Novel antibody with affinity maturation

Abstract

The present invention relates to a binding molecule which can bind specifically to Lrig-1 protein, which is a protein present on the surface of a regulatory T cell. The binding molecule provided by the present invention can effectively prevent, alleviate or treat cancer, particularly, solid cancer by suppressing the function of regulatory T cells, can effectively target Lrig-1 protein, compared with existing commercially available antibodies to Lrig-1, and has an excellent binding affinity force therefor.

Description

Novel antibodies with affinity maturation

[0001] The present invention relates to a novel binding molecule having superior binding affinity to the Lrig-1 antigen compared to existing antibodies, and its use for preventing, improving, or treating cancer, immune-related diseases, and neurological diseases.

[0002]

[0003] One of the most important characteristics of all normal organisms is the ability to recognize and eliminate non-self antigens while not reacting harmfully to antigenic substances that constitute the self. This lack of response of the body to self-antigens is called immunological unresponsiveness or tolerance. This tolerance is achieved by eliminating lymphocytes that may have specific receptors for self-antigens, or by inactivating the body's ability to respond to self-antigens after exposure to them. If there is a problem in inducing or maintaining tolerance, an immune response to self-antigens occurs, and the resulting disease is called an autoimmune disease.

[0004] To treat the above autoimmune diseases, the concept of suppressor T cells, which could control and suppress the effector function of conventional T cells, was first introduced by Gershon in the early 1970s. Since then, research has been conducted in many fields of immunology to elucidate the biological characteristics and functions of suppressor T cells, namely regulatory T cells (Treg). Accordingly, although regulatory T cells play an important role in naturally preventing the occurrence of excessive inflammation and immune responses, it has been reported that the function and number of regulatory T cells are significantly reduced in cases of autoimmune diseases and chronic inflammatory diseases. Therefore, it is important for patients with immune diseases and inflammatory diseases to produce regulatory T cells at normal levels, and this can be one of the treatments for the above diseases.

[0005]

[0006] One object of the present invention is to provide a binding molecule having superior binding ability to Lrig-1 protein present on the surface of regulatory T cells (Treg) compared to existing antibodies and having excellent effects in preventing, improving, or treating immune-related diseases.

[0007] Another object of the present invention is to provide a composition for preventing, improving or treating a neurological disease comprising a binding molecule according to the present invention.

[0008] Another object of the present invention is to provide a composition for preventing, improving or treating cancer comprising a binding molecule according to the present invention.

[0009] Another object of the present invention is to provide a composition for diagnosing cancer, immune-related diseases or neurological diseases; a kit and a method for providing information for diagnosis.

[0010]

[0011] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0012]

[0013] Various embodiments of the present invention are described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, 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 in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to an embodiment means that a 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 places throughout this specification does not necessarily indicate the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

[0014]

[0015] As used herein, a "binding molecule" means an intact immunoglobulin, such as a chimeric, humanized, or human monoclonal antibody, or a variable domain comprising an immunoglobulin fragment that competes with the intact immunoglobulin for binding to an antigen-binding immunoglobulin, for example, monomeric HA or trimeric HA of an influenza A virus. Regardless of structure, the antigen-binding fragment binds the same antigen recognized by the intact immunoglobulin. The antigen-binding fragment may comprise a peptide or polypeptide comprising an amino acid sequence of at least two consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 30 consecutive amino acid residues, at least 35 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues of the amino acid sequence of the binding molecule. "Antigen-binding fragments" include, in particular, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of an immunoglobulin sufficient to bind a specific antigen to the polypeptide, and the like. The fragments may be produced synthetically or by enzymatic or chemical degradation of complete immunoglobulins, or may be genetically engineered by recombinant DNA techniques.The method of production is well known in the art.

[0016] In the present invention, the "Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein" is a transmembrane protein consisting of 1091 amino acids present on the surface of regulatory T cells, and is composed of a leucine-rich repeat sequence (LRR) on the extracellular or lumen side, three immunoglobulin-like domains, a transmembrane sequence, and a cytoplasmic tail. The LRIG gene family consists of LRIG1, LRIG2, and LRIG3, and the amino acids between them are highly conserved. The LRIG1 gene is highly expressed in normal skin, and can regulate the proliferation of epithelial stem cells by being expressed in basal and hair follicle cells. Therefore, it plays an important role in maintaining epidermal homeostasis, and its absence can lead to psoriasis or skin cancer. It has been reported that when the chromosome 3p14.3 where LRIG1 is located is cut off, there is a possibility of developing into cancer cells, and in fact, it has been confirmed that the expression of LRIG1 is greatly reduced in renal cell carcinoma and cutaneous squamous cell carcinoma. However, it has recently been revealed that only about 20-30% of cancers express the Lrig-1. Meanwhile, for the purpose of the present invention, the Lrig-1 protein may be a protein existing in humans or mice, but is not limited thereto.

[0017] In the present invention, the binding molecule may further include an Fc region (Fragment crystallization region) or a constant region. In this case, the Fc region may be an Fc region of an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody, or may be derived therefrom, or may be a hybrid Fc region.

[0018] In the present invention, the Fc region may be an Fc region of a mammalian IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 antibody, and preferably, may be an Fc region of a human-derived IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 antibody, but is not limited thereto.

[0019] As an example of the present invention, the Fc region may be, but is not limited to, a human-derived immunoglobulin lambda constant region.

[0020] In the present invention, the "hybrid Fc" may be derived from a combination of human IgG subclasses or a combination of human IgD and IgG. When the hybrid Fc binds to a biologically active molecule, polypeptide, or the like, it not only increases the serum half-life of the biologically active molecule, but also has the effect of increasing the expression level of the polypeptide when a nucleotide encoding an Fc-polypeptide fusion protein is expressed.

[0021] In the binding molecule of the present invention, the Fc or constant region may be linked to the variable region by a linker. At this time, the linker may be linked to the C-terminus of the Fc or constant region, and the N-terminus of the binding molecule of the present invention may be linked to the linker, but is not limited thereto.

[0022] In the present invention, the "linker" may include a sequence that can be cleaved by an enzyme that is overexpressed in the tissue or cell of the target disease. In the case where the linker can be cleaved by the overexpressed enzyme as described above, the activity of the polypeptide can be effectively prevented from being reduced due to the Fc or constant region. In the present invention, a preferred example of the linker may be a peptide linker consisting of 33 amino acids located at positions 282 to 314 of human albumin, which is most abundant in blood, and more preferably a peptide linker consisting of 13 amino acids located at positions 292 to 304. Such a portion is a portion that is mostly exposed to the outside due to its three-dimensional structure and is a portion that has a minimal possibility of inducing an immune response in the body. However, the present invention is not limited thereto.

[0023] The binding molecule of the present invention is characterized by being, but is not limited to, an antibody or a fragment thereof. The antibody includes all antibody fragments capable of binding to the Lrig-1 protein as a monoclonal antibody, a full-length antibody, or a portion of an antibody and capable of competitively binding to the Lrig-1 antigenic determinant site with the binding molecule of the present invention.

[0024] In the present invention, the "antibody" refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically reactives with a specific antigen. For the purposes of the present invention, the antigen may be the Lrig-1 protein present on the surface of a regulatory T cell. Preferably, the antigen may specifically recognize the Leucine Rich Region or the immunoglobulin-like domain of the Lrig-1 protein, but is not limited thereto.

[0025] In the present invention, the "immunoglobulin" has a heavy chain and a light chain, and each of the heavy chain and the light chain includes a constant region and a variable region. The variable regions of the light chain and the heavy chain include three variably variable regions called complementarity determining regions (hereinafter referred to as "CDRs") and four framework regions. The CDRs mainly 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 also identified by the chain on which a specific CDR is located.

[0026] In addition, in the present invention, the "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and exhibits single binding specificity and affinity for a specific antigenic determinant (Epitope).

[0027] In the present invention, the "full-length antibody" has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond, and includes IgA, IgD, IgE, IgM, and IgG. The IgG includes IgG1, IgG2, IgG3, and IgG4 as its subtypes.

[0028] In addition, in the present invention, the "antibody fragment" means a fragment having an antigen-binding function, and includes Fab, Fab', F(ab')2, and Fv, etc. The Fab has one antigen-binding site in a structure having variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain. In addition, Fab' is different from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibody is produced when the cysteine ​​residues of the hinge region of Fab' form a disulfide bond. Fv (Variable fragment) means the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. Double-chain Fv (dsFv) has a heavy chain variable region and a light chain variable region linked by a disulfide bond, and single-chain Fv (scFv) has a heavy chain variable region and a light chain variable region covalently linked, typically via a peptide linker. The antibody fragments can be obtained as Fab or F(ab')2 fragments using a proteolytic enzyme, such as papain or pepsin, and can be produced through genetic recombination technology.

[0029] In addition, in the present invention, the antibody may be, but is not limited to, a chimeric antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody, a tetrabody, or a fragment thereof.

[0030] In the present invention, the "chimeric antibody" is an antibody in which the variable region of a mouse antibody and the constant region of a human antibody are recombined, and is an antibody in which the immune response is greatly improved compared to a mouse antibody.

[0031] Additionally, 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 is modified to resemble an antibody variant naturally produced in humans. For example, the humanized antibody can be produced by recombining a mouse-derived CDR with a human antibody-derived FR to produce a humanized variable region, which is then recombined with a desired human antibody constant region.

[0032] In the present invention, the binding molecule can be provided as a bispecific antibody or bispecific antigen-binding fragment that can bind to Lrig-1 protein and also bind to other proteins.

[0033] In the present invention, the bispecific antibody and bispecific antigen-binding fragment may comprise a binding molecule according to the present invention. As an example in the present invention, the bispecific antibody and bispecific antigen-binding fragment may comprise an antigen-binding domain capable of binding to the Lrig-1 protein, wherein the antigen-binding domain capable of binding to the Lrig-1 protein may comprise or consist of a binding molecule according to the present invention.

[0034] The bispecific antibody and bispecific antigen-binding fragment provided by the present invention comprise an antigen-binding domain, which is a binding molecule capable of binding to the Lrig-1 protein according to the present invention, and an antigen-binding domain capable of binding to another target protein. Here, the antigen-binding domain capable of binding to another target protein is a protein other than the Lrig-1 protein, and may be, but is not limited to, an antigen-binding domain capable of binding to PD-1 or a cell surface receptor, for example, but is not limited thereto.

[0035] The bispecific antibodies and bispecific antigen-binding fragments according to the present invention may be provided in any suitable format, for example, as described in the literature incorporated herein by reference. For example, a bispecific antibody or bispecific antigen-binding fragment may be a bispecific antibody conjugate (e.g., IgG2, F(ab')2 or CovX-body), a bispecific IgG or IgG-like molecule (e.g., IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, or 2 in 1-IgG, mAb2, or Tandemab common LC), an asymmetric bispecific IgG or IgG-like molecule (e.g., kih IgG, kih IgG common LC, CrossMab, kih 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), bispecific Fc and CH3 fusion proteins (e.g., taFv-Fc, d-diabody, scDb-CH3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-CH3), or bispecific fusion proteins (e.g., scFv2-albumin, scDb-albumin, taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine2). Those skilled in the art can design and prepare bispecific antibodies and bispecific antigen-binding fragments according to the present invention.

[0036] The method for producing the bispecific antibody of the present invention comprises chemical cross-linking of antibodies or antibody fragments with reducing disulfide or non-reducing 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 produce disulfide-linked bispecific F(ab)2 heterodimers.

[0037] Additionally, another method for producing the bispecific antibody in the present invention comprises fusing the antibody-producing hybridoma with, for example, polyethylene glycol to produce quadroma cells capable of secreting the bispecific antibody.

[0038] Bispecific antibodies and bispecific antigen-binding fragments according to the present invention can be produced recombinantly, for example, by expression from a nucleic acid construct encoding a polypeptide for an antigen-binding molecule.

[0039] For example, a DNA construct comprising sequences 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 to PD-1, etc., and light and heavy chain variable domains for an antigen binding domain capable of binding to another target protein), and encoding a suitable linker or dimerization domain between the antigen binding domains, can be prepared by molecular cloning techniques. A recombinant bispecific antibody 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 antibody can then optionally be purified.

[0040] Antibodies can be produced by an affinity maturation process, which produces a modified antibody with improved affinity for an antigen compared to the unmodified parent antibody. Affinity-matured antibodies can be produced by procedures known in the art.

[0041] Additionally, the binding molecule provided in the present invention may include variants of the amino acid sequence, as long as they can specifically bind to the Lrig-1 protein. For example, the amino acid sequence of the antibody may be altered to improve its binding affinity and / or other biological properties. Such alterations include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody.

[0042] These amino acid mutations are based on the relative similarity of the amino acid side-chain substituents, such as hydrophobicity, hydrophilicity, charge, and size. Analysis of the size, shape, and type of amino acid side-chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine are similar in size; and phenylalanine, tryptophan, and tyrosine are similar in shape. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be considered biologically functional equivalents.

[0043] When introducing mutations, the hydrophobicity index of an amino acid can be considered. Each amino acid is assigned a hydrophobicity 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 amino acid index is crucial for imparting interactive biological functions to proteins. It is well known that amino acids with similar hydrophobic indices must be substituted to retain similar biological activity. When introducing mutations based on hydrophobic indices, substitutions are preferably made between amino acids with a difference in hydrophobicity index of ±2, more preferably ±1, or even ±0.5.

[0044] Meanwhile, it is also well known that substitutions between amino acids having 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 with reference to hydrophilicity values, substitutions can be made between amino acids that exhibit a difference in hydrophilicity values ​​of preferably within ± 2, more preferably within ± 1, and even more preferably within ± 0.5.

[0045] Amino acid exchanges in proteins that do not alter the overall activity of the molecule are well known in the art. The most common 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.

[0046] Considering the mutations having biologically equivalent activity described above, the binding molecule of the present invention is interpreted to also include sequences showing substantial identity with the sequences listed in the sequence listing.

[0047] The term "substantial identity" as used herein means 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 and any other sequence are paralleled to the greatest extent possible and the parallel sequences are analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are accessible from the NCBI Basic Local Alignment Search Tool (BLAST), NBCI (National Center for Biological Information), etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST can be accessed at this address (ncbi.nlm.nih.gov / BLAST / ). A method for comparing sequence homology using this program can be found online (ncbi.nlm.nih.gov / BLAST / blast_help.html).

[0048] In the present invention, the binding molecule, preferably the antibody, may be produced by a conventional method for producing antibodies, but may be produced by affinity maturation.

[0049] In the present invention, the “H6 antibody” or “GTC110-04” antibody is a monoclonal antibody identical to a known antibody that has a preventive and therapeutic effect on cancer or neurological diseases.

[0050] In the present invention, the “GTC310-01 antibody” or “1C07” antibody is a monoclonal antibody identical to a known antibody having a preventive and therapeutic effect on brain and nervous system diseases.

[0051]

[0052] 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, i.e., antibodies with enhanced antigen binding ability, during an immune response. For the purposes of the present invention, the affinity maturation can produce antibodies or antibody fragments produced by affinity maturation based on the principles of mutation and selection, similar to processes occurring in nature.

[0053] The ten affinity-matured antibody clones (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01) provided in the present invention are affinity-matured antibodies from the previously known H6 or GTC310-01 antibody, in which only one of the six CDRs of the GTC310-01 antibody is partially modified (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8 and MH6) or an antibody (6F01) in which only three CDRs included in the light chain are modified, and have the characteristic of higher binding affinity to Lrig-1 than the H6 or GTC310-01 antibody. Therefore, compared to the H6 or GTC310-01 antibodies, these antibodies have improved binding affinity for binding to the antigen Lrig-1, while retaining the pharmacological effects of the existing H6 or GTC310-01 antibodies because only a portion of the entire 6CDR has been modified.

[0054] Generally, the effectiveness of an antibody or antigen-binding fragment is determined by the antigen to which it binds. Therefore, it is widely known in the art that an antibody with superior binding affinity for the same antigen compared to an antibody with a known pharmacological effect will have the same pharmacological effect, but with an effect equal to or superior to that of a previously known antibody.

[0055] In particular, the ten affinity-matured antibody clones (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01) provided in the present invention have a KD value that is 2 to 737 times lower than that of the GTC310-01 antibody or H6 antibody, which have previously been shown to have pharmacological effects, indicating a higher binding affinity, or a binding affinity for CD4+FoxP3+Lrig1+ regulatory T cells that is 90% or higher when 5 ug of the antibodies are administered.

[0056] Therefore, a person skilled in the art can readily predict that the antibodies provided in the present invention, which have a KD value that is 2 to 737 times lower than that of the GTC310-01 antibody, which has a known pharmacological effect, and exhibit a high binding affinity, or have a binding affinity for CD4+FoxP3+Lrig1+ regulatory T cells that is 90% or higher when 5 ug of the antibodies are administered, have pharmacological effects that are the same as or superior to those of the existing H6 or GTC310-01 antibodies.

[0057]

[0058] The binding molecule, preferably an antibody, provided in the present invention can effectively prevent, improve or treat immune-related diseases, neurological diseases and cancer.

[0059] According to another embodiment of the present invention, a nucleic acid molecule encoding the binding molecule provided in the present invention is provided.

[0060] The nucleic acid molecule of the present invention includes all nucleic acid molecules translated into polynucleotide sequences based on the amino acid sequence of the binding molecule provided in the present invention, as known to those skilled in the art. Therefore, various polynucleotide sequences can be produced by ORF (open reading frame), and all of these are also included in the nucleic acid molecule of the present invention.

[0061] According to another embodiment of the present invention, an expression vector into which the isolated nucleic acid molecule provided by the present invention is inserted is provided.

[0062] In the present invention, the "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 into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, into which additional DNA segments can be ligated to the viral genome. Some vectors are capable of autonomous replication in the host cell 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 host cell genome upon introduction, thereby replicating along with the host genome. Furthermore, some vectors can direct the expression of genes to which they are linked in an operational manner. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." In general, expression vectors useful in recombinant DNA techniques often exist in the form of plasmids. In this specification, “plasmid” and “vector” may be used interchangeably, as the plasmid is the most commonly used form of vector.

[0063] In the present invention, specific examples of the expression vector may be selected from the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμμ, T-even, T2, T3, T7, etc.; and plant viruses, but are not limited thereto, and all expression vectors known to those skilled in the art as expression vectors can be used in the present invention, and when selecting an expression vector, it depends on the properties of the target host cell. When introducing a vector into a host cell, it may be performed by calcium phosphate transfection, viral infection, DEAE-dextran controlled transfection, lipofectamine transfection, or electroporation, but is not limited thereto, and those skilled in the art can select and use an introduction method appropriate for the expression vector and host cell to be used. Preferably, the vector contains one or more selectable markers, but this is not limited to this. Selection can be made based on whether a product is produced using a vector that does not contain a selectable marker. The selectable marker is selected based on the target host cell, and since this is done using methods already known to those skilled in the art, the present invention is not limited thereto.

[0064] To facilitate purification of the nucleic acid molecule of the present invention, a tag sequence can be inserted into an expression vector and fused thereto. Such tags include, but are not limited to, a hexahistidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag known to those skilled in the art that facilitates purification can be utilized in the present invention.

[0065] According to another embodiment of the present invention, a host cell line transformed with an expression vector provided by the present invention is provided.

[0066] In the present invention, the term "host cell" includes an individual cell or cell culture that may be or was the recipient of the vector(s) for incorporating the polypeptide insert. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (morphologically or in genomic DNA complement) to the original parent cell due to natural, accidental, or intentional mutation. Host cells include cells that have been transfected in vivo with the polypeptide(s) of the present disclosure.

[0067] In the present invention, the host cell may include a mammalian, plant, insect, fungal or cellular cell, and 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 Drozophylla and Spodoptera Sf9 cells; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bow melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), or PERC.6 (human retinal cells); Alternatively, the host cell line may be a plant cell, but is not limited thereto, and any cell known to those skilled in the art that can be used as a host cell line may be used.

[0068] According to another embodiment of the present invention, an antibody-drug conjugate (ADC) comprising an antibody and a drug provided by the present invention is provided.

[0069] In the present invention, the "antibody-drug conjugate (ADC)" refers to a form in which a drug and an antibody are chemically linked without reducing the biological activity 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 an antibody, specifically, a form in which a drug is bound to an α-amine group at the N-terminus of the heavy chain and / or light chain of an antibody.

[0070] In the present invention, the term "drug" may refer to any substance having a specific biological activity in cells, including DNA, RNA, or peptides. The drug may be in a form that includes a reactive group capable of reacting with an α-amine group to form a cross-linking molecule, and also includes a form in which a linker is connected that includes a reactive group capable of reacting with an α-amine group to form a cross-linking molecule.

[0071] In the present invention, examples of reactive groups capable of reacting with the α-amine group to crosslink include, but are not limited to, any group that can react with the α-amine group at the N-terminus of the heavy or light chain of an antibody to crosslink, and include all groups known in the art that react with amine groups. Examples thereof include, but are not limited to, any one of isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester.

[0072] In the present invention, the drug may be included regardless of its type as long as it is a drug that can treat immune-related diseases, neurological diseases, and cancer.

[0073] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating a neurological disease is provided, which comprises, 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.

[0074] 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 coreceptor, 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 an extracellular antigen-binding domain fused or linked thereto. In the present invention, the binding domain may comprise a single-chain variable fragment (scFv) capable of specifically recognizing the Lrig-1 protein. As used herein, the "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable heavy (VH) chain and the variable light (VL) chain of an antibody via a peptide linker between the VL and VH chains.

[0075] Additionally, in the present invention, the VH domain and the VL domain may be connected via a flexible linker. In the present invention, the flexible linker may be a glycine / serine linker of about 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), and preferably may be 15 amino acids long. In the present invention, the linker length may serve as an important determining site of the chimeric antigen receptor, and a linker shorter than the above range may increase affinity, but may also cause intracellular multimer formation, thereby impairing CAR expression, whereas a linker longer than the above range may decrease antigen affinity by spatially moving the VL and VH CDRs further apart.

[0076] The chimeric antigen receptor of the present invention may further comprise at least one of a hinge region (or spacer) and a signal transduction domain. In the present invention, the hinge region is a portion that connects the antigen binding domain and the transmembrane domain, also called a "spacer," and has the purpose of extending the antigen binding domain from the T cell membrane or NK cell membrane. In the present invention, the hinge region may be obtained from any suitable sequence from any genus, including, for example, human or a part thereof, or may include a hinge region of a human protein, including all or part of CD8, CD28, 4-1BB, OX40, 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, but is not limited thereto. In addition, the hinge region in the present invention may include one selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD), but is not limited thereto.

[0077] In the present invention, the signal transduction domain refers to a portion of a chimeric antigen receptor that is found or engineered to be found within a T cell. In the present invention, the signal transduction domain may or may not include a transmembrane domain that serves to anchor the chimeric antigen receptor to the plasma membrane of the T cell. In the present invention, the transmembrane domain and the signal transduction domain may be derived from the same protein (e.g., a CD3 zeta (ζ) molecule), or the transmembrane domain and the signal transduction domain may be derived from different proteins (e.g., a transmembrane domain of CD28 and an intracellular signal transduction domain of a CD3 zeta (ζ) molecule, or vice versa).

[0078] In the present invention, the transmembrane domain may include, but is not limited to, for example, T cell receptor α or β chain, all or part of 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, 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 functional signaling domain derived from a polypeptide comprising a ligand that binds to LFA-1 (CD11a / CD18) or ICOS; an active fragment thereof; a functional derivative thereof; or a combination thereof, may be included, but is not limited thereto.

[0079] In the present invention, the signal transduction domain may include, but is not limited to, a functional signal transduction domain derived from a polypeptide comprising all or part of CD3 zeta (ζ), common FcR gamma (FcER1G), FcgammaRIIIa, FcRbeta (Fc epsilon lip), CD3gamma, CD3delta, CD3epsilon, CD79a, CD79b, DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), an active fragment thereof, a functional derivative thereof, or a combination thereof, and such signal transduction domains are known in the art.

[0080] According to another embodiment of the present invention, there is provided a composition for preventing, improving or treating a neurological disease, comprising a binding molecule of the present invention; a nucleic acid molecule; an expression vector; a host cell line; or an antibody-drug conjugate (ADC) as an active ingredient.

[0081] The "immune-related disease" of the present invention is a disease induced by excessive activation and expression of various immune cells and inflammatory cells, and may be, for example, autoimmune disease; graft-versus-host disease; organ transplant rejection; asthma; atopy; or acute or chronic inflammatory disease, but is not limited thereto.

[0082] The "autoimmune disease" of the present invention refers to a disease that can occur when an abnormality in the immune function occurs and attacks the organs or tissues of our body. For the purpose of the present invention, it can occur when the function of T cells, particularly regulatory T cells, does not function normally, resulting in an immune response to self-antigens. The autoimmune disease may be selected from the group consisting of, for example, rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjögren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa, but is not limited thereto.

[0083] The “neurological disease” that the composition provided in the present invention is intended to prevent, improve or treat may be a neurodegenerative disease or a neuroinflammatory disease.

[0084] In the present invention, the “neurodegenerative disease” may refer to a disease caused by a decrease or loss of function of nerve cells, and the “neuroinflammatory disease” may refer to a disease caused by an excessive inflammatory response of the nervous system. In the present invention, specific examples of the neurodegenerative disease or neuroinflammatory disease include multiple sclerosis, stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy. May be selected from the military, but is not limited to this.

[0085] It is becoming clear that neuroinflammatory responses are one of the major factors that cause neurodegeneration. That is, glial cells, which are immune cells present in the central nervous system, can be activated by various exogenous and endogenous substances, and activated glial cells produce and release substances such as inflammatory cytokines such as TNF-α and IL-1β, nitric oxide, prostaglandins, and superoxide (Gao et al., J Neurochem, 81, 1285-97, 2002; Nelson, PT. et al., Ann Med, 34, 491-500, 2002; Griffin, WS et al., J Neuroinflammation, 3, 5, 2006). The production of these substances induces an immune response in the short term, but excessive or continuous production induces the death of nearby neurons, ultimately leading to neurodegeneration.

[0086] Considering the importance of neuroinflammation in neurodegenerative diseases, it may be possible to treat neuroinflammation and the neurodegenerative diseases that may arise from it by reducing the expression level of proinflammatory mediators within these glial cells.

[0087] Activated glial cells have been reported to cause various central nervous system diseases such as multiple sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease by continuously inducing the activity of nitric oxide (NO), reactive oxygen species (ROS), inflammatory enzymes such as inducible nitric oxidesynthase (iNOS) and cyclooxygenase-2 (COX-2), and proinflammatory mediators such as IL-1β, IL-6, and tumor necrosis factor-α (Gao et al., 2002; Nelson et al., 2002; Eikelenboom and Van Gool, 2004).

[0088] At this time, if proinflammatory mediators are reduced within glial cells, the onset of these diseases can be suppressed (Matsumoto et al., 1992; Liu and Hong, 2003). It is widely known through mechanistic studies that various proinflammatory factors and neurotoxins induced by LPS in glial cells contribute to neuronal damage during the inflammatory process in cell-based models (Kim et al., 2004a,b). Furthermore, numerous reports have shown that glial cells activated by LPS increase neurotoxicity through proinflammatory mediators and cytotoxins (Dawson et al., 1994). Considering the importance of the inflammatory response in neuroinflammation, suppressing the production of these inflammatory factors in activated glial cells will be a goal for treating various neuroinflammatory diseases.

[0089] The "cancer" of the present invention refers to or represents a physiological condition typically characterized by uncontrolled cell growth in mammals. The cancer that is the target of prevention, improvement, or treatment of the present invention may be a solid tumor formed by a mass caused by abnormal cell growth in a solid organ, and depending on the location of the solid organ, may be gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer, and examples thereof include, but are not limited to, melanoma or colon cancer.

[0090] In addition, the composition provided in the present invention can be used as a pharmaceutical composition or a food composition, but is not limited thereto.

[0091] The above "prevention" of the present invention may include, without limitation, any act that can block, suppress or delay symptoms caused by a neurodegenerative disease or a neuroinflammatory disease by using the composition of the present invention.

[0092] The above “treatment” and “improvement” of the present invention may include, without limitation, any act that can improve or benefit symptoms caused by a neurodegenerative disease or a neuroinflammatory disease by using the composition of the present invention.

[0093] In the present invention, the pharmaceutical composition may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and the pharmaceutical composition may be characterized as being for human use.

[0094] The pharmaceutical composition of the present invention is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, aqueous suspensions, etc., external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. for oral administration, and buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. for injections. In the case of topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The formulation of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0095] Meanwhile, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, or mineral oil. In addition, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, and the like may be additionally included.

[0096] Routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or parenteral administration is preferred.

[0097] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.

[0098] 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, diet, administration time, administration route, excretion rate, drug combination, and severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may 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 administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a liquid, a gel, a syrup, a slurry, or a suspension.

[0099] A food composition comprising the composition of the present invention as an active ingredient can be manufactured in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc. Since the food composition of the present invention is composed of plant extracts with almost no toxicity or side effects, it can be used with confidence even when taken for long periods of time for preventive purposes.

[0100] When the composition of the present invention is included in a food composition, the amount may be added in a ratio of 0.1 to 50% of the total weight.

[0101] Here, when the food composition is manufactured in the form of a beverage, there are no special restrictions other than containing the food composition in the indicated ratio, and various flavoring agents or natural carbohydrates, etc. may be contained as additional ingredients like in a typical beverage. That is, as a natural carbohydrate, it may include a monosaccharide such as glucose, a disaccharide such as fructose, a polysaccharide such as sucrose, a dextrin, a cyclodextrin, a typical sugar such as dextrin, a cyclodextrin, a sugar alcohol such as xylitol, sorbitol, and erythritol. As the flavoring agent, there may be mentioned a natural flavoring agent (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and a synthetic flavoring agent (saccharin, aspartame, etc.).

[0102] In addition, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

[0103] These components can be used independently or in combination. The proportions of these additives are not particularly critical, but are typically selected within the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

[0104]

[0105] According to one embodiment of the present invention, a binding molecule is provided, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26; and a light chain CDR2 represented by a selected amino acid sequence represented by SEQ ID NO: 27.

[0106] According to another embodiment of the present invention, the binding molecule comprises a heavy chain variable region further comprising a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 43; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 and 79; a light chain CDR2 represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 and 80; and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 47, 51, 55, 59, 63, 67, 71, 75, and 81.

[0107] According to another embodiment of the present invention, the binding molecule comprises a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; and a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35.

[0108] According to another embodiment of the present invention, the binding molecule comprises a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 33 and 45; and a light chain variable region comprising a light chain CDR1 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 34 and 84; a light chain CDR2 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 35 and 85; and a light chain CDR3 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 36, 49, 53, 57, 61, 65, 69, 73, 77, and 86.

[0109] According to another embodiment of the present invention, there is provided a binding molecule comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 28; and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 28, and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 47, and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25;A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 51, and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 55, and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 59, and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25;A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 63, and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 67, and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 71, and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25;And a binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 75; and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 79, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 81; A binding molecule selected from the group consisting of;

[0110] According to another embodiment of the present invention, the binding molecule comprises a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 36; and a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 45; and a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34. A binding molecule comprising a light chain variable region comprising a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 36; and a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 49; and a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33;A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 53; and a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 57; and a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; A binding molecule comprising a heavy chain variable region comprising a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 61; and a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 65;A binding molecule comprising a light chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 69; and a light chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; A binding molecule comprising a light chain variable region comprising a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 73; and a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 77; and a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33;A binding molecule is provided, wherein the binding molecule comprises a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 84; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 85; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 86;

[0111] According to another embodiment of the present invention, a heavy chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 44 and 82; and

[0112] A binding molecule comprising a light chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 48, 52, 56, 60, 64, 68, 72, 76 and 83 is provided.

[0113] According to another embodiment of the present invention, a heavy chain variable region encoded by any one base sequence selected from the group consisting of SEQ ID NOs: 37, 46 and 87; and

[0114] A binding molecule comprising a light chain variable region encoded by any one base sequence selected from the group consisting of SEQ ID NOs: 38, 50, 54, 58, 62, 66, 70, 74, 78 and 88.

[0115] According to another embodiment of the present invention, the binding molecule further comprises an Fc region or a constant region.

[0116] According to another embodiment of the present invention, the binding molecule provides an Fc region of an IgG1, IgG2, IgG3 or IgG4 antibody, or a hybrid Fc region.

[0117] According to another embodiment of the present invention, the binding molecule further comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 7, 8, 9, 10 and 12.

[0118] According to another embodiment of the present invention, the binding molecule further comprises a light chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 6 and 11.

[0119] According to another embodiment of the present invention, a binding molecule is provided, further comprising: a heavy chain constant region encoded by a base sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 17; and a light chain constant region encoded by a base sequence selected from the group consisting of SEQ ID NOs: 14, 16, and 18.

[0120] According to another embodiment of the present invention, a binding molecule is provided, wherein the binding molecule is an antibody or a fragment thereof.

[0121] According to another embodiment of the present invention, the antibody provides a binding molecule that 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.

[0122] According to another embodiment of the present invention, a nucleic acid molecule encoding the binding molecule is provided.

[0123] According to another embodiment of the present invention, an expression vector into which the nucleic acid molecule is inserted is provided.

[0124] According to another embodiment of the present invention, a host cell line transfected with the expression vector is provided.

[0125] According to another embodiment of the present invention, an antibody-drug conjugate comprising the binding molecule and a drug is provided.

[0126] According to another embodiment of the present invention, a chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain, wherein the antigen-specific binding domain comprises a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26; and a light chain CDR2 represented by a selected amino acid sequence represented by SEQ ID NO: 27; and a chimeric antigen receptor which is a fusion protein comprising an antigen-specific binding domain; and an immunoglobulin Fc region.

[0127] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating cancer is provided, comprising the binding molecule as an active ingredient.

[0128] According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the cancer is a solid cancer.

[0129] According to another embodiment of the present invention, a pharmaceutical composition is provided wherein the cancer is gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer.

[0130] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating a neurological disease is provided, comprising the binding molecule as an active ingredient.

[0131] According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the neurological disease is a neurodegenerative disease or a neuroinflammatory disease.

[0132] According to another embodiment of the present invention, the neurodegenerative disease or neuroinflammatory disease is selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy. A pharmaceutical composition is provided.

[0133] According to another embodiment of the present invention, a pharmaceutical composition for preventing or treating an immune-related disease is provided, comprising the binding molecule as an active ingredient.

[0134] According to another embodiment of the present invention, a pharmaceutical composition is provided wherein the immune-related disease is an autoimmune disease; graft-versus-host disease; organ transplant rejection; asthma; atopy; or an acute or chronic inflammatory disease.

[0135] According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa.

[0136]

[0137] The binding molecule provided in the present invention has superior binding affinity to the Lrig-1 antigen, and thus can specifically prevent, improve, or treat various immune-related diseases, neurological diseases, and cancer.

[0138]

[0139] Figure 1 is a line graph showing the ELISA results of 11 antibodies that underwent affinity maturation with mLrig1-His.

[0140] Figure 2 is a line graph showing the ELISA results of 11 other antibodies that underwent affinity maturation with mLrig1-His.

[0141] Figure 3 shows the BLI results for the 10 selected TregL1-mFc types, focusing on the KD value.

[0142] Figure 4 is a graph comparing the binding affinity of 10 affinity-matured antibodies compared to the control H6 antibody and GTC310-01 antibody.

[0143] Figure 5 is a schematic diagram of an experimental design for cancer treatment using a monoclonal antibody (H6) specific for the Lrig-1 protein according to one embodiment of the present invention.

[0144] Figure 6 shows the results of analyzing the cancer treatment effect using a monoclonal antibody (H6) specific for the Lrig-1 protein according to one embodiment of the present invention.

[0145] Figure 7 is a schematic diagram of an experimental design for the cancer treatment effect using an affinity-matured monoclonal antibody (ML2) specific for the Lrig-1 protein according to one embodiment of the present invention.

[0146] Figure 8 shows the results of analyzing the cancer treatment effect using an affinity-matured monoclonal antibody (ML2) specific for the Lrig-1 protein according to one embodiment of the present invention.

[0147] Figure 9 shows an experimental design diagram for confirming the Alzheimer's treatment effect of a monoclonal antibody according to one embodiment of the present invention.

[0148] Figure 10 is a graph showing the results of calculating the ThS+ area ratio (%) compared to a normal mouse after staining the brain cortex and hippocampus tissue of the mouse obtained after each treatment including a monoclonal antibody according to one embodiment of the present invention in an Alzheimer's disease-induced mouse with thioflavin S (ThS).

[0149] Figure 11 is a graph showing the change in spontaneous alteration (%) values ​​in a Y-maze experiment after each treatment including a monoclonal antibody according to one embodiment of the present invention in an Alzheimer's disease-induced mouse.

[0150] Figure 12 is a graph showing changes in preference values ​​as a result of a novel object recognition experiment after each treatment including a monoclonal antibody according to one embodiment of the present invention in an Alzheimer's disease-induced mouse.

[0151] Figure 13 shows an experimental design diagram of 5xFAD mice and 6xTg mice, which are Alzheimer's disease-induced mice, to confirm the Alzheimer's treatment effect of a monoclonal antibody according to one embodiment of the present invention.

[0152] Figure 14 is a graph showing the change in spontaneous alteration (%) values ​​in a Y-maze experiment after each treatment including a monoclonal antibody according to one embodiment of the present invention in 5xFAD mice and 6xTg mice, which are Alzheimer's disease-induced mice.

[0153] Figure 15 is a graph showing the change in preference value as a result of a novel object recognition experiment after each treatment including a monoclonal antibody according to one embodiment of the present invention in 6xTg mice, which are Alzheimer's disease-induced mice.

[0154] Figure 16 is a graph showing the change in retention time as a result of a passive avoidance experiment after each treatment including a monoclonal antibody according to one embodiment of the present invention in 5xFAD mice and 6xTg mice, which are Alzheimer's disease-induced mice.

[0155] Figure 17 shows an experimental design diagram for confirming the therapeutic effect of a monoclonal antibody on neurological diseases and autoimmune diseases according to one embodiment of the present invention.

[0156] Figure 18 shows the therapeutic effects of MH6 and 6F01 antibodies on neurological and autoimmune diseases.

[0157] Figure 19 shows an experimental design diagram for confirming the therapeutic effect of a monoclonal antibody on neurological diseases and autoimmune diseases according to one embodiment of the present invention.

[0158] Figure 20 shows the therapeutic effect of ML4 antibody on neurological and autoimmune diseases.

[0159]

[0160] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0161]

[0162] Example

[0163]

[0164] [Manufacturing Example 1] Production of a monoclonal antibody (GTC310-01) specific for Lrig-1 protein

[0165] An antibody specific to the Lrig-1 protein was produced according to the present invention. This antibody was produced without targeting a specific antigenic determinant, but rather capable of binding to any site on the Lrig-1 protein.

[0166] To produce the above antibody, cells expressing the Lrig-1 protein were created. More specifically, the DNA fragment corresponding to sequence number 20 and pcDNA (hygro) were cleaved with a cleavage enzyme, then cultured at 37°C and ligated to produce pcDNA having the DNA sequence of the Lrig-1 protein inserted therein. The produced pcDNA having the inserted sequence number 20 was introduced into L cells through transfection, allowing the Lrig-1 protein to be expressed on the surface of L cells.

[0167] The sequences of light and heavy chain amino acids capable of binding to Lrig-1 expressed on the cell surface were selected from a human scFv library.

[0168] The selected heavy and light chain amino acid sequences were fused with the heavy chain constant region of SEQ ID NO: 1 or 4 and the light chain constant region of SEQ ID NO: 2 or 6, respectively, to produce a monoclonal antibody. The sequence of the monoclonal antibody is as shown in Table 1 below.

[0169] 구분클론위치아미노산 서열서열정보제조예 1GTC310-01 마우스 항체중쇄EVQLLESGGGLVQPGGSLRLSCAASGFTFS NYAMS WVRQAPGKGLEWVS VISHGGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR VISNCHLGVCYYSNGMDV WGQGTLVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK서열번호 39경쇄QLVLTQPPSVSAAPGQRVSISC SGSSSNIGDNYVS WYQQVPGSAPKLLIY DNNKRPS GIPDRFSASKSGTSATLGITGLQTGDEADYYCAT WDGSLSAGRV FGGGTKLTVLRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC서열번호 40제조예 2GTC310-01 인간화 항체중쇄EVQLLESGGGLVQPGGSLRLSCAASGFTFS NYAMS WVRQAPGKGLEWVS VISHGGGSTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR VISNCHLGVCYYSNGMDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK서열번호 41경쇄QLVLTQPPSVSAAPGQRVSISC SGSSSNIGDNYVS WYQQVPGSAPKLLIY DNNKRPS GIPDRFSASKSGTSATLGITGLQTGDEADYYCAT WDGSLSAGRV FGGGTKLTVLRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC서열번호 42제조예 3H6클론중쇄EVQLLESGGGLVQPGGSLRLSCAASGFTFSNYAMSWVRQAPGKGLEWVSVISHGGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVISNCHLGVCYYSNGMDVWGQGTLVTVSSTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQTHREDYNSTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK서열번호 89경쇄QSVLTQPPSASGTPGQRVTISCSGSSSNIGNNDVYWYQQLPGTAPKLLIYSDSQRPSGVPDRFSGSKSGTSASLAISGLRSEDEADYYCGTWDYSLSGYVFGGGTKLTVLRTVAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC서열번호 90

[0170]

[0171] [제조예 2] GTC310-01 항체의 친화도 성숙된 항체 제조

[0172] In order to produce antibodies that can bind specifically to Lrig-1 more than the H6, GTC310-01 antibodies produced in Table 1 above, 10 affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, ML6, and 6F01) were produced. The ML (modified light chain) clones were affinity-matured by modifying part of the sequence of the light chain CDR3 to better specifically bind to Lrig-1, and the MH (modified heavy chain) clones were affinity-matured by modifying part of the sequence of the heavy chain CDR3 to better specifically bind to Lrig-1. The 6F01 clone was affinity-matured by modifying only the light chain CDRs while having the same heavy chain to better specifically bind to Lrig-1.

[0173]

[0174] [Example 1] Binding affinity analysis of 10 new affinity-matured antibodies using ELISA

[0175] First, a single colony grown in a square plate after bead panning was inoculated into each 96-well well and grown to an OD600=0.5. IPTG induction was then performed overnight. Afterwards, the recombinant protein, His-tagged mouse Lrig-1 (antigen), was immobilized in an immunotube at 4°C overnight. The antigen-coated plate was washed three times with TBST and blocked with 3% skim milk. Then, the cells that had been IPTG-induced the previous day were down-regulated, and 50 μl of the supernatant was loaded onto the antigen-coated plate. After that, 3% skim milk and HA-HRP (secondary antibody) were mixed at a ratio of 1000:1 and loaded at 50ul / well, and then 50ul of substrate solution was added. After reaction for approximately 10 minutes, the reaction was stopped by adding 50ul of sulfuric acid or Stop buffer. Then, the absorbance at 450nm was measured using a plate reader.

[0176] As a result, as shown in Figures 1 to 2 and Table 2, the Kd value of the previously manufactured 6F01 antibody was about half that of the H6 antibody, so it was found that it binds to his-tagged mouse Lrig-1 (antigen) about twice as well as the H6 antibody.

[0177] mLRIG1-His(Good Cell)H6-mIgG2aH6H6_FRH6_FR_006A04H6_FR_006B06H6_FR_006F01 H6_FR_007A05H6_FR_007A06H6_FR_007A07H6_FR_007B05H6_FR_007B09Kd4.9x10 9 1.2x10 10 9.4x10 116.9x10 11 7.0x10 11 6.1x10 11 8.3x10 11 7.5x10 11 8.9x10 11 7.0x10 11 9.6x10 11 R square0.99140.99260.99440.98990.99180.99490.99210.99680.99580.98890.9867Cons(nM)1001.9432.4322.1662.0612.0962.232.1362.2782.3862.3492.04333.333331.6352.4372.1792.2372.0892.2032.2882.3232.2632.2791.93211.111111.252.2732.1471.9372.0062.2142.2092.212.172.0432.1223.7037040.8732.2981.9851.961.9452.0382.1762.3132.312.2072.1511.2345680.4372.0782.1262.0742.0282.0482.1012.0692.1962.2282.1430.4115230.2231.6681.6991.8221.8151.7781.6851.9121.9192.1151.8330.1371740.111.2181.2191.3131.2371.4611.2851.4511.3391.4541.1590.0457250.0690.760.7340.8080.8120.940.870.8450.770.8130.6020.0152420.0540.3520.3620.4270.460.4890.4330.4370.3880.4390.340.0050810.050.1740.170.2050.210.2220.20.2030.1830.2250.1690.0016940.0480.090.0940.1180.1470.1180.1020.1110.0980.1120.1040.0005650.0480.0630.0630.0830.0960.0750.070.070.0660.0710.068

[0178]

[0179] [Example 2] Binding affinity analysis of 10 novel affinity-matured antibodies using Octet®

[0180] [Example 2-1] Differentiation and staining method of human iTreg cells

[0181] Peripheral blood mononuclear cells (PBMCs) were counted and resuspended in 40 μl buffer per 10ⁿ (n=7). Then, 10 μl naive CD4+ T cell biotin-antibody cocktail Ⅱ per 10ⁿ (n=7) was added and incubated at 4℃ for 10 minutes. Tapping was also performed every minute. Again, resuspended in 30 μl buffer per 10ⁿ (n=7) and 20 μl naive CD4+ T cell microbead cocktail Ⅱ per 10ⁿ (n=7) was added and incubated at 4℃ for 15 minutes. Tapping was also performed every minute. Then, an LS column was prepared and the flow-through (naive CD4+ T cells) was collected. 2*10^6cell / 2ml in 24 wells, Dynabead® CD3 / CD28 2*10^ 5After preparing at a cell / 4 μl standard, naive CD4+ T cells were dispensed per well and treated with TGF-β 5 ng / mL and IL-2 20 ng / mL. The cells were then cultured in an incubator for 4 days. Each differentiated cell (200 μl) was pipetted and transferred to a round well. After centrifugation at 2500 rpm for 2 minutes, for staining of cell surface proteins, the medium was removed and 50 μl of PBS and antibodies were mixed and pipetted per well. The plate was wrapped in foil and incubated at 4°C for 30 minutes. Afterwards, 150 μl of PBS was added to each well and centrifuged at 2500 rpm for 2 minutes. For staining of intracellular proteins, the medium was removed, the fixative / permeabilizer concentration (4x) was diluted to 1x using the fixative / permeabilizer diluent, 80 μl per well was dissolved, the plate was wrapped in foil, and incubated at 4°C for 30 minutes. After that, 10x permeabilization buffer was diluted to 1x permeabilization buffer using DW, 120 μl per well was added, and centrifuged at 2500 rpm for 2 minutes. After that, 50 μl of 1x permeabilization buffer per well and the antibody were mixed and pipetted. The plate was wrapped in foil and incubated at 4°C for 30 minutes. 150 μl of 1x permeabilization buffer was additionally added, and centrifuged at 2500 rpm for 2 minutes. 200 μl of PBS was added, dissolved, and centrifuged at 2500 rpm for 2 minutes to wash.

[0182]

[0183] [Example 2-2] Binding affinity analysis using Octet®

[0184] First, equilibrate with water for 60 seconds. Then, activate with EDC / s-NHS buffer for 300 seconds, and immobilize in a buffer containing ligand (= anti-L1 antibody 20 μg / ml) in 10 mM acetate at pH 6.0 for 10 minutes. Then, stop by treating with ethanolamine for 5 minutes, treat with 1X Kinetics buffer for 2 minutes, and treat with regeneration buffer and 1X Kinetics buffer for 20 seconds. Repeat twice for 5 seconds for regeneration and 5 seconds for neutralization. Then, treat with 1X Kinetics buffer again for 2 minutes, and bind the analyte with 1X Kinetics buffer for 5 minutes. Then, wash with 1X Kinetics buffer for 5 minutes and treat with 1X Kinetics buffer again for 2 minutes.

[0185]

[0186] [Example 2-3] Results

[0187] The results are shown in Figs. 3 and 4. As shown in Fig. 3, the KD values, which indicate higher affinity for the antigen as they are lower, are at least 2-fold and at most 737-fold lower for the affinity-matured novel antibodies than for the GTC310-01 antibody, and thus it was confirmed that the binding affinity for the Lrig-1 antigen is at least 2-fold and at most 737-fold higher than that of the existing GTC310-01 antibody.

[0188] Finally, as shown in Fig. 4, it was confirmed that the affinity-matured novel antibodies had higher affinity for CD4+FoxP3+Lrig1+ cells than the existing H6 and GTC310-01 antibodies.

[0189]

[0190] [Example 3] Cancer therapeutic effect of the antibody according to the present invention

[0191] [Example 3-1] Cancer treatment effect of existing H6 antibodies

[0192] In order to confirm the therapeutic effect of the monoclonal antibody (H6) manufactured in the above Table 1 on solid tumors, as shown in Fig. 5, 3X10 B16F10 melanoma cells were injected into mice. 5 After subcutaneous injection of the cells, the antibody was injected intraperitoneally at a dose of 200 ug on days 4, 8, and 12. The change in tumor volume over time after the melanoma cell transplantation was measured, and the results are shown in Figure 8.

[0193] As shown in Figure 6, it was confirmed that the tumor size was significantly reduced when treated with the existing Lrig-1 protein-specific monoclonal antibody (H6) compared to the negative control group that was not treated with the antibody.

[0194] Through this, it can be seen that the existing Lrig-1 protein-specific monoclonal antibody can effectively prevent, improve, or treat various solid cancer cells by inhibiting their growth.

[0195]

[0196] [Example 3-2] Cancer therapeutic effect of affinity-matured antibodies

[0197] Among the affinity-matured antibodies manufactured in Manufacturing Example 2, based on Fig. 4, the ML2 antibody was selected to specifically bind to CD4+Foxp3+Lrig1+ cells at a concentration of 5 ug, that is, at least 9 times more than the existing H6 antibody, i.e., at least 90%, and its cancer treatment effect was confirmed.

[0198] As shown in Figure 7, a CT26 mouse model was prepared, and 3X10 CT26 cancer cells were injected into the back of 7-week-old female mice. 5After subcutaneous injection at a dose of 200 μl per cell, the size of the cancer cells was measured 7 days later. Thereafter, the ML2 antibody was injected intraperitoneally at concentrations of 100 μg, 200 μg, and 500 μg at 2-day intervals, and the size of the cancer cells was measured. The results are shown in Figure 8.

[0199] As a result, it was confirmed that the novel affinity-matured ML2 antibody provided by the present invention can treat cancer by reducing the size of cancer compared to the control group. Therefore, it will be clearly understood by those skilled in the art that other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01) that specifically bind to CD4+Foxp3+Lrig1+ cells at a concentration of 5 ug, that is, more than 9 times more, that is, more than 90%, compared to the existing H6 antibody, can treat cancer by activating effector T cells by specifically binding to the Lrig-1 protein of regulatory T cells and inhibiting the function of regulatory T cells.

[0200] Finally, as examined in Example 3-1 above, it was confirmed that the H6 antibody with relatively low binding affinity for the Lrig-1 protein also has a cancer therapeutic effect, and among other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01) that bind specifically to CD4+Foxp3+Lrig1+ cells about 9 times more, i.e., more than 90%, at a concentration of 5 ug compared to the existing H6 antibody, the anticancer effect of the ML2 antibody was confirmed, so it is possible that other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01) that bind specifically to CD4+Foxp3+Lrig1+ cells about 9 times more, i.e., more than 90%, at a concentration of 5 ug compared to the existing H6 antibody also have an anticancer effect on regulatory T cells. Those skilled in the art will readily understand that cancer can be treated by activating effector T cells by specifically binding to the Lrig-1 protein and inhibiting the function of regulatory T cells.

[0201]

[0202] [Example 4] Evaluation of the therapeutic ability of the antibody according to the present invention for neurological diseases

[0203] [Example 4-1] Evaluation of the therapeutic efficacy of the existing GTC310-01 antibody for neurological diseases - 5xFAD mice

[0204] In order to evaluate the therapeutic ability of the antibody according to the present invention for neurological diseases, a group was designed as shown in Fig. 9. Specifically, the GTC310-01 mouse antibody of the above preparation example was intravenously injected at a dose of 10 mpk for 3 weeks into male Alzheimer's disease-induced 5xFAD mice aged 5 to 6 months. However, for comparison, as a positive control, glatiramer acetate (glatiramer acetate, GA, Copaxone®) was subcutaneously injected at a dose of 100 ug for 3 weeks, or the H6 antibody (GTC110-04 antibody) of the above preparation example was intravenously injected at a dose of 10 mpk for 4 weeks.

[0205]

[0206] 1. Amyloid-β plaque reduction effect

[0207] For immunohistological analysis, the treated mice were euthanized, and the cortical and hippocampal tissues were obtained and fixed in 4% paraformaldehyde (pH 7.4) for 16 h. The fixed cortical and hippocampal brain tissues were immersed in 30% sucrose for cryoprotection and sliced ​​into 35 μm thick sections 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 min. Thioflavin S was purchased from Sigma-Aldrich (catalog no. T-1892). 500 μM of ThS was dissolved in 50% ethanol. After sequential washes 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 counterstaining was performed using 4'6'-diamino-2-phenylindole dihydrochloride hydrate (DAPI, 1 mg / mL, 1:2000, Sigma). Images were captured with a Leica DM2500 fluorescence microscope, and the results are shown in Figure 12. In addition, the ThS+ area ratio (%) compared to normal mice was calculated using the ImageJ software program, and the results are shown in Figure 12.

[0208] As shown in Fig. 10, the ThS+ area was significantly increased in Alzheimer's disease-induced mice, but when the existing GTC310-01 and H6 (GTC110-04) antibodies according to the above manufacturing example were administered, the ThS+ area was significantly reduced, especially in the cortex, indicating that the amyloid-β plaque reduction effect was very excellent.

[0209]

[0210] 2. Y-maze test

[0211] The Y-maze experiment used a maze frame made of three identical arms, each 40 cm long (with walls 15 cm high), arranged at a 120-degree angle. This experiment is a behavioral experiment that utilizes the instinctive exploration habits of rodents, and the method was based on the high possibility of exploring a new area. The animals remembered the arm they had explored just before, and the higher the memory value, the more they avoided entering the same arm. Each individual was given 8 minutes of exploration time, and the final results are expressed as the spontaneous alteration (%) value in Figure 13. The spontaneous alteration (%) value was calculated using the following equation 1. Here, the analysis of the behavioral pattern was conducted using SMART VIDEO TRACKING Software (Panlab, USA).

[0212] [Formula 1]

[0213] Spontaneous alternation (%) = The number of triplet / (total arm entry-2)

[0214] As shown in Fig. 11, in this experiment, which measured the relative frequency with which experimental animals sequentially entered the maze by recognizing surrounding clues, it was confirmed that when the existing GTC310-01 or H6 (GTC110-04) antibody of the above-mentioned manufacturing example was administered to Alzheimer's-induced mice, they had spontaneous alternation values ​​at a level similar to that of the normal control group.

[0215]

[0216] 3. Novel Object Recognition Experiment

[0217] The novel object recognition experiment assessed memory using two different objects in a 40 cm x 40 cm acrylic cage. After familiarizing the cage with the two objects, the animals were allowed to freely recognize them by placing them in fixed locations. The time it took for each object to be explored was measured. After a delay of 24 hours for each animal, only one object was replaced with a different object in the same location. The longer the exploration time, the better the memory. If the animals cannot remember the object from 24 hours ago, they may not be able to distinguish between the novel and old objects and may explore both equally. The animals were allowed to freely explore for a total of 10 minutes, and the results are presented in Figure 14 as a preference index (preference index = novel object exploring time / total exploring time). The analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA).

[0218] As shown in Figure 12, the preference index for novel objects was observed to be lower in the Alzheimer's-induced group than in the normal control group. However, when the existing GTC310-01 antibody or H6 (GTC110-04) antibody of the above-mentioned manufacturing example was administered, it was confirmed that the preference index increased above that of the normal control group.

[0219] Through these experiments, it was confirmed that the existing GTC310-01 antibody or H6 (GTC110-04) antibody of the above manufacturing example has an effect of preventing, improving, or treating brain nervous system diseases.

[0220]

[0221] [Example 4-2] Evaluation of the therapeutic efficacy of the existing GTC310-01 antibody for neurological diseases - 6xTg mice

[0222] In order to evaluate the Alzheimer's treatment ability of the existing GTC310-01 antibody, groups were designed and experiments were conducted as shown in Fig. 13. Specifically, the GTC310-01 antibody of Preparation Example 1 was intravenously injected at a dose of 10 mpk every other week for 2 months into female Alzheimer's-induced 5xFAD mice and female 6xTg mice that had undergone an adaptation period of 4.5 months. However, for comparison, glatiramer acetate (GA, Copaxone®) was intravenously injected at a dose of 100 ug every other week for 2 months as a positive control. After 2 months, the Y-maze test, novel object recognition test, and passive avoidance test were conducted.

[0223]

[0224] 1. Features of the 6xTg Mouse

[0225] 5xFAD mice are widely used as an Alzheimer's disease model, but the 6xTg mice used in the following experiments are a mouse model in which not only amyloid-β (Aβ42) but also tau protein (MAPT) is mutated and accumulated, and are thus closer to human disease models than the 5xFAD mice, making them an ideal animal model for neurological diseases including Alzheimer's disease.

[0226]

[0227] 2. Y-maze test

[0228] The Y-maze experiment used a maze frame made of three identical arms, each 40 cm long (with a wall height of 15 cm), arranged at a 120-degree angle. This experiment is a behavioral experiment that utilizes the instinctive exploration habits of rodents, and the method was based on the high possibility of exploring a new area. The animals remembered the arm they had explored just before, and the higher the memory value, the more they avoided entering the same arm. Each animal was given 8 minutes of exploration time, and the final results are expressed as the spontaneous alteration (%) value in Figure 14. The spontaneous alteration (%) value was calculated using the following equation 1. Here, the analysis of the behavioral pattern was conducted using SMART VIDEO TRACKING Software (Panlab, USA).

[0229] [Formula 1]

[0230] Spontaneous alternation (%) = The number of triplet / (total arm entry-2)

[0231] As shown in Fig. 14, in this experiment, which measured the relative frequency with which experimental animals sequentially entered the maze by recognizing surrounding clues, it was confirmed that when the GTC310-01 antibody according to the present invention was administered to Alzheimer's disease-induced mice, they had spontaneous alternation values ​​at a level similar to that of the normal control group. Furthermore, it was confirmed that the 6xTg model mice, which are closer to the human disease model, had spontaneous alternation values ​​at a level more similar to that of the normal control group than the 5xFAD mice.

[0232]

[0233] 3. Novel Object Recognition Experiment

[0234] The novel object recognition experiment assessed memory using two different objects in a 40 cm x 40 cm acrylic cage. After familiarizing the cage with the two objects, the animals were allowed to freely recognize them by placing them in fixed locations. The time it took for each object to be explored was measured. After a delay of 24 hours for each animal, only one object was replaced with a different object in the same location. The longer the exploration time, the better the memory. If the animal cannot remember the object from 24 hours ago, it is likely that the animal will not be able to distinguish between the novel and existing objects and will explore both equally. The animals were allowed to freely explore for a total of 10 minutes, and the results are presented in Figure 21 as a preference index (preference index = novel object exploring time / total exploring time). The analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA).

[0235] As shown in Figure 15, the preference index for novel objects was observed to be lower in the Alzheimer's-induced group compared to the normal control group. However, when the GTC310-01 antibody according to the present invention was administered, it was confirmed that the preference index increased above the normal control group in the 6xTg model, which is closer to the human disease model.

[0236]

[0237] 4. Passive Avoidance Test

[0238] The passive avoidance test experimental apparatus was divided into two areas: a bright chamber with lighting and a dark chamber, and the floor was made of wire mesh. On the first day, the mice were allowed to move freely and acclimate. The next day, each 5xFAD mouse and 6xTg mouse was allowed to acclimate for 1 minute in the lighted chamber with the light turned off. Then, the light was turned on and the mice were allowed to acclimate for 2 minutes. As soon as the mice moved to the dark chamber, an electric shock of 0.5 mA for 1 second was administered to conduct a learning test. The day after the learning test, a memory test (teat trial) was conducted for each mouse. The 5xFAD mouse and 6xTg mouse were placed in the chamber with the light on, and the time it took for the 5xFAD mouse and 6xTg mouse to enter the chamber with all four paws (latency time) was set to 300 seconds or less. As a result, as shown in Fig. 16, it was found that when GTC310-01 antibody was administered to not only 5xFAD mice but also 6xTg mice, recovery was achieved to a level close to that of the normal control group.

[0239]

[0240] [Example 4-3] Therapeutic effects of affinity-matured antibodies on autoimmune diseases and neurological diseases

[0241] Among the affinity-matured antibodies manufactured in Manufacturing Example 2, the antibodies with the lowest and highest affinities were selected based on affinity to determine their therapeutic effects on autoimmune diseases and neurological diseases. If the therapeutic effects of the above antibodies are confirmed, those skilled in the art will be able to clearly understand the therapeutic effects of other antibodies with intermediate binding affinities compared to the above antibodies without excessive experimentation or additional special knowledge.

[0242]

[0243] For this purpose, based on Figure 3, the MH6 antibody, which has a KD value about twice as low as that of the existing GTC310-01 antibody, and thus has the lowest Lrig-1 binding affinity among other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01), although it has a superior binding affinity to the Lrig-1 protein about twice as high as that of the GTC310-01 antibody, and conversely, the ML4 antibody, which has a KD value more than 737 times lower than that of the existing GTC310-01 antibody, and thus has the highest Lrig-1 binding affinity among other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01), and based on Table 2, the KD value of the existing H6 is 1.2x10 10 In comparison, the KD value is 6.1x10 11 Similarly, the treatment effect of 6F01 antibody, which has a KD value about twice as low, was confirmed.

[0244]

[0245] 1. Creation of an EAE (Experimental autoimmune encephalomyelitis) mouse disease model

[0246] To confirm the reduction in inflammatory responses, we used a mouse model of experimental autoimmune encephalomyelitis (EAE), a representative animal model. Given that neuroinflammation is widely recognized as a key factor in causing neurodegeneration, this model is also a representative mouse model of neurological diseases.

[0247] To create an EAE mouse model, experiments were performed according to the experimental design shown in Figure 17. On day 0, 9-week-old female C57Bl / 6J mice were injected subcutaneously with MOG peptide, and 260 ng of PTX was injected intraperitoneally. On day 1, 260 ng of PTX was injected once more to boost the immune system of the mice. On day 10, the mice were administered 50 ug of the 6F01 and MH6 antibodies produced in the above-mentioned manufacturing example, and 50 ug of anti-IL-17a antibody was administered as a positive control.

[0248] In addition, to confirm the therapeutic effect of the ML4 antibody, an experiment was performed according to the experimental design shown in Fig. 19. On day 0, 11-week-old female C57L / 6N mice were injected subcutaneously with MOG peptide, and 200 ng of PTX was injected intraperitoneally. On day 1, 200 ng of PTX was injected once more to strengthen the immune system of the mice. As shown in Table 3 below, the cases in which the ML4 antibody produced in the above manufacturing example was administered three times at 2.5 mg / kg to mice on days 12, 14, and 16, and the case in which the ML4 antibody was administered once at 10 mg / kg on day 12 were confirmed. As a positive control, 200 ug of anti-IL-17a antibody was administered.

[0249] GroupsConc.nInjection time1mIgG2a control200 ug / 100 uL103 times2ML4 10 mg / kg200 ug / 100 uL101 times3ML4 2.5 mg / kg50 ug / 100 uL103 times4Anti-IL17A200 ug / 100 uL103 times

[0250]

[0251] 2. Evaluation of the therapeutic efficacy of multiple sclerosis - 6F01 and MH6

[0252] From the 7th day, the clinical score was calculated according to the evaluation criteria below to evaluate the therapeutic effect on multiple sclerosis in mice, and the results are shown in Figure 18.

[0253] <Evaluation Criteria>

[0254] 0 points: No symptoms and moves normally

[0255] 1 point: Part of the tip of the tail is paralyzed and hangs down.

[0256] 2 points: The entire tail is paralyzed and the entire tail is drooping.

[0257] 3 points: Partial paralysis of one hind leg, but able to respond to stimulation

[0258] 4 points: One hind leg is completely paralyzed, does not respond to stimuli, and drags the leg.

[0259] 5 points: All hind legs are paralyzed, so the body is dragged around with the front legs.

[0260] 6 points: The animal has difficulty dragging itself with its front legs, but the front legs still respond to stimulation.

[0261] 7 points: The mouse cannot move, but one forelimb responds to stimulation.

[0262] 8 points: The mouse is immobile and neither forelimb responds to stimulation.

[0263] 9 points: Mouse cannot move and breathing is irregular.

[0264] 10 points: Mouse dies

[0265] As a result, we confirmed that 6F01 and MH6 antibodies, which had the lowest binding affinity for Lrig-1 among other affinity-matured antibodies in the multiple sclerosis-induced model, could significantly treat multiple sclerosis.

[0266]

[0267] 3. Evaluation of the effectiveness of multiple sclerosis treatment - ML4

[0268] From the 12th day, the clinical score was calculated according to the evaluation criteria below to evaluate the therapeutic effect on multiple sclerosis in mice, and the results are shown in Figure 20.

[0269] <Evaluation Criteria>

[0270] 0 points: No symptoms and moves normally

[0271] 1 point: Part of the tip of the tail is paralyzed and hangs down.

[0272] 2 points: The entire tail is paralyzed and the entire tail is drooping.

[0273] 3 points: Partial paralysis of one hind leg, but able to respond to stimulation

[0274] 4 points: One hind leg is completely paralyzed, does not respond to stimuli, and drags the leg.

[0275] 5 points: All hind legs are paralyzed, so the body is dragged around with the front legs.

[0276] 6 points: The animal has difficulty dragging itself with its front legs, but the front legs still respond to stimulation.

[0277] 7 points: The mouse cannot move, but one forelimb responds to stimulation.

[0278] 8 points: The mouse is immobile and neither forelimb responds to stimulation.

[0279] 9 points: Mouse cannot move and breathing is irregular.

[0280] 10 points: Mouse dies

[0281] As a result, we confirmed that the ML4 antibody, which has the highest binding affinity for Lrig-1 among other affinity-matured antibodies, can significantly treat multiple sclerosis in a multiple sclerosis-induced model.

[0282]

[0283] 4. Conclusion

[0284] As previously discussed in Examples 4-1 and 4-2, it was confirmed that the H6 antibody and GTC310-01 antibody, which have relatively low binding affinity for the Lrig-1 protein, also have a therapeutic effect on brain and nervous system diseases.

[0285] In addition, as examined in Example 4-3 above, among the affinity-matured antibodies manufactured in Manufacturing Example 2, the KD value is about 2 times lower than that of the existing GTC310-01 antibody based on FIG. 4, so although the binding affinity for the Lrig-1 protein is about 2 times higher than that of the GTC310-01 antibody, the MH6 antibody has the lowest Lrig-1 binding affinity among other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01), and thus has the highest Lrig-1 binding affinity among other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01). Based on the ML4 antibody and Table 2, the KD value of the existing H6 is 1.2x10 10 In comparison, the KD value is 6.1x10 11Similarly, in order to confirm the reduction of inflammatory response targeting the 6F01 antibody with a KD value about twice as low, it was confirmed that it treats autoimmune diseases and neurological diseases by reducing inflammatory response in the experimental autoimmune encephalomyelitis (EAE) mouse disease model, which is a representative animal model.

[0286] Therefore, a person skilled in the art will be able to understand that other affinity matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01) having a higher binding affinity for the Lrig-1 protein also bind better to Lrig-1 present on the surface of regulatory T cells and regulate the function of regulatory T cells, thereby reducing inflammatory responses and treating autoimmune diseases and neurological diseases.

[0287]

[0288] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. (a) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24; and (b) a binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26; and a light chain CDR2 represented by a selected amino acid sequence represented by SEQ ID NO:

27.

2. In paragraph 1, The above binding molecule is, A heavy chain variable region further comprising a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 43; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 and 79; a light chain CDR2 represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 and 80; and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 47, 51, 55, 59, 63, 67, 71, 75, and 81.

3. In paragraph 1, The above binding molecule is, (a) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and (b) a binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; and a light chain CDR2 encoded by a base sequence represented by SEQ ID NO:

35.

4. In paragraph 1, The above binding molecule is, (a) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 33 and 45; and (b) a binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 34 and 84; a light chain CDR2 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 35 and 85; and a light chain CDR3 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 36, 49, 53, 57, 61, 65, 69, 73, 77, and 86.

5. In paragraph 1, The above binding molecule is, (a) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 28; (b) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 28; (c) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 47; (d) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 51; (e) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 55; (f) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 59; (g) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 63; (h) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 67; (i) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 71; (j) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 75; (k) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 79, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 80, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 81; A binding molecule selected from the group consisting of:

6. In paragraph 1, The above binding molecule is, (a) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 36; (b) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 45; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 36; (c) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 49; (d) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 53; (e) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 57; (f) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a nucleotide sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a nucleotide sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a nucleotide sequence represented by SEQ ID NO: 61; (g) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 65; (h) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 69; (i) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 73; (j) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and A binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 77; (k) a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and A binding molecule selected from the group consisting of a binding molecule comprising a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 84; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 85; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO:

86.

7. In paragraph 1, The above binding molecule is, A heavy chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 44, and 82; and A binding molecule comprising a light chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 48, 52, 56, 60, 64, 68, 72, 76 and 83.

8. In paragraph 1, The above binding molecule is, A heavy chain variable region encoded by any one of the base sequences selected from the group consisting of SEQ ID NOs: 37, 46, and 87; and A binding molecule comprising a light chain variable region encoded by any one of the base sequences selected from the group consisting of SEQ ID NOs: 38, 50, 54, 58, 62, 66, 70, 74, 78 and 88.

9. In paragraph 1, A binding molecule further comprising an Fc region or a constant region.

10. In paragraph 9, The above Fc region is a binding molecule, which is an Fc region of an IgG1, IgG2, IgG3 or IgG4 antibody, or a hybrid Fc region.

11. In paragraph 1, The binding molecule further comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 7, 8, 9, 10 and 12.

12. In paragraph 1, A binding molecule further comprising a light chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 6 and 11.

13. In paragraph 1, The above binding molecule is, A heavy chain constant region encoded by a base sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 17; and A binding molecule further comprising a light chain constant region encoded by a base sequence selected from the group consisting of SEQ ID NOs: 14, 16 and 18.

14. In paragraph 1, The binding molecule is an antibody or an antigen-binding fragment.

15. In paragraph 14, The antibody is a binding molecule that 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.

16. A nucleic acid molecule encoding a binding molecule according to any one of claims 1 to 15.

17. An expression vector into which the nucleic acid molecule of item 16 has been inserted.

18. A host cell line transfected with the expression vector of Article 17.

19. The binding molecule of paragraph 5; and an antibody-drug conjugate comprising a drug.

20. In a chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain, The above antigen-specific binding domain is (a) a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24; and (b) an antigen-specific binding domain comprising a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26; and a light chain CDR2 represented by a selected amino acid sequence represented by SEQ ID NO: 27; A chimeric antigen receptor, which is a fusion protein comprising an antigen-specific binding domain selected from the group consisting of; and an immunoglobulin Fc region.

21. A pharmaceutical composition for preventing or treating cancer, comprising the binding molecule of any one of claims 1 to 20 as an active ingredient.

22. In paragraph 21, A pharmaceutical composition wherein the cancer is a solid cancer.

23. In paragraph 21, A pharmaceutical composition wherein the cancer is gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma or lung cancer.

24. A pharmaceutical composition for preventing or treating a brain nervous system disease, comprising the binding molecule of any one of claims 1 to 20 as an active ingredient.

25. In paragraph 24, A pharmaceutical composition wherein the above-mentioned brain and nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

26. In paragraph 25, A pharmaceutical composition wherein the neurodegenerative disease or neuroinflammatory disease is selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy.

27. A pharmaceutical composition for preventing or treating an immune-related disease, comprising the binding molecule of any one of claims 1 to 20 as an active ingredient.

28. In paragraph 27, A pharmaceutical composition wherein the immune-related disease is an autoimmune disease; graft-versus-host disease; organ transplant rejection; asthma; atopy; or an acute or chronic inflammatory disease.

29. In paragraph 28, A pharmaceutical composition wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa.

30. A method for preventing or treating cancer by administering to a subject a binding molecule of any one of claims 1 to 20.

31. A method for preventing or treating a neurological disease by administering to a subject a binding molecule of any one of claims 1 to 20.

32. A method for preventing or treating an immune-related disease by administering to a subject a binding molecule of any one of claims 1 to 20.

33. A method for preventing or treating cancer, comprising administering to a subject an antibody-drug conjugate comprising the binding molecule of paragraph 5 and a drug.

34. A method for preventing or treating a neurological disease, comprising administering to a subject an antibody-drug conjugate comprising the binding molecule of Article 5 and a drug.

35. A method for preventing or treating an immune-related disease, comprising administering to a subject an antibody-drug conjugate comprising the binding molecule of paragraph 5 and a drug.

Images