes the process of decomposing sphingomyelin into ceramide and phosphorylcholine, and can be classified as alkaline, neutral or acidic according to the pH at which optimal enzymatic activity is exhibited.

[0022] The acid sphingomyelinase protein may include all types of acid sphingomyelinase proteins known in the art of conventional technology. Specifically, the acid sphingomyelinase protein may be derived from mammals, and more specifically, may be derived from humans, monkeys, rats or mice. And, the acid sphingomyelinase protein may include all amino acid sequences known in the art of conventional technology as acid sphingomyelinase proteins. For example, the acid sphingomyelinase protein may be a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 139 or a nucleic acid encoding the same.

[0023] One or more amino acids may be added, deleted or substituted in the amino acid sequence shown in SEQ ID NO: 139 in the acid sphingomyelinase protein, as long as it maintains the same or corresponding biological activity. In this case, the amino acid substitution may be a conservative substitution within the range that does not affect or slightly affects the charge, i.e., polarity or hydrophobicity, of the entire protein. And, the acid sphingomyelinase 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 shown in SEQ ID NO: 139.

[0024] As used herein, the term "antibody" refers to an immunoprotein that binds to an antigen to interfere with the action of the antigen or remove the antigen. The antibody may include all types of antibodies included in the art of conventional technology. Specifically, the antibody may include IgM, IgD, IgG, IgA and IgE, which respectively include heavy chains composed of genes μ, δ, γ, α and ε encoding the constant regions of the heavy chains. Generally, IgG is mainly used in antibody technology, which is again composed of isotypes of IgG1, IgG2, IgG3 or IgG4, and their respective structural and functional characteristics may be different. The antibody may simultaneously include humanized antibodies containing the minimum sequence derived from non-human antibodies, human antibodies composed of human-derived sequences, or chimeric antibodies composed of sequences mixed from different species.

[0025] The IgG can form a very stable Y-shaped structure (about 150 kDa) composed of two heavy chain proteins of about 50 kDa and two light chain proteins of about 25 kDa. The light and heavy chains that make up the antibody can be divided into a variable region with different amino acid sequences between antibodies and a constant region with the same amino acid sequence. In this case, the heavy chain constant region contains CH1, hinge (H), CH2, and CH3 domains, each domain consisting of two β-sheets and being connected by an intramolecular disulfide bond. In this case, the two variable regions of the heavy and light chains combine to form an antigen binding site, which can be present in each of the two Y-shaped arms. The part of the full-length antibody that can bind to the antigen is called the antibody binding fragment (Fab), and the part that cannot bind to the antigen is called the crystallizable fragment (Fc). Fab and Fc can be connected by a hinge.

[0026] In one embodiment of the present invention, the IgG can be a human IgG. Specifically, it can include a human IgG heavy chain constant region composed of the amino acid sequence shown in SEQ ID NO: 137 and a human light chain lambda constant region composed of the amino acid sequence shown in SEQ ID NO: 138. Moreover, the human IgG can include a human IgG heavy chain constant region composed of the nucleotide sequence shown in SEQ ID NO: 140 and a human light chain lambda constant region composed of the nucleotide sequence shown in SEQ ID NO: 141.

[0027] The antibody of the present invention includes not only the full-length antibody but also its antigen binding fragments. Specifically, the antigen binding fragment can mean the part except for Fc that functions to transmit the binding stimulus between the antigen to the cell or complement. As an example, the antigen binding fragments of the antibody can include all of Fab, scFv, F(ab)2, and Fv, etc., and can also include third-generation antibody fragments such as single-domain antibodies or minibodies.

[0028] For example, the antibody or its antigen-binding fragment may comprise: a heavy-chain variable region consisting of a heavy-chain CDR1 having the amino acid sequence shown in Sequence 89, a heavy-chain CDR2 in which no more than 5 amino acids in the polypeptide having the amino acid sequence shown in Sequence 90 are substituted, and a heavy-chain CDR3 having the amino acid sequence shown in Sequence 91; and a light-chain variable region consisting of a light-chain CDR1 having the amino acid sequence shown in Sequence 92, a light-chain CDR2 in the polypeptide having the amino acid sequence shown in Sequence 93 or in which no more than 5 amino acids in said polypeptide are substituted, and a light-chain CDR3 in the polypeptide having the amino acid sequence shown in Sequence 94 or in which no more than 2 amino acids in said polypeptide are substituted.

[0029] The term "CDR (complementarity determining region)" refers to the hypervariable region, which is the site where each antibody has a different amino acid sequence within the variable regions of the heavy and light chains of the antibody, and means the site that actually binds to the antigen. In the three-dimensional structure of the antibody, the CDRs are located on the surface of the antibody in loops, and there may be an FR (framework region) that structurally supports them below the loops. Both the heavy and light chains each have three loop structures, and these six loop structures can combine to directly contact the antigen. For convenience, the CDRs, which are the antigen-binding sites having the six loop structures, can be referred to as heavy-chain CDR1, heavy-chain CDR2, heavy-chain CDR3, light-chain CDR1, light-chain CDR2, or light-chain CDR3, respectively.

[0030] In the heavy-chain CDR2 of the antibody or its antigen-binding fragment of the present invention, in which no more than 5 amino acids in the polypeptide having the amino acid sequence shown in Sequence 90 are substituted, no more than 5 amino acids can be substituted from among those selected from the group consisting of the 2nd, 4th to 10th, 12th, 13th, and 17th amino acids starting from the N-terminus of the polypeptide.

[0031] For example, the amino acids at the 2nd, 4th, 6th, 7th, 9th, 12th, or 13th positions starting from the N-terminus of the polypeptide composed of the amino acid sequence shown in Sequence 90 can be replaced by neutral amino acids. Specifically, the neutral amino acids may include alanine, glycine, leucine, isoleucine, proline, valine, phenylalanine, tryptophan, tyrosine, serine, threonine, cysteine, methionine, asparagine, and glutamine. More specifically, isoleucine at the 2nd position starting from the N-terminus can be replaced by leucine, valine, or threonine; tyrosine at the 4th position can be replaced by phenylalanine or tryptophan; serine at the 6th position can be replaced by glycine or asparagine; glycine at the 7th position can be replaced by valine; isoleucine at the 9th position can be replaced by proline, alanine, or valine; alanine at the 12th position can be replaced by serine; and aspartic acid at the 13th position can be replaced by asparagine or alanine. On the other hand, the amino acid at the 10th position starting from the N-terminus of the polypeptide composed of the amino acid sequence shown in Sequence 90 can be replaced by basic amino acids. Specifically, the basic amino acids may include arginine, histidine, and lysine. More specifically, tyrosine at the 10th position starting from the N-terminus can be replaced by arginine. Also, the amino acid at the 17th position starting from the N-terminus of the polypeptide composed of the amino acid sequence shown in Sequence 90 can be replaced by acidic amino acids. Specifically, the acidic amino acids may include aspartic acid and glutamic acid. More specifically, glycine at the 17th position starting from the N-terminus can be replaced by aspartic acid or glutamic acid. And the amino acid at the 5th position starting from the N-terminus of the polypeptide composed of the amino acid sequence shown in Sequence 90 can be a neutral or acidic amino acid, and the neutral or acidic amino acids can be as described above. More specifically, glycine at the 5th position starting from the N-terminus can be replaced by alanine, aspartic acid, serine, or proline. Additionally, the amino acid at the 8th position starting from the N-terminus of the polypeptide composed of the amino acid sequence shown in Sequence 90 can be a neutral, acidic, or basic amino acid, and the neutral, acidic, or basic amino acids can be as described above.More specifically, asparagine, which is the 8th amino acid from the N-terminus, can be replaced with aspartic acid, lysine, isoleucine, threonine, valine, glycine, or tyrosine.

[0032] In one embodiment of the present invention, the heavy chain CDR2 in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 90 with 5 or fewer amino acids replaced can be a polypeptide consisting of the amino acid sequences shown in SEQ ID NOs: 95 to 111, respectively.

[0033] In the antibody or antigen-binding fragment thereof of the present invention, in the light chain CDR2 in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 93 with 5 or fewer amino acids replaced, 4 or fewer amino acids selected from the group consisting of the 3rd to 7th amino acids from the N-terminus of the polypeptide can be replaced.

[0034] For example, the 3rd, 4th, and 5th amino acids from the N-terminus of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 93 can be neutral amino acids, and the neutral amino acids can be as described above. More specifically, serine, which is the 3rd amino acid from the N-terminus, can be replaced with isoleucine, leucine, methionine, glutamine, threonine, valine, tryptophan, or tyrosine; histidine, which is the 4th amino acid, can be replaced with phenylalanine or tyrosine; and arginine, which is the 5th amino acid, can be replaced with glutamine, proline, leucine, threonine, or serine. On the other hand, the 6th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 93 can be a basic or neutral amino acid, and the basic or neutral amino acid can be as described above. More specifically, proline, which is the 6th amino acid from the N-terminus, can be replaced with alanine, glutamine, arginine, serine, or histidine. Additionally, the 7th amino acid from the N-terminus of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 93 can be a neutral, acidic, or basic amino acid, and the neutral, acidic, or basic amino acid can be as described above. More specifically, serine, which is the 7th amino acid from the N-terminus, can be replaced with aspartic acid, tyrosine, glycine, asparagine, threonine, phenylalanine, tryptophan, or arginine.

[0035] In one embodiment of the present invention, the light chain CDR2 in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 93 with 5 or fewer amino acids replaced can be a polypeptide consisting of the amino acid sequences shown in SEQ ID NOs: 112 to 135, respectively.

[0036] In the antibody or antigen-binding fragment thereof of the present invention, in the light chain CDR2 in which 2 or fewer amino acids in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 94 are substituted, 2 or fewer amino acids selected from the group consisting of the 6th to 8th amino acids can be substituted starting from the N-terminus of the polypeptide.

[0037] For example, the 6th and 8th amino acids starting from the N-terminus of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 94 can be neutral amino acids, and the neutral amino acids can be as described above. More specifically, serine, which is the 6th and 8th amino acids starting from the N-terminus, can be substituted with tryptophan and glycine, respectively.

[0038] In one embodiment of the present invention, the light chain CDR3 in which 2 or fewer amino acids in the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 94 are substituted can be a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 136.

[0039] Moreover, the antibody or antigen-binding fragment thereof of the present invention can be obtained by deformation as needed. Specifically, the antibody or antigen-binding fragment thereof can be deformed by conjugation, glycosylation, labeling, or a combination thereof. Specifically, the antibody or antigen-binding fragment thereof can be deformed by horseradish peroxidase (HRP), alkaline phosphatase, hapten, biotin, streptavidin, fluorescent substance, radioactive substance, quantum dot, polyethylene glycol (PEG), histidine tag, etc. In addition, the antibody or antigen-binding fragment thereof can also be conjugated with other drugs as needed.

[0040] The antibody or antigen-binding fragment thereof can be prepared according to the monoclonal antibody preparation method well-known in the art, and the preparation method can be appropriately deformed by those skilled in the art. As an example, the antibody can be prepared by preparing a hybridoma using B lymphocytes obtained from an animal immunized with an antigen, or can be prepared using phage display technology.

[0041] Furthermore, the present invention provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0042] The antibody or antigen-binding fragment thereof encoded by the nucleic acid of the present invention may have the characteristics described above. The amino acid sequence constituting the antibody or antigen-binding fragment thereof of the present invention is known, and thus the nucleic acid sequence encoding the same is also well-known to those of ordinary skill in the art. Further, as long as the nucleic acid sequence maintains the activity of the antibody translated therefrom or the antigen fragment of the antibody, one or more bases may be added, deleted or substituted.

[0043] Furthermore, the present invention provides an expression vector containing the nucleic acid.

[0044] The nucleic acid contained in the expression vector of the present invention may encode an antibody or antigen-binding fragment thereof having the characteristics described above.

[0045] The term "expression vector" used in the present specification may include all of plasmid vectors, cosmid vectors, phage vectors, viral vectors, etc. as means for expressing a target gene in a host cell. The expression vector may contain necessary elements for generating a peptide from the 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, etc. In this case, the nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention may be operably linked to the promoter.

[0046] As an example, an expression vector for prokaryotic cells may contain a promoter for transcription, a ribosome binding site for starting translation, and termination sequences for transcription and translation. On the other hand, an expression vector for eukaryotic cells may contain a promoter derived from a mammal or mammalian virus and a polyadenylation sequence.

[0047] Furthermore, the marker gene contained in the expression vector may be any marker gene well-known in the art of ordinary skill, and specifically, may be an antibiotic resistance gene. Specifically, the antibiotic resistance gene may be a gene showing resistance to antibiotics including ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, neomycin, tetracycline, etc.

[0048] Furthermore, the present invention provides a host cell containing the nucleic acid or expression vector.

[0049] The nucleic acid or expression vector contained in the host cell of the present invention may have the characteristics described above. As an example, the nucleic acid may encode an antibody or antigen-binding fragment thereof that specifically binds to the acid sphingomyelinase protein of the present invention, and the expression vector may contain the nucleic acid described above.

[0050] The host cell can be any type of cell that can be used in the general technical field for the production of antibodies or their antigen-binding fragments. Specifically, the host cell can be a prokaryotic cell, yeast, or eukaryotic cell. The prokaryotic cell can include Escherichia coli, Bacillus strains, Streptomyces strains, Pseudomonas strains, Staphylococcus strains, etc., and the yeast can include Saccharomyces cerevisiae, etc. On the other hand, the eukaryotic cell can 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, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PERC6, SP2 / 0, NS-0, U20S, and HT1080, etc.

[0051] Moreover, the host cell can be transfected with the nucleic acid or expression vector as described above according to methods well-known in the general technical field. Specifically, the transfection can be carried out by methods such as transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, etc. And the methods can be appropriately modified by those of ordinary skill in the art.

[0052] Furthermore, the present invention provides a method for producing an antibody or its antigen-binding fragment that specifically binds to the acid sphingomyelinase protein, which includes the step of producing the antibody or its antigen-binding fragment by culturing the host cell.

[0053] The antibody or its antigen-binding fragment produced by the production method of the present invention can have the characteristics as described above.

[0054] The culturing can be carried out using a suitable culture medium according to the type of host cell used in the production, and can contain appropriate supplements as needed. And the culturing can be carried out in a suitable environment according to the type of host cell.

[0055] The production method of the present invention may further include the step of recovering the antibody or its antigen-binding fragment produced by the host cell. The recovery can be carried out according to methods well-known in the art, which can be appropriately modified by those skilled in the art according to requirements. As an example, the recovery can be carried out by removing debris using centrifugation or ultrafiltration and further purifying the resulting product by chromatography or the like. The chromatography can include affinity chromatography, anion chromatography, hydrophobic interaction chromatography, etc.

[0056] Furthermore, the present invention provides a composition and a kit for detecting acid sphingomyelinase protein, which contain the antibody or its antigen-binding fragment.

[0057] The antibody or its antigen-binding fragment contained in the composition and kit for detecting acid sphingomyelinase protein of the present invention may have the above-mentioned characteristics.

[0058] Furthermore, the composition may contain a ligand that specifically binds to the antibody or its antigen-binding fragment of the present invention. The ligand can be a conjugate labeled with a detection body such as a chromogenic enzyme, a fluorescent substance, a radioisotope, or a colloid, and a ligand treated with streptavidin or avidin. In addition to the reagents described above, the detection composition of the present invention may further contain distilled water or a buffer solution that can stably maintain their structures.

[0059] Furthermore, the kit can be combined with a solid matrix to facilitate subsequent steps such as washing the antibody or its antigen-binding fragment contained therein or separating the complex. In this case, the solid matrix can use synthetic resin, nitrocellulose, glass substrate, metal substrate, glass fiber, microspheres, or microbeads. And the synthetic resin can use polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, or nylon, etc.

[0060] Furthermore, the kit can be manufactured by existing manufacturing methods well-known to those skilled in the art, and may also contain a buffer solution, a stabilizer, an inactive protein, etc.

[0061] Furthermore, the present invention provides a method for detecting acid sphingomyelinase protein, which includes the step of reacting the antibody or its antigen-binding fragment with a sample.

[0062] The antibody or its antigen-binding fragment used in the method for detecting acid sphingomyelinase protein of the present invention may have the above-mentioned characteristics.

[0063] As long as it is a sample for detecting acid sphingomyelinase protein, the sample can include all kinds of samples. And the method of detecting a target protein using an antibody or its antigen-binding fragment is well-known in the art, which can be appropriately modified and carried out by those skilled in the art according to requirements.

[0064] Embodiments of the present invention

[0065] Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are only illustrative of the present invention, and the present invention is not limited thereto. As long as a structure that is actually the same as the technical idea in the claimed scope of the present invention is formed and the same effects are provided, it will fall within the technical scope of the present invention.

[0066] Example 1. Preparation of an antibody that specifically binds to acid sphingomyelinase (ASM) protein

[0067] The #9104 antibody is an antibody that specifically binds to human acid sphingomyelinase protein (Sequence 139), and variants thereof are prepared from the #9104 antibody.

[0068] First, the VH and VL genes of the #9104 antibody are amplified by a conventional method using random primers to induce random mutations in the heavy-chain and light-chain CDR sequences of the #9104 antibody. Each amplified VH and VL gene is ligated to the mouse heavy-chain constant region 1 (CH1, Sequence 142) and the light-chain constant region (CL, Sequence 143) and inserted into the pComb3xss phagemid vector in the form of scFab. A random mutagenesis library of #9104 is prepared using it, and the library is screened by a conventional method to select scFab that specifically binds to human acid sphingomyelinase protein. Expression vectors are prepared at the carboxy terminus of the heavy-chain variable region and the light-chain variable region of the selected scFab, respectively, such that the human heavy-chain constant region and the human light-chain lambda constant region composed of the base sequences shown in Sequence 137 or Sequence 138, respectively, are expressed in a linked form. As a result, the amino acid sequences and nucleic acid sequences of the heavy-chain variable region of the selected scFab 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.

[0069] Table 1

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077]

[0078] Table 2

[0079]

[0080]

[0081]

[0082]

[0083]

[0084]

[0085]

[0086]

[0087]

[0088]

[0089]

[0090]

[0091] In this case, a mammalian expression vector was used as the expression vector. The prepared expression vector was transfected into the ExpiCHO cell line to prepare a full-length antibody that binds to the human acid sphingomyelinase protein.

[0092] Example 2. Determination of the complementarity determining region (CDR)

[0093] The complementarity determining regions in the prepared scFv were confirmed by a conventional method. 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.

[0094] Table 3

[0095]

[0096]

[0097]

[0098] Table 4

[0099]

[0100]

[0101]

[0102]

[0103] As shown in Table 3 and Table 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 the #9104 antibody. On the contrary, CDR2 of the heavy chain variable region, CDR2 and CDR3 of the light chain variable region have sequences in which some of the constituent amino acids are substituted.

[0104] Experimental Example 1. Confirmation of binding affinity to acid sphingomyelinase protein

[0105] Using the QK384 system (Pall Life Sciences) to determine the binding affinity and interaction kinetics of the antibody specifically binding to acid sphingomyelinase protein prepared in the above with acid sphingomyelinase protein.

[0106] First, capture the antibody specifically binding to acid sphingomyelinase protein prepared in Example 1 using an anti-human IgG Fc capture (AHC) biosensor, and then add a recombinant human acid sphingomyelinase protein solution at 1.25 nM, 2.5 nM, 5 nM, 10 nM or 20 nM. Add the human acid sphingomyelinase protein solution, and observe the binding phase of the reactants for about 1200 seconds, then add 1× kinetics buffer (ForteBio), and observe the dissociation phase of the reactants for about 1500 seconds. Using analysis software (Pall Life Sciences) to determine the association constant (K a ), dissociation constant (K d ), and equilibrium dissociation constant (K D) As a result, the binding affinities of the antibodies with mutations in the heavy-chain CDR regions of the #9104 antibody as shown in Table 1 for human acid sphingomyelinase protein are shown in Table 5, and the binding affinities of the antibodies with mutations in the light-chain CDR regions as shown in Table 2 for human acid sphingomyelinase protein are shown in Table 6.

[0107] Table 5

[0108]

[0109]

[0110] Table 6

[0111]

[0112]

[0113] As shown in Table 5 and Table 6, the antibodies prepared in Example 1 bind to human acid sphingomyelinase protein with a binding affinity at the level of 10 -10 M to 10 -9 M.

[0114] Experimental Example 2. Combining the heavy and light chain variable regions

[0115] Forty antibodies (Table 7) were prepared by combining 8 heavy-chain variable regions and 5 light-chain variable regions of the #9104 antibody selected as described above.

[0116] Table 7

[0117]

[0118]

[0119]

[0120] As described above, after small-scale expression in the ExpiCHO cell line, the binding affinity for human acid sphingomyelinase protein was confirmed as described in Example 1. In this case, the antibody prepared from the combination of the #9104v-H33 heavy-chain variable region and the #9104v-L23 light-chain variable region was not expressed, and thus was excluded from the analysis of the binding affinity for acid sphingomyelinase protein. As a result, the binding affinities of the antibodies for human acid sphingomyelinase protein are shown in Table 8.

[0121] Table 8

[0122]

[0123]

[0124] As shown in Table 8, the prepared antibody binds to human acid sphingomyelinase protein with a binding capacity at the level of 10 -10 M to 10 -9 M.

Claims

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

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

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein The antibody or antigen-binding fragment thereof comprises: A heavy chain variable region consisting of a heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 89, a heavy chain CDR2 in which 5 or fewer amino acids in the polypeptide composed of the amino acid sequence shown in SEQ ID NO: 90 are substituted, and a heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 91; And A light chain variable region consisting of a light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 92, a light chain CDR2 in which 5 or fewer amino acids in the polypeptide composed of the amino acid sequence shown in SEQ ID NO: 93 or the polypeptide are substituted, and a light chain CDR3 in which 2 or fewer amino acids in the polypeptide composed of the amino acid sequence shown in SEQ ID NO: 94 or the polypeptide are substituted.

4. The antibody or antigen-binding fragment thereof according to claim 3, wherein, In the heavy chain CDR2 in which 5 or fewer amino acids in the polypeptide composed of the amino acid sequence shown in SEQ ID NO: 90 are substituted, 5 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.

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

6. The antibody or antigen-binding fragment thereof according to claim 3, wherein In the light chain CDR2 in which 5 or fewer amino acids in the polypeptide composed of the amino acid sequence shown in SEQ ID NO: 93 are substituted, 4 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.

7. The antibody or antigen-binding fragment thereof according to claim 3, wherein The light chain CDR2 in which 5 or fewer amino acids in the polypeptide composed of the amino acid sequence shown in SEQ ID NO: 93 are substituted is a polypeptide composed of the amino acid sequences shown in SEQ ID NOs: 112 to 135, respectively.

8. The antibody or antigen-binding fragment thereof according to claim 3, wherein In the light chain CDR3 in which 2 or fewer amino acids in the polypeptide composed of the amino acid sequence shown in SEQ ID NO: 94 are substituted, 2 or fewer amino acids selected from the group consisting of the 6th to 8th amino acids from the N-terminus of the polypeptide are substituted.

9. The antibody or antigen-binding fragment thereof according to claim 3, wherein The light chain CDR3 in which 2 or fewer amino acids in the polypeptide composed of the amino acid sequence shown in SEQ ID NO: 94 are substituted is a polypeptide composed of the amino acid sequence shown in SEQ ID NO:

136.

10. A nucleic acid, characterized in that, Encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

11. An expression vector, characterized in that, Comprising the nucleic acid according to claim 10.

12. A host cell, characterized in that, Comprising the nucleic acid according to claim 10 or the expression vector according to claim 11.

13. A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to acid sphingomyelinase protein, characterized in that, Including the step of culturing the host cell according to claim 12 to produce an antibody or antigen-binding fragment thereof.

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