CN117396517A - Antibodies that specifically bind to acid sphingomyelinase proteins - Google Patents

Antibodies that specifically bind to acid sphingomyelinase proteins Download PDF

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Publication number
CN117396517A
CN117396517A CN202280037975.4A CN202280037975A CN117396517A CN 117396517 A CN117396517 A CN 117396517A CN 202280037975 A CN202280037975 A CN 202280037975A CN 117396517 A CN117396517 A CN 117396517A
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amino acid
heavy chain
light chain
ser
gly
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洪承范
洪美琳
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Lishu Aibosi Co ltd
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Lishu Aibosi Co ltd
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Priority claimed from KR1020220047295A external-priority patent/KR20220160474A/en
Application filed by Lishu Aibosi Co ltd filed Critical Lishu Aibosi Co ltd
Priority claimed from PCT/KR2022/095105 external-priority patent/WO2022250520A1/en
Publication of CN117396517A publication Critical patent/CN117396517A/en
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Abstract

The present invention relates to antibodies that specifically bind to acidic sphingomyelinase (ASM, acid sphingomyelinase) proteins. Specifically, the antibody or antigen-binding fragment thereof of the present invention can be effectively used for detection of an acid sphingomyelinase protein, diagnosis of a disease caused by overexpression of an acid sphingomyelinase protein, or the like by specifically binding to an acid sphingomyelinase protein with high binding force.

Description

Antibodies that specifically bind to acid sphingomyelinase proteins
Technical Field
The present invention relates to antibodies that specifically bind to acidic sphingomyelinase (ASM, acid sphingomyelinase) proteins.
Background
Sphingolipid metabolism regulates normal cellular signaling, and abnormal changes in sphingolipid metabolism can affect a variety of neurodegenerative diseases including Alzheimer's disease. Acid sphingomyelinase (ASM, acid sphingomyelinase) protein, which is an enzyme regulating sphingolipid metabolism, is a protein expressed in almost all kinds of cells, and plays an important role in sphingolipid metabolism (turn over).
In the brains of patients suffering from neurodegenerative diseases such as Alzheimer's disease, the activity of acid sphingomyelinase protein is markedly increased over normal. Related thereto is korean patent laid-open No. 10-1521117, which discloses that neurodegenerative diseases can be treated by inhibiting the activity of over-expressed acid sphingomyelinase protein or inhibiting the expression of acid sphingomyelinase protein to inhibit the accumulation of beta-amyloid and improve learning ability and memory. Further, since an increase in the activity of an acidic sphingomyelinase protein has recently been shown in neurological diseases such as depression, depression can be ameliorated by inhibiting the expression or activity of the acidic sphingomyelinase protein.
However, substances that directly inhibit the expression or activity of acid sphingomyelinase proteins have not been developed, and only several inhibitors that indirectly inhibit the expression of acid sphingomyelinase proteins have been developed. For example, tricyclic antidepressants (tricyclic antidepressant) are used in the treatment of depression, among which are amitriptyline (amitriptyline), desipramine (desipramine), mipramine (mipramine), and the like. The development of these tricyclic antidepressants is not aimed at acid sphingomyelinase protein inhibitors, but has been demonstrated to exhibit acid sphingomyelinase protein inhibition by a variety of research results. The main pharmacological mechanism of tricyclic antidepressants is to increase their activity by inhibiting neurotransmitter re-uptake in nerve cells, acting as an acid sphingomyelinase inhibitor as an adjunct. However, tricyclic antidepressants can induce side effects such as blurred vision, increased photoreceptors, vomiting, etc. by acting on the nervous system and nerve cells.
Disclosure of Invention
Technical problem
It is an object of the present invention to provide antibodies or antigen-binding fragments thereof that specifically bind to acid sphingomyelinase proteins.
It is still another object of the present invention to provide a method for producing the above antibody or antigen-binding fragment thereof.
It is still another object of the present invention to provide the use of the above antibody or antigen binding fragment thereof for detecting an acidic sphingomyelinase protein.
Means for solving the problems
To achieve the above object, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to an acidic sphingomyelinase (acid sphingomyelinase) protein.
The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.
The present invention also provides an expression vector comprising the nucleic acid.
Also, the present invention provides a host cell comprising the above nucleic acid or expression vector.
Also, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof, which comprises the step of producing an antibody or an antigen-binding fragment thereof by culturing the above-described host cell, wherein the antibody or the antigen-binding fragment thereof specifically binds to an acidic sphingomyelinase protein.
The present invention also provides a composition for detecting an acidic sphingomyelinase protein, which comprises the antibody or an antigen binding fragment thereof, and a kit.
Further, the present invention provides a method for detecting an acidic sphingomyelinase protein, comprising the step of reacting the antibody or antigen-binding fragment thereof with a sample.
Effects of the invention
The antibody or antigen-binding fragment thereof of the present invention specifically binds to an acid sphingomyelinase protein with high binding force, and thus can be effectively used for detection of an acid sphingomyelinase protein, diagnosis of a disease caused by overexpression of an acid sphingomyelinase protein, or the like.
Drawings
Fig. 1 is a schematic diagram showing the amino acid sequences of the heavy and light chain variable regions of an antibody prepared in an embodiment of the present invention.
FIG. 2 is a graph showing the results of confirming the binding between an antibody prepared in an embodiment of the present invention and an acidic sphingomyelinase protein by ELISA assay.
FIG. 3 is a schematic diagram showing binding sites of an antibody to an acid sphingomyelinase protein in a model of the structure of the acid sphingomyelinase protein.
Fig. 4 is a graph showing the results of heavy hydrogen substitution in hot spot for the acid sphingomyelinase protein saporin (saposin) domain of an antibody prepared in an embodiment of the invention.
Detailed Description
The present invention will be described in detail below.
The present invention provides antibodies or antigen binding fragments thereof that specifically bind to an acidic sphingomyelinase (ASM, acid sphingomyelinase) protein.
The term "acid sphingomyelinase (ASM, acid sphingomyelinase) protein" as used in the present specification means an enzyme of one of the SMase (sphingomyelinase) families which regulates sphingolipid metabolism. The above acidic sphingomyelinase proteins facilitate the process of decomposing sphingomyelin (sphingosine) into ceramide (ceramide) and phosphorylcholine (phosphorylcholine), which can be classified as alkaline, neutral or acidic depending on the pH indicating optimal enzymatic activity.
The above-mentioned acid sphingomyelinase proteins may include all kinds of acid sphingomyelinase proteins known in the general technical field. Specifically, the above acidic sphingomyelinase protein may be derived from a mammal, more specifically, from a human, monkey, rat or mouse. Also, the above-mentioned acid sphingomyelinase protein may contain all amino acid sequences known in the art as acid sphingomyelinase proteins. The acidic sphingomyelinase protein may be, for example, a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 66 or SEQ ID NO. 67, or a nucleic acid encoding the polypeptide.
The acidic sphingomyelinase protein may be one or more amino acids added, deleted or substituted in the amino acid sequence shown by SEQ ID NO. 66 or 67, as long as the same or corresponding biological activity is maintained. In this case, the substitution of the amino acid may be a conservative substitution performed in a range where the charge of the entire protein, that is, polarity or hydrophobicity is not affected or the degree of influence is low. The acidic 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 represented by SEQ ID NO. 66 or 67, respectively. The acidic sphingomyelinase protein may contain only a part of the acidic sphingomyelinase protein, as long as the same or corresponding biological activity is maintained.
The term "antibody" as used in the present specification means an immune protein that impedes the action of an antigen or removes an antigen by binding to an antigen. Such antibodies may include all kinds of antibodies encompassed in the general technical field. Specifically, the above antibody may include IgM, igD, igG, igA and IgE, respectively, including heavy chains composed of μ, δ, γ, α, and ε as genes encoding heavy chain constant regions. Generally, igG is mainly used in antibody technology, which again consists of isotypes (isotype) of IgG1, igG2, igG3 or IgG4, which may differ in their respective structural and functional properties. The above-mentioned antibodies may include all of humanized antibodies in which the minimum sequences derived from non-human antibodies are contained, human antibodies composed of sequences derived from human, chimeric antibodies in which sequences derived from different species are mixed, or the like.
The IgG may form a Y-shaped very stable structure (about 150 kDa) from 2 heavy chain (heavy chain) proteins of about 50kDa and 2 light chain (light chain) proteins of about 25 kDa. The light chain and heavy chain constituting an antibody can be divided into variable regions (variable regions) having different amino acid sequences and constant regions (constant regions) having the same amino acid sequences among antibodies. In this case, the heavy chain constant region includes a CH1 domain, a hinge (H) domain, a CH2 domain, and a CH3 domain, each consisting of two β -sheets, which may be linked by an intramolecular disulfide bond. Also, the light chain constant region may comprise a CL domain. On the other hand, the antigen binding site (antigen binding site) is formed by combining the variable region of the heavy chain and the variable region of the light chain, and the antigen binding site may be present in one of the two branches of the Y-shape. In a full-length antibody, the portion that can bind to an antigen is called Fab (antibody binding fragment), the portion that does not bind to an antigen is called Fc (crystallizable fragment), and Fab and Fc can be linked by stranding. In one embodiment of the present invention, the IgG may be a mouse-derived IgG, and specifically, may include a mouse IgG heavy chain constant region composed of the amino acid sequence shown in sequence number 74 and a mouse light chain lambda constant region composed of the amino acid sequence shown in sequence number 76. The IgG derived from the mouse may include a mouse IgG heavy chain constant region composed of the base sequence shown in SEQ ID NO. 75 and a mouse light chain lambda constant region composed of the base sequence shown in SEQ ID NO. 77.
The antibodies of the invention include not only full length antibodies, but also antigen binding fragments thereof. In particular, the antigen binding fragment described above may mean a portion of Fc that functions in addition to delivering to cells or complement a binding stimulus with an antigen. As an example, an antigen-binding fragment of an antibody may include Fab, scFv, F (ab) 2 And Fv, etc., and may also include third generation antibody sections such as single domain antibodies (singledomain antibody) or minibodies (minibodies).
In one embodiment of the present invention, the antibody or antigen-binding fragment binds to one or more epitopes selected from the group consisting of an amino acid fragment at position 53 to an amino acid fragment at position 72, an amino acid fragment at position 101 to an amino acid fragment at position 123, an amino acid fragment at position 135 to an amino acid fragment at position 159, an amino acid fragment at position 135 to an amino acid fragment at position 155, an amino acid fragment at position 218 to an amino acid fragment at position 228, and an amino acid fragment at position 259 to an amino acid fragment at position 269 from the N-terminus of the acid sphingomyelinase protein. In this case, the above-mentioned acidic sphingomyelinase protein may have the characteristics as described above. In an embodiment of the present invention, the 53 rd to 72 th amino acid fragments from the N-terminal of the acid sphingomyelinase protein may be a polypeptide consisting of an amino acid sequence shown by SEQ ID NO. 68 (TAINLGLKKEPNVARVGSVA), the 101 rd to 123 rd amino acid fragments may be a polypeptide consisting of an amino acid sequence shown by SEQ ID NO. 69 (VWRRSVLSPSEACGLLLGSTCGH), the 135 th to 159 th amino acid fragments may be a polypeptide consisting of an amino acid sequence shown by SEQ ID NO. 70 (PTVPKPPPKPPSPPAPGAPVSRILF), the 135 th to 155 th amino acid fragments may be a polypeptide consisting of an amino acid sequence shown by SEQ ID NO. 71 (PTVPKPPPKPPSPPAPGAPVS), the 218 th to 228 th amino acid fragments may be a polypeptide consisting of an amino acid sequence shown by SEQ ID NO. 72 (SGLGPAGPFDM), and the 259 th to 269 th amino acid fragments may be a polypeptide consisting of an amino acid sequence shown by SEQ ID NO. 73 (VRKFLGPVPVY). That is, in another embodiment of the present invention, the above antibody or antigen-binding fragment may bind to one or more epitopes selected from the group consisting of polypeptides represented by SEQ ID Nos. 68 to 73, respectively.
The term "epitope" as used herein refers to a specific site of an antigen that is recognized by an antibody, and means a protein-determining site that allows the antibody to specifically bind. The above epitopes are used in the same sense as the terms of determinants or epitopes (antigenic determinant) and the like. The epitopes mentioned above consist of a group of chemically active surface molecules, for example amino acids or sugar side chains, which generally have specific three-dimensional structural features, and furthermore have specific charge characteristics.
In one embodiment of the present invention, the antibody or antigen-binding fragment thereof may comprise: a heavy chain variable region comprising a heavy chain CDR1 (X) consisting of the amino acid sequence shown in SEQ ID NO. 61 1 YX 2 MS), heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID No. 62 (X) 3 IX 4 X 5 X 6 X 7 X 8 X 9 X 10 YYADSVKG) and a heavy chain CDR3 selected from the group consisting of the amino acid sequences shown in seq id nos. 27, 30, 33, 36, 39 and 42, respectively; and a light chain variable region comprising a sequence represented by SEQ ID NO. 63Light chain CDR1 (X) consisting of amino acid sequences 11 GSSSNIGX 12 NX 13 VX 14 ) Light chain CDR2 (X) consisting of the amino acid sequence depicted in seq id No. 64 15 X 16 X 17 X 18 RPS) and a light chain CDR3 (X) consisting of an amino acid sequence represented by SEQ ID No. 65 19 X 20 WDX 21 SLX 22 X 23 YV)。
The term "CDR (complementarity determining region)" as used above is a hypervariable region (hypervariable region) which is the site of each antibody having a different amino acid sequence within the heavy and light chain variable regions of the antibody, meaning the site of actual binding to an antigen. In the stereo structure of an antibody, CDRs are located on the surface of the antibody in loops (loops) under which FR (framework region) can exist which structurally support them. The heavy and light chains each have three loop structures that can be combined and brought into direct contact with antigen. For convenience, CDRs that are antigen binding sites having the six loop structures described above may be referred to as heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, or light chain CDR3, respectively.
As an example, in the heavy chain CDR1 (X) consisting of the amino acid sequence shown in SEQ ID NO. 61 1 YX 2 MS), X 1 X is as follows 2 Can be selected from acidic amino acids and neutral amino acids, respectively. Specifically X is as described above 1 Can be an acidic amino acid or a neutral amino acid, X 2 May be a neutral amino acid. For example, X is as described above 1 X is as follows 2 Can be selected from the group consisting of asparagine (Asn), glycine (Gly), serine (Ser), aspartic acid (Asp), alanine (Ala), and tyrosine (Tyr), respectively. Specifically, the above X 1 Can be asparagine, glycine, serine or aspartic acid, X 2 May be alanine or tyrosine. In one embodiment of the present invention, the heavy chain CDR1 may consist of the amino acid sequence of SEQ ID Nos. 25, 28, 31, 34, 37 or 40.
And, in the above sequence number 62 shows the amino acid sequence groupSynthetic heavy chain CDR2 (X 3 IX 4 X 5 X 6 X 7 X 8 X 9 X 10 YYADSVKG), X 3 To X 10 Can be selected from neutral amino acids and basic amino acids, respectively. Specifically, the above X 3 X is as follows 4 To X 9 Can be neutral amino acids, X 10 Can be neutral amino acids or basic amino acids. For example, X is as described above 3 To X 10 Can be selected from the group consisting of glycine, leucine (Leu), alanine, serine, tyrosine, proline (Pro), asparagine, lysine (Lys), and isoleucine (Ile), respectively. Specifically, the above X 3 Can be glycine, leucine, alanine or serine, X 4 Can be tyrosine or serine, X 5 Can be proline or tyrosine, X 6 Can be asparagine or glycine, X 7 X is as follows 8 Can be glycine or serine, respectively, X 9 May be asparagine or serine, X 10 Lysine or isoleucine. In one embodiment of the present invention, the heavy chain CDR2 may consist of the amino acid sequence shown in seq id nos. 26, 29, 32, 35, 38 or 41.
Furthermore, in the light chain CDR1 (X) consisting of the amino acid sequence shown in SEQ ID NO. 63 11 GSSSNIGX 12 NX 13 VX 14 ) Wherein X is 11 To X 14 Can be selected from neutral amino acids respectively. For example X as described above 11 To X 14 Can be selected from the group consisting of threonine (Thr), serine, asparagine, alanine, proline, and tyrosine, respectively. Specifically, the above X 11 Can be threonine or serine, X 12 May be asparagine or serine, X 13 Can be alanine, proline, threonine or tyrosine, X 14 May be asparagine, tyrosine or serine. In one embodiment of the present invention, the light chain CDR1 may consist of the amino acid sequence of SEQ ID Nos. 43, 46, 49, 52, 55 or 58.
And, at the amino acid represented by the above-mentioned SEQ ID NO. 64Sequence composed light chain CDR2 (X 15 X 16 X 17 X 18 RPS), X 15 To X 18 Can be selected from acidic amino acids, basic amino acids and neutral amino acids, respectively. Specifically, the above X 15 X is as follows 16 Can be an acidic amino acid or a neutral amino acid, respectively, X 17 Can be neutral amino acid, X 18 May be a basic amino acid or a neutral amino acid. For example, X is as described above 15 To X 18 Can be selected from the group consisting of tyrosine, alanine, aspartic acid, serine, asparagine, histidine (His), glutamine (gin) and lysine, respectively. Specifically, X 15 Can be tyrosine, alanine, aspartic acid or serine, X 16 Can be aspartic acid or asparagine, X 17 Can be serine or asparagine, X 18 May be histidine, glutamine or lysine. In one embodiment of the present invention, the light chain CDR2 is comprised of the amino acid sequence of SEQ ID Nos. 44, 47, 50, 53, 56 or 59.
Furthermore, in the light chain CDR3 (X) consisting of the amino acid sequence shown in SEQ ID NO. 65 19 X 20 WDX 21 SLX 22 X 23 YV), X 19 To X 23 May be selected from neutral amino acids, respectively. For example, X is as described above 19 To X 23 Selected from the group consisting of glycine, alanine, serine, threonine, tyrosine, aspartic acid, and asparagine. Specifically, X 19 Can be glycine or alanine, X 20 Can be alanine, serine or threonine, X 21 Can be tyrosine, serine, alanine or aspartic acid, X 22 Can be serine or asparagine, X 23 May be alanine or glycine. In one embodiment of the present invention, the light chain CDR3 consists of the amino acid sequence depicted in seq id No. 45, 48, 51, 54, 57 or 60.
In another embodiment of the present invention, the above antibody or antigen binding fragment thereof may comprise: a heavy chain variable region comprising a heavy chain CDR1 selected from the group consisting of the amino acid sequences shown in seq id nos. 25, 28, 31, 34, 37 and 40, respectively, a heavy chain CDR2 selected from the group consisting of the amino acid sequences shown in seq id nos. 26, 29, 32, 35, 38 and 41, respectively, and a heavy chain CDR3 selected from the group consisting of the amino acid sequences shown in seq id nos. 27, 30, 33, 36, 39 and 42, respectively; and a light chain variable region comprising a light chain CDR1 selected from the group consisting of the amino acid sequences shown in seq id nos. 43, 46, 49, 52, 55 and 58, respectively, a light chain CDR2 selected from the group consisting of the amino acid sequences shown in seq id nos. 44, 47, 50, 53, 56 and 59, respectively, and a light chain CDR3 selected from the group consisting of the amino acid sequences shown in seq id nos. 45, 48, 51, 54, 57 and 60, respectively.
Also, the above antibody or antigen-binding fragment thereof may include: a heavy chain variable region selected from the group consisting of a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 25, 26, and 27, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 28, 29, and 30, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 31, 32, and 33, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 34, 35, and 36, respectively, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 37, 38, and 39, respectively, and a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 40, 41, and 42, respectively, and a heavy chain CDR2 comprising the heavy chain CDR3 comprising the heavy chain variable region; and a light chain variable region selected from the group consisting of a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 43, 44, and 45, respectively, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 46, 47, and 48, respectively, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 49, 50, and 51, respectively, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 52, 53, and 54, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 55, 56, and 57, respectively, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 49, 50, and 51, respectively, and a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 52, 53, and 54, respectively.
Further, the antibody or antigen-binding fragment thereof may include: a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in sequence numbers 25, 26, and 27, respectively, and a light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in sequence numbers 43, 44, and 45, respectively; a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in seq id nos 28, 29, and 30, respectively, and a light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in seq id nos 46, 47, and 48, respectively; a heavy chain variable region in which a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in sequence numbers 31, 32, and 33, respectively, are contained, and a light chain variable region in which a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in sequence numbers 49, 50, and 51, respectively, are contained; a heavy chain variable region in which a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in sequence numbers 34, 35, and 36, respectively, are contained, and a light chain variable region in which a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in sequence numbers 52, 53, and 54, respectively, are contained; a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in seq id nos. 37, 38, and 39, respectively, and a light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in seq id nos. 55, 56, and 57, respectively; or a heavy chain variable region in which a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in sequence numbers 40, 41, and 42, respectively, are contained, and a light chain variable region in which a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in sequence numbers 58, 59, and 60, respectively, are contained.
As an example, the heavy chain variable region may be a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 1, 3, 5, 7, 9 or 11, and the light chain variable region may be a polypeptide comprising the amino acid sequence shown in SEQ ID NO. 13, 15, 17, 19, 21 or 23.
The antibody or antigen-binding fragment thereof of the present invention may be modified as required. Specifically, the above-described antibodies or antigen-binding fragments thereof may be deformed by conjugation (conjugation), glycation (glycation), labeling, or a combination thereof. Specifically, the above antibody or antigen-binding fragment thereof may be deformed by horseradish peroxidase (HRP, horseradish peroxidase), alkaline phosphatase (alkaline phosphatase), hapten (hapten), biotin, streptavidin, a fluorescent substance, a radioactive substance, quantum dots, polyethylene glycol (PEG, polyethylene glycol), a histidine tag, or the like. Furthermore, the above-described antibodies or antigen-binding fragments thereof may be conjugated to other drugs as desired.
The above-mentioned antibody or antigen-binding fragment thereof may be prepared according to a monoclonal antibody preparation method well known in the art, and the above-mentioned preparation method may be appropriately modified by a person of ordinary skill. As an example, the above antibody can be produced by preparing hybridomas using B lymphocytes obtained from an animal immunized with the antigen, or can be produced by using phage display technology.
The present invention also provides a nucleic acid encoding the antibody or antigen-binding fragment thereof.
The antibodies or antigen binding fragments thereof encoded by the nucleic acids of the invention may have the characteristics described above. The amino acid sequences constituting the antibodies or antigen-binding fragments thereof of the present invention are known, and thus the nucleic acid sequences encoding them are also known to the ordinarily skilled artisan. The nucleic acid sequence may be added, deleted or substituted with one or more bases as long as the activity of the antibody or antigen fragment of the antibody translated thereby is maintained.
As an example, the nucleic acid encoding the heavy chain variable region included in the antibody or antigen-binding fragment thereof of the present invention may be a polynucleotide composed of the base sequence shown in SEQ ID NO. 2, 4, 6, 8, 10 or 12, and the nucleic acid encoding the light chain variable region may be a polynucleotide composed of the base sequence shown in SEQ ID NO. 14, 16, 18, 20, 22 or 24.
The present invention also provides an expression vector comprising the nucleic acid.
The nucleic acid included in the expression vector of the present invention may encode an antibody or antigen-binding fragment thereof having the characteristics described above.
The term "expression vector (expression vector)" used in the present specification as a means for expressing a target gene in a host cell may include all of a plasmid vector, a cosmid vector, a phage vector, a viral vector, and the like. The expression vector may contain an essential element for producing a peptide from the nucleic acid contained therein. Specifically, the above expression vector may contain a signal sequence, a replication source, 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 of the invention may be operatively linked to a promoter.
As an example, an expression vector for a prokaryotic cell may comprise a promoter for transcription, a ribosome binding site for initiation of reading, and a termination sequence for transcription and reading. In another aspect, an expression vector for eukaryotic cells may comprise a promoter derived from a mammalian or mammalian virus, and a polyadenylation sequence.
The marker gene contained in the expression vector may be any marker gene known in the art, and specifically, may be an antibiotic resistance gene. Specifically, the antibiotic resistance gene may be a gene exhibiting resistance to an antibiotic including ampicillin, gentamycin, carboxyicillin, chloramphenicol, streptomycin, kanamycin, neomycin, tetracycline, and the like.
Also, the present invention provides a host cell comprising the above nucleic acid or expression vector.
The nucleic acid or expression vector of the invention contained in a host cell may have the characteristics as described above. As an example, the nucleic acid may encode the antibody or antigen-binding fragment thereof that specifically binds to the acid sphingomyelinase protein of the present invention, and the expression vector may comprise the nucleic acid as described above.
The above-mentioned host cells may use all kinds of cells that are useful in the production of antibodies or antigen-binding fragments thereof in the general art. Specifically, the host cell may be a prokaryotic cell, a yeast cell, or a eukaryotic cell. The prokaryotic cells may include E.coli (E.coli), bacillus, streptomyces, pseudomonas, staphylococcus, etc., and the yeasts may include Saccharomyces cerevisiae, etc. In another aspect, the eukaryotic cells described 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, HT1080, and the like.
Also, the above-mentioned host cell may be transfected with the nucleic acid or the expression vector as described above according to methods well known in the art. Specifically, the above transfection may be performed by methods of transient transfection (transient transfection), microinjection, transduction (transduction), cell fusion, calcium phosphate precipitation, liposome-mediated transfection (liponame-mediated transfection), DEAE dextran-mediated transfection (DEAE dextran-mediated transfection), polybrene-mediated transfection, electroporation, gene gun, etc. And, the above method may be appropriately modified by one of ordinary skill.
Furthermore, the present invention provides a method for producing an antibody or an antigen-binding fragment thereof that specifically binds to an acid sphingomyelinase protein, comprising the step of producing the antibody or the antigen-binding fragment thereof by culturing the above-described host cell.
Antibodies or antigen binding fragments thereof produced by the production methods of the invention may possess the characteristics described above.
The above-described cultivation may be performed using a suitable medium according to the kind of host cell used in the production, and may contain a suitable supplement according to the need. Further, the above-described culture may be performed under a suitable environment according to the kind of host cell.
The production methods of the invention may further comprise the step of recovering the antibodies or antigen-binding fragments thereof produced by the host cells. The above recovery may be performed according to a method well known in the art, which may be appropriately modified by one of ordinary skill in the art according to the need. For example, the recovery may be performed by removing impurities by centrifugation or ultrafiltration and further purifying the obtained product by chromatography or the like. The chromatography may include affinity chromatography, anion chromatography, hydrophobic interaction chromatography, and the like.
The present invention also provides a composition for detecting an acidic sphingomyelinase protein, which comprises the antibody or an antigen binding fragment thereof, and a kit.
The antibody or antigen-binding fragment thereof contained in the composition for detecting an acidic sphingomyelinase protein according to the present invention may have the above-described features.
Further, the above composition may comprise a ligand that specifically binds to the antibody or antigen-binding fragment thereof of the present invention. The ligand may be a conjugate in which a detection body such as a chromogenic enzyme, a fluorescent substance, a radioisotope, or a colloid is labeled, or a ligand in which streptavidin or avidin is treated. In addition to the reagents described above, the detection composition of the present invention may further comprise distilled water or a buffer capable of stably maintaining their structure.
Also, the above-described kit may be combined with a solid substrate to facilitate the subsequent steps of washing of the antibody or antigen-binding fragment thereof contained therein, separation of the complex, or the like. In this case, the solid matrix may use synthetic resin, nitrocellulose, a glass substrate, a metal substrate, glass fibers, microspheres, or microbeads. Also, as the synthetic resin, polyester, polyvinyl chloride, polystyrene, polypropylene, PVDF, nylon, or the like can be used.
The kit may be manufactured by a conventional manufacturing method known to those of ordinary skill, and may further include a buffer, a stabilizer, an inactive protein, and the like.
Further, the present invention provides a method for detecting an acidic sphingomyelinase protein, comprising a step of reacting the antibody or an antigen binding fragment thereof with a sample.
The antibodies or antigen binding fragments thereof used in the acidic sphingomyelinase protein detection method according to the invention may be characterised as described above.
The above-mentioned sample may include all kinds of samples as long as it is a sample for detecting acidic sphingomyelinase protein. Also, 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 one of ordinary skill with appropriate modifications as required.
Description of the embodiments
The present invention is described in detail below by way of examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited thereto. It is to be considered that the present invention is not limited to the above-described embodiments, and may be embodied as follows.
Example 1 preparation of antibodies specifically binding to acid sphingomyelinase (ASM, acid sphingomyelinase) protein
Antibodies that specifically bound to human acid sphingomyelinase protein (SEQ ID NO: 66) and mouse acid sphingomyelinase protein (SEQ ID NO: 67) were prepared by the following methods.
Specifically, recombinant human and mouse acid sphingomyelinase proteins and human synthetic scFv-phage display libraries were screened (panning) to select phages with scFv that specifically bind to human acid sphingomyelinase protein or mouse acid sphingomyelinase protein in the usual manner. The nucleic acid sequence encoding the selected scFv was analyzed and the amino acid sequence thus generated was confirmed. Expression vectors were prepared in such a manner that a mouse heavy chain constant region (constant region) and a mouse light chain lambda constant region composed of the base sequences shown in SEQ ID Nos. 75 and 77 were linked at the carboxy terminus (carboxy terminal) of each of the heavy chain variable region and the light chain variable region of the confirmed scFv sequence. In this case, the amino acid sequence and the nucleic acid sequence of the heavy chain variable region constituting the scFv contained in the prepared expression vector are shown in table 1, and the amino acid sequence and the nucleic acid sequence of the light chain variable region are shown in table 2.
TABLE 1
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TABLE 2
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In this case, a mammalian expression vector (mammalian expression vector) was used as the expression vector. The prepared expression vector was transformed in CHO or HEK293 cell lines, thereby preparing full-length antibodies that bind to human acid sphingomyelinase protein or to both human acid sphingomyelinase protein and mouse acid sphingomyelinase protein.
Example 2 determination of complementarity determining regions (complementarity determining region, CDR)
Complementarity determining regions in scfvs prepared according to the above were confirmed by conventional methods, and finally, CDR sequences of the heavy chain variable region are shown in table 3, and CDR sequences of the light chain variable region are shown in table 4.
TABLE 3 Table 3
TABLE 4 Table 4
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Experimental example 1 confirmation of binding to acid sphingomyelinase protein
Binding between the acid sphingomyelinase protein to the antibody specifically binding to the acid sphingomyelinase protein prepared according to the above was confirmed by ELISA assay.
First, 100. Mu.l of 1. Mu.g/ml recombinant human acid sphingomyelinase protein (R) was added to a multi-square 96-well plate (Thermo scientific)&D systems) and left at a temperature of 4 ℃ for 16 hours, thereby coating the plate. 200 μl of PBS containing 5% BSA was added to the coated plate to allow for 2 hours at 37℃temperatureThe reaction was pre-treated, followed by 3 washes with PBS containing 0.05% Tween 20 (Tween 20). Wherein, the anti-acid sphingomyelinase antibody treatment is performed at 0.0001nM, 0.001nM, 0.01nM, 0.1nM, 1nM, 10nM or 100nM, and the reaction is performed at 37℃for 1 hour. After the reaction, the plate was washed 3 times with PBS containing 0.05% Tween, and 100. Mu.l of goat anti-mouse IgG-HRP (Jackson Immunoresearch) antibody was added as the 2-time antibody. The reaction was again carried out at 37℃for 1 hour and washed 3 times with PBS containing 0.05% Tween. In this, 100. Mu.l of TMB matrix (tetramethylzidine) was added thereto, the reaction was further carried out at room temperature for 5 minutes, and thereafter, the reaction was stopped with 100. Mu.l of 2N sulfuric acid solution, and the absorption brightness was measured at a wavelength of 450 nm. Finally, the measured absorbance is shown in FIG. 2, and EC of the antibody is calculated from the absorbance value 50 The values are shown in table 5.
TABLE 5
Antibody # EC 50 (nM)
#9101 0.0815
#9102 0.0954
#9104 1.948
#9108 0.3269
#9123 0.7578
As shown in FIG. 2, 5 antibodies prepared according to the above were all bound to acid sphingomyelinase protein in a concentration-dependent manner. In particular, as shown in table 5, antibody #9101 exhibited the lowest EC 50 The binding force with the acid sphingomyelinase protein was highest.
Experimental example 2 confirmation of binding affinity with acid sphingomyelinase protein
UsingThe QK384 system (Pall Life Sciences) measured the binding affinity and interaction kinetics between the acid sphingomyelinase protein of antibodies that specifically bind to the acid sphingomyelinase protein prepared according to the above.
First, carboxyl groups were activated by treatment of 20mM 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and 40mM N-hydroxysuccinimide (sulfo-NHS) solution in an AR2G sensor (forteBio). On the other hand, 10. Mu.g/ml of recombinant human acid sphingomyelinase protein or 2.5. Mu.g/ml of recombinant mouse acid sphingomyelinase protein was diluted in 10mM sodium acetate (pH 5.0, forteBio) solution. The above-mentioned acidic sphingomyelinase protein solution was diluted to the carboxyl-activated AR2G sensor to obtain a recombinant human or mouse acidic sphingomyelinase protein-immobilized AR2G sensor. To the obtained acid sphingomyelinase protein immobilized sensor 1M ethanolamine (ForteBio) was added to deactivate the unreacted remaining carboxyl groups. Wherein the antibodies prepared in example 1 were added at a concentration of 0.4nM, 2nM or 10nM and the binding phase of the reactants was observed until about 900 seconds. Thereafter, 1 Xkinetic buffer (kinetic buffer, forteBio) was added and the separated phases of the reactants were observed for about 1200 seconds. Using Analysis software (Pall Life Sciences) determines association constants (associates) for individual antibodiesn constant: kon), dissociation constant (dissociation constant: kdis) and equilibrium dissociation constant (equilibrium dissociation constant: KD). Finally, the results of the analysis for recombinant human acid sphingomyelinase protein are shown in Table 6, and the results of the analysis for recombinant mouse acid sphingomyelinase protein are shown in Table 7.
TABLE 6
Antibody # KD(M) Kon(1/Ms) Kdis(1/s) RMax Full R 2
#9101 1.16E-09 6.26E+05 7.28E-04 0.4527 0.9861
#9102 2.52E-10 4.16E+05 1.05E-04 0.8652 0.997
#9104 2.09E-10 3.92E+05 8.18E-05 0.3324 0.9759
#9108 1.19E-10 2.49E+05 2.96E-05 0.45 0.999
#9113 5.64E-10 1.16E+06 6.56E-04 0.2868 0.9635
TABLE 7
Antibody # KD(M) Kon(1/Ms) Kdis(1/s) RMax Full R2
#9101 4.25E-11 3.76E+06 1.59E-04 0.0185 0.5527
#9102 4.98E-10 4.89E+05 2.43E-04 0.4281 0.9944
#9104 6.38E-10 3.10E+05 1.98E-04 0.7027 0.9971
#9108 3.39E-09 4.59E+05 1.56E-03 0.3921 0.9865
#9113 <1.0E-12 1.00E+06 <1.0E-07 0 0
As shown in Table 6, the 5 antibodies prepared in example 1 were used in an amount of 10 -10 To 10 -9 The M level of binding force binds to human acid sphingomyelinase protein. On the other hand, as shown in Table 7, only antibodies #9102, #9104 or #9108 exhibited 10 for mouse acid sphingomyelinase protein -10 To 10 -9 M level binding force.
Experimental example 3 determination of the epitope of acid sphingomyelinase protein
The epitope which is the binding site of the antibody of the present invention to the acid sphingomyelinase protein was confirmed by the HDX-MS (hydrogen deuterium exchange mass spectrometry) method.
First, 1000. Mu.g/ml of acid sphingomyelinase protein, #9101mIgG1, #9102mIgG2, #9104mIgG4, #9108mIgG1, #9113mIgG1, # 3300. Mu.g/ml, or #9123mIgG1 antibody, #9123mIgG1, # 1250. Mu.g/ml was serially diluted 2-fold, thereby preparing a 128-fold dilution.
Mu.l of each of the prepared dilutions was mixed with an equal amount of 10mg/ml sinapic acid matrix (K200 MALDI kit, covalX) to form a mixture, and 1. Mu.l of the mixture was poured into SCOUT384MALDI plates, and crystallization was performed at room temperature to form crystals. Thereafter, the crystallization was measured using a MALDI mass analyzer (mass spectrometer). On the other hand, to obtain cross-linking (cross-link) data for non-covalent interaction analysis, 1. Mu.l of 128-fold dilution was mixed with an equivalent amount of 2mg/ml of K200 stabilizing solution (K200 stabilizer reagent, K200MALDI kit, covalX) and reacted at room temperature for 3 hours. After the completion of the reaction, 1. Mu.l of the mixture was measured by a MALDI mass analyzer in the same manner as described above, and the measurement result was analyzed in a high-quality MALDI MS (high-mass MALDI MS) mode. Finally, the amino acid sequence at the site where a significant difference from the insertion of deuterium into the acid sphingomyelinase protein was confirmed is shown in Table 8, and the binding site to the acid sphingomyelinase protein, which binds to the antibody in the structural model of the acid sphingomyelinase protein, is shown in FIG. 3. Further, a graph of the heavy hydrogen substitution rate for each antibody in the acidic sphingomyelinase protein and the saposin (saposin) domain is shown in fig. 4.
TABLE 8
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As shown in Table 8 and FIG. 3, the antibodies of the present invention bind to the 53 rd amino acid fragment to the 72 th amino acid fragment (SEQ ID NO: 68: TAINLGLKKEPEPNVARVGASVA), the 101 st amino acid fragment to the 123 rd amino acid fragment (SEQ ID NO: 69: VWRRRSVLSPSEACGLLLGSTCGH), the 135 th amino acid fragment to the 159 th amino acid fragment (SEQ ID NO: 70: PTVPKPPPKPPSPPAPGAPVSRILF), the 135 th amino acid fragment to the 155 th amino acid fragment (SEQ ID NO: 71: PTVPKPPPKPPSPGAPVS), the 218 th amino acid fragment to the 228 th amino acid fragment (SEQ ID NO: 72: SGLGPAGPFDM) or the 259 th amino acid fragment to the 269 th amino acid fragment (SEQ ID NO: 73: VRKFGPVY) from the N-terminus of the acid sphingomyelinase protein. In particular, as shown in FIG. 4, the antibodies of the present invention bind predominantly to the alpha-helix 1 and alpha-helix 2 sites in the saporin domain of proteins.
<110> pear tree love Bosi Co., ltd (ISU ABXIS CO., LTD.)
<120> antibody specifically binding to acid sphingomyelinase protein
<130> FP22-0025-PCT
<150> KR 10-2021-0068075
<151> 2021-05-27
<150> KR 10-2022-0047295
<151> 2022-04-18
<160> 77
<170> KoPatentIn 3.0
<210> 1
<211> 116
<212> PRT
<213> artificial sequence
<220>
<223> #9101 heavy chain variable region amino acid sequence
<400> 1
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Tyr Pro Asn Gly Gly Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Asn Ala Tyr Arg Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 2
<211> 348
<212> DNA
<213> artificial sequence
<220>
<223> #9101 heavy chain variable region nucleotide sequence
<400> 2
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctccggatt cacctttagc aattatgcta tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaggg atctatccta atggtggtaa taaatattac 180
gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaaaatgct 300
tatcgtttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348
<210> 3
<211> 116
<212> PRT
<213> artificial sequence
<220>
<223> #9102 heavy chain variable region amino acid sequence
<400> 3
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Leu Ile Ser Pro Gly Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ser Trp His His Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 4
<211> 348
<212> DNA
<213> artificial sequence
<220>
<223> #9102 heavy chain variable region nucleotide sequence
<400> 4
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctccggatt cacctttagc ggttattata tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcattg atctctcctg gtagtggtag tatatattac 180
gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaatcttgg 300
catcatttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348
<210> 5
<211> 116
<212> PRT
<213> artificial sequence
<220>
<223> #9104 heavy chain variable region amino acid sequence
<400> 5
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Gly Ile Tyr Tyr Gly Ser Gly Asn Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Thr Pro Gly Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 6
<211> 348
<212> DNA
<213> artificial sequence
<220>
<223> #9104 heavy chain variable region nucleotide sequence
<400> 6
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctccggatt cacctttagc aattattata tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaggg atctattatg gtagtggtaa tatatattac 180
gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagatacg 300
cctgggttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348
<210> 7
<211> 121
<212> PRT
<213> artificial sequence
<220>
<223> #9108 heavy chain variable region amino acid sequence
<400> 7
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Tyr Pro Gly Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Val Leu Gly Leu Thr Pro Lys Pro Phe Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 8
<211> 363
<212> DNA
<213> artificial sequence
<220>
<223> #9108 heavy chain variable region nucleotide sequence
<400> 8
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctccggatt cacctttagc aattatgcta tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagcg atctatcctg gtggtggtag tatatattac 180
gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagatgtt 300
ttgggtctga ctcctaagcc gttcgactac tggggccagg gtacactggt caccgtgagc 360
tca 363
<210> 9
<211> 116
<212> PRT
<213> artificial sequence
<220>
<223> #9113 heavy chain variable region amino acid sequence
<400> 9
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ser Ile Ser Pro Gly Gly Ser Ser Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Gly Ala Ser Leu Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val
100 105 110
Thr Val Ser Ser
115
<210> 10
<211> 348
<212> DNA
<213> artificial sequence
<220>
<223> #9113 heavy chain variable region nucleotide sequence
<400> 10
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctccggatt cacctttagc agttattata tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcatcg atctctcctg gtggtagtag tatatattac 180
gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gaaaggggcg 300
tctctgttcg actactgggg ccagggtaca ctggtcaccg tgagctca 348
<210> 11
<211> 127
<212> PRT
<213> artificial sequence
<220>
<223> #9123 heavy chain variable region amino acid sequence
<400> 11
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Ser Tyr Gly Gly Gly Asn Ile Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Val Gly Gly Met Cys Thr Arg Arg Gln Cys Tyr Tyr Asp Tyr
100 105 110
Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 12
<211> 381
<212> DNA
<213> artificial sequence
<220>
<223> #9123 heavy chain variable region nucleotide sequence
<400> 12
gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60
tcctgtgcag cctccggatt cacctttagc gattatgcta tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcagcg atctcttatg gtggtggtaa tatatattac 180
gctgattctg taaaaggtcg gttcaccatc tccagagaca attccaagaa cacgctgtat 240
ctgcaaatga acagcctgag agccgaggac acggccgtgt attactgtgc gagagttggt 300
ggtatgtgta ctaggcgtca gtgttattat gattatggta tggacgtctg gggccagggt 360
acactggtca ccgtgagctc a 381
<210> 13
<211> 110
<212> PRT
<213> artificial sequence
<220>
<223> #9101 light chain variable region amino acid sequence
<400> 13
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn
20 25 30
Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Ala Asp Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Gly Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Tyr Ser Leu
85 90 95
Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 14
<211> 330
<212> DNA
<213> artificial sequence
<220>
<223> #9101 light chain variable region nucleotide sequence
<400> 14
cagtctgtgc tgactcagcc accctcagct agcgggaccc ccgggcagag ggtcaccatc 60
tcttgtagtg gctcttcatc caatattggc aataattatg tctcctggta ccagcagctc 120
ccaggaacgg cccccaaact cctcatctat gctgatagta agcggccaag cggggtccct 180
gaccgattct ctggctccaa gtctggcacc tcagcctcgc tggccatcgg tgggctccgg 240
tccgaggatg aggctgatta ttactgtggt tcttgggatt atagcctgaa tgcttatgtc 300
ttcggcggag gcaccaagct tacggtccta 330
<210> 15
<211> 110
<212> PRT
<213> artificial sequence
<220>
<223> #9102 light chain variable region amino acid sequence
<400> 15
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn
20 25 30
Pro Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Ala Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Ser Ser Leu
85 90 95
Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 16
<211> 330
<212> DNA
<213> artificial sequence
<220>
<223> #9102 light chain variable region nucleotide sequence
<400> 16
cagtctgtgc tgactcagcc accctcagct agcgggaccc ccgggcagag ggtcaccatc 60
tcttgtagtg gctcttcatc taatattggc aataatcctg tctactggta ccagcagctc 120
ccaggaacgg cccccaaact cctcatctat gctaataatc agcggccaag cggggtccct 180
gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240
tccgaggatg aggctgatta ttactgtgct gcttgggatt ctagcctgag tggttatgtc 300
ttcggcggag gcaccaagct tacggtccta 330
<210> 17
<211> 110
<212> PRT
<213> artificial sequence
<220>
<223> #9104 light chain variable region amino acid sequence
<400> 17
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn
20 25 30
Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Tyr Asp Ser His Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ala Trp Asp Tyr Ser Leu
85 90 95
Ser Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 18
<211> 330
<212> DNA
<213> artificial sequence
<220>
<223> #9104 light chain variable region nucleotide sequence
<400> 18
cagtctgtgc tgactcagcc accctcagct agcgggaccc ccgggcagag ggtcaccatc 60
tcttgtactg gctcttcatc taatattggc aataatgctg tcaactggta ccagcagctc 120
ccaggaacgg cccccaaact cctcatctat tatgatagtc atcggccaag cggggtccct 180
gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240
tccgaggatg aggctgatta ttactgtggt gcttgggatt atagcctgag tgcttatgtc 300
ttcggcggag gcaccaagct tacggtccta 330
<210> 19
<211> 110
<212> PRT
<213> artificial sequence
<220>
<223> #9108 light chain variable region amino acid sequence
<400> 19
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Leu Ser Cys Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn
20 25 30
Thr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Ala Asn Ser Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Trp Asp Tyr Ser Leu
85 90 95
Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 20
<211> 330
<212> DNA
<213> artificial sequence
<220>
<223> #9108 light chain variable region nucleotide sequence
<400> 20
cagtctgtgc tgactcagcc accctcagct agcgggaccc ccgggcagag ggtcaccctc 60
tcttgtactg gctcttcatc taatattggc agtaatactg tctactggta ccagcagctc 120
ccaggaacgg cccccaaact cctcatctat gctaatagtc agcggccaag cggggtccct 180
gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240
tccgaggatg aggctgatta ttactgtggt tcttgggatt atagcctgag tggttatgtc 300
ttcggcggag gcaccaagct tacggtccta 330
<210> 21
<211> 110
<212> PRT
<213> artificial sequence
<220>
<223> #9113 light chain variable region amino acid sequence
<400> 21
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn
20 25 30
Ala Val Ser Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Ser Asp Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ala Ser Leu
85 90 95
Asn Ala Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 22
<211> 330
<212> DNA
<213> artificial sequence
<220>
<223> #9113 light chain variable region nucleotide sequence
<400> 22
cagtctgtgc tgactcagcc accctcagct agtgggaccc ccgggcagag ggtcaccatc 60
tcttgtagtg gctcttcatc taatattggc aataatgctg tctcctggta ccagcagctc 120
ccaggaacgg cccccaaact cctcatctat tctgataata agcggccaag cggggtccct 180
gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240
tccgaggatg aggctgatta ttactgtggt acttgggatg ctagcctgaa tgcttatgtc 300
ttcggcggag gcaccaagct tacggtccta 330
<210> 23
<211> 110
<212> PRT
<213> artificial sequence
<220>
<223> #9123 light chain variable region amino acid sequence
<400> 23
Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln
1 5 10 15
Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn
20 25 30
Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45
Ile Tyr Asp Asn Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe Ser
50 55 60
Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg
65 70 75 80
Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Asp Ser Leu
85 90 95
Ser Gly Tyr Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
100 105 110
<210> 24
<211> 330
<212> DNA
<213> artificial sequence
<220>
<223> #9123 light chain variable region nucleotide sequence
<400> 24
cagtctgtgc tgactcagcc accctcagct agcgggaccc ccgggcagag ggtcaccatc 60
tcttgtagtg gctcttcatc taatattggc agtaatactg tcaactggta ccagcagctc 120
ccaggaacgg cccccaaact cctcatctat gataatagta agcggccaag cggggtccct 180
gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240
tccgaagatg aggctgatta ttactgtggt acttgggatg atagcctgag tggttatgtc 300
ttcggcggag gcaccaagct tacggtccta 330
<210> 25
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> #9101 heavy chain CDR1
<400> 25
Asn Tyr Ala Met Ser
1 5
<210> 26
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> #9101 heavy chain CDR2
<400> 26
Gly Ile Tyr Pro Asn Gly Gly Asn Lys Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 27
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> #9101 heavy chain CDR3
<400> 27
Asn Ala Tyr Arg Phe Asp Tyr
1 5
<210> 28
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> #9102 heavy chain CDR1
<400> 28
Gly Tyr Tyr Met Ser
1 5
<210> 29
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> #9102 heavy chain CDR2
<400> 29
Leu Ile Ser Pro Gly Ser Gly Ser Ile Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 30
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> #9102 heavy chain CDR3
<400> 30
Ser Trp His His Phe Asp Tyr
1 5
<210> 31
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> #9104 heavy chain CDR1
<400> 31
Asn Tyr Tyr Met Ser
1 5
<210> 32
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> #9104 heavy chain CDR2
<400> 32
Gly Ile Tyr Tyr Gly Ser Gly Asn Ile Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 33
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> #9104 heavy chain CDR3
<400> 33
Asp Thr Pro Gly Phe Asp Tyr
1 5
<210> 34
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> #9108 heavy chain CDR1
<400> 34
Asn Tyr Ala Met Ser
1 5
<210> 35
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> #9108 heavy chain CDR2
<400> 35
Ala Ile Tyr Pro Gly Gly Gly Ser Ile Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 36
<211> 12
<212> PRT
<213> artificial sequence
<220>
<223> #9108 heavy chain CDR3
<400> 36
Asp Val Leu Gly Leu Thr Pro Lys Pro Phe Asp Tyr
1 5 10
<210> 37
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> #9113 heavy chain CDR1
<400> 37
Ser Tyr Tyr Met Ser
1 5
<210> 38
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> #9113 heavy chain CDR2
<400> 38
Ser Ile Ser Pro Gly Gly Ser Ser Ile Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 39
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> #9113 heavy chain CDR3
<400> 39
Gly Ala Ser Leu Phe Asp Tyr
1 5
<210> 40
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> #9123 heavy chain CDR1
<400> 40
Asp Tyr Ala Met Ser
1 5
<210> 41
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> #9123 heavy chain CDR2
<400> 41
Ala Ile Ser Tyr Gly Gly Gly Asn Ile Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 42
<211> 18
<212> PRT
<213> artificial sequence
<220>
<223> #9123 heavy chain CDR3
<400> 42
Val Gly Gly Met Cys Thr Arg Arg Gln Cys Tyr Tyr Asp Tyr Gly Met
1 5 10 15
Asp Val
<210> 43
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> #9101 light chain CDR1
<400> 43
Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser
1 5 10
<210> 44
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> #9101 light chain CDR2
<400> 44
Ala Asp Ser Lys Arg Pro Ser
1 5
<210> 45
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> #9101 light chain CDR3
<400> 45
Gly Ser Trp Asp Tyr Ser Leu Asn Ala Tyr Val
1 5 10
<210> 46
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> #9102 light chain CDR1
<400> 46
Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Pro Val Tyr
1 5 10
<210> 47
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> #9102 light chain CDR2
<400> 47
Ala Asn Asn Gln Arg Pro Ser
1 5
<210> 48
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> #9102 light chain CDR3
<400> 48
Ala Ala Trp Asp Ser Ser Leu Ser Gly Tyr Val
1 5 10
<210> 49
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> #9104 light chain CDR1
<400> 49
Thr Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn
1 5 10
<210> 50
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> #9104 light chain CDR2
<400> 50
Tyr Asp Ser His Arg Pro Ser
1 5
<210> 51
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> #9104 light chain CDR3
<400> 51
Gly Ala Trp Asp Tyr Ser Leu Ser Ala Tyr Val
1 5 10
<210> 52
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> #9108 light chain CDR1
<400> 52
Thr Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Tyr
1 5 10
<210> 53
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> #9108 light chain CDR2
<400> 53
Ala Asn Ser Gln Arg Pro Ser
1 5
<210> 54
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> #9108 light chain CDR3
<400> 54
Gly Ser Trp Asp Tyr Ser Leu Ser Gly Tyr Val
1 5 10
<210> 55
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> #9113 light chain CDR1
<400> 55
Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Ser
1 5 10
<210> 56
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> #9113 light chain CDR2
<400> 56
Ser Asp Asn Lys Arg Pro Ser
1 5
<210> 57
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> #9113 light chain CDR3
<400> 57
Gly Thr Trp Asp Ala Ser Leu Asn Ala Tyr Val
1 5 10
<210> 58
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> #9123 light chain CDR1
<400> 58
Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Thr Val Asn
1 5 10
<210> 59
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> #9123 light chain CDR2
<400> 59
Asp Asn Ser Lys Arg Pro Ser
1 5
<210> 60
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> #9123 light chain CDR3
<400> 60
Gly Thr Trp Asp Asp Ser Leu Ser Gly Tyr Val
1 5 10
<210> 61
<211> 5
<212> PRT
<213> artificial sequence
<220>
<223> heavy chain CDR1
<400> 61
Xaa Tyr Xaa Met Ser
1 5
<210> 62
<211> 17
<212> PRT
<213> artificial sequence
<220>
<223> heavy chain CDR2
<400> 62
Xaa Ile Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Tyr Ala Asp Ser Val Lys
1 5 10 15
Gly
<210> 63
<211> 13
<212> PRT
<213> artificial sequence
<220>
<223> light chain CDR1
<400> 63
Xaa Gly Ser Ser Ser Asn Ile Gly Xaa Asn Xaa Val Xaa
1 5 10
<210> 64
<211> 7
<212> PRT
<213> artificial sequence
<220>
<223> light chain CDR2
<400> 64
Xaa Xaa Xaa Xaa Arg Pro Ser
1 5
<210> 65
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> light chain CDR3
<400> 65
Xaa Xaa Trp Asp Xaa Ser Leu Xaa Xaa Tyr Val
1 5 10
<210> 66
<211> 584
<212> PRT
<213> artificial sequence
<220>
<223> human ASM protein
<400> 66
Leu Ala Leu Ser Asp Ser Arg Val Leu Trp Ala Pro Ala Glu Ala His
1 5 10 15
Pro Leu Ser Pro Gln Gly His Pro Ala Arg Leu His Arg Ile Val Pro
20 25 30
Arg Leu Arg Asp Val Phe Gly Trp Gly Asn Leu Thr Cys Pro Ile Cys
35 40 45
Lys Gly Leu Phe Thr Ala Ile Asn Leu Gly Leu Lys Lys Glu Pro Asn
50 55 60
Val Ala Arg Val Gly Ser Val Ala Ile Lys Leu Cys Asn Leu Leu Lys
65 70 75 80
Ile Ala Pro Pro Ala Val Cys Gln Ser Ile Val His Leu Phe Glu Asp
85 90 95
Asp Met Val Glu Val Trp Arg Arg Ser Val Leu Ser Pro Ser Glu Ala
100 105 110
Cys Gly Leu Leu Leu Gly Ser Thr Cys Gly His Trp Asp Ile Phe Ser
115 120 125
Ser Trp Asn Ile Ser Leu Pro Thr Val Pro Lys Pro Pro Pro Lys Pro
130 135 140
Pro Ser Pro Pro Ala Pro Gly Ala Pro Val Ser Arg Ile Leu Phe Leu
145 150 155 160
Thr Asp Leu His Trp Asp His Asp Tyr Leu Glu Gly Thr Asp Pro Asp
165 170 175
Cys Ala Asp Pro Leu Cys Cys Arg Arg Gly Ser Gly Leu Pro Pro Ala
180 185 190
Ser Arg Pro Gly Ala Gly Tyr Trp Gly Glu Tyr Ser Lys Cys Asp Leu
195 200 205
Pro Leu Arg Thr Leu Glu Ser Leu Leu Ser Gly Leu Gly Pro Ala Gly
210 215 220
Pro Phe Asp Met Val Tyr Trp Thr Gly Asp Ile Pro Ala His Asp Val
225 230 235 240
Trp His Gln Thr Arg Gln Asp Gln Leu Arg Ala Leu Thr Thr Val Thr
245 250 255
Ala Leu Val Arg Lys Phe Leu Gly Pro Val Pro Val Tyr Pro Ala Val
260 265 270
Gly Asn His Glu Ser Thr Pro Val Asn Ser Phe Pro Pro Pro Phe Ile
275 280 285
Glu Gly Asn His Ser Ser Arg Trp Leu Tyr Glu Ala Met Ala Lys Ala
290 295 300
Trp Glu Pro Trp Leu Pro Ala Glu Ala Leu Arg Thr Leu Arg Ile Gly
305 310 315 320
Gly Phe Tyr Ala Leu Ser Pro Tyr Pro Gly Leu Arg Leu Ile Ser Leu
325 330 335
Asn Met Asn Phe Cys Ser Arg Glu Asn Phe Trp Leu Leu Ile Asn Ser
340 345 350
Thr Asp Pro Ala Gly Gln Leu Gln Trp Leu Val Gly Glu Leu Gln Ala
355 360 365
Ala Glu Asp Arg Gly Asp Lys Val His Ile Ile Gly His Ile Pro Pro
370 375 380
Gly His Cys Leu Lys Ser Trp Ser Trp Asn Tyr Tyr Arg Ile Val Ala
385 390 395 400
Arg Tyr Glu Asn Thr Leu Ala Ala Gln Phe Phe Gly His Thr His Val
405 410 415
Asp Glu Phe Glu Val Phe Tyr Asp Glu Glu Thr Leu Ser Arg Pro Leu
420 425 430
Ala Val Ala Phe Leu Ala Pro Ser Ala Thr Thr Tyr Ile Gly Leu Asn
435 440 445
Pro Gly Tyr Arg Val Tyr Gln Ile Asp Gly Asn Tyr Ser Gly Ser Ser
450 455 460
His Val Val Leu Asp His Glu Thr Tyr Ile Leu Asn Leu Thr Gln Ala
465 470 475 480
Asn Ile Pro Gly Ala Ile Pro His Trp Gln Leu Leu Tyr Arg Ala Arg
485 490 495
Glu Thr Tyr Gly Leu Pro Asn Thr Leu Pro Thr Ala Trp His Asn Leu
500 505 510
Val Tyr Arg Met Arg Gly Asp Met Gln Leu Phe Gln Thr Phe Trp Phe
515 520 525
Leu Tyr His Lys Gly His Pro Pro Ser Glu Pro Cys Gly Thr Pro Cys
530 535 540
Arg Leu Ala Thr Leu Cys Ala Gln Leu Ser Ala Arg Ala Asp Ser Pro
545 550 555 560
Ala Leu Cys Arg His Leu Met Pro Asp Gly Ser Leu Pro Glu Ala Gln
565 570 575
Ser Leu Trp Pro Arg Pro Leu Phe
580
<210> 67
<211> 582
<212> PRT
<213> artificial sequence
<220>
<223> mouse ASM protein
<400> 67
Leu Phe Asp Ser Thr Val Leu Trp Val Pro Ala Arg Ala Tyr Pro Leu
1 5 10 15
Pro Ser Glu Gly His Ser Val Lys Phe Ser Ala Ile Ala Pro Pro Leu
20 25 30
Gln Ser Ala Phe Gly Trp Gln Asn Leu Thr Cys Pro Ala Cys Lys Val
35 40 45
Leu Phe Thr Ala Leu Asn His Gly Leu Lys Lys Glu Pro Asn Val Ala
50 55 60
Arg Val Gly Ser Val Ala Ile Lys Ile Cys Lys Met Leu Asn Ile Ala
65 70 75 80
Pro Leu Asp Val Cys Gln Ser Ala Val His Leu Phe Glu Asp Asp Val
85 90 95
Val Glu Val Trp Thr Arg Ser Val Leu Ser Pro Ser Glu Ala Cys Gly
100 105 110
Leu Leu Leu Gly Ser Ser Cys Gly His Trp Asp Ile Phe Ser Thr Trp
115 120 125
Asn Ile Ser Leu Pro Ser Val Pro Lys Pro Pro Pro Lys Pro Pro Ser
130 135 140
Pro Pro Ala Pro Gly Ala Pro Val Ser Arg Val Leu Phe Leu Thr Asp
145 150 155 160
Leu His Trp Asp His Glu Tyr Leu Glu Gly Thr Asp Pro Tyr Cys Ala
165 170 175
Asp Pro Leu Cys Cys Arg Arg Gly Ser Gly Trp Pro Pro Asn Ser Gln
180 185 190
Lys Gly Ala Gly Phe Trp Gly Glu Tyr Ser Lys Cys Asp Leu Pro Leu
195 200 205
Arg Thr Leu Glu Ser Leu Leu Lys Gly Leu Gly Pro Ala Gly Pro Phe
210 215 220
Glu Met Val Tyr Trp Thr Gly Asp Ile Pro Ala His Asp Val Trp Gln
225 230 235 240
Gln Ser Arg Gln Asp Gln Leu Arg Ala Leu Thr Thr Ile Thr Asp Leu
245 250 255
Val Arg Lys Phe Leu Gly Pro Val Pro Val Tyr Pro Ala Val Gly Asn
260 265 270
His Glu Ser Thr Pro Val Asn Gly Phe Pro Pro Pro Phe Ile Lys Gly
275 280 285
Asn Gln Ser Ser Gln Trp Leu Tyr Glu Ala Met Ala Lys Ala Trp Glu
290 295 300
Pro Trp Leu Pro Ala Asp Ala Leu His Thr Leu Arg Ile Gly Gly Phe
305 310 315 320
Tyr Ala Leu Thr Pro Arg Pro Gly Leu Arg Leu Ile Ser Leu Asn Met
325 330 335
Asn Phe Cys Ser Arg Glu Asn Phe Trp Leu Leu Ile Asn Ser Thr Asp
340 345 350
Pro Ala Gly Gln Leu Gln Trp Leu Val Glu Glu Leu Gln Ala Ala Glu
355 360 365
Asn Arg Gly Asp Lys Val His Ile Ile Gly His Ile Pro Pro Gly His
370 375 380
Cys Leu Lys Ser Trp Ser Trp Asn Tyr Tyr Lys Ile Ile Ala Arg Tyr
385 390 395 400
Glu Asn Thr Leu Ala Gly Gln Phe Phe Gly His Thr His Val Asp Glu
405 410 415
Phe Glu Ile Phe Tyr Asp Glu Glu Thr Leu Ser Arg Pro Leu Ala Val
420 425 430
Ala Phe Leu Ala Pro Ser Ala Thr Thr Phe Ile Asn Leu Asn Pro Gly
435 440 445
Tyr Arg Val Tyr Gln Ile Asp Gly Asn Tyr Pro Gly Ser Ser His Val
450 455 460
Val Leu Asp His Glu Thr Tyr Ile Leu Asn Leu Thr Gln Ala Asn Ala
465 470 475 480
Ala Gly Gly Thr Pro Ser Trp Lys Arg Leu Tyr Arg Ala Arg Glu Thr
485 490 495
Tyr Gly Leu Pro Asp Ala Met Pro Ala Ser Trp His Asn Leu Val Tyr
500 505 510
Arg Met Arg Asp Asp Glu Gln Leu Phe Gln Thr Phe Trp Phe Leu Tyr
515 520 525
His Lys Gly His Pro Pro Ser Glu Pro Cys Gly Thr Pro Cys Arg Leu
530 535 540
Ala Thr Leu Cys Ala Gln Leu Ser Ala Arg Ala Asp Ser Pro Ala Leu
545 550 555 560
Cys Arg His Leu Met Pro Asn Gly Ser Leu Pro Asp Ala Asn Arg Leu
565 570 575
Trp Ser Arg Pro Leu Leu
580
<210> 68
<211> 20
<212> PRT
<213> artificial sequence
<220>
<223> epitope of ASM protein (53-72)
<400> 68
Thr Ala Ile Asn Leu Gly Leu Lys Lys Glu Pro Asn Val Ala Arg Val
1 5 10 15
Gly Ser Val Ala
20
<210> 69
<211> 23
<212> PRT
<213> artificial sequence
<220>
<223> epitope of ASM protein (101-123)
<400> 69
Val Trp Arg Arg Ser Val Leu Ser Pro Ser Glu Ala Cys Gly Leu Leu
1 5 10 15
Leu Gly Ser Thr Cys Gly His
20
<210> 70
<211> 25
<212> PRT
<213> artificial sequence
<220>
<223> epitope of ASM protein (135-159)
<400> 70
Pro Thr Val Pro Lys Pro Pro Pro Lys Pro Pro Ser Pro Pro Ala Pro
1 5 10 15
Gly Ala Pro Val Ser Arg Ile Leu Phe
20 25
<210> 71
<211> 21
<212> PRT
<213> artificial sequence
<220>
<223> epitope of ASM protein (135-155)
<400> 71
Pro Thr Val Pro Lys Pro Pro Pro Lys Pro Pro Ser Pro Pro Ala Pro
1 5 10 15
Gly Ala Pro Val Ser
20
<210> 72
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> epitope of ASM protein (218-228)
<400> 72
Ser Gly Leu Gly Pro Ala Gly Pro Phe Asp Met
1 5 10
<210> 73
<211> 11
<212> PRT
<213> artificial sequence
<220>
<223> epitope of ASM protein (259-269)
<400> 73
Val Arg Lys Phe Leu Gly Pro Val Pro Val Tyr
1 5 10
<210> 74
<211> 324
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of heavy chain constant region of mouse IgG1
<400> 74
Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala
1 5 10 15
Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu
50 55 60
Ser Ser Ser Val Thr Val Pro Ser Ser Pro Arg Pro Ser Glu Thr Val
65 70 75 80
Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys
85 90 95
Ile Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro
100 105 110
Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu
115 120 125
Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser
130 135 140
Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu
145 150 155 160
Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr
165 170 175
Phe Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn
180 185 190
Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro
195 200 205
Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln
210 215 220
Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val
225 230 235 240
Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val
245 250 255
Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln
260 265 270
Pro Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn
275 280 285
Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val
290 295 300
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His
305 310 315 320
Ser Pro Gly Lys
<210> 75
<211> 972
<212> DNA
<213> artificial sequence
<220>
<223> mouse IgG1 heavy chain constant region nucleotide sequence
<400> 75
gccaagacaa cacctcctag cgtgtaccct ctggctcctg gatctgccgc tcagaccaat 60
agcatggtca ccctgggctg tctggtcaag ggctactttc ctgagcctgt gaccgtgacc 120
tggaacagcg gatctctgtc tagcggcgtg cacacctttc cagccgtgct gcagagcgat 180
ctgtacaccc tgagcagcag cgtgaccgtg cctagctctc ctagacctag cgagacagtg 240
acctgcaacg tggcccatcc tgccagcagc accaaggtgg acaagaaaat cgtgcccaga 300
gactgcggct gcaagccctg catctgtacc gtgcctgaag tgtccagcgt gttcatcttc 360
ccacctaagc ctaaggacgt gctgaccatc acactgaccc ctaaagtgac ctgtgtggtg 420
gtggacatca gcaaggacga ccccgaggtg cagttcagtt ggttcgtgga cgacgtggaa 480
gtgcacacag cccagacaca gcctagagag gaacagttca acagcacctt cagaagcgtg 540
tccgagctgc ccatcatgca ccaggattgg ctgaacggca aagaattcaa gtgcagagtg 600
aacagcgccg cctttcctgc tcctatcgag aaaaccatct ccaagaccaa gggcagaccc 660
aaggctcccc aggtgtacac aatccctcca cctaaagaac agatggccaa ggacaaggtg 720
tccctgacct gcatgatcac cgatttcttc ccagaggaca tcaccgtgga atggcagtgg 780
aatggacagc ccgccgagaa ctacaagaat acccagccta tcatgaacac caacggcagc 840
tacttcgtgt acagcaagct gaacgtgcag aagtccaact gggaggccgg caacaccttc 900
acctgttctg tgctgcacga gggcctgcac aatcaccaca ccgagaagtc cctgagccac 960
tctcctggca ag 972
<210> 76
<211> 106
<212> PRT
<213> artificial sequence
<220>
<223> amino acid sequence of mouse light chain lambda constant region
<400> 76
Gly Gln Pro Lys Ser Ser Pro Ser Val Thr Leu Phe Pro Pro Ser Ser
1 5 10 15
Glu Glu Leu Glu Thr Asn Lys Ala Thr Leu Val Cys Thr Ile Thr Asp
20 25 30
Phe Tyr Pro Gly Val Val Thr Val Asp Trp Lys Val Asp Gly Thr Pro
35 40 45
Val Thr Gln Gly Met Glu Thr Thr Gln Pro Ser Lys Gln Ser Asn Asn
50 55 60
Lys Tyr Met Ala Ser Ser Tyr Leu Thr Leu Thr Ala Arg Ala Trp Glu
65 70 75 80
Arg His Ser Ser Tyr Ser Cys Gln Val Thr His Glu Gly His Thr Val
85 90 95
Glu Lys Ser Leu Ser Arg Ala Asp Cys Ser
100 105
<210> 77
<211> 318
<212> DNA
<213> artificial sequence
<220>
<223> mouse light chain lambda constant region nucleotide sequence
<400> 77
ggccagccta agagcagccc tagcgtgacc ctgtttcctc caagcagcga ggaactggaa 60
acaaacaagg ccacactcgt gtgcaccatc accgacttct atcccggcgt ggtcaccgtg 120
gattggaagg tggacggaac ccctgtgaca caaggcatgg aaaccacaca gcccagcaag 180
cagagcaaca acaagtacat ggccagcagc tacctgacac tgaccgccag agcctgggag 240
agacacagct cctacagctg ccaagtgaca cacgagggcc acaccgtgga aaagtctctg 300
agcagagccg actgcagc 318

Claims (18)

1. An antibody or antigen-binding fragment thereof, which specifically binds to an acidic sphingomyelinase (ASM, acid sphingomyelinase) protein.
2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof binds to one or more epitopes selected from the group consisting of an amino acid fragment at position 53 to an amino acid fragment at position 72, an amino acid fragment at position 101 to an amino acid fragment at position 123, an amino acid fragment at position 135 to an amino acid fragment at position 159, an amino acid fragment at position 135 to an amino acid fragment at position 155, an amino acid fragment at position 218 to an amino acid fragment at position 228, and an amino acid fragment at position 259 to an amino acid fragment at position 269 from the N-terminus of an acid sphingomyelinase protein.
3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody or antigen-binding fragment thereof binds to one or more epitopes selected from the group consisting of polypeptides represented by sequence numbers 68 to 73, respectively.
4. 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 comprising a heavy chain CDR1 (X) consisting of the amino acid sequence shown in SEQ ID NO. 61 1 YX 2 MS), heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID No. 62 (X) 3 IX 4 X 5 X 6 X 7 X 8 X 9 X 10 YYADSVKG) and a heavy chain CDR3 selected from the group consisting of the amino acid sequences shown in seq id nos. 27, 30, 33, 36, 39 and 42, respectively; and
Light chain variable regionComprising a light chain CDR1 (X) consisting of the amino acid sequence shown in SEQ ID No. 63 11 GSSSNIGX 12 NX 13 VX 14 ) Light chain CDR2 (X) consisting of the amino acid sequence depicted in seq id No. 64 15 X 16 X 17 X 18 RPS) and a light chain CDR3 (X) consisting of an amino acid sequence represented by SEQ ID No. 65 19 X 20 WDX 21 SLX 22 X 23 YV)。
5. The antibody or antigen-binding fragment thereof according to claim 4, wherein,
above X 1 X is as follows 2 Respectively selected from the group consisting of asparagine (Asn), glycine (Gly), serine (Ser), aspartic acid (Asp), alanine (Ala), and tyrosine (Tyr),
above X 3 To X 10 Respectively selected from the group consisting of glycine, leucine (Leu), alanine, serine, tyrosine, proline (Pro), asparagine, lysine (Lys), isoleucine (Ile),
above X 11 To X 14 Respectively selected from the group consisting of threonine (Thr), serine, asparagine, alanine, proline and tyrosine,
above X 15 To X 18 Respectively selected from the group consisting of tyrosine, alanine, aspartic acid, serine, asparagine, histidine, glutamine (Gln),
Above X 19 To X 23 Respectively selected from the group consisting of glycine, alanine, serine, threonine, tyrosine, aspartic acid, and asparagine.
6. The antibody or antigen-binding fragment thereof according to claim 5, wherein,
above X 1 Is asparagine, glycine, serine or aspartic acid,
above X 2 Is an amino acid of alanine or tyrosine,
above X 3 Is glycine, leucine, alanine or serine,
above X 4 Is a compound which is tyrosine or serine,
above X 5 Is a compound which is proline or tyrosine,
above X 6 Is an amino acid selected from the group consisting of asparagine and glycine,
above X 7 X is as follows 8 Glycine or serine, respectively, is used as the main component,
above X 9 Is an amino acid such as asparagine or serine,
above X 10 Is a lysine or an isoleucine, and is preferably selected from the group consisting of lysine and isoleucine,
above X 11 Is threonine or serine, and is preferably selected from the group consisting of,
above X 12 Is an amino acid such as asparagine or serine,
above X 13 Is alanine, proline, threonine or tyrosine,
above X 14 Is asparagine, tyrosine or serine, and is preferably selected from the group consisting of asparagine, tyrosine or serine,
above X 15 Is tyrosine, alanine, aspartic acid or serine,
above X 16 Is an amino acid such as aspartic acid or asparagine,
above X 17 Is serine or asparagine, and is preferably selected from the group consisting of serine and asparagine,
above X 18 Is histidine, glutamine or lysine, and is preferably selected from the group consisting of,
above X 19 Is glycine or alanine, and is preferably selected from the group consisting of glycine or alanine,
Above X 20 Is alanine, serine or threonine, and is preferably selected from the group consisting of,
above X 21 Is tyrosine, serine, alanine or aspartic acid,
above X 22 Is serine or asparagine, and is preferably selected from the group consisting of serine and asparagine,
above X 23 Is alanine or glycine.
7. 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 comprising a heavy chain CDR1 selected from the group consisting of the amino acid sequences shown in seq id nos. 25, 28, 31, 34, 37 and 40, respectively, a heavy chain CDR2 selected from the group consisting of the amino acid sequences shown in seq id nos. 26, 29, 32, 35, 38 and 41, respectively, and a heavy chain CDR3 selected from the group consisting of the amino acid sequences shown in seq id nos. 27, 30, 33, 36, 39 and 42, respectively; and
a light chain variable region comprising a light chain CDR1 selected from the group consisting of the amino acid sequences shown in seq id nos. 43, 46, 49, 52, 55 and 58, respectively, a light chain CDR2 selected from the group consisting of the amino acid sequences shown in seq id nos. 44, 47, 50, 53, 56 and 59, respectively, and a light chain CDR3 selected from the group consisting of the amino acid sequences shown in seq id nos. 45, 48, 51, 54, 57 and 60, respectively.
8. 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 selected from the group consisting of a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 25, 26, and 27, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 28, 29, and 30, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 31, 32, and 33, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 34, 35, and 36, respectively, a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 37, 38, and 39, respectively, and a heavy chain CDR2, and a heavy chain CDR3 comprising the amino acid sequences shown by sequence numbers 40, 41, and 42, respectively, and a heavy chain CDR2 comprising the heavy chain CDR3 comprising the heavy chain variable region; and
A light chain variable region selected from the group consisting of a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 43, 44, and 45, respectively, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 46, 47, and 48, respectively, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 49, 50, and 51, respectively, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 52, 53, and 54, respectively, a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 55, 56, and 57, respectively, and a light chain CDR1, a light chain CDR2, and a light chain CDR3 comprising the amino acid sequences shown by sequence numbers 58, and a light chain CDR1, and a CDR3 comprising the amino acid sequences shown by sequence numbers 59, respectively.
9. 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 comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in sequence numbers 25, 26, and 27, respectively, and a light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in sequence numbers 43, 44, and 45, respectively;
a heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in seq id nos 28, 29, and 30, respectively, and a light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in seq id nos 46, 47, and 48, respectively;
a heavy chain variable region in which a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in sequence numbers 31, 32, and 33, respectively, are contained, and a light chain variable region in which a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in sequence numbers 49, 50, and 51, respectively, are contained;
a heavy chain variable region in which a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in sequence numbers 34, 35, and 36, respectively, are contained, and a light chain variable region in which a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in sequence numbers 52, 53, and 54, respectively, are contained;
A heavy chain variable region comprising a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in seq id nos. 37, 38, and 39, respectively, and a light chain variable region comprising a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in seq id nos. 55, 56, and 57, respectively; or (b)
A heavy chain variable region in which a heavy chain CDR1, a heavy chain CDR2, and a heavy chain CDR3 consisting of the amino acid sequences shown in sequence numbers 40, 41, and 42, respectively, are contained, and a light chain variable region in which a light chain CDR1, a light chain CDR2, and a light chain CDR3 consisting of the amino acid sequences shown in sequence numbers 58, 59, and 60, respectively, are contained.
10. The antibody or antigen-binding fragment thereof of claim 1, wherein the acidic sphingomyelinase protein is mammalian-derived.
11. The antibody or antigen-binding fragment thereof according to claim 1, wherein the acidic sphingomyelinase protein is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 66 or 67.
12. A nucleic acid, wherein the antibody or antigen-binding fragment thereof of claim 1 is encoded.
13. An expression vector comprising the nucleic acid of claim 12.
14. A host cell comprising the nucleic acid of claim 12 or the expression vector of claim 13.
15. A method of producing an antibody or antigen-binding fragment thereof that specifically binds to an acidic sphingomyelinase protein, comprising the step of producing the antibody or antigen-binding fragment thereof by culturing the host cell of claim 14.
16. An acidic sphingomyelinase protein detection composition, comprising an antibody or antigen binding fragment thereof according to any one of claims 1 to 11.
17. A kit for the detection of an acidic sphingomyelinase protein, comprising the antibody or antigen binding fragment thereof according to any one of claims 1 to 11.
18. A method for detecting an acidic sphingomyelinase protein, comprising the step of reacting the antibody or antigen binding fragment thereof according to any one of claims 1 to 11 with a sample.
CN202280037975.4A 2021-05-27 2022-05-26 Antibodies that specifically bind to acid sphingomyelinase proteins Pending CN117396517A (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2021-0068075 2021-05-27
KR1020220047295A KR20220160474A (en) 2021-05-27 2022-04-18 Antibodies specifically binding to asm protein
KR10-2022-0047295 2022-04-18
PCT/KR2022/095105 WO2022250520A1 (en) 2021-05-27 2022-05-26 Antibody specifically binding to asm protein

Publications (1)

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CN117396517A true CN117396517A (en) 2024-01-12

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