Epitopes on regulatory T cell surface antigens and antibodies that specifically bind to them

Abstract

The present invention relates to an epitope of the Lrig-1 (leucine-rich and immunoglobulin-like domain 1) protein, an antigen present on the surface of regulatory T cells, and an antibody or antigen-binding fragment that specifically binds to the epitope. The antibody or antigen-binding fragment that specifically binds to the epitope of the Lrig-1 protein according to the present invention specifically binds to the epitope of the present invention present on regulatory T cells and regulates the function of the regulatory T cells, thereby maintaining or increasing the activity of effector T cells and can be used very effectively in the prevention, amelioration, or treatment of various diseases.

Description

[Technical Field]

[0001] The present invention relates to an epitope of the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein, an antigen present on the surface of regulatory T cells, and an antibody or antigen-binding fragment that specifically binds to the epitope. [Background technology]

[0002] In degenerative neurological diseases such as stroke, dementia, and Alzheimer's disease, declines in memory, attention, cognitive ability, and emotional regulation are observed, which are the result of neuronal death and atrophy of nerve branches. Neuronal branch elongation plays an important role in the memory and learning functions of neural circuits by increasing neuroplasticity. Therefore, active ingredients that promote neuronal branch elongation and neural regeneration are expected to be developed as new therapeutic agents for degenerative neurological diseases.

[0003] As the elderly population rapidly increases, the incidence of degenerative nervous system diseases is also on the rise. Despite innovative advances in medicine, the prevention and treatment methods for degenerative nervous system diseases are still unclear, and no definitively effective drugs have been found. Currently, although drugs and treatments for degenerative nervous system diseases have been developed, they often cause side effects and toxicity due to long-term use, and are only effective in alleviating symptoms rather than treating them. Therefore, there is an urgent need to develop materials that can reduce side effects and toxicity and provide treatment. Summary of the Invention [Problem to be solved by the invention]

[0004] One object of the present invention is to provide an epitope of the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present on the surface of regulatory T cells (Treg cells).

[0005] Another object of the present invention is to provide an antibody or antigen-binding fragment capable of specifically binding to said epitope.

[0006] It is yet another object of the present invention to provide a nucleic acid molecule encoding the epitope of the present invention; an expression vector into which the nucleic acid molecule has been inserted; and a host cell line transfected with the expression vector.

[0007] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating various diseases, which comprises, as an active ingredient, an antibody or antigen-binding fragment capable of specifically binding to the epitope.

[0008] It is yet another object of the present invention to provide an antibody-drug conjugate (ADC) in which the antibody according to the present invention is conjugated with a drug for preventing or treating various diseases.

[0009] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating cranial nervous system diseases, comprising the chimeric antigen receptor (CAR) according to the present invention as an active ingredient.

[0010] A further object of the present invention is to provide a diagnostic composition and diagnostic kit for cranial nervous system diseases, which contain the binding molecule of the present invention, and a method for providing information for diagnosing cranial nervous system diseases.

[0011] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating various diseases, such as immune-related diseases, neurodegenerative diseases, or neuroinflammatory diseases, which comprises an antibody-drug conjugate as an active ingredient.

[0012] It is yet another object of the present invention to provide an antibody or antigen-binding fragment capable of specifically binding to the epitope; and a method for preventing or treating various diseases using the antibody-drug conjugate.

[0013] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0014] Various embodiments according to the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and steps, are set forth to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details or with other known methods and configurations. In other instances, known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily or obscure the present invention. Reference throughout this specification to an embodiment implies that the particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearance of an embodiment in various locations throughout this specification does not necessarily refer to the same embodiment of the present invention. Additionally, particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0015] The present invention relates to an epitope of the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein, or an antibody or antigen-binding fragment that specifically binds to said epitope.

[0016] As used herein, "epitope" refers to a sequence of amino acids within an antigen, the amino acids (or a subset thereof) of which are specifically recognized by an antibody or binding fragment, such as an antibody or binding fragment described herein. An epitope can comprise one or more antigenic determinants. For example, an antibody raised against an isolated peptide corresponding to a topographical epitope will recognize part or all of the epitope sequence.

[0017] In the present invention, the "Lrig-1 protein" is a transmembrane protein present on the surface of regulatory T cells. It consists of an extracellular or lumenal leucine-rich repeat (LRR), three immunoglobulin-like domains, a transmembrane sequence, and a cytoplasmic tail. The LRIG gene family consists of LRIG1, LRIG2, and LRIG3, and the amino acid sequences constituting each family are highly conserved. The LRIG1 gene is highly expressed in normal skin, where it is expressed in basal and hair follicle cells and can regulate the proliferation of epidermal stem cells. Therefore, it plays an important role in maintaining epidermal homeostasis, and its absence can lead to the development of psoriasis and skin cancer. It has been reported that disruption of the chromosome 3p14.3 region where LRIG1 is located may lead to the development of cancer cells. Indeed, it has been confirmed that LRIG1 expression is significantly reduced in renal cell carcinoma and cutaneous squamous cell carcinoma. However, it has recently been found that only about 20 to 30% of cancers express the Lrig-1 protein. Meanwhile, for purposes of the present invention, the Lrig-1 protein may be derived from mammals or mice, but is not limited thereto.

[0018] In one example of the present invention, the Lrig-1 protein may be an Lrig-1 protein derived from a mammal, for example, a primate such as a human or a monkey, or a rodent such as a mouse or a rat.

[0019] In one example of the present invention, the Lrig-1 protein may be a human-derived Lrig-1 protein represented by SEQ ID NO: 1, which may be encoded by the nucleic acid sequence represented by SEQ ID NO: 2, but is not limited thereto.

[0020] In another example of the present invention, the Lrig-1 protein may be a mouse-derived Lrig-1 protein represented by SEQ ID NO: 3, which may be encoded by the nucleic acid sequence represented by SEQ ID NO: 4, but is not limited thereto.

[0021] In yet another embodiment of the present invention, the Lrig-1 protein may be, but is not limited to, the extracellular domain of the Lrig-1 protein.

[0022] The Lrig-1 extracellular domain of the present invention may be an extracellular domain of an Lrig-1 protein derived from a mammal, for example, a primate such as a human or a monkey, or a rodent such as a mouse or a rat. For purposes of the present invention, the extracellular domain of an Lrig-1 protein may be, but is not limited to, an extracellular domain of an Lrig-1 protein derived from a human or a mouse.

[0023] In one example of the present invention, the extracellular domain of the Lrig-1 protein may be represented by sequence number 5, which corresponds to the amino acid sequence from positions 35 to 794 of the human Lrig-1 protein, but is not limited to this.

[0024] In another example of the present invention, the extracellular domain of the Lrig-1 protein may be represented by sequence number 6, which corresponds to the amino acid sequence from positions 35 to 794 of the mouse-derived Lrig-1 protein, but is not limited thereto.

[0025] The present invention will be described in more detail below.

[0026] According to one embodiment of the present invention, there is provided an epitope of the Lrig-1 protein, which is selected from the group consisting of polypeptides comprising the amino acid sequences represented by SEQ ID NOs: 24, 27, 30, 31, 34, 38, 42, and 51.

[0027] As an example of the present invention, the epitope of the Lrig-1 protein may be an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 23 to 60, but is not limited thereto.

[0028] In another example of the present invention, the epitope of the Lrig-1 protein may be an epitope comprising at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NO: 41 to SEQ ID NO: 45, but is not limited thereto.

[0029] The epitope of the present invention may be a conformational epitope.

[0030] The "conformational epitope" of the present invention is composed of a discontinuous amino acid sequence, unlike a linear epitope, which is composed of a continuous sequence. Such a conformational epitope reacts with the three-dimensional structure of the antigen-binding site of an antibody.

[0031] According to another embodiment of the present invention, there are provided nucleic acid molecules encoding the epitopes provided herein.

[0032] The nucleic acid molecules of the present invention include all nucleic acid molecules in which the amino acid sequence of the polypeptide provided by the present invention has been translated into a polynucleotide sequence, as known to those skilled in the art. Therefore, various polynucleotide sequences can be produced using ORFs (open reading frames), and all of these are also included in the nucleic acid molecules of the present invention.

[0033] In yet another embodiment of the present invention, there is provided an expression vector into which the isolated nucleic acid molecule provided by the present invention is inserted.

[0034] As used herein, a "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA to which additional DNA segments can be ligated. Another type of vector is a phage vector. Yet another type of vector is a viral vector, to which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors are episomal mammalian vectors with a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) can integrate into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

[0035] In the present invention, specific examples of the expression vector can be selected from the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμμ, T-even, T2, T3, and T7; and plant viruses, but are not limited thereto. All expression vectors known to those skilled in the art can be used in the present invention, and the selection of an expression vector depends on the properties of the intended host cell. Introduction of the vector into the host cell can be performed by calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, lipofectamine transfection, or electroporation, but is not limited thereto. Those skilled in the art can select an introduction method appropriate for the expression vector and host cell used. Preferably, the vector contains one or more selectable markers, but is not limited thereto. Vectors without selectable markers can be used to allow selection based on the presence or absence of product production. The selection of the selectable marker is determined by the desired host cell, and this is done using methods already known to those skilled in the art, so the present invention is not limited thereto.

[0036] To facilitate purification of the protein encoded by the nucleic acid molecule of the present invention, a tag sequence can be inserted into the expression vector to fuse it. Examples of such tags include, but are not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag. Any tag known to those skilled in the art that facilitates purification can be used in the present invention.

[0037] According to yet another embodiment of the present invention, there is provided a host cell line transformed with the expression vector provided herein.

[0038] As used herein, the term "host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector for incorporation of a polypeptide insert. A host cell includes the progeny of a single host cell, which may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. A host cell includes cells transformed in vivo with a polypeptide of the present application.

[0039] In the present invention, the host cells may include cells of mammalian, plant, insect, fungal, or cellular origin, for example, bacterial cells such as Escherichia coli, Streptomyces, and Salmonella Typhimurium; fungal cells such as yeast cells and Pichia pastoris; insect cells such as Drosophila and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bowes melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), and PERC.6 (human retinal cells); or plant cells. Any cell known to those skilled in the art that can be used as a host cell line may be used, but is not limited to these.

[0040] The transformation method of the present invention may be any method for injecting a target vector into the host cell, and may include any known method for injecting a vector into the host cell, such as, but not limited to, a method using CaCl2, electroporation, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, and virus-based transformation.

[0041] According to yet another embodiment of the present invention, there is provided an antibody-drug conjugate (ADC) comprising an antibody provided by the present invention and a drug.

[0042] In the present invention, the term "antibody-drug conjugate (ADC)" refers to a form in which a drug and an antibody are chemically linked without reducing the biological activities of the antibody and the drug. In the present invention, the antibody-drug conjugate refers to a form in which a drug is bound to an amino acid residue at the N-terminus of the heavy chain and / or light chain of the antibody, specifically, a form in which a drug is bound to the α-amine group at the N-terminus of the heavy chain and / or light chain of the antibody.

[0043] In the present invention, the term "drug" refers to any substance that has a specific biological activity in cells, including DNA, RNA, and peptides. The drug may be in a form containing a reactive group capable of reacting with an α-amine group to crosslink, or may be linked to a linker containing a reactive group capable of reacting with an α-amine group to crosslink.

[0044] In the present invention, examples of the reactive group capable of reacting with the α-amine group to crosslink include, but are not limited to, any group known in the art that reacts with an amine group, as long as it can react with the α-amine group at the N-terminus of an antibody heavy or light chain to crosslink, including, but not limited to, any one of isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters.

[0045] In the present invention, the drug may be any drug capable of treating a cranial nervous system disease, particularly a neurodegenerative disease or a neuroinflammatory disease, regardless of its type.

[0046] According to yet another embodiment of the present invention, there are provided binding molecules that specifically bind to an epitope of the present invention.

[0047] The "binding molecule" of the present invention may be, but is not limited to, an antibody or an antigen-binding fragment.

[0048] In the present invention, the "antibody" includes all antibody fragments, which are full-length antibodies or portions of antibodies, have the ability to bind to Lrig-1 protein, and bind to the Lrig-1 antigenic determinant site competitively with the binding molecule of the present invention.

[0049] In the present invention, the term "antibody" refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule immunologically reactive with a specific antigen. For purposes of the present invention, the antigen may be the Lrig-1 protein present on the surface of regulatory T cells. Preferably, the antibody may specifically recognize the leucine-rich region or immunoglobulin-like domain of the Lrig-1 protein, but is not limited thereto.

[0050] In the present invention, the "immunoglobulin" has a heavy chain and a light chain, each of which contains a constant region and a variable region. The variable regions of the light and heavy chains contain three variable regions called complementarity determining regions (hereinafter referred to as "CDRs") and four framework regions. The CDRs primarily play a role in binding to an epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, sequentially, starting from the N-terminus, and are identified by the chain in which the particular CDR is located.

[0051] In the present invention, the antibodies or antigen-binding fragments thereof provided by the present invention are chimeric antibodies or fragments comprising heavy chain CDRs and light chain CDRs selected from the CDRs provided by the present invention, or comprising conservative variants of the CDRs provided by the present invention.

[0052] For purposes of the present invention, "variants" of an amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. Amino acid deletion variants, including deletions at the N-terminus and / or C-terminus of a protein, are also referred to as N-terminal and / or C-terminal truncation variants.

[0053] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible.

[0054] Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0055] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletions can be at any position in the protein.

[0056] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Preferably, the alterations occur at positions in the amino acid sequence that are not conserved among homologous proteins or peptides and / or involve the substitution of an amino acid with another amino acid with similar properties. Preferably, the amino acid changes in the protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of a member of a family of amino acids with related side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified jointly as aromatic amino acids.

[0057] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, preferably the degree of similarity or identity is given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, may be performed using tools known in the art, preferably the best sequence alignment, for example, using Align using standard settings, preferably EMBOSS::Needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0058] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of identical amino acids between the sequences.

[0059] The term "percent identity" is intended to indicate the percentage of identical amino acid residues between the two sequences being compared after optimal alignment; this percentage is purely statistical, and the differences between the two sequences are distributed randomly and throughout their entire length. Sequence comparison between two amino acid sequences is usually performed by optimally aligning these sequences and then comparing them, and the comparison is performed by segments or "comparison windows" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison can be generated manually or by local homology algorithms or similarity search methods, or by computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA).

[0060] The percentage identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared, and multiplying the result by 100 to obtain the percentage identity between the two sequences.

[0061] Homologous amino acid sequences according to the present invention exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% of the amino acid residues.

[0062] The amino acid sequence variants described herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to produce proteins and peptides with substitutions, additions, insertions, or deletions is well known. The peptides and amino acid variants described herein can also be readily prepared thanks to well-known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

[0063] In the present invention, the antibody or antigen-binding fragment thereof provided by the present invention is a humanized antibody or fragment comprising a heavy chain CDR and a light chain CDR selected from the CDRs provided by the present invention, or comprising conservative variants of the CDRs provided by the present invention.

[0064] In the present invention, the antibody or antigen-binding fragment thereof provided by the present invention comprises a light chain and / or a heavy chain comprising a sequence provided by the present invention, or a conservative variant thereof. In one embodiment, the conservative variant has a sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the reference sequence provided by the present invention. In one embodiment, the conservative variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 10 or more amino acid substitutions, insertions, or deletions.

[0065] In the present invention, antibodies that specifically bind to the Lrig-1 protein provided by the present invention include the sequences provided herein or conservative variants thereof. As used herein, "conservative variants" include conservative amino acid substitutions, insertions, or deletions. Those skilled in the art will recognize that a conservative amino acid substitution is the substitution of one amino acid with another amino acid that has similar structural or chemical properties, such as a similar side chain, while maintaining the biological activity of the reference sequence. Exemplary conservative substitutions are well known in the art.

[0066] In the present invention, the "full length antibody" has a structure having two full length light chains and two full length heavy chains, each of which is linked to a heavy chain by a disulfide bond, and includes IgA, IgD, IgE, IgM, and IgG. The IgG subtypes include IgG1, IgG2, IgG3, and IgG4.

[0067] In the present invention, the term "antigen-binding fragment" refers to a fragment that retains antigen-binding function. Examples of antigen-binding fragments include: (1) a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL), and the first heavy chain constant region (CH1); (2) an Fd fragment consisting of a VH and CH1 domain; (3) an Fv fragment consisting of the VL and VH domains of a single antibody; (4) a dAb fragment consisting of a VH domain; (5) isolated CDR regions; (6) an F(ab')2 fragment, which is a bivalent fragment containing two linked Fab fragments; (7) a single-chain Fv molecule (scFv) in which the VH and VL domains are linked by a peptide linker to form an antigen-binding site; (8) a bispecific single-chain Fv dimer; and (9) a diabody, which is a multivalent or multispecific fragment produced by gene fusion. The antigen-binding fragment can be produced by using proteolytic enzymes such as papain or pepsin to obtain Fab or F(ab')2 fragments, or by genetic recombination techniques.

[0068] Furthermore, in the present invention, the antibody and fragment thereof may be, but is not limited to, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bivalent antibody, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimic, a unibody, a diabody, a triabody, a tetrabody, or a fragment thereof.

[0069] In the present invention, the "chimeric antibody" is an antibody obtained by recombining the variable region of a mouse antibody and the constant region of a human antibody, and exhibits a significantly improved immune response compared to mouse antibodies.

[0070] In the present invention, the term "humanized antibody" refers to an antibody in which the protein sequence of an antibody derived from a non-human species has been modified to resemble an antibody variant naturally produced in humans. For example, the humanized antibody can be prepared by combining mouse-derived CDRs with human-derived FRs to prepare humanized variable regions, which can then be combined with a preferred human antibody constant region to prepare a humanized antibody.

[0071] In the present invention, the binding molecule can also be provided as a bispecific antibody or bispecific antigen-binding fragment that can bind to the Lrig-1 protein and also to other proteins.

[0072] In the present invention, the bispecific antibodies and bispecific antigen-binding fragments may comprise binding molecules according to the present invention. In one example, the bispecific antibodies and bispecific antigen-binding fragments according to the present invention comprise an antigen-binding domain capable of binding to Lrig-1 protein, and the antigen-binding domain capable of binding to Lrig-1 protein may comprise or consist of a binding molecule according to the present invention.

[0073] The bispecific antibodies and bispecific antigen-binding fragments provided herein comprise an antigen-binding domain that is a binding molecule capable of binding to the Lrig-1 protein of the present invention, and an antigen-binding domain that can bind to another target protein, where the antigen-binding domain that can bind to another target protein is a protein other than the Lrig-1 protein, including, but not limited to, an antigen-binding domain that can bind to PD-1 or a cell surface receptor.

[0074] Bispecific antibodies and bispecific antigen-binding fragments according to the invention may be provided in any suitable format, such as those described in the documents incorporated herein by reference in their entirety. For example, the bispecific antibodies or bispecific antigen-binding fragments may be provided as bispecific antibody conjugates (e.g., IgG2, F(ab')2, or CovX-bodies), bispecific IgG or IgG-type molecules (e.g., IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, or 2in1-IgG, mAb2, or Tandemab common LC), asymmetric bispecific IgG or IgG-type molecules (e.g., kih IgG, kih IgG common LC, CrossMab, kih The antibody may be an IgG-scFab, mAb-Fv, charge pair or SEED-body), a small bispecific antibody molecule (e.g., diabody (Db), dsDb, DART, scDb, tandAbs, tandem scFv (taFv), tandem dAb / VHH, triple body, triple head, Fab-scFv, or F(ab')2-scFv2), a bispecific Fc and CH3 fusion protein (e.g., taFv-Fc, di-diabody, scDb-CH3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-CH3), or a bispecific fusion protein (e.g., scFv2-albumin, scDb-albumin, taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine2). A person skilled in the art can design and produce bispecific antibodies and bispecific antigen-binding fragments according to the invention.

[0075] In the present invention, methods for producing the bispecific antibodies include chemical cross-linking of antibodies or antibody fragments with reducible disulfide or non-reducible thioether bonds. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically cross-link Fab fragments, for example, via hinge region SH-groups, to generate disulfide-linked bispecific F(ab)2 heterodimers.

[0076] Also, in the present invention, another method for producing the bispecific antibody includes fusing an antibody-producing hybridoma with, for example, polyethylene glycol, to generate a quadroma cell capable of secreting the bispecific antibody.

[0077] Bispecific antibodies and bispecific antigen-binding fragments according to the invention can be produced recombinantly, for example, by expression from nucleic acid constructs encoding the polypeptides for the antigen-binding molecules.

[0078] For example, a DNA construct encoding light and heavy chain variable domains for two antigen-binding domains (i.e., light and heavy chain variable domains for an antigen-binding domain capable of binding, e.g., PD-1, and light and heavy chain variable domains for an antigen-binding domain capable of binding to another target protein), and containing sequences encoding a suitable linker or dimerization domain between the antigen-binding domains, can be produced by molecular cloning techniques. Recombinant bispecific antibodies can then be produced by expression (e.g., in vitro) of the construct in a suitable host cell (e.g., a mammalian host cell), and the expressed recombinant bispecific antibodies can then, optionally, be purified.

[0079] Antibodies may also be produced by an affinity maturation process, which produces modified antibodies that have improved affinity for the antigen compared to the unmodified parent antibody. Affinity matured antibodies can be produced by procedures known in the art.

[0080] In addition, the binding molecules provided by the present invention may include variants of the amino acid sequences, as long as they are able to specifically bind to the Lrig-1 protein. For example, the amino acid sequence of the antibody may be altered to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion, and / or substitution of residues in the amino acid sequence of the antibody.

[0081] Such amino acid mutations are made based on the relative similarity of the amino acid side chain substitutes, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substitutes reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0082] When introducing mutations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydrophobic index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The hydrophobic index is crucial for conferring interactive biological functions of proteins. It is well known that similar biological activity can be maintained only by substituting amino acids with similar hydrophobic indexes. When introducing mutations based on the hydrophobic index, substitutions are made between amino acids showing a difference in hydrophobic index within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0083] On the other hand, it is also well known that substitutions between amino acids with similar hydrophilicity values ​​result in proteins with equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). When introducing mutations based on the hydrophilicity value, substitutions can be made between amino acids showing a difference in hydrophilicity value preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0084] Amino acid exchanges in proteins that do not overall alter the activity of the molecule are known in the art. The most commonly occurring exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, and Gln / Glu.

[0085] In view of the above-mentioned variations with bioequivalent activity, the binding molecules of the present invention are also understood to include sequences that show substantial identity to the sequences set forth in the sequence listing.

[0086] As used herein, the term "substantial identity" refers to a sequence that exhibits at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology when the sequence of the present invention is aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using algorithms commonly used in the art. Alignment methods for sequence comparison are well known in the art. Various alignment methods and algorithms are available through the NCBI Basic Local Alignment Search Tool (BLAST), the National Center for Biological Information (NBCI), and other sources, and can be used online in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx. BLSAT can be accessed at the following address (www.ncbi.nlm.nih.gov / BLAST / ). A method for comparing sequence homology using this program can be found online (www.ncbi.nlm.nih.gov / BLAST / blast_help.html).

[0087] In the present invention, the binding molecule, preferably the antibody, can be produced by a conventional method for producing an antibody, but may also be produced by affinity maturation.

[0088] In the present invention, the term "affinity maturation" refers to the process by which activated B cells produce antibodies with increased affinity for an antigen during an immune response. For the purposes of the present invention, affinity maturation can be performed based on the principles of mutation and selection, similar to the process that occurs in nature, to produce antibodies or antibody fragments produced by affinity maturation.

[0089] In addition, in the present invention, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and exhibits a single binding specificity and affinity for a specific antigenic determinant (epitope).

[0090] In the present invention, the term "binding" or "specific binding" refers to the affinity of the antibody or antibody composition of the present application for an antigen. "Specific binding" in antigen-antibody binding typically refers to a dissociation constant (Kd) of 1×10 -5 Less than M or 1 x 10 -6 Less than M or 1 x 10 -7 If the binding is less than M, it can be distinguished from nonspecific background binding. Specific binding can be detected by methods known in the art, such as ELISA, surface plasmon resonance (SPR), immunoprecipitation, coprecipitation, etc., and includes appropriate controls that can distinguish specific binding from nonspecific binding.

[0091] The antibodies or antigen-binding fragments of the present invention can exist in multimers, such as dimers, trimers, tetramers, and pentamers, that contain at least a portion of the antigen-binding ability of the monomers. Such multimers also include homomultimers and heteromultimers. Antibody multimers contain multiple antigen-binding sites and therefore have superior antigen-binding ability compared to monomers. Antibody multimers also facilitate the production of multifunctional (bifunctional, trifunctional, and tetrafunctional) antibodies.

[0092] In the present invention, the term "multifunctional" refers to an antibody or antigen-binding fragment having two or more activities or functions (e.g., antigen-binding ability, enzymatic activity, ligand- or receptor-binding ability). For example, the antibody of the present invention can bind to a polypeptide with enzymatic activity, such as luciferase, acetyltransferase, or galactosidase. Multifunctional antibodies also include multivalent or multispecific (bispecific, trispecific, etc.) antibodies.

[0093] According to yet another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating a cranial nervous system disease, comprising as an active ingredient a chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain.

[0094] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor comprising an extracellular antigen-binding domain and an intracellular signaling domain. While the most common type of CAR comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the transmembrane and intracellular domains of a T cell co-receptor, such as the CD3 zeta (ζ) chain, the invention described herein is not limited to these domains. Rather, as used herein, "chimeric antigen receptor" or "CAR" refers to any receptor engineered to express any intracellular signaling molecule and its extracellular antigen-binding domain fused or linked thereto. In the present invention, the binding domain can comprise a single-chain variable fragment (scFv) capable of specifically recognizing the Lrig-1 protein. In the present invention, the "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable heavy chain (VH) and variable light chain (VL) of an antibody via a peptide linker between the VL and VH.

[0095] In addition, in the present invention, the VH domain and VL domain can be linked via a flexible linker. In the present invention, the flexible linker may be a glycine / serine linker of approximately 10 to 30 amino acids (e.g., 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids), preferably 15 amino acids in length. In the present invention, the length of the linker can act as an important determining site for the chimeric antigen receptor. Linkers shorter than this range can increase affinity but may cause intracellular multimer formation, impairing CAR expression. In contrast, linkers longer than this range may reduce antigen affinity by moving the VL and VH CDRs farther in space.

[0096] The chimeric antigen receptor of the present invention may further comprise at least one of a hinge region (or spacer) and a signaling domain. In the present invention, the hinge region, also referred to as a "spacer," connects the antigen-binding domain and the transmembrane domain and serves to extend the antigen-binding domain from the T cell membrane or NK cell membrane. In the present invention, the hinge region may be derived from any suitable sequence from any genus, including humans or portions thereof, or may include, but is not limited to, hinge regions of human proteins commonly used in the art, including CD8, CD28, 4-1BB, OX40, all or a portion of the CD3 zeta (ζ) chain, T cell receptor α or β chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof. Furthermore, in the present invention, the hinge region is not limited to immunoglobulins and may include one selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD), but is not limited thereto.

[0097] In the present invention, the signaling domain refers to a portion of a chimeric antigen receptor that is found or engineered to be found inside a T cell. In the present invention, the signaling domain may or may not include a transmembrane domain that serves to anchor the chimeric antigen receptor in the plasma membrane of a T cell. In the present invention, the transmembrane domain and the signaling domain can be derived from the same protein (e.g., a CD3 zeta (ζ) molecule), or the transmembrane domain and the signaling domain can be derived from different proteins (e.g., the transmembrane domain of CD28 and the intracellular signaling domain of a CD3 zeta (ζ) molecule, or vice versa).

[0098] In the present invention, the transmembrane domain may include, but is not limited to, for example, the T cell receptor α or β chain, all or part of the CD3 zeta (ζ) chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof. In the present invention, the costimulatory domain may include, but is not limited to, a functional signaling domain derived from a polypeptide comprising 4-1BB (CD137); OX40; CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; natural killer group 2 member C (NKG2C); natural killer group 2 member (NKG2D); B7-H3; CD83; ICAM-1; a ligand that binds to LFA-1 (CD11a / CD18) or ICOS; an active fragment thereof; a functional derivative thereof; or a combination thereof.

[0099] In the present invention, the signaling domain can include, but is not limited to, a functional signaling domain derived from a polypeptide including all or a portion of CD3 zeta (ζ), common FcR gamma (FcER1G), Fc gamma RIIIa, FcR beta (Fc epsilon RIP), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX-activation protein 10 (DAP10), DNAX-activation protein 12 (DAP12), an active fragment thereof, a functional derivative thereof, or a combination thereof, and such signaling domains are known in the art.

[0100] The cranial nervous system disease to be prevented, improved or treated by the composition provided by the present invention may be a neurodegenerative disease or a neuroinflammatory disease.

[0101] In the present invention, the "neurodegenerative disease" may mean a disease caused by a decrease or loss of nerve cell function, and the "neuroinflammatory disease" may mean a disease caused by an excessive inflammatory response in the nervous system. In the present invention, specific examples of the neurodegenerative disease or neuroinflammatory disease include stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and the like. The present invention may be selected from the group consisting of, but not limited to, tauopathy, tauopathy, and tauopathy.

[0102] Furthermore, the composition provided by the present invention can be used as, but is not limited to, a pharmaceutical composition or a food composition.

[0103] The "prevention" of the present invention may include, without limitation, any action that can block, suppress, or delay symptoms caused by a neurodegenerative disease or a neuroinflammatory disease using the composition of the present invention.

[0104] The "treatment" and "improvement" of the present invention may include, without limitation, any action that can improve or benefit symptoms caused by neurodegenerative diseases or neuroinflammatory diseases using the composition of the present invention.

[0105] In the present invention, the pharmaceutical composition may be in the form of a capsule, tablet, granule, injection, ointment, powder or drink, and may be intended for humans.

[0106] The pharmaceutical composition of the present invention can be formulated into oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, using conventional methods, but is not limited thereto. The pharmaceutical composition of the present invention can also contain a pharmaceutically acceptable carrier. For oral administration, pharmaceutically acceptable carriers can include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavorings, etc.; for injections, buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, etc.; and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The pharmaceutical composition of the present invention can be formulated into various dosage forms by mixing with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the compound may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, the compound may be prepared in the form of unit-dose ampoules or multiple doses. It may also be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0107] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition, fillers, anti-agglomerating agents, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc. may also be included.

[0108] The routes of administration of the pharmaceutical compositions of the present invention include, but are not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal, with oral or parenteral administration being preferred.

[0109] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration.

[0110] The dosage of the pharmaceutical composition of the present invention may vary depending on various factors, including the activity of the specific compound used, age, body weight, general health, sex, dietary requirements, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease being prevented or treated. The dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, severity of disease, drug form, administration route, and duration, but can be appropriately selected by those skilled in the art and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The dosage may be administered once or in several divided doses per day. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition of the present invention may be formulated into pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, or suspensions.

[0111] Food compositions containing the composition of the present invention as an active ingredient may be prepared in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, sweets, rice cakes, bread, etc. The food compositions of the present invention are composed of plant extracts with little toxicity or side effects, and therefore can be safely taken for prophylactic purposes over a long period of time.

[0112] When the composition of the present invention is contained in a food composition, the amount added may be 0.1 to 50% of the total weight.

[0113] When the food composition is prepared in the form of a beverage, there are no particular limitations other than that the food composition be contained in the indicated proportions, and various flavorings or natural carbohydrates may be added as additional ingredients, as in conventional beverages. Natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. Examples of flavorings include natural flavorings (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavorings (saccharin, aspartame, etc.).

[0114] In addition, the food compositions of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavorings and natural flavorings, coloring agents, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, etc.

[0115] These components can be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

[0116] According to yet another embodiment of the present invention, there is provided a method for preventing, ameliorating, or treating a cranial nervous system disease, comprising the step of administering to an individual in need thereof a binding molecule provided by the present invention; a nucleic acid molecule encoding the binding molecule; an expression vector into which the nucleic acid molecule has been inserted; a host cell line transfected with the expression vector; or an antibody-drug conjugate (ADC) provided by the present invention.

[0117] In the present invention, the "individual" refers to an individual suspected of developing a cranial nervous system disease, and the individual suspected of developing the disease refers to mammals including rodents, including humans, and livestock that have developed or may develop the disease, but includes, without limitation, individuals that can be treated with the effective substance provided by the present invention.

[0118] The cranial nervous system disease to be treated by the method of the present invention may be a neurodegenerative disease or a neuroinflammatory disease, and specific examples thereof include stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and the like. The present invention may be selected from the group consisting of, but not limited to, tauopathy, tauopathy, and tauopathy.

[0119] The methods of the present invention may comprise administering a pharmaceutically effective amount of a binding molecule provided by the present invention; a nucleic acid molecule encoding the binding molecule; an expression vector into which the nucleic acid molecule has been inserted; a host cell line transfected with the expression vector; or an antibody-drug conjugate (ADC) provided by the present invention.

[0120] The appropriate total daily dose can be determined by the treating physician within the scope of sound medical judgment and can be administered in one or several doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will preferably vary depending on various factors, including the type and degree of response to be achieved, the specific composition containing the active ingredient, including whether or not other preparations are used, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, the excretion rate of the composition containing the active ingredient, the duration of treatment, drugs used together with or simultaneously with the specific composition, and similar factors well known in the pharmaceutical arts.

[0121] Meanwhile, the method for preventing or treating a cranial nervous system disease may be, but is not limited to, a combination therapy that further includes administering a compound or substance having therapeutic activity against one or more diseases.

[0122] In the present invention, the term "combination" should be understood to refer to simultaneous, separate or sequential administration. If the administration is sequential or separate, the interval between administrations of the secondary components should be such that the beneficial effect of the combination is not lost.

[0123] In the present invention, the administration dose of the binding molecule or antibody-drug conjugate may be, but is not limited to, about 0.0001 μg to 500 mg per kg of patient body weight.

[0124] According to yet another embodiment of the present invention, there is provided a diagnostic composition for immune-related diseases, comprising a preparation for measuring the expression level of Lrig-1 protein or its extracellular domain present on the surface of T cells, or the gene encoding the same, using the binding molecule.

[0125] The T cells of the present invention may be regulatory T cells.

[0126] The diagnostic composition of the present invention may comprise, but is not limited to, a formulation for measuring the expression level of the Lrig-1 protein or its extracellular domain present on the surface of activated regulatory T cells, i.e., regulatory T cells whose suppressive ability on effector T cells has been activated; or a gene encoding the same.

[0127] The diagnostic composition of the present invention may further comprise an agent for measuring the expression level of one or more proteins present on the surface of T cells selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and Lrig-1 protein; or the gene encoding the same. In this way, when the expression levels of various proteins present on the surface of T cells or the gene encoding them are additionally measured, a significant synergistic effect can be achieved in diagnosing the target disease compared to measuring the Lrig-1 protein or its extracellular domain or the gene encoding it alone.

[0128] The formulation for measuring the expression level of the Lrig-1 protein or its extracellular domain of the present invention and the expression level of one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16 and Lrig-1 protein is not particularly limited, and may include, for example, one or more proteins selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid) and aptamers that specifically bind to the proteins.

[0129] A formulation for measuring the expression level of the gene encoding the Lrig-1 protein of the present invention (i.e., LAIR1) or the gene encoding the extracellular domain of the Lrig-1 protein, and a gene encoding one or more proteins selected from the group consisting of CCD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and the Lrig-1 protein, can contain one or more selected from the group consisting of primers, probes, LNAs, and antisense nucleotides that specifically bind to the genes.

[0130] The "primer" of the present invention is a fragment that recognizes a target gene sequence and includes a forward and reverse primer pair, but is preferably a primer pair that provides analytical results with specificity and sensitivity. High specificity can be achieved when the nucleic acid sequence of the primer is a sequence that does not match non-target sequences present in the sample, so that only the target gene sequence containing the complementary primer binding site is amplified and non-specific amplification is not induced.

[0131] The term "probe" as used herein refers to a substance capable of specifically binding to a target substance to be detected in a sample and capable of specifically confirming the presence of the target substance in the sample through this binding. The type of probe may be any substance commonly used in the art, but is preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA, or DNA, with PNA being most preferred. More specifically, the probe may be a biological substance derived from an organism, a substance similar thereto, or a substance produced in vitro. For example, the probe may be an enzyme, protein, antibody, microorganism, animal or plant cell or organ, nerve cell, DNA, or RNA. DNA includes cDNA, genomic DNA, and oligonucleotides; RNA includes genomic RNA, mRNA, and oligonucleotides; and examples of proteins include antibodies, antigens, enzymes, and peptides.

[0132] Yet another embodiment of the present invention relates to a kit for diagnosing cranial nervous system diseases, which comprises the binding molecule provided by the present invention.

[0133] The kit of the present invention may be, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0134] The kits of the present invention may further comprise one or more other component compositions, solutions or devices suitable for the analytical method.

[0135] The kit of the present invention may further include essential elements necessary for performing a reverse transcription polymerase reaction, for example. The reverse transcription polymerase reaction kit includes a primer pair specific to a gene encoding a marker protein. The primer pair is a nucleotide having a sequence specific to the nucleic acid sequence of the gene and may have a length of, for example, about 7 bp to 50 bp, or about 10 bp to 30 bp. It may also include a primer pair specific to the nucleic acid sequence of a control gene. Other reverse transcription polymerase reaction kits may further include test tubes or other suitable containers, reaction buffers (with various pHs and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0136] The kit of the present invention can include essential elements required for DNA chip generation. The DNA chip kit can include a substrate to which cDNA or oligonucleotides corresponding to genes or their fragments are attached, as well as reagents, formulations, enzymes, and the like for preparing fluorescently labeled probes. The substrate can also include cDNA or oligonucleotides corresponding to control genes or their fragments.

[0137] The kit of the present invention can include essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific to the protein. The antibody has high specificity and affinity for the marker protein and little cross-reactivity with other proteins, and can be a monoclonal antibody, polyclonal antibody, or recombinant antibody. The ELISA kit can also include an antibody specific to a control protein. Other ELISA kits can include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies) and their substrates, or other substances capable of binding to antibodies.

[0138] According to yet another embodiment of the present invention, the present invention relates to a method for providing information for diagnosing immune-related diseases, comprising the step of measuring the expression level of Lrig-1 protein or its extracellular domain present on the surface of T cells in a biological sample isolated from a target individual using the binding molecule; or the expression level of a gene encoding the same.

[0139] The T cells of the present invention may be regulatory T cells.

[0140] In the present invention, the step of measuring the expression level may further measure the expression level of one or more proteins present on the surface of T cells selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and Lrig-1 protein; or the genes encoding same. In this way, when the expression levels of various proteins present on the surface of T cells or the genes encoding same are further measured, a significant synergistic effect can be achieved in diagnosing the target disease compared to measuring the Lrig-1 protein or the gene encoding it alone.

[0141] The "target individual" of the present invention refers to an individual whose onset of the disease is uncertain, but who has a high probability of developing the disease.

[0142] The "biological sample" of the present invention means any substance, biological fluid, tissue, or cell obtained from or derived from an individual, such as whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph, etc. The fluid may include, but is not limited to, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0143] The Lrig-1 protein of the present invention or its extracellular domain may also be expressed on the cell surface of T cells, particularly regulatory T cells, for example, activated regulatory T cells, i.e., regulatory T cells whose suppressive ability over effector T cells has been activated.

[0144]

[0023] Methods for measuring or comparing the expression levels of the Lrig-1 protein or its extracellular domain and one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and Lrig-1 protein of the present invention include protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Surface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), and the like. Examples of suitable methods include, but are not limited to, immunoblotting (ELISA), spectrometry (Spectrometry), Western blotting, and enzyme-linked immunosorbent assay (ELISA).

[0145] To confirm the presence or absence and expression level of the gene encoding the Lrig-1 protein or its extracellular domain of the present invention and the gene encoding one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and Lrig-1 protein, analytical methods for measuring the expression level of the gene include, but are not limited to, reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, and DNA chip.

[0146] The method of the present invention may include predicting that there is a high possibility of developing an immune-related disease when the expression level of the Lrig-1 protein or its extracellular domain; or the gene encoding the same, measured in a biological sample of the target individual, is reduced compared to a normal control group.

[0147] The method of the present invention may further include predicting the likelihood of onset of an immune-related disease when the expression level of one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16 and Lrig-1 proteins present on the surface of the T cells or genes encoding the same changes (increases or decreases) compared to a normal control group.

[0148] In another embodiment of the present invention, the method may include predicting that there is a high possibility of developing an immune-related disease if the expression level of the Lrig-1 protein or the gene encoding it is decreased compared to a normal control group.

[0149] Furthermore, in the present invention, when the expression level of the Lrig-1 protein or its extracellular domain; or the gene encoding it, measured in a biological sample of a target individual as described above, is measured to predict or diagnose a high possibility of developing an immune-related disease, the method may additionally include a step of administering a drug for the disease to the target individual.

[0150] In one embodiment of the present invention, an epitope of the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein is provided, which is selected from the group consisting of polypeptides comprising the amino acid sequences represented by SEQ ID NOs: 22, 25, 28, 30, 33, 38, 41, 43, 44, 47, 51 and 60.

[0151] In another embodiment of the present invention, the polypeptide provides an epitope consisting of the amino acid sequence represented by SEQ ID NO: 21, 23, 24, 26, 27, 31, 32, 34 to 40, 42, 45, 46, 48 to 50, 52 to 59, or 61 to 69.

[0152] In one embodiment of the present invention, a nucleic acid molecule encoding said epitope is provided.

[0153] In one embodiment of the present invention, an expression vector is provided into which the nucleic acid molecule is inserted.

[0154] In one embodiment of the present invention, a host cell line transfected with said expression vector is provided.

[0155] One embodiment of the present invention provides a binding molecule that specifically binds to at least one epitope selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 21 to 69.

[0156] In another embodiment of the invention, the binding molecule is provided wherein the binding molecule is an antibody or an antigen-binding fragment.

[0157] In yet another embodiment of the present invention, the binding molecule is provided wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, a tetrabody, or a fragment thereof.

[0158] One embodiment of the present invention provides a chimeric antigen receptor (CAR) that is a fusion protein comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain, wherein the antigen-specific binding domain specifically binds to at least one epitope selected from the group consisting of polypeptides consisting of the amino acid sequences set forth in SEQ ID NOs: 21 to 69; and an immunoglobulin Fc region.

[0159] In one embodiment of the present invention, an antibody-drug conjugate is provided that comprises a binding molecule and a drug.

[0160] In one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating a neurological disease, comprising at least one of the binding molecule, the chimeric antigen receptor, and the antibody-drug conjugate as an active ingredient.

[0161] In yet another embodiment of the present invention, there is provided a pharmaceutical composition, wherein the cranial nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

[0162] In yet another embodiment of the present invention, the neurodegenerative or neuroinflammatory disease is selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and the like. The present invention provides a pharmaceutical composition for preventing or treating a neurodegenerative disease or a neuroinflammatory disease, which is at least one selected from the group consisting of neurodegenerative diseases, neuroinflammatory disorders, neuroinflammatory diseases, and tauopathy.

[0163] In one embodiment of the present invention, a method is provided for screening for a binding molecule that is an antibody or a fragment thereof that specifically binds to said epitope.

[0164] In one embodiment of the present invention, a method for screening epitopes of Lrig-1 protein using the binding molecules is provided.

[0165] In one embodiment of the present invention, a method is provided for binding said epitope with a binding molecule that is an antibody or an antigen-binding fragment thereof.

[0166] One embodiment of the present invention provides a binding molecule that is an antibody or a fragment thereof that specifically binds to an Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein, wherein (a) the heavy chain variable region of the binding molecule comprises a heavy chain CDR1 represented by SEQ ID NO: 7; a heavy chain CDR2 represented by SEQ ID NO: 8; and a heavy chain CDR3 represented by SEQ ID NO: 9; or (b) the light chain variable region of the binding molecule comprises a light chain CDR1 represented by SEQ ID NO: 10; a light chain CDR2 represented by SEQ ID NO: 11; and a light chain CDR3 represented by SEQ ID NO: 12; and the binding molecule specifically binds to an epitope comprising at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 21 to 69.

[0167] In another embodiment of the invention, a binding molecule is provided wherein the heavy chain variable region of the binding molecule is represented by SEQ ID NO:13 or the light chain variable region is represented by SEQ ID NO:14.

[0168] In yet another embodiment of the invention, a binding molecule is provided wherein the heavy chain of said binding molecule is represented by SEQ ID NO: 15 or 17, or the light chain is represented by SEQ ID NO: 16 or 18. [Effects of the Invention]

[0169] The antibodies or antigen-binding fragments of the present invention that specifically bind to the epitope of the Lrig-1 protein specifically bind to the epitope of the present invention present on regulatory T cells and regulate the function of the regulatory T cells, thereby maintaining or increasing the activity of effector T cells, and can be used very efficiently in the prevention, amelioration, or treatment of various diseases. [Brief explanation of the drawings]

[0170] [Figure 1] FIG. 1 shows an experimental design diagram for confirming the therapeutic effect of a monoclonal antibody according to one embodiment of the present invention on Alzheimer's disease. [Figure 2]FIG. 1 shows photographs of the results of staining the brain cortex and hippocampus tissues of Alzheimer's-induced mice with thioflavin S (ThS) after each treatment containing a monoclonal antibody according to one embodiment of the present invention, taken with a fluorescence microscope. [Figure 3] This figure shows a graph of the results of staining the cerebral cortex and hippocampus tissues of Alzheimer's-induced mice with thioflavin S (ThS) after each treatment containing a monoclonal antibody according to one embodiment of the present invention, and then calculating the ThS+ area ratio (%) compared to normal mice. [Figure 4] 1 is a graph showing changes in spontaneous alteration (%) in a Y-maze experiment after treatment of Alzheimer's disease-induced mice with a monoclonal antibody according to an embodiment of the present invention. [Figure 5] 1 is a graph showing the change in preference score in a novel object recognition experiment after treatment of Alzheimer's disease-induced mice with a monoclonal antibody according to an embodiment of the present invention. [Figure 6] FIG. 10 is a graph showing the change in the time taken to reach the location of the platform in a water maze experiment after treatment of Alzheimer's disease-induced mice with a monoclonal antibody according to one embodiment of the present invention. [Figure 7] 1 is a graph showing the change in the number of times the target was passed in a water maze experiment after treatment of Alzheimer's disease-induced mice with a monoclonal antibody according to an embodiment of the present invention. [Figure 8] This figure shows a graph of the results of measuring the change in the time it took for Alzheimer's-induced mice to reach the location of the platform during a water maze experiment after each treatment, including a monoclonal antibody according to one embodiment of the present invention. [Figure 9] FIG. 1 shows an experimental design for Alzheimer's-induced mice, 5xFAD mice and 6xTg mice, to confirm the therapeutic effect of a monoclonal antibody according to one embodiment of the present invention on Alzheimer's disease. [Figure 10]FIG. 1 is a graph showing the change in spontaneous alteration (%) in the Y-maze experiment after treatment of Alzheimer's induced mice, 5xFAD mice and 6xTg mice, with a monoclonal antibody according to one embodiment of the present invention. [Figure 11] 1 is a graph showing the change in preference score in a novel object recognition experiment after treatment of 6xTg mice, which are Alzheimer's disease-induced mice, with a monoclonal antibody according to an embodiment of the present invention. [Figure 12] FIG. 1 is a graph showing the change in latency time in a passive avoidance experiment after treatment of Alzheimer's disease-induced mice, 5xFAD mice and 6xTg mice, with a monoclonal antibody according to one embodiment of the present invention. [Figure 13] FIG. 1 shows tryptic peptide positions (% tryptic peptide positions) in tryptic CL-MS labeled peptides. [Figure 14] FIG. 1 shows tryptic CL-MS labeled peptide % (point tryptic peptide positions). [Figure 15] FIG. 1 shows tryptic peptide positions and labeling residues in tryptic CL-MS. [Figure 16] FIG. 1 shows chymotryptic peptide positions (% of chymotryptic peptides labeled by CL-MS). [Figure 17] FIG. 1 shows chymotryptic peptide positions and labeling residues in chymotrypsin CL-MS analysis. [Figure 18] FIG. 1 shows a CL-MS histogram. [Figure 19] FIG. 1 shows a trypsin XL-MS graph (detected peptides). [Figure 20] FIG. 1 shows a trypsin XL-MS graph (decreased peptides). [Figure 21] FIG. 1 shows a chymotrypsin XL-MS graph (detected peptides). [Figure 22] FIG. 1 shows a chymotrypsin XL-MS graph (decreased peptides). [Figure 23] FIG. 1 shows an XL-MS histogram. [Figure 24] FIG. 1 shows affinity-MS histograms. [Figure 25] FIG. 1 shows epitope mapping histograms. DETAILED DESCRIPTION OF THE INVENTION

[0171] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of explaining the present invention in more detail, and that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0172] Example [Manufacturing example] Production of monoclonal antibodies specific to Lrig-1 protein According to the present invention, we have produced an antibody specific to the Lrig-1 protein. This antibody was not produced by determining a specific antigenic determinant, but rather was produced to be able to bind to any site on the Lrig-1 protein.

[0173] To produce the antibody, cells expressing Lrig-1 protein were generated. More specifically, a DNA fragment corresponding to SEQ ID NO: 2 and pcDNA (hygro) were digested with a cleavage enzyme and then cultured at 37°C for ligation to generate pcDNA into which the DNA sequence of Lrig-1 protein had been inserted. The pcDNA containing SEQ ID NO: 2 was then transfected into T cells to allow expression of Lrig-1 protein on the surface of the T cells.

[0174] Light and heavy chain amino acid sequences capable of binding to Lrig-1 expressed on the surface of the cells were selected from a human scFv library.

[0175] The selected heavy and light chain amino acid sequences were fused to heavy chain constant regions and light chain constant regions to produce monoclonal antibodies, the sequences of which are shown in Table 1 below.

[0176] [Table 1(1)] [Table 1(2)]

[0177] [Example 1] Evaluation of the therapeutic potential of the antibody according to the present invention for Alzheimer's disease - 5xFAD mice To evaluate the therapeutic ability of the antibodies of the present invention to treat Alzheimer's disease, groups were designed as shown in Figure 1. Specifically, 5- to 6-month-old male Alzheimer's-induced 5xFAD mice were intravenously injected with the GTC310-01 mouse antibody of Preparation Example 1 at a dose of 10 mpk for 3 weeks. For comparison, a positive control group was administered with subcutaneous injections of glatiramer acetate (GA, Copaxone®) at a dose of 100 μg for 3 weeks, or with intravenous injections of the H6 antibody shown in Table 2 below at a dose of 10 mpk for 4 weeks.

[0178] [Table 2]

[0179] 1. Amyloid-β plaque reduction effect For immunohistological analysis, the treated mice were euthanized, and the brain cortex and hippocampus tissues were obtained and fixed in 4% paraformaldehyde (pH 7.4) for 16 hours. The fixed cortical and hippocampal brain tissues were cryoprotected in 30% sucrose and sliced ​​at 35 μm thickness using a cryostat (Microm HM 525, Thermo Scientific, Waltham, MA, USA). To visualize Aβ plaques, the sliced ​​brain sections were stained with thioflavin S (ThS) for 7 minutes. Thioflavin S was purchased from Sigma-Aldrich (catalog number T-1892). 500 μM ThS was dissolved in 50% ethanol. After sequential washing with 100%, 95%, and 70% ethanol, the sections were transferred to PBS. For immunofluorescence, relatively thick free-floating brain sections were incubated with goat anti-GFAP antibody at 4°C for 7 days. The sections were then incubated overnight at 4°C with Alexa Fluor 594-conjugated donkey anti-goat IgG (1:200, Abcam, ab150132). Nuclear contrast staining was performed using 4'6'-diamino-2-phenylindole dihydrochloride hydrate (DAPI, 1 mg / mL, 1:2000, Sigma). Images were taken with a Leica DM2500 fluorescence microscope, and the results are shown in Figure 10. The ThS+ area ratio (%) relative to normal mice was calculated using ImageJ software, and the results are shown in Figure 3.

[0180] As shown in Figures 2 and 3, the ThS+ area significantly increased in Alzheimer's-induced mice, but when the GTC310-01 antibody of the present invention was administered, the ThS+ area significantly decreased, particularly in the cortex, indicating a highly effective reduction in amyloid-β plaques.

[0181] 2. Y-maze test The Y-maze experiment used a maze frame with three identical arms, 40 cm long (wall height 15 cm), arranged at a 120-degree angle. This behavioral experiment utilizes rodents' instinctive exploratory behavior and focuses on their high likelihood of exploring new areas. The more a rodent remembers the arm it has explored most recently and refuses to enter the same arm, the higher its memory score is. Each animal was given 8 minutes to explore, and the final results are expressed as spontaneous alternation (%) in Figure 4. Spontaneous alternation (%) was calculated using the following equation: Here, behavioral pattern analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA).

[0182] [Formula 1] Spontaneous alternation(%)=The number of triplet / (total arm entry-2) As shown in Figure 4, in this experiment, in which the relative frequency with which experimental animals grasped peripheral cues and sequentially entered the maze was measured, it was confirmed that when the GTC310-01 antibody of the present invention was administered to Alzheimer's-induced mice, the spontaneous alternation value was at a similar level to that of the normal control group.

[0183] 3. Novel Object Recognition Experiment The novel object recognition experiment assessed memory ability using two different objects in a 40 cm x 40 cm acrylic cage. After habituation to the acrylic cage, the two objects were placed in fixed locations and allowed to be freely recognized. The time spent exploring each object was measured. A 24-hour delay was given to each individual, and then one object was replaced with a different object in the same location. The longer the exploration time, the better the memory. If the animal recognized the replaced object as a novel object, it may be unable to distinguish between the novel and the existing object and explore both objects equally. The animals were allowed to freely explore for a total of 10 minutes, and the results are shown in Figure 5 as a preference index (novel object exploring time / total exploring time). Analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA).

[0184] As shown in Figure 5, the preference index for a novel object was lower in the Alzheimer's-induced group than in the normal control group. However, it was confirmed that administration of the GTC310-01 antibody according to the present invention increased the preference index to a level greater than that of the normal control group.

[0185] 4. Water maze test The underwater maze test was performed based on the method devised by Morris. A stainless steel pool (90 cm diameter, 50 cm high) was filled with water (22 ± 1°C) and the water surface was set to a height of 30 cm. A hidden platform (5 cm diameter) was located 1 cm below the water surface. Three to four training sessions were conducted on the first day of evaluation. The total swimming time per animal was set to 60 seconds, and animals that found the platform within 60 seconds were required to remain on the platform for 10 seconds to induce memory. Animals that failed to find the platform within 60 seconds were artificially guided to the platform and required to remain on the platform for 10 seconds, with an escape time of 60 seconds. After the swimming practice, a probe test was conducted on the sixth day to measure spatial perception (latency to target, sec) and target crossing numbers. The results are shown in Figures 6 and 7. An acquisition test was also conducted to measure the time to platform (time to platform, sec) during the test. The results are shown in Figure 8. Analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA). The criterion for exclusion from the analysis of the results was set to individuals that did not explore by swimming but only circled a specific area.

[0186] As shown in Figures 6 to 8, when the GTC310-01 antibody according to the present invention was administered, the number of times the target was passed significantly increased, and the time taken to reach the position where the platform was located significantly decreased.

[0187] These experiments confirmed that the antibody according to the present invention has the effect of preventing, improving or treating diseases of the central nervous system.

[0188] [Example 2] Evaluation of the therapeutic potential of the antibody according to the present invention for Alzheimer's disease - 6xTg mice To evaluate the therapeutic potential of the antibodies of the present invention for Alzheimer's disease, groups were designed as shown in Figure 9 and an experiment was conducted. Specifically, female Alzheimer's-induced 5xFAD mice and female 6xTg mice, which had undergone a 4.5-month adaptation period, were intravenously injected with 10 mpg of the GTC310-01 antibody (Preparation Example) every other week for two months. For comparison, a positive control group was administered glatiramer acetate (GA, Copaxone®) intravenously at 100 μg every other week for two months. After two months, the mice were subjected to a Y-maze test, a novel object recognition test, and a passive avoidance test.

[0189] 1.6xTg Mouse Characteristics 5xFAD mice are widely used as an Alzheimer's disease induction model, but the 6xTg mice used in the experiments described below are a mouse model in which not only amyloid beta (Aβ42) but also tau protein (MAPT) has mutated and accumulated. This model is closer to a human disease model than the 5xFAD mouse, and therefore represents an ideal animal model for neurological diseases, including Alzheimer's disease.

[0190] 2. Y-maze test The Y-maze experiment used a maze frame with three identical arms, 40 cm long (wall height 15 cm), arranged at a 120-degree angle. This behavioral experiment utilizes rodents' instinctive exploratory behavior and focuses on their high likelihood of exploring new areas. The animals demonstrated high memory, remembering the arm they had just explored and refusing to enter the same arm. Each animal was given 8 minutes of exploration time, and the final results are expressed as spontaneous alteration (%) in Figure 10. Spontaneous alteration (%) was calculated using the following equation: Here, behavioral pattern analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA).

[0191] [Formula 1] Spontaneous alternation(%)=The number of triplet / (total arm entry-2) 10, in this experiment, in which the relative frequency of experimental animals grasping peripheral cues and sequentially entering the maze was measured, it was confirmed that when the GTC310-01 antibody of the present invention was administered to Alzheimer's-induced mice, the spontaneous alternation value was similar to that of the normal control group. Furthermore, it was confirmed that the 6xTg model mice, which are closer to a human disease model, had a spontaneous alternation value similar to that of the normal control group compared to the 5xFAD mice.

[0192] 3. Novel Object Recognition Experiment The novel object recognition experiment assessed memory ability using two different objects in a 40 cm x 40 cm acrylic cage. After habituation to the acrylic cage, the two objects were placed in fixed locations and allowed to be freely recognized. The time spent exploring each object was measured. A 24-hour delay was given to each individual, and then one object was replaced with a different object in the same location. The longer the exploration time, the better the memory. If the rats were unable to remember the object from 24 hours earlier, they may be unable to distinguish between the novel and the existing object and may explore both objects equally. The rats were allowed to freely explore for a total of 10 minutes, and the results are shown in Figure 11 as a preference index (novel object exploring time / total exploring time). Analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA).

[0193] As shown in Figure 11, the preference index for a novel object was lower in the Alzheimer's disease-induced group than in the normal control group. However, when the GTC310-01 antibody according to the present invention was administered, the preference index increased more than that of the normal control group in the 6xTg model, which is closer to a human disease model.

[0194] 4. Passive Avoidance Test The passive avoidance experiment was conducted in an experimental apparatus divided into two areas, a lighted chamber and a dark chamber, with a wire mesh bottom. On the first day, mice were allowed to move freely and adapt. The following day, each 5xFAD mouse and 6xTg mouse was allowed to adapt for 1 minute without the lights on in the lighted chamber, then for 2 minutes with the lights on. As soon as the mouse entered the dark chamber, a 0.5 mA electric shock was administered for 1 second, conducting a learning test. Following this learning test, each mouse was subjected to a memory test (teat trial). The 5xFAD and 6xTg mice were placed in the lighted chamber, and the latency time for the 5xFAD and 6xTg mice to enter the chamber was set to within 300 seconds. As shown in Figure 12, administration of GTC310-01 antibody resulted in a recovery similar to that of normal controls in both 5xFAD and 6xTg mice.

[0195] [Example 3] Identification of epitopes of Lrig-1 protein In order to identify an epitope in the extracellular domain of the Lrig-1 protein to which the GTC310-01 antibody of the above Preparation Example can bind, the following experiment was carried out.

[0196] [3-1] Analytical samples, reagents and materials [3-1-1]Antigen Substance name: hTregL1-his Ingredients: Protein Molecular weight: Approximately 83.9 kDa (excluding carbohydrate chains) Storage conditions: -20℃ Test substance: hTregL1-his (0.5 mg / ml) [3-1-2]Antibody Substance name: GTC310-01-hlgG1 Ingredients: Protein Molecular weight: Approximately 159 kDa (excluding carbohydrate chains) Storage conditions: -20℃ Test substance: GTC310-01-hlgG1 (2.7 mg / ml) [3-1-3] Reagents and materials CNBr-activated Sepharose 4B TM 4B) (GE, 17-0430-01) Diethylpyrocarbonate (DEPC, Sigma, 159220) Imidazole (Acros, 301870010) Sequencing grade modified trypsin (Promega, V5117) Chymotrypsin, sequencing grade (Promega, V1062) Disuccinimidyl dibutyric urea (DSBU; Thermo, A35459) Dimethyl sulfoxide (DMSO, Sigma, D5879) Sodium bicarbonate (NaHCO3, Sigma, 792519) Sodium chloride (NaCl, Sigma, S3014) Sodium acetate (NaOAc, Sigma, S2889) Tris (GE, 17-1321-01), Glycine (GE, 17-1323-01) Acetone (Merck, 1.07021.2521) Dithiothreitol (DTT; GE, 17-1318-02) Iodoacetamide (IAA; Sigma, I-6125) Sodium phosphate monobasic dihydrate (NaH2PO4, Sigma, 71505) Sodium phosphate dibasic hepta-hydrate(Na2HPO4, Sigma, S9390) Syringe filter 0.2 μm (Sartorius stedim, 16534) Acetonitrile, water (HPLC grade, JT Baker) Acetic acid (Sigma, 695092) Formic acid (Sigma, 33015) Urea (GE, 17-1319-01) [3-2] Experimental method [3-2-1] Covalent Labeling MS (CL-MS) An antigen sample was prepared with an antigen to antibody ratio of 2:1. Then, DEPC was added so that it did not exceed 1%, and the reaction was allowed to proceed at 37°C for 1 minute. The reaction was then terminated by adding imidazole. After acetone was added to carry out a precipitation reaction, the supernatant was removed by centrifugation, and the precipitated protein was dissolved using urea. The dissolved precipitate was digested with trypsin and chymotrypsin, and then the glycans attached to the protein were removed using PNGase-F. Finally, the disulfide bonds between the cysteines of the protein were cleaved using DTT and IAA, and the resulting protein was analyzed by LC-MS and MS. 2 The analysis was carried out.

[0197] [3-2-2] Cross-linking MS (XL-MS) Two antigen samples were prepared with an antigen to antibody ratio of 2:1. Then, a crosslinker (DSBU) was thoroughly dissolved in DMSO. Then, one sample was added to the other so that the sample to DSBU ratio was 1:100, and DMSO was added to the other, followed by a reaction at 25°C for 1 hour. After the reaction was complete, the sample was decomposed using trypsin or chymotrypsin, and then the glycans attached to the protein were removed using PNGase-F. Finally, the disulfide bonds between the cysteines in the protein were cleaved using DTT and IAA, and the resulting protein was analyzed by LC-MS, MS, and other methods. 2 The analysis was carried out.

[0198] [3-2-3] Peptide Screening CNBr activation (cyanogen bromide activation) was performed. Specifically, 1 mM HCl was added to the CNBr resin. Next, the antibody was added and incubated at 25°C for 1 hour. This was followed by 10 column volume (CV) washing with coupling buffer (0.1 M NaHCO3, 0.5 M NaCl, pH 8.3). Next, 5-10 CV of 0.1 M Tris-HCl, pH 8.0 was added and incubated at 25°C for 1 hour. After the reaction was complete, 5 column volumes of washing with coupling buffer were performed. Next, 5 column volumes of washing with 0.1 M sodium acetate / acetic acid, 0.5 M NaCl, pH 4.0 were performed. 5 column volumes of washing with 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.0 were performed. This acetic acid / acetic acid Tris-HCl, and NaCl washing process was repeated three times.

[0199] Next, immunoaffinity purification was performed. Specifically, 50 μg of antigen was digested with trypsin and chymotrypsin, respectively, and then the digested antigen was added to a CNBr-antibody column and reacted at room temperature for 1 hour. Then, washing with 1X PBS was repeated three times, each with 5 column volumes. Five column volumes of elution buffer (0.1 M glycine pH 2.4) were added, and the elution fraction was concentrated and analyzed by LC-MS and MS. 2 The analysis proceeded.

[0200] [3-2-4]LC-MS, MS analysis conditions 1. Liquid chromatography (LC) conditions Column: ACQUITY UPLC CSH C18 (1.7 μm, 2.1 × 100 mm, water) Flow rate: 100μl / min Mobile phase A: DW / 0.1%Formic acid Mobile phase B: ACN / 0.1%Formic acid Temperature: 25℃ LC condition (gradient)

[0201] [Table 3]

[0202] 2. MS, MS 2 conditions

[0203] [Table 4]

[0204] [Table 5]

[0205] [Table 6]

[0206] [Table 7]

[0207] [Table 8]

[0208] [3-3]Result [3-3-1] Covalent labeling MS (CL-MS) Covalent labeling MS (CL-MS) is a method for labeling amino acid residues on the solvent-accessible surface of proteins and then examining changes in protein structure through peptide mass fingerprinting and MS / MS analysis. Specifically, the test involves labeling the antigen and the antigen-antibody complex, respectively, and analyzing the MS analysis results to determine the labeling ratio of the antigen surface structure. This method also allows for the analysis of conformational epitopes (discontinuous antigenic determinants) that form closely spaced epitopes. This test used DEPC, which induces carbethoxy labeling (COOC2H4) at specific amino acid residues (H, Y, S, T, K, C, and N-terminal of the protein). The ratio of the labeled peptide (modification; +72.021 Da) to the non-labeled peptide (normal) was examined to identify peptides that were reduced in the antigen-antibody (Ag+Ab, experimental group) conjugate compared to the antigen (Ag, control group) sample.

[0209] hTregL1-his (Ag, control group) and hTregL1-his / 1C07-hlgG1 (Ag + Ab, experimental group) samples were prepared, treated with DEPC, and digested with protease. Each sample was analyzed in triplicate. MS analysis results for the control group (DEPC-labeled Ag) and the experimental group (DEPC-labeled Ag-Ab complex) were simultaneously matched to the hTregL1-his sequence using the BioPharma finder program, and the labeled peptides were compared. We identified non-labeled and labeled peptides in the control and experimental samples treated with trypsin (T) or chymotrypsin (Y). The results were then combined and the labeling ratios (percentages) of the control and experimental groups were compared. The results were summarized by amino acid sequence position (Table 9, Table 12), and the results were plotted in bar graphs (Figures 13, 16). The main sections where a percentage decrease in DEPC labeling was observed were enlarged and re-plotted (Table 10, Figure 14). To determine which amino acids in the hydrolyzed fragments in the decreased sequences contributed significantly to the decrease in DEPC labeling, we examined the DEPC labeling reduction ratios around DEPC-labeled residues (Tables 11, 13, and Figures 15, 17). The results were then substituted into the hTregL1-his sequence (figure not shown). A histogram was created by plotting the antigen sequence number on the x-axis and representing the degree of DEPC labeling reduction as a color spectrum from 0-20% (Figure 18). From blue to red, fragments with a relatively high percentage reduction in DEPC labeling are represented in red, while fragments with a small percentage reduction are represented in blue. Combining the results of the two protease tests used in this experiment, matching sequences were highlighted in red (figure omitted).

[0210] In trypsin, the 477-498, 480-498, 480-531, 532-558, and 541-558 sequences showed a 4% or greater reduction in DEPC labeling (Table 11, Figure 13). Lysine 497 and lysine 556 showed reductions ranging from a minimum of 5% to a maximum of 19% (Table 12, Figure 14). In chymotrypsin, the Y495-524 sequence showed a reduction in DEPC labeling of approximately 3% (Table 12, Figure 16), and DEPC labeling of lysine 495 showed a 5.12% reduction compared to the control (Table 13, Figure 17).

[0211] Based on the CL-MS analysis results of hTregL1-his / GTC310-01-hlgG1, the sequence containing lysine 497 was found to be exposed externally, and the sequence 495-524, which showed a repeated decrease in the DEPC labeling rate in both trypsin and chymotrypsin experiments, was determined to be the most likely epitope.

[0212] [Table 9(1)] [Table 9(2)]

[0213] [Table 10]

[0214] [Table 11]

[0215] [Table 12(1)]

[0216] [Table 12(2)]

[0217] [Table 13]

[0218] [Table 14]

[0219] [3-3-2] Cross-linking MS (XL-MS) Chemical cross-linking combined with mass spectrometry (MS) is used for analyzing protein interactions, epitope mapping, and conjugates between peptides, DNA, and RNA. This study was performed using a chemical cross-linker (disuccinimidyl dibutyric urea; DSBU) that binds to primary amines in proteins. It is generally known to bind best to the N-terminus and lysine (K) of proteins, and experimentally also binds to serine (S) and trypsin (Y). The peptide-linker bond patterns identified by MS analysis are commonly known to those skilled in the art as mono-links, loop-links, and cross-links. The cross-link analysis strategy for epitope mapping (EP) tests involves comparing control (without crosslinker) and experimental (with crosslinker) samples to identify peptides in the experimental group where the antigen EP site and antibody are linked by a linker, either not detected or reduced (<10%) compared to the control group. When only the linker is attached to the peptide, the XL% was calculated to additionally check that it was not an EP site.

[0220] hTregL1-his / 1C07-hlgG1 mixture samples were prepared, and only the experimental sample was treated with a crosslinker (DSBU) and then digested with protease. Peptides detected in the experimental group at 10% or less (Test%; T%) compared to the chymotrypsin (Y) and trypsin (T) control groups were summarized (Tables 15 and 17). Peptide sequences that met the overall requirement of Test+XL% being 10% or less in the experimental sample were examined, including modifications of the peptides that only contained a linker (XL%) (Tables 16 and 18 and Figures 19-22).

[0221] Chymotrypsin (Y) and trypsin (T) analyses of three regions (216-222, 447-524, and 557-577) of the hTregL1-his sequence confirmed that the percentage of peptides (T%) reduced in the experimental group compared to the control group was less than 10% (Table 19, Figure 23). Based on the crosslinker binding residues, K415 and K566 are interpreted as peptide reduction due to linker binding alone, while K460, 476, 497, and 498 were analyzed to still show a reduction rate of less than 10%. XL-MS analysis confirmed the epitope site at 425-531. Of the less than 10% commonly recognized by trypsin and chymotrypsin, the remaining sequences, excluding 425-531, are considered to be due to non-specific crosslinking.

[0222] [Table 15(1)] [Table 15(2)]

[0223] [Table 16]

[0224] [Table 17]

[0225] [Table 18]

[0226] [Table 19(1)] [Table 19(2)]

[0227] [3-3-3] Peptide screening (Affinity-MS) The most common epitope mapping analysis method using mass spectrometry (MS) is "epitope excision" or "epitope extraction." This study applied the epitope extraction method. The hTregL1-his antigen (Ag) peptide mixture generated by proteolytic degradation (trypsin, chymotrypsin) was reacted with the prepared GTC310-01-hlgG1 antibody (Ab) and CNBr sepharose (cyanogen bromide) coupled column. Peptides that do not bind to the antibody were removed, and the hTregL1-his peptides that interact with GTC310-01-hlgG1 were extracted. LC-MS, MS 2 The peptides detected in the analysis and their amino acid sequences were confirmed.

[0228] After confirming the binding of hTregL1-his(Ag) to a CNBr column conjugated with GTC310-01-hlgG1(Ab), Ag peptides digested with chymotrypsin or trypsin were loaded. Immuno-affinity elution fractions were analyzed by LC-MS / MS to detect Ag-derived peptides. Trypsin analysis revealed the highest abundances in the 499-531 and 630-668 regions. Chymotrypsin analysis also revealed high abundances in the 402-416 and 430-440 regions. However, as summarized in Table 20, the 402-471 region contains complex disulfide bonds, which may have led to epitope-independent trapping. In addition, T103-121, Y113-134, T630-668, and Y637-656, which contain N-glycosylated portions at N116 and N650, were commonly identified, but their possibility as epitopes was reduced. Taking into account the possibility that the Ag epitope is a structural epitope, the results were derived by combining all test results.

[0229] [Table 20]

[0230] [Table 21]

[0231] [3-4] Conclusion To identify the epitope of hTregL1-his, peptide screening, covalent labeling (CL-MS), and cross-linking (XL-MS) studies were performed. The results of all trypsin samples confirmed the 477-531 (498-531) region as the epitope. Chymotrypsin peptides were used to break down this region into 10 fragments, which were used as supplementary data to show the difference in the region detected by each test method. Because the results of CL-MS and peptide screening were highly accurate, these two data sets were used as the primary data points.

[0232] CL-MS (protein surface modification analysis) results revealed distinct epitopes. The T480-498 (15.42%) and T532-558 (19.02%) regions were labeled with K497 and K556. The Y495-524 (2.99%) region differed in sequence, but the K497 and K498 residues were identical (Table 14, Figure 15). Peptide screening (Affinity-MS) results confirmed the identity of T499-531 (Tables 20 and 21). The Affinity-MS results indicated that the central sequence, not 480-498 or 532-558, which are considered to be adjacent regions of the antigen and antibody in CL-MS, was identified. This result suggests that the antibody recognizes the 499-531 sequence, adjacent to the exposed K498 and K556 regions of the antigen. The absence of this region in the chymotrypsin analysis in Affinity-MS analysis is thought to be due to the theoretical generation of 12-14 chymotryptic peptides in the 480-558 (79 amino acids) region, resulting in the cleavage of the affinity region and making it undetectable. Contrary to Affinity-MS analysis, the undetected region is predicted to be the epitope in the XL-MS results. However, the T425-497 region was detected at less than 1% in various sequence forms (Tables 20 and 21). Furthermore, the T477-531, Y447-494, and Y495-524 regions were also detected at levels less than 10%, consistent with the results of the other two analyses (Figure 25).

[0233] The K497-Y524 (KKDNEVLTNADMENFVHVHAQDGEVMEY: SEQ ID NO: 29) sequence was confirmed using a molecular structure prediction model (figure omitted). The front part of the b-sheet structure model, 497-506 (KKDNEVLTNA: SEQ ID NO: 28) and 514-522 (VHAQDGEVM: SEQ ID NO: 30), which correspond to the CL-MS and PS results, are likely to be loop structures that are more exposed than the other parts and bind to antigens. In particular, K497 and K566 are sterically close to each other, showing a high correlation with the CL-MS labeling reduction results.

[0234] In conclusion, the epitope of the hTregL1-his antigen recognized by GTC310-10-hlgG1 Ab is the 497-506 sequence. We also concluded that the 514-522 sequence and the sequence containing K566 are also recognized by the antigen or are adjacent to it.

[0235] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. An epitope of Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein, An epitope selected from the group consisting of polypeptides comprising the amino acid sequences represented by SEQ ID NOs: 22, 25, 28, 30, 33, 38, 41, 43, 44, 47, 51 and 60.

2. The epitope according to claim 1, wherein the polypeptide consists of an amino acid sequence represented by SEQ ID NO: 21, 23, 24, 26, 27, 31, 32, 34-40, 42, 45, 46, 48-50, 52-59, or 61-69.

3. A nucleic acid molecule encoding the epitope of claim 1 or claim 2.

4. An expression vector into which the nucleic acid molecule of claim 3 has been inserted.

5. A host cell line transfected with the expression vector of claim 4.

6. A binding molecule that specifically binds to at least one epitope selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 21 to 69.

7. The binding molecule of claim 6, wherein the binding molecule is an antibody or an antigen-binding fragment.

8. 8. The binding molecule of claim 7, wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, a tetrabody, or a fragment thereof.

9. A chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain, The antigen-specific binding domain is an antigen-specific binding domain that specifically binds to at least one epitope selected from the group consisting of polypeptides consisting of the amino acid sequences represented by SEQ ID NOs: 21 to 69; and a chimeric antigen receptor that is a fusion protein comprising an immunoglobulin Fc region.

10. An antibody-drug conjugate comprising the binding molecule of claim 6 and a drug.

11. A pharmaceutical composition for preventing or treating a brain and nervous system disease, comprising as an active ingredient one or more of the binding molecule according to any one of claims 6 to 8, the chimeric antigen receptor according to claim 9, and the antibody-drug conjugate according to claim 10.

12. The pharmaceutical composition according to claim 11, wherein the cranial nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

13. The neurodegenerative or neuroinflammatory disease may be stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis (Alzheimer's disease), paraneoplastic neurological syndrome (PNS), or other neurodegenerative disorders.

13. The pharmaceutical composition for preventing or treating a neurodegenerative disease or a neuroinflammatory disease according to claim 12, wherein the neurodegenerative disease or the neuroinflammatory disease is at least one selected from the group consisting of progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, neurological autoimmune diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy.

14. A method for screening for a binding molecule that is an antibody or a fragment thereof that specifically binds to the epitope of claim 1 or claim 2.

15. A method for screening epitopes of Lrig-1 protein using the binding molecule according to any one of claims 6 to 8.

16. A method for binding the epitope of claim 1 or claim 2 with a binding molecule that is an antibody or an antigen-binding fragment thereof.

17. A binding molecule that is an antibody or a fragment thereof that specifically binds to Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein, (a) the heavy chain variable region of the binding molecule comprises a heavy chain CDR1 represented by SEQ ID NO:7; a heavy chain CDR2 represented by SEQ ID NO:8; and a heavy chain CDR3 represented by SEQ ID NO:9; or (b) the light chain variable region of the binding molecule comprises a light chain CDR1 represented by SEQ ID NO: 10; a light chain CDR2 represented by SEQ ID NO: 11; and a light chain CDR3 represented by SEQ ID NO: 12; The binding molecule specifically binds to an epitope comprising at least one polypeptide selected from the group consisting of polypeptides having the amino acid sequences represented by SEQ ID NOs: 21 to 69.

18. 18. The binding molecule of claim 17, wherein the heavy chain variable region of the binding molecule is represented by SEQ ID NO: 13 or the light chain variable region is represented by SEQ ID NO:

14.

19. 19. The binding molecule of claim 18, wherein the heavy chain of the binding molecule is represented by SEQ ID NO: 15 or 17, or the light chain is represented by SEQ ID NO: 16 or 18.

20. A method for preventing or treating a cranial nervous system disease, comprising administering to an individual, as an active ingredient, any one or more of the binding molecule according to any one of claims 6 to 8, the chimeric antigen receptor according to claim 9, the antibody-drug conjugate according to claim 10, and the binding molecule according to any one of claims 17 to 19.

21. The method for prevention or treatment according to claim 20, wherein the cranial nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

22. The neurodegenerative or neuroinflammatory disease may be stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis (Alzheimer's disease), paraneoplastic neurological syndrome (PNS), or other neurodegenerative disorders.

22. The method for preventing or treating a neurodegenerative disease or a neuroinflammatory disease according to claim 21, wherein the neurodegenerative disease or the neuroinflammatory disease is at least one selected from the group consisting of progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, neurological autoimmune diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy.

Images