Novel binding molecules and uses thereof

Abstract

The present invention relates to a novel binding molecule having excellent preventive, ameliorative or therapeutic effects on cranial nervous system diseases and its use. The binding molecule provided by the present invention can effectively prevent or treat cranial nervous system diseases, particularly neurodegenerative diseases or neuroinflammatory diseases.

Description

[Technical field]

[0001] The present invention relates to a novel binding molecule having excellent prophylactic, ameliorative or therapeutic effects on central nervous system diseases, and uses thereof. [Background technology]

[0002] In degenerative neurological diseases such as stroke, cognition, and Alzheimer's disease, declines in memory, attention, cognitive ability, and emotional regulation are observed, which are the result of neuronal death and atrophy of neural branches. The extension of neural branches of neural cells increases neuroplasticity and plays an important role in the memory and learning functions of neural circuits. Therefore, it is predicted that active ingredients that promote the extension of neural branches of neural cells and nerve regeneration may be developed as new therapeutic agents for degenerative neurological diseases.

[0003] The incidence of degenerative nervous system diseases is increasing with the rapid aging population, and despite the innovative development of medicine, the prevention and treatment methods for degenerative nervous system diseases are still unclear, and no definitively effective drugs have been found. Currently, drugs and treatment methods for degenerative nervous system diseases have been developed, but in many cases side effects and toxicity are observed due to long-term use, and they only have the effect of alleviating symptoms rather than treating the disease, so there is an urgent need to develop materials that can reduce side effects and toxicity and treat the disease. Summary of the Invention [Problem to be solved by the invention]

[0004] One object of the present invention is to provide a binding molecule that has excellent binding ability to Lrig-1 protein present on the surface of regulatory T cells (Treg) and has excellent effects on preventing, improving, or treating brain and nervous system diseases.

[0005] Another object of the invention is to provide a nucleic acid molecule encoding said binding molecule according to the invention.

[0006] It is yet another object of the present invention to provide an expression vector into which the nucleic acid molecule according to the present invention is inserted.

[0007] It is yet another object of the present invention to provide a host cell line transfected with said expression vector according to the present invention.

[0008] It is yet another object of the present invention to provide an antibody-drug conjugate according to the present invention.

[0009] It is still another object of the present invention to provide a composition for preventing, ameliorating or treating a brain and nervous system disease, comprising the binding molecule according to the present invention.

[0010] It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating a brain and nervous system disease, which comprises the antibody-drug conjugate according to the present invention as an active ingredient.

[0011] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating brain and nervous system diseases, comprising the chimeric antigen receptor (CAR) according to the present invention as an active ingredient.

[0012] A further object of the present invention is to provide a diagnostic composition and diagnostic kit for central nervous system diseases, which contain the binding molecule of the present invention, and a method for providing information for diagnosing central nervous system diseases.

[0013] It is yet another object of the present invention to provide a method for preventing or treating a brain and nervous system disease, which comprises using the binding molecule, antibody or fragment thereof, antibody-drug conjugate (ADC), or chimeric antigen receptor (CAR) according to the present invention.

[0014] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those having ordinary skill in the art from the following description. [Means for solving the problem]

[0015] According to one embodiment of the present invention, the present invention relates to a binding molecule that specifically binds to the leucine-rich and immunoglobulin-like domains 1 (Lrig-1) protein present in regulatory T cells (Treg cells).

[0016] As used herein, a "binding molecule" refers to an intact immunoglobulin, including a monoclonal antibody, such as a chimeric, humanized or human monoclonal antibody, or an immunoglobulin that binds an antigen, e.g., a variable domain containing immunoglobulin fragment that competes with the intact immunoglobulin for binding to monomeric or trimeric HA of influenza A virus. Regardless of structure, the antigen-binding fragment binds to the same antigen recognized by the intact immunoglobulin. Antigen-binding fragments can include peptides or polypeptides that include an amino acid sequence of two or more contiguous groups of the amino acid sequence of the binding molecule, 20 or more contiguous amino acid residues, 25 or more contiguous amino acid residues, 30 or more contiguous amino acid residues, 35 or more contiguous amino acid residues, 40 or more contiguous amino acid residues, 50 or more contiguous amino acid residues, 60 or more contiguous amino acid residues, 70 or more contiguous amino acid residues, 80 or more contiguous amino acid residues, 90 or more contiguous amino acid residues, 100 or more contiguous amino acid residues, 125 or more contiguous amino acid residues, 150 or more contiguous amino acid residues, 175 or more contiguous amino acid residues, 200 or more contiguous amino acid residues, or 250 or more contiguous amino acid residues. "Antigen-binding fragments" include, inter alia, 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 that contain one or more fragments of an immunoglobulin sufficient to bind a specific antigen to a polypeptide. The fragments may be produced synthetically or by enzymatic or chemical degradation of intact immunoglobulins, or may be produced genetically by recombinant DNA technology. Methods of production are well known in the art.

[0017] 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 an extracellular or lumenal leucine repeat (LRR), three immunoglobulin-like domains, a transmembrane sequence, and a cytoplasmic tail. The LRIG gene family includes LRIG1, LRIG2, and LRIG3, and the amino acids between them are highly conserved. The LRIG1 gene is highly expressed in normal skin, and is expressed in basal and hair follicle cells to regulate the proliferation of epithelial stem cells. Therefore, it plays an important role in maintaining epidermal homeostasis, and its absence may lead to psoriasis or skin cancer. It has been reported that when the chromosome 3p14.3 region where LRIG1 is located is broken, it may develop into cancer cells, and in fact, it has been confirmed that the expression of LRIG1 is significantly reduced in renal cell carcinoma and cutaneous squamous cell carcinoma. However, it has been recently found that only about 20 to 30% of cancers express Lrig-1. Meanwhile, for the purposes of the present invention, the Lrig-1 protein may be a protein present in humans or mice, but is not limited thereto.

[0018] In the present invention, the Lrig-1 protein may be a polypeptide represented by SEQ ID NO: 1 derived from humans, or a polypeptide represented by SEQ ID NO: 3 derived from mice, but is not limited thereto.

[0019] Furthermore, in the present invention, the Lrig-1 protein represented by SEQ ID NO: 1 may be encoded by a polynucleotide represented by SEQ ID NO: 2, but is not limited thereto.

[0020] Furthermore, in the present invention, the Lrig-1 protein represented by SEQ ID NO: 3 may be encoded by a polynucleotide represented by SEQ ID NO: 4, but is not limited thereto.

[0021] In the present invention, the binding molecule is

[0022] A heavy chain variable region comprising: a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and

[0023] The binding molecule may comprise a light chain variable region including: a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 8; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 9; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 10.

[0024] In the present invention, the binding molecule comprises a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 11; and

[0025] The binding molecule may comprise a light chain variable region consisting of the amino acid sequence shown in SEQ ID NO:12.

[0026] In the present invention, the binding molecule is

[0027] A heavy chain variable region comprising: a heavy chain CDR1 encoded by the base sequence represented by SEQ ID NO: 13; a heavy chain CDR2 encoded by the base sequence represented by SEQ ID NO: 14; and a heavy chain CDR3 encoded by the base sequence represented by SEQ ID NO: 15; and

[0028] The binding molecule may comprise a light chain variable region including: a light chain CDR1 encoded by the base sequence represented by SEQ ID NO: 16; a light chain CDR2 encoded by the base sequence represented by SEQ ID NO: 17; and a light chain CDR3 encoded by the base sequence represented by SEQ ID NO: 18.

[0029] In the present invention, the binding molecule comprises a heavy chain variable region encoded by the base sequence represented by SEQ ID NO: 19; and

[0030] The binding molecule may comprise a light chain variable region encoded by the base sequence shown in SEQ ID NO:20.

[0031] In the present invention, the binding molecule may further comprise an Fc region (fragment crystallization region) or a constant region, which may be or be derived from an Fc region of an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 antibody, or may be a hybrid Fc region.

[0032] In the present invention, the Fc region may be an Fc region of a mammalian-derived IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody, and preferably an Fc region of a human-derived IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3, or IgG4 antibody, but is not limited thereto.

[0033] As an example of the present invention, the constant region may be a mouse-derived IgG2a constant region represented by SEQ ID NO: 21, but is not limited thereto.

[0034] As an example of the present invention, the constant region may be, but is not limited to, a mouse-derived immunoglobulin kappa constant region represented by SEQ ID NO: 22.

[0035] As an example of the present invention, the constant region may be a human-derived IgG1 constant region represented by SEQ ID NO: 23 or 24, but is not limited thereto.

[0036] As an example of the present invention, the constant region may be a human-derived immunoglobulin kappa constant region represented by SEQ ID NO: 25 or 26, but is not limited thereto.

[0037] As an example of the present invention, the constant region may be a human-derived IgG2 constant region represented by SEQ ID NO: 27, but is not limited thereto.

[0038] As an example of the present invention, the constant region may be a human-derived IgG3 constant region represented by SEQ ID NO: 28, but is not limited thereto.

[0039] As an example of the present invention, the constant region may be a human-derived IgG4 constant region represented by SEQ ID NO: 29, but is not limited thereto.

[0040] In one embodiment of the present invention, the constant region may be, but is not limited to, the constant region represented by SEQ ID NO: 30.

[0041] In one embodiment of the present invention, the constant region may be the constant region represented by SEQ ID NO: 31, but is not limited thereto.

[0042] As an example of the present invention, the Fc region may be, but is not limited to, a human-derived immunoglobulin lambda constant region.

[0043] In the present invention, the "hybrid Fc" is derived from a combination of human IgG subclasses or a combination of human IgD and IgG. When the hybrid Fc is bound to a biologically active molecule, such as a polypeptide, 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.

[0044] As an example of the present invention, the hybrid Fc region may be a hybrid Fc region represented by SEQ ID NO: 32, but is not limited thereto.

[0045] In the binding molecule of the present invention, the Fc or constant region may be linked to the variable region via a linker, in which the linker is linked to the C-terminus of the Fc or constant region, and the N-terminus of the binding molecule of the present invention is linked to the linker, but is not limited thereto.

[0046] In the present invention, the "linker" may include a sequence that can be cleaved by an enzyme that is overexpressed in tissues or cells of a target disease. When the linker is cleavable by an enzyme that is overexpressed as described above, it is possible to effectively prevent the activity of the polypeptide from being reduced by the Fc or invariant region. In the present invention, a preferred example of the linker may be a peptide linker consisting of 33 amino acids located at the 282nd to 314th positions of human albumin, which is the most abundant in blood, and more preferably a peptide linker consisting of 13 amino acids located at the 292nd to 304th positions, and such a portion is a portion that is mostly exposed to the outside in a three-dimensional structure, and is a portion that minimizes the possibility of inducing an immune response in the body. However, the linker is not limited thereto.

[0047] A binding molecule of the invention can further comprise a heavy chain constant region represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 23, 24, 27, 28, 29, 30 and 32.

[0048] A binding molecule of the invention can further comprise a light chain constant region represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 25, 26 and 31.

[0049] The binding molecule of the invention comprises A heavy chain constant region consisting of the amino acid sequence represented by SEQ ID NO: 21; and It may further comprise a light chain constant region consisting of the amino acid sequence shown in SEQ ID NO:22.

[0050] The binding molecule of the invention comprises A heavy chain constant region represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 24, 27, 28, 29 and 30; and It may further comprise a light chain constant region represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 25, 26 and 31.

[0051] The binding molecule of the invention comprises A heavy chain constant region consisting of the amino acid sequence represented by SEQ ID NO: 24; and It may further comprise a light chain constant region consisting of the amino acid sequence shown in SEQ ID NO:26.

[0052] The binding molecule of the invention comprises A heavy chain constant region consisting of the amino acid sequence represented by SEQ ID NO: 30; and It may further comprise a light chain constant region consisting of the amino acid sequence shown in SEQ ID NO:31.

[0053] The binding molecule of the invention comprises It may further comprise a heavy chain constant region consisting of the amino acid sequence shown in SEQ ID NO:32.

[0054] The binding molecule of the invention comprises A heavy chain constant region encoded by the base sequence represented by SEQ ID NO: 33 or 35; and It may further comprise a light chain constant region encoded by the base sequence represented by SEQ ID NO:34 or 36.

[0055] The binding molecule of the present invention may be a binding molecule comprising a heavy chain consisting of the amino acid sequence shown in SEQ ID NO:37 or 38, and a light chain consisting of the amino acid sequence shown in SEQ ID NO:39 or 40.

[0056] The binding molecule of the present invention may be a binding molecule comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO:37 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:39.

[0057] The binding molecule of the present invention may be a binding molecule comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO:38 and a light chain consisting of the amino acid sequence represented by SEQ ID NO:40.

[0058] The binding molecule of the present invention may be a binding molecule comprising a heavy chain encoded by the base sequence shown in SEQ ID NO:41 or 43, and a light chain encoded by the base sequence shown in SEQ ID NO:42 or 44.

[0059] The binding molecule of the present invention may be a binding molecule comprising a heavy chain encoded by the base sequence shown in SEQ ID NO:41 and a light chain encoded by the base sequence shown in SEQ ID NO:42.

[0060] The binding molecule of the present invention may be a binding molecule comprising a heavy chain encoded by the base sequence shown in SEQ ID NO:43 and a light chain encoded by the base sequence shown in SEQ ID NO:44.

[0061] The binding molecule of the present invention is characterized in that it is an antibody or a fragment thereof, but is not limited thereto. The antibody includes all of the antibody fragments that have the ability to bind to the Lrig-1 protein as a monoclonal antibody, a full-length antibody, or a portion of an antibody and can bind to the Lrig-1 antigenic determinant site competitively with the binding molecule of the present invention.

[0062] In the present invention, the "antibody" refers to a protein molecule that plays the role of a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that is immunologically reactive with a specific antigen. For the purposes of the present invention, the antigen may be the Lrig-1 protein present on the surface of regulatory T cells. Preferably, the antibody may specifically recognize the leucine rich region or immunoglobulin-like domain of the Lrig-1 protein, but is not limited thereto.

[0063] In the present invention, the "immunoglobulin" has a heavy chain and a light chain, each of which contains a constant region and a variable region. The variable regions of the light chain and the heavy chain contain three 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 in which the particular CDR is located.

[0064] In addition, in the present invention, the "monoclonal antibody" is a term referring to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and exhibits a single binding specificity and affinity for a specific antigenic determinant (epitope).

[0065] In the present invention, the "full-length antibody" has a structure having two full-length light chains and two full-length heavy chains, each of which is linked to a heavy chain by a disulfide bond, and includes IgA, IgD, IgE, IgM, and IgG. The IgG subtypes include IgG1, IgG2, IgG3, and IgG4.

[0066] In the present invention, the "antibody fragment" refers to a fragment having an antigen-binding function, and includes Fab, Fab', F(ab')2, and Fv. The Fab has a structure including a light chain and a heavy chain variable region, a light chain constant region, and the first constant region (CH1 domain) of the heavy chain, and has one antigen-binding site. 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. The F(ab')2 antibody is generated by disulfide bonds between the cysteine ​​residues in the hinge region of Fab'. Fv (variable fragment) refers to the smallest antibody fragment having only a heavy chain variable region and a light chain variable region. In a double-chain Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond, and in a single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are generally linked by a covalent bond via a peptide linker. The antibody fragment can be produced by recombinant gene technology, such as when proteolytic enzymes, such as papain or pepsin, are used to obtain Fab or F(ab')2 fragments.

[0067] 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 mimic, a unibody, a diabody, a triabody, a tetrabody, or a fragment thereof. In the present invention, the "chimeric antibody" is an antibody obtained by recombining the variable region of a mouse antibody and the constant region of a human antibody, and has a greatly improved immune response compared to a mouse antibody.

[0068] In the present invention, the "humanized antibody" refers to an antibody in which the protein sequence of an antibody derived from a non-human species has been modified to resemble an antibody mutant naturally produced by humans. For example, the humanized antibody can be produced by recombining mouse-derived CDRs with human antibody-derived FRs to produce a humanized variable region, which can then be recombined with a preferred human antibody constant region to produce a humanized antibody.

[0069] In the present invention, the binding molecule can also be provided as a bispecific antibody or bispecific antigen-binding fragment capable of binding to the Lrig-1 protein and also to other proteins.

[0070] In the present invention, the bispecific antibodies and bispecific antigen-binding fragments may comprise a binding molecule according to the present invention. In one example, in the present invention, the bispecific antibodies and bispecific antigen-binding fragments comprise an antigen-binding domain capable of binding to the Lrig-1 protein, where the antigen-binding domain capable of binding to the Lrig-1 protein may comprise or be constituted by a binding molecule according to the present invention.

[0071] The bispecific antibodies and bispecific antigen-binding fragments provided herein comprise an antigen-binding domain that is a binding molecule capable of binding to the Lrig-1 protein according to the present invention, and an antigen-binding domain that is capable of binding to another target protein, where the antigen-binding domain that is capable of binding to another target protein may be, but is not limited to, an antigen-binding domain that is capable of binding to a protein other than the Lrig-1 protein, for example, but is not limited to, PD-1 or a cell surface receptor.

[0072] Bispecific antibodies and bispecific antigen-binding fragments according to the invention can be provided in any suitable format. For example, the bispecific antibodies or bispecific antigen-binding fragments can be provided as bispecific antibody conjugates (e.g. IgG2, F(ab')2 or CovX-bodies), bispecific IgG or IgG-type molecules (e.g. IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, or 2in1-IgG, mAb2, or Tandemab common LC), asymmetric bispecific IgG or IgG-type molecules (e.g. kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair or SEED-body), small bispecific antibody molecules (e.g. diabodies (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, di-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). A person skilled in the art can design and produce bispecific antibodies and bispecific antigen-binding fragments according to the invention.

[0073] In the present invention, the method for producing the bispecific antibodies includes chemical cross-linking of antibodies or antibody fragments with reducible disulfide or non-reducible thioether bonds. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically cross-link Fab fragments, for example, through hinge region SH-groups, to generate disulfide-linked bispecific F(ab)2 heterodimers.

[0074] Also in accordance with the present invention, another method for producing the bispecific antibody includes fusing an antibody-producing hybridoma with, for example, polyethylene glycol, to generate a quadroma cell capable of secreting the bispecific antibody.

[0075] Bispecific antibodies and bispecific antigen-binding fragments according to the invention can further be produced recombinantly, for example, by expression from nucleic acid constructs encoding the polypeptides for the antigen-binding molecules.

[0076] For example, a DNA construct encoding light and heavy chain variable domains for two antigen binding domains (i.e., light and heavy chain variable domains for an antigen binding domain capable of binding, e.g., PD-1, and light and heavy chain variable domains for an antigen binding domain capable of binding to another target protein), and comprising sequences encoding suitable linkers or dimerization domains between the antigen binding domains, can be produced by molecular cloning techniques. Recombinant bispecific antibodies can then be produced by expression (e.g., in vitro) of the construct in a suitable host cell (e.g., a mammalian host cell), and the expressed recombinant bispecific antibodies can then, optionally, be purified.

[0077] Antibodies can be generated by an affinity maturation process that produces modified antibodies that have improved affinity for the antigen compared to the unmodified parent antibody. Affinity matured antibodies can be produced by procedures known in the art.

[0078] In addition, the binding molecule provided by the present invention may include variants of the amino acid sequence as long as it can specifically bind to the Lrig-1 protein. For example, the amino acid sequence of the antibody may be altered to improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion and / or substitution of residues in the amino acid sequence of the antibody.

[0079] Such amino acid mutations are made based on the relative similarity of the amino acid side chain substitutes, e.g., hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substitutes reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are biologically functional equivalents.

[0080] When making mutations, the hydropathic index of amino acids is considered. Each amino acid is assigned a hydrophobic index according to its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+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 hydropathic amino acid index is very important in conferring interactive biological functions of proteins. It is a well-known fact that only by substituting amino acids with similar hydrophobicity indexes can similar biological activities be retained. When introducing mutations with reference to the hydrophobicity index, substitutions are made between amino acids showing a hydrophobicity index difference of preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0081] On the other hand, it is also well known that substitutions between amino acids with similar hydrophilicity values ​​result in proteins with equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). When introducing a mutation with reference to the hydrophilicity value, substitutions can be made between amino acids showing a difference in hydrophilicity value within preferably ±2, more preferably within ±1, and even more preferably within ±0.5.

[0082] Amino acid exchanges in proteins that do not globally alter the activity of the molecule are known in the art. The most commonly occurring exchanges are between the 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, Gln / Glu.

[0083] In view of the above mentioned biologically equivalent variants, the binding molecules of the present invention are also intended to include sequences that show substantial identity to the sequences set out in the sequence listing.

[0084] In the present invention, the term "substantial identity" refers to a sequence that shows at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology when the sequence of the present invention is aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using algorithms commonly used in the art. Alignment methods for sequence comparison are known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is accessible at NBCI (National Center for Biological Information) and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. BLSAT can be accessed at this link (http: / / www.ncbi.nlm.nih.gov / BLAST / ). A method for comparing sequence homology using this program can be confirmed online (http: / / www.ncbi.nlm.nih.gov / BLAST / blast_help.html).

[0085] In the present invention, the binding molecule, preferably the antibody, can be produced by conventional methods for producing antibodies, but can also be produced by affinity maturation.

[0086] In the present invention, the term "affinity maturation" refers to a process in which activated B cells produce antibodies with increased affinity to an antigen during an immune response. For the purposes of the present invention, affinity maturation can be based on the principles of mutation and selection, as in the natural process, to produce antibodies or antibody fragments produced by affinity maturation.

[0087] The binding molecules, preferably antibodies, provided by the present invention can effectively prevent, ameliorate or treat brain and nervous system diseases, in particular neurodegenerative diseases or neuroinflammatory diseases.

[0088] According to another embodiment of the invention there is provided a nucleic acid molecule encoding the binding molecule provided herein.

[0089] The nucleic acid molecules of the present invention include all nucleic acid molecules in which the amino acid sequence of the binding molecule provided by the present invention has been translated into a polynucleotide sequence, as known to those skilled in the art. Therefore, various polynucleotide sequences can be produced by ORF (open reading frame), and all of these are included in the nucleic acid molecules of the present invention.

[0090] The nucleic acid molecule of the present invention can comprise a heavy chain encoded by the base sequence represented by SEQ ID NO:41 or 43, and a light chain encoded by the base sequence represented by SEQ ID NO:42 or 44.

[0091] The nucleic acid molecule of the present invention can comprise a heavy chain encoded by the base sequence shown in SEQ ID NO:41 and a light chain encoded by the base sequence shown in SEQ ID NO:42.

[0092] The nucleic acid molecule of the present invention can comprise a heavy chain encoded by the base sequence represented by SEQ ID NO:43 and a light chain encoded by the base sequence represented by SEQ ID NO:44.

[0093] In yet another embodiment of the present invention, there is provided an expression vector into which the isolated nucleic acid molecule provided by the present invention is inserted.

[0094] In the present invention, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA to which additional DNA segments can be ligated. Another type of vector is a phage vector. Yet another type of vector is a viral vector, to which additional DNA segments can be ligated 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) are integrated into the genome of the host cell upon introduction into the host cell, and are thereby replicated along with the host genome. Moreover, some vectors are capable of directing the expression of genes to which they are linked at an operational level. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector.

[0095] In the present invention, specific examples of the expression vector may be selected from the group consisting of pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors, which are widely used commercially; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμμ, T-even, T2, T3, and T7; and plant viruses, but are not limited thereto. All expression vectors known to those skilled in the art as expression vectors can be used in the present invention, and the selection of an expression vector depends on the properties of the intended host cell. When a vector is introduced into a host cell, it is carried out by calcium phosphate transfection, virus infection, DEAE-dextran regulated transfection, lipofectamine transfection, or electroporation, but is not limited thereto, and those skilled in the art can select and use an introduction method suitable for the expression vector and host cell used. Preferably, the vector contains one or more selection markers, but is not limited thereto, and a vector without a selection marker can be used to select whether or not it produces a product. The selection of the selectable marker is determined by the intended host cell, and this is done using methods already known to those skilled in the art, and therefore the present invention is not limited thereto.

[0096] The nucleic acid molecule of the present invention can be fused to an expression vector by inserting a tag sequence to facilitate purification. The tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag known to those skilled in the art to facilitate purification can be used in the present invention.

[0097] According to yet another embodiment of the present invention, there is provided a host cell line transformed with an expression vector provided herein.

[0098] In the present invention, the "host cell" includes an individual cell or cell culture that can be or has been a recipient of a vector for incorporation of a polypeptide insert. A host cell includes the progeny of a single host cell, which may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental or deliberate mutations. A host cell includes a cell that has been transfected in vivo with a polypeptide of the present application.

[0099] In the present invention, the host cell may include cells of mammalian, plant, insect, fungal or cellular origin, for example, bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; fungal cells such as yeast cells and Pichia pastoris, etc.; insect cells such as Drosophila and Spodoptera Sf9 cells, etc.; animal cells such as CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, Bowes melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), or PERC.6 (human retinal cells); or plant cells, but are not limited thereto, and any cells that can be used as a host cell line known to those skilled in the art can be used.

[0100] According to yet another embodiment of the present invention, there is provided an antibody-drug conjugate (ADC) comprising an antibody provided by the present invention and a drug.

[0101] 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 the α-amine group at the N-terminus of the heavy chain and / or light chain of an antibody.

[0102] In the present invention, examples of reactive groups capable of reacting with the α-amine group to crosslink include, but are not limited to, all types known in the art that react with amine groups, as long as they can react with the α-amine group at the N-terminus of the heavy or light chain of an antibody to crosslink. Examples of reactive groups include, but are not limited to, any one of isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters.

[0103] In the present invention, the "drug" may refer to any substance having a specific biological activity in cells, including DNA, RNA, or peptide. The drug may be in a form containing a reactive group capable of reacting with an α-amine group to crosslink, or may be in a form linked to a linker containing a reactive group capable of reacting with an α-amine group to crosslink.

[0104] According to yet another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating a brain and nervous system disease, comprising as an active ingredient a chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain.

[0105] In the present invention, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor that includes an extracellular antigen-binding domain and an intracellular signaling domain. Although the most common type of CAR includes a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the transmembrane and intracellular domains of a T-cell co-receptor such as the CD3 zeta (ζ) chain, the invention described herein is not limited to these domains. Rather, as used herein, "chimeric antigen receptor" or "CAR" refers to any receptor engineered to express any intracellular signaling molecule and an extracellular antigen-binding domain fused or linked thereto. In the present invention, the binding domain can include a single-chain variable fragment (scFv) that can specifically recognize the Lrig-1 protein. In the present invention, the "single-chain variable fragment" or "scFv" refers to a fusion protein of the variable heavy chain (VH) and variable light chain (VL) of an antibody with a peptide linker between the VL and VH.

[0106] In addition, in the present invention, the VH domain and the VL domain can be linked 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), preferably 15 amino acids in length. In the present invention, the linker length can act as an important determining site of the chimeric antigen receptor, and a linker shorter than the above range can increase affinity, but may also cause the formation of intracellular multimers and impair the expression of CAR, while a linker longer than the above range may reduce antigen affinity by moving the VL and VH CDRs farther in space.

[0107] 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, which is also called "spacer" and is a part that connects the antigen binding domain and the transmembrane domain, has the purpose of extending the antigen binding domain from the T cell membrane or the NK cell membrane. In the present invention, the hinge region may be obtained from any suitable sequence from any genus, including human or a part thereof, or may include, but is not limited to, hinge regions of human proteins, including CD8, CD28, 4-1BB, OX40, all or a part of the CD3 zeta (ζ) chain, T cell receptor α or β chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof, which are commonly used in the art. Furthermore, in the present invention, the hinge region is not limited to immunoglobulins and may include one selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD), but is not limited thereto.

[0108] In the present invention, the signaling domain refers to a portion of a chimeric antigen receptor that is found or engineered to be found inside a T cell. In the present invention, the signaling domain may or may not include a transmembrane domain that serves to anchor the chimeric antigen receptor at the plasma membrane of a T cell. In the present invention, the transmembrane domain and the signaling domain can be derived from the same protein (e.g., the CD3 zeta (ζ) molecule), or the transmembrane domain and the signaling domain can be derived from different proteins (e.g., the transmembrane domain of CD28 and the intracellular signaling domain of the CD3 zeta (ζ) molecule, or vice versa).

[0109] In the present invention, the membrane-transducing domain may include, for example, a T cell receptor α or β chain, all or a part of the CD3 zeta (ζ) chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof, but is not limited thereto. In the present invention, the costimulatory domain can include, but is not limited to, a functional signaling domain derived from a polypeptide comprising 4-1BB (CD137); OX40; CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; natural killer group 2 member C (NKG2C); natural killer group 2 member (NKG2D); B7-H3; CD83; ICAM-1; a ligand that binds to LFA-1 (CD11a / CD18) or ICOS; active fragments thereof; functional derivatives thereof; or combinations thereof.

[0110] In the present invention, the signaling domain can include, but is not limited to, a functional signaling domain derived from a polypeptide including all or a portion of CD3 zeta (ζ), common FcR gamma (FcER1G), Fc gamma RIIIa, FcR beta (Fc epsilon lip), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX-activation protein 10 (DAP10), DNAX-activation protein 12 (DAP12), active fragments thereof, functional derivatives thereof, or combinations thereof, and such signaling domains are known in the art.

[0111] According to one specific example of the present invention, there is provided a chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain and a CD3 zeta (ζ) signaling domain, wherein the specific binding domain is a heavy chain variable region comprising: a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and a light chain variable region comprising: a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10.

[0112] In the present invention, the drug includes any drug capable of treating a brain and nervous system disease, particularly a neurodegenerative disease or a neuroinflammatory disease, regardless of its type.

[0113] According to yet another embodiment of the present invention, the present invention relates to a composition for preventing, ameliorating or treating a brain and nervous system disease, comprising as an active ingredient a binding molecule; a nucleic acid molecule; an expression vector; a host cell line; or an antibody-drug conjugate (ADC) of the present invention.

[0114] The cranial nervous system disease to be prevented, ameliorated or treated by the composition provided by the present invention may be a neurodegenerative disease or a neuroinflammatory disease.

[0115] In the present invention, the "neurodegenerative disease" may mean a disease caused by a decrease or loss of function of nerve cells, and the "neuroinflammatory disease" may mean a disease caused by an excessive inflammatory response in the nervous system. In the present invention, specific examples of the neurodegenerative disease or neuroinflammatory disease include stroke, cognitive impairment, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and the like. The present invention may be selected from the group consisting of, but not limited to, tauopathy, pulmonary arterial disease, and tauopathy.

[0116] Furthermore, the composition provided by the present invention can be used as a pharmaceutical composition or a food composition, but is not limited thereto.

[0117] The "prevention" of the present invention includes, without limitation, all actions that can block, suppress or delay symptoms caused by a neurodegenerative disease or a neuroinflammatory disease using the composition of the present invention.

[0118] The "treatment" and "improvement" of the present invention include, without limitation, any action of improving or favoring symptoms caused by a neurodegenerative disease or a neuroinflammatory disease using the composition of the present invention.

[0119] In the present invention, the pharmaceutical composition is characterized in that it is in the form of a capsule, tablet, granule, injection, ointment, powder or drink, and that the pharmaceutical composition is intended for humans.

[0120] The pharmaceutical composition of the present invention can be formulated into oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions by a conventional method, but is not limited thereto. The pharmaceutical composition of the present invention can contain a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier may be a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a dye, a flavoring, etc., when administered orally, and may be mixed with a buffer, a preservative, a soothing agent, a solubilizer, an isotonic agent, a stabilizer, etc., when administered locally, and may be a base, an excipient, a lubricant, a preservative, etc., when administered locally. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing with the above-mentioned pharmaceutically acceptable carrier. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, it can be prepared in unit dose ampoules or multiple dose forms. In addition, it can be formulated into solutions, suspensions, tablets, capsules, sustained release preparations, etc.

[0121] On the other hand, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil, etc. In addition, fillers, anti-agglomerating agents, lubricants, wetting agents, flavors, emulsifiers, preservatives, etc. may be additionally included.

[0122] The routes of administration of the pharmaceutical compositions according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or rectal. Oral or parenteral administration is preferred.

[0123] In the present invention, "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional and intracranial injection or infusion techniques. The pharmaceutical compositions of the present invention can also be administered in the form of suppositories for rectal administration.

[0124] 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 formulation, and the severity of the specific disease to be prevented or treated. The dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, and can be appropriately selected by those skilled in the art, and can be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. The administration may be once or divided into several times a day. The dosage does not limit the scope of the present invention in any aspect. The pharmaceutical composition according to the present invention can be formulated into pills, sugar-coated tablets, capsules, liquids, gels, syrups, slurries, and suspensions.

[0125] The food composition containing the composition of the present invention as an active ingredient can be manufactured into various food forms, such as beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, sweets, rice cakes, bread, etc. The food composition of the present invention is composed of plant extracts that have almost no toxicity or side effects, so it can be safely taken for prophylactic purposes over a long period of time.

[0126] When the composition of the present invention is included in a food composition, the amount of the composition added may be 0.1 to 50% of the total weight.

[0127] Here, when the food composition is manufactured in the form of a beverage, there is no particular limitation other than containing the food composition in the indicated ratio, and various flavorings or natural carbohydrates, etc., can be contained as additional ingredients, like ordinary beverages. That is, natural carbohydrates can include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, ordinary sugars such as dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, erythritol, etc. Examples of the flavorings include natural flavorings (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.), and synthetic flavorings (saccharin, aspartame, etc.).

[0128] In addition, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavorings such as synthetic flavors and natural flavors, colorants, pectinic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonation agents used in carbonated beverages, and the like.

[0129] Such components can be used independently or in combination. The ratio of such additives is not critical, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

[0130] According to yet another embodiment of the present invention, there is provided a method for preventing, ameliorating or treating a brain and nervous system disease, comprising the step of administering to an individual in need thereof a binding molecule provided by the present invention; a nucleic acid molecule encoding the binding molecule; an expression vector into which the nucleic acid molecule has been inserted; a host cell line transfected with the expression vector; or an antibody-drug conjugate (ADC) provided by the present invention.

[0131] In the present invention, the "individual" refers to an individual suspected of developing a cranial nervous system disease, and the individual suspected of developing the disease refers to mammals including mice, including humans, that have developed or may develop the disease, and livestock, but includes, without limitation, individuals that can be treated with the effective substance provided by the present invention.

[0132] The cranial nervous system disease to be treated by the method of the present invention may be a neurodegenerative disease or a neuroinflammatory disease, and specific examples thereof include stroke, cognitive disorders, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and the like. The present invention may be selected from the group consisting of, but not limited to, tauopathy, pulmonary arterial disease, and tauopathy.

[0133] The methods of the invention may comprise administering a pharma- tically effective amount of a binding molecule provided by the invention; a nucleic acid molecule encoding the binding molecule; an expression vector into which the nucleic acid molecule has been inserted; a host cell line transfected with the expression vector; or an antibody-drug conjugate (ADC) provided by the invention.

[0134] The total amount of the suitable daily use can be determined by the treating physician within the scope of sound medical judgment, and can be administered in one or several doses. However, for the purposes of the present invention, the specific therapeutically effective amount for a particular patient is preferably applied differently depending on various factors including the type and degree of response to be achieved, the specific composition containing the active ingredient, including whether other formulations are used, the age, weight, general health condition, sex and diet of the patient, the administration time, administration route and secretion rate of the composition containing the active ingredient, the duration of treatment, drugs used together with the specific composition or used simultaneously, and similar factors well known in the pharmaceutical art.

[0135] On the other hand, the method for preventing or treating a central nervous system disease may be, but is not limited to, a combination therapy that further includes administering a compound or substance having therapeutic activity against one or more diseases.

[0136] In the present invention, the term "combination" should be understood to indicate simultaneous, separate or sequential administration. If the administration is sequential or separate, the interval between administrations of the second components should be such that the beneficial effect of the combination is not lost.

[0137] In the present invention, the administration dose of the binding molecule or antibody-drug conjugate may be about 0.0001 μg to 500 mg per kg of patient body weight, but is not limited thereto.

[0138] According to yet another embodiment of the present invention, there is provided a diagnostic composition for immune-related diseases, comprising a preparation that uses the binding molecule to measure the expression level of Lrig-1 protein or its extracellular domain present on the surface of T cells; or a gene encoding the same.

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

[0140] The diagnostic composition of the present invention may comprise, but is not limited to, a formulation for measuring the expression level of a regulatory T cell, for example, an Lrig-1 protein or its extracellular domain present on the surface of an activated regulatory T cell, i.e., a regulatory T cell whose inhibitory ability on effector T cells has been activated; or a gene encoding the same.

[0141] The diagnostic composition of the present invention may further comprise a preparation for measuring the expression level of one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16 and Lrig-1 protein present on the surface of T cells; or the gene encoding the same. In this way, when the expression levels of various proteins present on the surface of T cells; or the gene encoding the same are further measured, a significant synergistic effect can be exhibited in diagnosing the target disease, compared to the case where the Lrig-1 protein or its extracellular domain; or the gene encoding the same is measured alone.

[0142] The formulation for measuring the expression level of the Lrig-1 protein or its extracellular domain of the present invention, and the expression level of one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16 and Lrig-1 protein is not particularly limited, and may include, for example, one or more proteins selected from the group consisting of antibodies, oligopeptides, ligands, peptide nucleic acids (PNAs) and aptamers that specifically bind to the proteins.

[0143] A preparation for measuring the expression level of a gene encoding the Lrig-1 protein of the present invention (i.e., LAIR1) or a gene encoding the extracellular domain of the Lrig-1 protein, and a gene encoding one or more proteins selected from the group consisting of CCD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16 and the Lrig-1 protein, may contain one or more types selected from the group consisting of primers, probes, LNA and antisense nucleotides that specifically bind to the genes.

[0144] The "primer" of the present invention is a fragment that recognizes a target gene sequence and includes forward and reverse primer pairs, but is preferably a primer pair that provides analysis results with specificity and sensitivity. Since the nucleic acid sequence of the primer is a sequence that does not match the non-target sequence present in the sample, only the target gene sequence containing the complementary primer binding site is amplified, and high specificity can be obtained when the primer does not induce non-specific amplification.

[0145] The "probe" of the present invention means a substance capable of specifically binding to a target substance to be detected in a sample, and a substance capable of specifically confirming the presence of the target substance in a sample by the binding. The type of probe is not limited to a substance commonly used in the art, but may be preferably PNA (peptide nucleic acid), LNA (locked nucleic acid), peptide, polypeptide, protein, RNA or DNA, and most preferably PNA. More specifically, the probe may be derived from an organism as a biological material, or may be similar to the organism or may be produced in vitro, and may be, for example, an enzyme, a protein, an antibody, a microorganism, an animal or plant cell and organ, a nerve cell, DNA, and RNA, and DNA may include cDNA, genomic DNA, and oligonucleotide, RNA may include genomic RNA, mRNA, and oligonucleotide, and examples of proteins may include antibodies, antigens, enzymes, peptides, etc.

[0146] A further embodiment of the present invention relates to a diagnostic kit for brain and nervous system diseases, comprising the binding molecule provided by the present invention.

[0147] The kit of the present invention may be, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0148] The kits of the invention may further comprise one or more other component compositions, solutions or devices suitable for the analytical method.

[0149] The kit of the present invention may further include essential elements required for performing a reverse transcription polymerase reaction, for example. The reverse transcription polymerase reaction kit includes a primer pair specific to a gene encoding a marker protein. The primer pair is a nucleotide having a sequence specific to the nucleic acid sequence of the gene, and may have a length of, for example, about 7 bp to 50 bp, or about 10 bp to 30 bp. It may also include a primer pair specific to the nucleic acid sequence of a control gene. Other reverse transcription polymerase reaction kits may further include a test tube or other suitable container, a reaction buffer (varies in pH and magnesium concentration), deoxynucleotides (dNTPs), enzymes such as Taq-polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC water, sterile water, etc.

[0150] The kit of the present invention can include essential elements required for performing DNA chips. The DNA chip kit can include a substrate to which cDNA or oligonucleotides corresponding to genes or fragments thereof are attached, and reagents, preparations, enzymes, etc. for producing fluorescently labeled probes. The substrate can also include cDNA or oligonucleotides corresponding to control genes or fragments thereof.

[0151] The kit of the present invention may include essential elements required for performing ELISA. The ELISA kit includes an antibody specific to the protein. The antibody is an antibody that has high specificity and affinity for the marker protein and has little cross-reactivity with other proteins, and may be a monoclonal antibody, a polyclonal antibody, or a recombinant antibody. The ELISA kit may also include an antibody specific to a control protein. Other ELISA kits may include reagents capable of detecting the bound antibody, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to the antibody) and their substrates or other substances capable of binding to the antibody.

[0152] According to yet another embodiment of the present invention, the present invention relates to a method for providing information for diagnosing immune-related diseases, comprising a step of measuring the expression level of Lrig-1 protein or its extracellular domain present on the surface of T cells in a biological sample isolated from an individual of interest, or a gene encoding the same, using the binding molecule.

[0153] The T cells of the present invention may be regulatory T cells.

[0154] The step of measuring the expression level of the present invention may further measure the expression level of one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16 and Lrig-1 proteins present on the surface of T cells; or the gene encoding the same. In this way, when the expression levels of various proteins present on the surface of T cells; or the gene encoding the same are further measured, a significant synergistic effect can be exhibited in diagnosing the target disease, compared to the case where the Lrig-1 protein; or the gene encoding the same is measured alone.

[0155] The "target individual" of the present invention refers to an individual for whom the presence or absence of onset of the disease is uncertain, and who has a high possibility of onset of the disease.

[0156] The "biological sample" of the present invention means any substance, biological fluid, tissue or cell obtained from or derived from an individual, such as whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymphatic fluid, and the like. These may include, but are not limited to, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0157] The Lrig-1 protein of the present invention; or its extracellular domain, may also be expressed in T cells, particularly on the cell surface of regulatory T cells, for example, activated regulatory T cells, i.e., regulatory T cells whose suppressive ability on effector T cells has been activated.

[0158] The method for measuring or comparing the expression level of the Lrig-1 protein or its extracellular domain and one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16 and Lrig-1 protein of the present invention includes protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Surface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis analysis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (Liquid chromatography-Mass Spectrometry / Mass Spectrometry) analysis, and the like. Examples of such techniques include, but are not limited to, immunofluorescence spectrometry, Western blotting, and ELISA (Enzyme linked immunosorbent assay).

[0159] In order to confirm the presence or absence and the expression level of the gene encoding the Lrig-1 protein or its extracellular domain of the present invention and the gene encoding one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16 and Lrig-1 protein, analytical methods for measuring the expression level of the gene include, but are not limited to, reverse transcription polymerase reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, or DNA chip.

[0160] The present invention may include a step of predicting that there is a high possibility of developing an immune-related disease when the expression level of the Lrig-1 protein or its extracellular domain; or the gene encoding the same, measured in a biological sample of the target individual, is decreased compared to a normal control group.

[0161] The method of the present invention may further include a step of predicting that there is a high possibility of developing an immune-related disease, when the expression level of one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16 and Lrig-1 proteins present on the surface of the T cells, or genes encoding the same, is changed (increased or decreased) compared to a normal control group.

[0162] In another embodiment of the present invention, the method may include predicting that there is a high possibility of developing an immune-related disease if the expression level of the Lrig-1 protein or the gene encoding it is decreased compared to a normal control group.

[0163] Furthermore, in the present invention, when the expression level of the Lrig-1 protein or its extracellular domain; or the gene encoding the same is measured in a biological sample of a target individual as described above to predict or diagnose a high possibility of developing an immune-related disease, the method may additionally include a step of administering a drug for the disease to the target individual.

[0164] According to one embodiment of the present invention, there is provided a binding molecule comprising a heavy chain variable region comprising: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and a light chain variable region comprising: light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8; light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9; and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10.

[0165] According to one embodiment of the present invention, the binding molecule comprises a heavy chain variable region comprising a heavy chain CDR1 encoded by the base sequence represented by SEQ ID NO: 13; a heavy chain CDR2 encoded by the base sequence represented by SEQ ID NO: 14; a heavy chain CDR3 encoded by the base sequence represented by SEQ ID NO: 15; and a light chain variable region comprising a light chain CDR1 encoded by the base sequence represented by SEQ ID NO: 16; a light chain CDR2 encoded by the base sequence represented by SEQ ID NO: 17; and a light chain CDR3 encoded by the base sequence represented by SEQ ID NO: 18.

[0166] According to one embodiment of the present invention, the binding molecule comprises a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO:11; and a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO:12.

[0167] According to one embodiment of the present invention, the binding molecule comprises a heavy chain variable region encoded by the base sequence represented by SEQ ID NO: 19; and a light chain variable region encoded by the base sequence represented by SEQ ID NO: 20.

[0168] According to one embodiment of the present invention, the binding molecule further comprises an Fc region (fragment crystallization region) or a constant region.

[0169] According to one embodiment of the invention, the binding molecule is provided, wherein the Fc region is an Fc region of an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 antibody, or a hybrid Fc region.

[0170] According to one embodiment of the invention, the binding molecule is provided, further comprising a heavy chain constant region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 23, 24, 27, 28, 29, 30 and 32.

[0171] According to one embodiment of the invention, the binding molecule is provided, further comprising a light chain constant region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 25, 26 and 31.

[0172] According to one embodiment of the present invention, the binding molecule further comprises a heavy chain constant region encoded by the base sequence represented by SEQ ID NO: 33 or 35; and a light chain constant region encoded by the base sequence represented by SEQ ID NO: 34 or 36.

[0173] According to one embodiment of the present invention, the binding molecule is provided comprising: a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 37 or 38; and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 39 or 40.

[0174] According to one embodiment of the present invention, the binding molecule is provided comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO:37; and a light chain consisting of the amino acid sequence represented by SEQ ID NO:39.

[0175] According to one embodiment of the present invention, the binding molecule is provided comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 38; and a light chain consisting of the amino acid sequence represented by SEQ ID NO: 40.

[0176] According to one embodiment of the present invention, the binding molecule comprises a heavy chain encoded by the base sequence represented by SEQ ID NO: 41 or 43; and a light chain encoded by the base sequence represented by SEQ ID NO: 42 or 44.

[0177] According to one embodiment of the present invention, the binding molecule comprises a heavy chain encoded by the base sequence represented by SEQ ID NO: 41; and a light chain encoded by the base sequence represented by SEQ ID NO: 42.

[0178] According to one embodiment of the present invention, the binding molecule is provided comprising a heavy chain encoded by the base sequence represented by SEQ ID NO: 43; and a light chain encoded by the base sequence represented by SEQ ID NO: 44.

[0179] According to one embodiment of the invention there is provided a binding molecule, wherein said binding molecule is an antibody or a fragment thereof.

[0180] According to one embodiment of the invention, the binding molecule is provided, wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody, a tetrabody or a fragment thereof.

[0181] According to one embodiment of the invention, there is provided a nucleic acid molecule encoding said binding molecule.

[0182] According to one embodiment of the present invention, there is provided an expression vector into which the nucleic acid molecule is inserted.

[0183] According to one embodiment of the present invention, there is provided a host cell line transfected with said expression vector.

[0184] According to another embodiment of the present invention, there is provided an antibody-drug conjugate (ADC) comprising: a heavy chain variable region comprising heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and an antibody comprising a light chain variable region comprising light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8; light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9; and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10; and a drug.

[0185] According to one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating a brain and nervous system disease, comprising the binding molecule as an active ingredient.

[0186] According to one embodiment of the present invention, there is provided a pharmaceutical composition, wherein the cranial nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

[0187] According to one embodiment of the present invention, the neurodegenerative or neuroinflammatory disease is selected from the group consisting of stroke, cognitive, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and the like. The present invention provides a pharmaceutical composition for treating tauopathy, comprising the steps of:

[0188] According to yet another embodiment of the present invention, there is provided a chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain, wherein the specific binding domain is a heavy chain variable region comprising: a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and a light chain variable region comprising: a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10.

[0189] According to one embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating brain and nervous system diseases, comprising the chimeric antigen receptor as an active ingredient.

[0190] According to one embodiment of the present invention, there is provided a composition for diagnosing a brain and nervous system disease, comprising the binding molecule.

[0191] According to one embodiment of the present invention, there is provided a diagnostic kit for a central nervous system disease, comprising the composition.

[0192] According to one embodiment of the present invention, there is provided a method for providing information for diagnosing nervous system diseases, comprising a step of measuring the expression level of Lrig-1 protein present on the surface of T cells in a biological sample isolated from a target individual using the binding molecule.

[0193] According to another embodiment of the present invention, there is provided a method for preventing or treating a brain and nervous system disease, characterized by a binding molecule comprising a heavy chain variable region including a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and a light chain variable region including a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10.

[0194] According to one embodiment of the present invention, the binding molecule is an antibody or a fragment thereof, and the antibody is a chimeric antibody, a humanized antibody, a bivalent antibody, 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.

[0195] According to another embodiment of the present invention, there is provided a method for preventing or treating a brain and nervous system disease, characterized by an antibody comprising a heavy chain variable region including a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and a light chain variable region including a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10; and an antibody-drug conjugate comprising a drug.

[0196] According to another embodiment of the present invention, there is provided a method for preventing or treating a brain and nervous system disease characterized by a chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain, wherein the specific binding domain is a heavy chain variable region comprising: a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and a light chain variable region comprising: a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10. Effect of the Invention

[0197] The binding molecules provided by the present invention can specifically prevent, ameliorate or treat various neurodegenerative or neuroinflammatory diseases of the brain and nervous system. [Brief description of the drawings]

[0198] [Figure 1] FIG. 1 shows the structure of Lrig-1 protein according to one embodiment of the present invention. [Diagram 2] FIG. 1 shows the structure of Lrig-1 protein according to one embodiment of the present invention. [Diagram 3] A graph showing the expression level of Lrig-1 mRNA according to one embodiment of the present invention. [Figure 4] A graph showing the expression level of Lrig-1 mRNA according to one embodiment of the present invention. [Diagram 5] A graph showing the expression level of Lrig-1 mRNA according to one embodiment of the present invention. [Figure 6] A figure showing the expression levels of Lrig-1, Lrig-2 and Lrig-3 mRNA according to one embodiment of the present invention. [Figure 7] FIG. 1 shows a comparison of the expression levels of Lrig-1 protein in regulatory T cells and non-regulatory T cells according to one embodiment of the present invention. [Figure 8] FIG. 1 shows expression of Lrig-1 protein on the surface of regulatory T cells according to one embodiment of the present invention. [Figure 9] FIG. 2 is an experimental design diagram for confirming the therapeutic effect of a monoclonal antibody according to one embodiment of the present invention on Alzheimer's disease. [Figure 10] FIG. 1 shows photographs of the results of staining with thioflavin S (ThS) of mouse brain cortex and hippocampal tissues obtained from Alzheimer's-induced mice after each treatment containing a monoclonal antibody according to one embodiment of the present invention, taken with a fluorescence microscope. [Figure 11] This is a graph showing the results of staining the brain cortex and hippocampus tissues of Alzheimer's-induced mice with thioflavin S (ThS) after each treatment containing a monoclonal antibody according to one embodiment of the present invention, and calculating the ratio (%) of ThS+ area compared to normal mice. [Figure 12] FIG. 1 is a graph showing changes in spontaneous alteration (%) in Alzheimer's disease-induced mice after treatment with a monoclonal antibody according to an embodiment of the present invention in a Y-maze experiment. [Figure 13] 1 is a graph showing the change in preference score as a result of a novel object recognition experiment after each treatment of Alzheimer's-induced mice containing a monoclonal antibody according to an embodiment of the present invention. [Figure 14]FIG. 13 is a graph showing the change in the time taken to reach the location of the platform in an underwater maze experiment after each treatment of Alzheimer's-induced mice containing a monoclonal antibody according to one embodiment of the present invention. [Figure 15] FIG. 13 is a graph showing the change in the number of times the target was passed in a water maze experiment after each treatment of Alzheimer's-induced mice, including a monoclonal antibody according to an embodiment of the present invention. [Figure 16] This is a graph showing the results of measuring the change in the time it took for Alzheimer's-induced mice to reach the location of the platform during a water maze experiment after each treatment, including a monoclonal antibody according to one embodiment of the present invention. [Figure 17] FIG. 2 is an experimental design diagram of Alzheimer's-induced mice, 5xFAD mice and 6xTg mice, for confirming the therapeutic effect of a monoclonal antibody according to one embodiment of the present invention on Alzheimer's disease. [Figure 18] FIG. 1 is a graph showing the change in spontaneous alteration (%) in a Y-maze experiment after treatment of Alzheimer's-induced mice, 5xFAD mice and 6xTg mice, with a monoclonal antibody according to one embodiment of the present invention. [Figure 19] FIG. 1 is a graph showing the change in preference score as a result of a novel object recognition experiment after each treatment of 6xTg mice, which are Alzheimer's induced mice, including a monoclonal antibody according to one embodiment of the present invention. [Figure 20] FIG. 1 is a graph showing the change in time spent in a passive avoidance experiment after treatment of Alzheimer's disease-induced mice, 5xFAD mice and 6xTg mice, with a monoclonal antibody according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0199] According to one embodiment of the present invention, there is provided a binding molecule comprising a heavy chain variable region comprising: heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and a light chain variable region comprising: light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8; light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9; and light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:10. EXAMPLES

[0200] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are merely intended to more specifically illustrate the present invention, and that the scope of the present invention is not limited to these examples according to the gist of the present invention.

[0201] Working Example [Preparation Example 1] Cultivation of T cell subtypes To confirm whether Lrig-1 protein is expressed only in regulatory T cells (Treg), T cell subtypes Th0, Th1, Th2, Th17, and iTreg were prepared. The iTreg is different from naturally isolated nTreg, and refers to cells whose differentiation was artificially induced in a medium containing the following composition.

[0202] To determine the subtypes of T cells, naive T cells were first isolated from the mouse spleens and then differentiated into each type of cell by culturing for 72 hours in a 37°C, 5% CO2 incubator in RPMI1640 (Invitrogen Gibco, Grand Island, NY) nutrient medium containing 10% fetal bovine serum (FBS; hyclone, logan, UT) and the additional components listed in Table 1 below.

[0203] [Table 1]

[0204] [Example 1] Lrig-1 structural analysis In order to generate antibodies specific to Lrig-1 protein, a surface protein of regulatory T cells, the three-dimensional structure of the extracellular domain of Lrig-1 protein was predicted.

[0205] First, to confirm the structure of the extracellular domain (ECD) of the Lrig-1 protein for predicting the base sequence of the antigenic determinant (epitope), the three-dimensional structure was predicted using Uniprot (http: / / www.unipro.org) and RCSB Protein Data Bank (http: / / www.rcsb.org / pdb) tools, and the results are shown in Figures 1 and 2.

[0206] As shown in Figure 1, the Lrig-LRR domain (amino acid sequence 41 to 494) in the extracellular domain of the Lrig-1 protein contained a total of 15 leucine-rich regions, LRR1 to LRR15. Each of the LRR domains consisted of 23 to 27 amino acids and contained 3 to 5 leucines.

[0207] Furthermore, as shown in FIG. 2, the extracellular domain of the Lrig-1 protein contained three immunoglobulin-like domains in the amino acid sequence 494 to 781 of the Lrig-1 protein.

[0208] [Example 2] Prediction of the amino acid sequence of the Lrig-1 antigenic determinant (epitope) The base sequence was predicted using the Ellipro server (http: / / tools.iedb.org / ellipro / ), which is an epitope prediction software based on the structure of the Lrig-1 protein. The Ellipro search engine was used because it is known to be the most reliable of the currently available algorithms for predicting antigenic determinants.

[0209] After inputting the extracellular domain analyzed in Example 1 into the antigenic determinant prediction software, a total of 22 predicted contiguous or discontinuous amino acid sequences of predicted antigenic determinants were predicted, and a total of 8 predicted amino acid sequences of discontinuous antigenic determinants were predicted.

[0210] [Production Examples 1 and 2] Preparation of monoclonal antibodies specific to Lrig-1 protein The present invention produced an antibody specific to the Lrig-1 protein. This antibody was produced without specifying a specific antigenic determinant, but was capable of binding to any site on the Lrig-1 protein.

[0211] To prepare the antibody, cells expressing Lrig-1 protein were prepared. More specifically, the DNA fragment corresponding to SEQ ID NO: 2 and pcDNA (hygro) were cleaved with a cleavage enzyme, then cultured at 37°C for ligation to prepare pcDNA into which the DNA sequence of Lrig-1 protein was inserted. The prepared pcDNA into which SEQ ID NO: 2 was inserted was introduced into L cells by transfection to allow the expression of Lrig-1 protein on the surface of the L cells.

[0212] Light chain and heavy chain amino acid sequences capable of binding to Lrig-1 expressed on the cell surface were selected from a human scFv library.

[0213] The selected heavy and light chain amino acid sequences were fused to the heavy chain constant region of SEQ ID NO: 24 or 30 and the light chain constant region of SEQ ID NO: 26 or 31, respectively, to prepare monoclonal antibodies. The sequences of the monoclonal antibodies are shown in Table 2 below.

[0214] [Table 2]

[0215] [Example 3] Confirmation of specific expression of Lrig-1 mRNA in regulatory T cells We examined whether Lrig-1 protein could act as a specific biomarker for regulatory T cells.

[0216] For this verification, CD4+ T cells were isolated from mouse spleens using a magnet-activated cell sorting (MACS) with CD4 beads. Then, regulatory T (CD4+CD25+ T) cells and non-regulatory T (CD4+CD25- T) cells were isolated using a fluorescence activated cell sorting (FACS) with CD25 antibodies. After extracting mRNA from each cell and the cells differentiated in Preparation Example 1 using Trizol, gDNA was removed from the genomic RNA using a gDNA extraction kit (Qiagen) according to the protocol provided by the company. The gDNA-removed mRNA was synthesized into cDNA using the BDsprint cDNA synthesis kit (Clonetech).

[0217] To quantitatively confirm the expression level of Lrig-1 mRNA using the cDNA, real-time polymerase chain reaction (real-time PCR) was performed.

[0218] The real-time polymerase chain reaction was performed using SYBR Green (Molecular Probes) according to the protocol provided by the company under the conditions of 95°C for 3 minutes, 61°C for 15 seconds, and 72°C for 30 seconds, for 40 cycles, using the primers in Table 3 below. The relative gene expression levels were calculated using the ΔCT method and normalized using HPRT. The results are shown in Figures 3 to 6.

[0219] [Table 3]

[0220] As shown in Figure 3, the expression of Lrig-1 was 18.1 times higher in regulatory T (CD4+CD25+ T) cells than in non-regulatory T (CD4+CD25- T) cells. This was about 10 times higher than the expression levels of Lag3 and Ikzf4, which are conventionally known regulatory T cell markers. In addition, as shown in Figures 4 and 5, the expression of Lrig-1 mRNA was significantly higher in naturally isolated regulatory T cells (nTreg) than in other types of immune cells, especially in induced regulatory T cells (iTreg). Furthermore, as shown in Figure 6, the expression of Lrig-1 was the highest among Lrig-1, Lrig-2, and Lrig-3, which correspond to the Lrig family.

[0221] From the above results, it can be seen that the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells, particularly in naturally occurring regulatory T cells.

[0222] [Example 4] Confirmation of specific expression of Lrig-1 protein in regulatory T cells We confirmed whether Lrig-1 protein expressed from Lrig-1 mRNA was specifically expressed only in regulatory T cells.

[0223] Using FOXP3-RFP knock-in mice, in which RFP (red fluorescence protein) is bound to the FOXP3 promoter, a transcription factor specific to regulatory T cells, CD4+ T cells were isolated from the spleen of the mice using a magnet-activated cell sorting (MACS) with CD4 beads. Then, using RFP protein, regulatory T (CD4+RFP+ T) cells and non-regulatory T (CD4+RFP- T) cells were isolated using a fluorescence-activated cell sorting (FACS) to obtain them. Each of the cells was stained with the purchased Lrig-1 antibody and the negative control group with an isotype, and the expression level of Lrig-1 was measured using a fluorescence-activated cell sorting machine, and the results are shown in Figure 7.

[0224] As shown in Figure 7, non-regulatory T cells, represented by the dotted line, showed Lrig-1 expression levels almost the same as those of the negative control group, but regulatory T cells contained a large number of cells with high Lrig-1 expression levels.

[0225] From the above results, it can be seen that the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells.

[0226] [Example 5] Confirmation of specific expression of Lrig-1 protein on the surface of regulatory T cells In order for the Lrig-1 protein to be a target for cell therapy, it must be expressed on the surface of regulatory T cells for effective targeted therapy, so we confirmed whether the Lrig-1 protein is expressed on the surface.

[0227] Each differentiated T cell subtype from Preparatory Example 1 was stained with anti-CD4-APC and anti-Lrig-1-PE antibodies, and the expression level of Lrig-1 on the cell surface was measured using a Fluorescence-Activated Cell Sorter (FACS). The results are shown in Figure 8.

[0228] As shown in Figure 8, Lrig-1 was expressed at levels of 0.77 to 15.3 in activated T cells, Th1 cells, Th2 cells, Th17 cells, and naive T cells, whereas it was highly expressed at 83.9 in differentiated T cells (iTreg).

[0229] From the above results, it can be seen that the Lrig-1 protein according to the present invention is not only specifically expressed in regulatory T cells (Treg), but is particularly highly expressed on the surface of Treg cells.

[0230] [Example 6] Evaluation of the therapeutic ability of the antibody according to the present invention for Alzheimer's disease - 5xFAD mice In order to evaluate the therapeutic ability of the antibody according to the present invention against Alzheimer's disease, groups were designed as shown in FIG. 9. Specifically, the GTC310-01 mouse antibody of Preparation Example 1 was intravenously injected at a dose of 10 mpk for 3 weeks into 5-6 month-old male Alzheimer's-induced 5xFAD mice. For comparison, a positive control group was also administered glatiramer acetate (GA, Copaxone). (R) ) was injected subcutaneously at a dose of 100 ug for three weeks, or the H6 antibody shown in Table 4 below was injected intravenously at a dose of 10 mpk for four weeks.

[0231] [Table 4]

[0232] 1. Reduction of amyloid-β plaque For immunohistological analysis, the treated mice were euthanized, and the brain cortex and hippocampus tissues were obtained and then 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 ​​to a thickness of 35 μm 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 number T-1892). 500 μM of thioflavin S (ThS) was dissolved in 50% ethanol. After successive washing with 100%, 95% and 70% ethanol, the sections were transferred to PBS. For immunofluorescence, relatively thick free-floating brain sections were incubated with goat anti-GFAP antibody at 4°C for 7 days. Sections were then incubated with Alexa Fluor594-conjugated donkey anti-goat IgG (1:200, Abcam, ab150132) overnight at 4°C. Nuclear control staining was performed with 4'6'-diamino-2-phenylindole dihydrochloride hydrate (DAPI, 1mg / mL, 1:2000, Sigma). Images were taken with a Leica DM2500 fluorescent microscope, and the results are shown in Figure 10. The ratio (%) of ThS+ area to normal mice was also calculated using the ImageJ software program, and the results are shown in Figure 11.

[0233] As shown in Figures 10 and 11, the ThS+ area significantly increased in Alzheimer's-induced mice, but when the GTC310-01 antibody according to the present invention was administered, the ThS+ area significantly decreased, especially in the cortex, indicating that the effect of reducing amyloid-β plaques was very excellent.

[0234] 2. Y-maze test The Y-maze experiment used a maze frame with three identical arms, each 40 cm long (wall height 15 cm), arranged at a 120-degree angle. This experiment is a behavioral experiment that utilizes the instinctive exploration behavior of rodents, focusing on the high possibility of exploring new areas. The animals showed high memory scores, as they memorized the arm they had just explored and did not enter the same arm. Each animal was given 8 minutes of exploration time, and the final results are shown in Figure 12 as spontaneous alteration (%) values. Spontaneous alteration (%) values ​​were calculated using the following formula 1. Here, behavioral pattern analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA).

[0235]

number

[0236] As shown in FIG. 12, in this experiment, in which the relative frequency with which the experimental animals grasped the surrounding cues and sequentially entered the maze was measured, it was confirmed that when the GTC310-01 antibody according to the present invention was administered to Alzheimer's-induced mice, the spontaneous alternation value was at a level similar to that of the normal control group.

[0237] 3. Novel Object Recognition In the novel object recognition experiment, memory was evaluated using two different objects in a 40 cm × 40 cm acrylic cage. After getting used to the acrylic cage, the two objects were placed in fixed positions and the rats were allowed to freely recognize them, and the time spent exploring each object was measured. A 24-hour delay was given to each rat, and then only one object was replaced with another object in the same position. At this time, the longer the exploration time after recognizing the replaced object as a novel object, the better the memory is considered to be. If the rats cannot remember the object from 24 hours ago, they may not be able to distinguish between the novel object and the existing object, and may explore both objects equally. The rats were allowed to freely explore for a total of 10 minutes, and the results are shown in Figure 13 as a preference index (novel object exploring time / total exploring time). Analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA).

[0238] As shown in Figure 13, the preference index for novel objects was lower in the Alzheimer's-induced group than in the normal control group. However, it was confirmed that the preference index increased more than that of the normal control group when the GTC310-01 antibody according to the present invention was administered.

[0239] 4. Water maze test The underwater maze test was performed based on the method devised by Morris. A stainless steel pool (diameter 90 cm, height 50 cm) was filled with water (22 ± 1°C) and the water surface was set to 30 cm high. A hidden platform (diameter 5 cm) was placed 1 cm below the water surface. On the first day of evaluation, three to four training sessions were performed. The total swimming time for each animal was set to 60 seconds, and animals that found the platform within 60 seconds were made to stay on the platform for 10 seconds to induce memory. Animals that could not find the platform after 60 seconds were artificially guided to the platform and made to stay on the platform for 10 seconds, with an escape time of 60 seconds. After swimming practice, a probe test was conducted on the sixth day to measure spatial perception ability (time to reach the platform location (latency to target, sec) and number of times the target was crossed (target crossing numbers, number)). The results are shown in Figures 14 and 15. An acquisition test was conducted to measure the time to reach the platform location during the test (time to platform, sec). The results are shown in Figure 16. Analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA). The criterion for exclusion from the analysis of the results was set to individuals that did not explore by swimming and only turned around a specific area.

[0240] As shown in Figures 14 to 16, it was confirmed that when the GTC310-01 antibody according to the present invention was administered, the number of times the target was passed significantly increased and the time it took to reach the position where the platform was located significantly decreased.

[0241] From these experiments, it was confirmed that the antibody according to the present invention has the effect of preventing, improving or treating diseases of the central nervous system.

[0242] [Example 7] Evaluation of the therapeutic ability of the antibody according to the present invention for Alzheimer's disease - 6xTg mice In order to evaluate the therapeutic ability of the antibody according to the present invention for Alzheimer's disease, the experiment was carried out by designing groups as shown in Figure 17. Specifically, the GTC310-01 antibody of Preparation Example 1 was intravenously injected at 10 mpk every other week for two months into female Alzheimer's-induced 5xFAD mice and female 6xTg mice that had undergone an adaptation period of 4.5 months. For comparison, a positive control group was also administered glatiramer acetate (GA, Copaxone (R) ) was intravenously injected at 100ug every other week for two months. After two months, the rats were subjected to Y-maze, novel object recognition, and passive avoidance tests.

[0243] 1. Characteristics of 6xTg mice 5xFAD mice are widely used as an induced Alzheimer's model, but the 6xTg mice used in the experiments described below are a mouse model in which not only amyloid-β (Aβ42) but also tau protein (MAPT) is mutated and accumulated, and are therefore closer to the human disease model than 5xFAD mice, making them an ideal animal model for brain and nervous system diseases, including Alzheimer's.

[0244] 2. Y-maze test The Y-maze experiment used a maze frame with three identical arms, each 40 cm long (wall height 15 cm), arranged at a 120 degree angle. This experiment is a behavioral experiment that utilizes the instinctive exploration behavior of rodents, focusing on the high possibility of exploring new areas. The animals showed high memory scores, as they memorized the arm they had just explored and did not enter the same arm. Each animal was given 8 minutes of exploration time, and the final results are shown in Figure 20 as spontaneous alteration (%) values. Spontaneous alteration (%) values ​​were calculated using the following formula 1. Here, behavioral pattern analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA).

[0245]

number

[0246] 18, in the present experiment, in which the relative frequency of experimental animals grasping peripheral cues and sequentially entering the maze was measured, it was confirmed that when the GTC310-01 antibody according to the present invention was administered to Alzheimer's-induced mice, the spontaneous alternation value was at a level similar to that of the normal control group. Furthermore, it was confirmed that the spontaneous alternation value was at a level similar to that of the normal control group in the 6xTg model mice, which are closer to the human disease model, than in the 5xFAD mice.

[0247] 3. Novel Object Recognition In the novel object recognition experiment, memory was evaluated using two different objects in a 40 cm × 40 cm acrylic cage. After getting used to the acrylic cage, the two objects were placed in fixed positions and the rats were allowed to freely recognize them, and the time spent exploring each object was measured. A 24-hour delay was given to each rat, and only one object was replaced with another object in the same position. The longer the exploration time after recognizing the replaced object as a novel object, the better the memory is considered to be. If the rats cannot remember the object from 24 hours ago, they may not be able to distinguish between the novel object and the existing object, and may explore both objects equally. The rats were allowed to freely explore for a total of 10 minutes, and the results are shown in Figure 19 as a preference index (novel object exploring time / total exploring time). Analysis was performed using SMART VIDEO TRACKING Software (Panlab, USA).

[0248] As shown in Figure 19, the preference index for novel objects was lower in the Alzheimer's-induced group than in the normal control group. However, it was confirmed that administration of the GTC310-01 antibody according to the present invention increased the preference index in the 6xTg model, which is closer to a human disease model, to a level higher than that of the normal control group.

[0249] 4. Passive Avoidance Test The experimental apparatus for the passive avoidance test was divided into two areas, a lighted chamber and a dark chamber, and the bottom was made of wire mesh. First, on the first day, the mice were allowed to move freely and adapted. On the next day, each 5xFAD mouse and 6xTg mouse was adapted for 1 minute in the lighted chamber without the light on, then the light was turned on and they were adapted for 2 minutes. As soon as the mouse moved to the dark chamber, an electric shock of 0.5 mA was applied for 1 second 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 the 6xTg mouse were placed in the lighted chamber, and the time it took for the 5xFAD mouse and the 6xTg mouse to enter all four paws (latency time) was set to within 300 seconds. As a result, as shown in Figure 20, when the GTC310-01 antibody was administered to not only the 5xFAD mouse but also the 6xTg mouse, it was found that the recovery was close to that of the normal control group.

[0250] Although the present invention has been described in detail above, the scope of the invention is not limited thereto, and it will be obvious to those having ordinary skill in the art that various modifications and variations are possible within the scope that does not deviate from the technical idea of ​​the invention described in the claims.

[0251] [Industrial Applicability] The present invention relates to a novel binding molecule having excellent preventive, ameliorative or therapeutic effects on nervous system diseases, and its use for preventing or treating nervous system diseases, particularly neurodegenerative diseases or neuroinflammatory diseases.

[0252] [Sequence list free text] <110> Good T Cells, Inc. <120> Novel binding molecule and use thereof <130> POPB214473PCT <160> 54 <170> KoPatentIn 3.0 <210> 1 <211> 759 <212> PRT <213> Homo sapiens <400> 1 Gly Pro Arg Ala Pro Cys Ala Ala Ala Cys Thr Cys Ala Gly Asp Ser 1 5 10 15 Leu Asp Cys Gly Gly Arg Gly Leu Ala Ala Leu Pro Gly Asp Leu Pro 20 25 30 Ser Trp Thr Arg Ser Leu Asn Leu Ser Tyr Asn Lys Leu Ser Glu Ile 35 40 45 Asp Pro Ala Gly Phe Glu Asp Leu Pro Asn Leu Gln Glu Val Tyr Leu 50 55 60 Asn Asn Asn Glu Leu Thr Ala Val Pro Ser Leu Gly Ala Ala Ser Ser 65 70 75 80 His Val Val Ser Leu Phe Leu Gln His Asn Lys Ile Arg Ser Val Glu 85 90 95 Gly Ser Gln Leu Lys Ala Tyr Leu Ser Leu Glu Val Leu Asp Leu Ser 100 105 110 Leu Asn Asn Ile Thr Glu Val Arg Asn Thr Cys Phe Pro His Gly Pro 115 120 125 Pro Ile Lys Glu Leu Asn Leu Ala Gly Asn Arg Ile Gly Thr Leu Glu 130 135 140 Leu Gly Ala Phe Asp Gly Leu Ser Arg Ser Leu Leu Thr Leu Arg Leu 145 150 155 160 Ser Lys Asn Arg Ile Thr Gln Leu Pro Val Arg Ala Phe Lys Leu Pro 165 170 175 Arg Leu Thr Gln Leu Asp Leu Asn Arg Asn Arg Ile Arg Leu Ile Glu 180 185 190 Gly Leu Thr Phe Gln Gly Leu Asn Ser Leu Glu Val Leu Lys Leu Gln 195 200 205 Arg Asn Asn Ile Ser Lys Leu Thr Asp Gly Ala Phe Trp Gly Leu Ser 210 215 220 Lys Met His Val Leu His Leu Glu Tyr Asn Ser Leu Val Glu Val Asn 225 230 235 240 Ser Gly Ser Leu Tyr Gly Leu Thr Ala Leu His Gln Leu His Leu Ser 245 250 255 Asn Asn Ser Ile Ala Arg Ile His Arg Lys Gly Trp Ser Phe Cys Gln 260 265 270 Lys Leu His Glu Leu Val Leu Ser Phe Asn Asn Leu Thr Arg Leu Asp 275 280 285 Glu Glu Ser Leu Ala Glu Leu Ser Ser Leu Ser Val Leu Arg Leu Ser 290 295 300 His Asn Ser Ile Ser His Ile Ala Glu Gly Ala Phe Lys Gly Leu Arg 305 310 315 320 Ser Leu Arg Val Leu Asp Leu Asp His Asn Glu Ile Ser Gly Thr Ile 325 330 335 Glu Asp Thr Ser Gly Ala Phe Ser Gly Leu Asp Ser Leu Ser Lys Leu 340 345 350 Thr Leu Phe Gly Asn Lys Ile Lys Ser Val Ala Lys Arg Ala Phe Ser 355 360 365 Gly Leu Glu Gly Leu Glu His Leu Asn Leu Gly Gly Asn Ala Ile Arg 370 375 380 Ser Val Gln Phe Asp Ala Phe Val Lys Met Lys Asn Leu Lys Glu Leu 385 390 395 400 His Ile Ser Ser Asp Ser Phe Leu Cys Asp Cys Gln Leu Lys Trp Leu 405 410 415 Pro Pro Trp Leu Ile Gly Arg Met Leu Gln Ala Phe Val Thr Ala Thr 420 425 430 Cys Ala His Pro Glu Ser Leu Lys Gly Gln Ser Ile Phe Ser Val Pro 435 440 445 Pro Glu Ser Phe Val Cys Asp Asp Phe Leu Lys Pro Gln Ile Ile Thr 450 455 460 Gln Pro Glu Thr Thr Met Ala Met Val Gly Lys Asp Ile Arg Phe Thr 465 470 475 480 Cys Ser Ala Ala Ser Ser Ser Ser Ser Pro Met Thr Phe Ala Trp Lys 485 490 495 Lys Asp Asn Glu Val Leu Thr Asn Ala Asp Met Glu Asn Phe Val His 500 505 510 Val His Ala Gln Asp Gly Glu Val Met Glu Tyr Thr Thr Ile Leu His 515 520 525 Leu Arg Gln Val Thr Phe Gly His Glu Gly Arg Tyr Gln Cys Val Ile 530 535 540 Thr Asn His Phe Gly Ser Thr Tyr Ser His Lys Ala Arg Leu Thr Val 545 550 555 560 Asn Val Leu Pro Ser Phe Thr Lys Thr Pro His Asp Ile Thr Ile Arg 565 570 575 Thr Thr Thr Val Ala Arg Leu Glu Cys Ala Ala Thr Gly His Pro Asn 580 585 590 Pro Gln Ile Ala Trp Gln Lys Asp Gly Gly Thr Asp Phe Pro Ala Ala 595 600 605 Arg Glu Arg Arg Met His Val Met Pro Asp Asp Asp Val Phe Phe Ile 610 615 620 Thr Asp Val Lys Ile Asp Asp Ala Gly Val Tyr Ser Cys Thr Ala Gln 625 630 635 640 Asn Ser Ala Gly Ser Ile Ser Ala Asn Ala Thr Leu Thr Val Leu Glu 645 650 655 Thr Pro Ser Leu Val Val Pro Leu Glu Asp Arg Val Val Ser Val Gly 660 665 670 Glu Thr Val Ala Leu Gln Cys Lys Ala Thr Gly Asn Pro Pro Pro Arg 675 680 685 Ile Thr Trp Phe Lys Gly Asp Arg Pro Leu Ser Leu Thr Glu Arg His 690 695 700 His Leu Thr Pro Asp Asn Gln Leu Leu Val Val Gln Asn Val Val Ala 705 710 715 720 Glu Asp Ala Gly Arg Tyr Thr Cys Glu Met Ser Asn Thr Leu Gly Thr 725 730 735 Glu Arg Ala His Ser Gln Leu Ser Val Leu Pro Ala Ala Gly Cys Arg 740 745 750 Lys Asp Gly Thr Thr Val Gly 755 <210> 2 <211> 2397 <212> DNA <213> Homo sapiens <400> 2 ggcccgcggg cgccctgcgc ggccgcctgc acttgcgctg gggactcgct ggactgcggt 60 gggcgcgggc tggctgcgtt gcccggggac ctgccctcct ggacgcggag cctaaacctg 120 agttacaaca aactctctga gattgaccct gctggttttg aggacttgcc gaacctacag 180 gaagtgtacc tcaataataa tgagttgaca gcggtaccat ccctgggcgc tgcttcatca 240 catgtcgtct ctctctttct gcagcacaac aagattcgca gcgtggaggg gagccagctg 300 aaggcctacc tttccttaga agtgttagat ctgagtttga acacac ggaagtgcgg 360 aacacctgct ttccacgg accgcctata aaggagctca acctggcagg caatcggatt 420 ggcaccctgg agttgggagc atttgatggt ctgtcacggt cgctgctaac tcttcgcctg 480 agcaaaaaca ggatcacca gcttcctgta agagcattca agctacccag gctgacacaa 540 ctggacctca atcggaacag gattcggctg atagagggcc tcaccttcca ggggctcaac 600 agcttggagg tgctgaagct tcagcgaaac aacatcagca aactgacaga tggggccttc 660 tggggactgt ccaagatgca tgtgctgcac ctggagtaca acagcctggt agaagtgaac 720 agcggctcgc tctacggcct cacggccctg catcagctcc acctcagcaa caattccatc 780 gctcgcattc accgcaaggg ctggagcttc tgccagaagc tgcatgagtt ggtcctgtcc 840 ttcaacaacc tgacacggct ggacgaggag agcctggccg agctgagcag cctgagtgtc 900 ctgcgtctca gccacaattc catcagccac attgcggagg gtgccttcaa gggactcagg 960 agcctgcgag tcttggatct ggaccataac gagatttcgg gcacaataga ggacacgagc 1020 ggcgccttct cagggctcga cagcctcagc aagctgactc tgtttggaaa caagatcaag 1080 tctgtggcta agagagcatt ctcggggctg gaaggcctgg agcacctgaa ccttggaggg 1140 aatgcgatca gatctgtcca gtttgatgcc tttgtgaaga tgaagaatct taaagagctc 1200 catatcagca gcgacagctt cctgtgtgac tgccagctga agtggctgcc cccgtggcta 1260 attggcagga tgctgcaggc ctttgtgaca gccacctgtg cccacccaga atcactgaag 1320 ggtcagagca ttttctctgt gccaccagag agtttcgtgt gcgatgactt cctgaagcca 1380 cagatcatca cccagccaga aaccaccatg gctatggtgg gcaaggacat ccggtttaca 1440 tgctcagcag ccagcagcag cagctccccc atgacctttg cctggaagaa agacaatgaa 1500 gtcctgacca atgcagacat ggagaacttt gtccacgtcc acgcgcagga cggggaagtg 1560 atggagtaca ccaccatcct gcacctccgt caggtcactt tcgggcacga gggccgctac 1620 caatgtgtca tcaccaacca ctttggctcc acctattcac ataaggccag gctcaccgtg 1680 aatgtgttgc catcattcac caaaacgccc cacgacataa ccatccggac caccaccgtg 1740 gcccgcctcg aatgtgctgc cacaggtcac ccaaaccctc agattgcctg gcagaaggat 1800 ggaggcacgg atttccccgc tgcccgtgag cgacgcatgc atgtcatgcc ggatgacgac 1860 gtgtttttca tcactgatgt gaaaatagat gacgcagggg tttacagctg tactgctcag 1920 aactcagccg gttctatttc agctaatgcc accctgactg tcctagagac cccatccttg 1980 gtggtcccct tggaagaccg tgtggtatct gtgggagaaa cagtggccct ccaatgcaaa 2040 gccacgggga accctccgcc ccgcatcacc tggttcaagg gggaccgccc gctgagcctc 2100 actgagcggc accacctgac ccctgacaac cagctcctgg tggttcagaa cgtggtggca 2160 gaggatgcgg gccgatatac ctgtgagatg tccaacaccc tgggcacgga gcgagctcac 2220 agccagctga gcgtcctgcc cgcagcaggc tgcaggaagg atgggaccac ggtaggcatc 2280 ttcaccattg ctgtcgtgag cagcatcgtc ctgacgtcac tggtctgggt gtgcatcatc 2340 taccagacca ggaagaagag tgaagagtac agtgtcacca acacagatga aaccgtc 2397 <210> 3 <211> 761 <212> PRT <213> Mus musculus <400> 3 Gln Ala Gly Pro Arg Ala Pro Cys Ala Ala Ala Cys Thr Cys Ala Gly 1 5 10 15 Asp Ser Leu Asp Cys Ser Gly Arg Gly Leu Ala Thr Leu Pro Arg Asp 20 25 30 Leu Pro Ser Trp Thr Arg Ser Leu Asn Leu Ser Tyr Asn Arg Leu Ser 35 40 45 Glu Ile Asp Ser Ala Ala Phe Glu Asp Leu Thr Asn Leu Gln Glu Val 50 55 60 Tyr Leu Asn Ser Asn Glu Leu Thr Ala Ile Pro Ser Leu Gly Ala Ala 65 70 75 80 Ser Ile Gly Val Val Ser Leu Phe Leu Gln His Asn Lys Ile Leu Ser 85 90 95 Val Asp Gly Ser Gln Leu Lys Ser Tyr Leu Ser Leu Glu Val Leu Asp 100 105 110 Leu Ser Ser Asn Asn Ile Thr Glu Ile Arg Ser Ser Cys Phe Pro Asn 115 120 125 Gly Leu Arg Ile Arg Glu Leu Asn Leu Ala Ser Asn Arg Ile Ser Ile 130 135 140 Leu Glu Ser Gly Ala Phe Asp Gly Leu Ser Arg Ser Leu Leu Thr Leu 145 150 155 160 Arg Leu Ser Lys Asn Arg Ile Thr Gln Leu Pro Val Lys Ala Phe Lys 165 170 175 Leu Pro Arg Leu Thr Gln Leu Asp Leu Asn Arg Asn Arg Ile Arg Leu 180 185 190 Ile Glu Gly Leu Thr Phe Gln Gly Leu Asp Ser Leu Glu Val Leu Arg 195 200 205 Leu Gln Arg Asn Asn Ile Ser Arg Leu Thr Asp Gly Ala Phe Trp Gly 210 215 220 Leu Ser Lys Met His Val Leu His Leu Glu Tyr Asn Ser Leu Val Glu 225 230 235 240 Val Asn Ser Gly Ser Leu Tyr Gly Leu Thr Ala Leu His Gln Leu His 245 250 255 Leu Ser Asn Asn Ser Ile Ser Arg Ile Gln Arg Asp Gly Trp Ser Phe 260 265 270 Cys Gln Lys Leu His Glu Leu Ile Leu Ser Phe Asn Asn Leu Thr Arg 275 280 285 Leu Asp Glu Glu Ser Leu Ala Glu Leu Ser Ser Leu Ser Ile Leu Arg 290 295 300 Leu Ser His Asn Ala Ile Ser His Ile Ala Glu Gly Ala Phe Lys Gly 305 310 315 320 Leu Lys Ser Leu Arg Val Leu Asp Leu Asp His Asn Glu Ile Ser Gly 325 330 335 Thr Ile Glu Asp Thr Ser Gly Ala Phe Thr Gly Leu Asp Asn Leu Ser 340 345 350 Lys Leu Thr Leu Phe Gly Asn Lys Ile Lys Ser Val Ala Lys Arg Ala 355 360 365 Phe Ser Gly Leu Glu Ser Leu Glu His Leu Asn Leu Gly Glu Asn Ala 370 375 380 Ile Arg Ser Val Gln Phe Asp Ala Phe Ala Lys Met Lys Asn Leu Lys 385 390 395 400 Glu Leu Tyr Ile Ser Ser Glu Ser Phe Leu Cys Asp Cys Gln Leu Lys 405 410 415 Trp Leu Pro Pro Trp Leu Met Gly Arg Met Leu Gln Ala Phe Val Thr 420 425 430 Ala Thr Cys Ala His Pro Glu Ser Leu Lys Gly Gln Ser Ile Phe Ser 435 440 445 Val Leu Pro Asp Ser Phe Val Cys Asp Asp Phe Pro Lys Pro Gln Ile 450 455 460 Ile Thr Gln Pro Glu Thr Thr Met Ala Val Val Gly Lys Asp Ile Arg 465 470 475 480 Phe Thr Cys Ser Ala Ala Ser Ser Ser Ser Ser Pro Met Thr Phe Ala 485 490 495 Trp Lys Lys Asp Asn Glu Val Leu Ala Asn Ala Asp Met Glu Asn Phe 500 505 510 Ala His Val Arg Ala Gln Asp Gly Glu Val Met Glu Tyr Thr Thr Ile 515 520 525 Leu His Leu Arg His Val Thr Phe Gly His Glu Gly Arg Tyr Gln Cys 530 535 540 Ile Ile Thr Asn His Phe Gly Ser Thr Tyr Ser His Lys Ala Arg Leu 545 550 555 560 Thr Val Asn Val Leu Pro Ser Phe Thr Lys Ile Pro His Asp Ile Ala 565 570 575 Ile Arg Thr Gly Thr Thr Ala Arg Leu Glu Cys Ala Ala Thr Gly His 580 585 590 Pro Asn Pro Gln Ile Ala Trp Gln Lys Asp Gly Gly Thr Asp Phe Pro 595 600 605 Ala Ala Arg Glu Arg Arg Met His Val Met Pro Asp Asp Asp Val Phe 610 615 620 Phe Ile Thr Asp Val Lys Ile Asp Asp Met Gly Val Tyr Ser Cys Thr 625 630 635 640 Ala Gln Asn Ser Ala Gly Ser Val Ser Ala Asn Ala Thr Leu Thr Val 645 650 655 Leu Glu Thr Pro Ser Leu Ala Val Pro Leu Glu Asp Arg Val Val Thr 660 665 670 Val Gly Glu Thr Val Ala Phe Gln Cys Lys Ala Thr Gly Ser Pro Thr 675 680 685 Pro Arg Ile Thr Trp Leu Lys Gly Gly Arg Pro Leu Ser Leu Thr Glu 690 695 700 Arg His His Phe Thr Pro Gly Asn Gln Leu Leu Val Val Gln Asn Val 705 710 715 720 Met Ile Asp Asp Ala Gly Arg Tyr Thr Cys Glu Met Ser Asn Pro Leu 725 730 735 Gly Thr Glu Arg Ala His Ser Gln Leu Ser Ile Leu Pro Thr Pro Gly 740 745 750 Cys Arg Lys Asp Gly Thr Thr Val Gly 755 760 <210> 4 <211> 2283 <212> DNA <213> Mus musculus <400> 4 caggctggcc cgcgggcccc ctgcgcggcc gcctgcactt gcgccgggga ctcgctggac 60 tgcagtgggc gcgggctggc gacgctgccc cgggacctgc cctcctggac gcgcagccta 120 aacctgagtt ataacagact ctccgagatc gactctgctg cttttgagga cttgacgaat 180 ctgcaggaag tgtacctcaa cagcaatgag ctgacagcca taccatcact gggcgctgct 240 tccataggag ttgtctctct ctttttgcag cacaacaaga tccttagtgt ggatgggagc 300 cagctgaagt cgtacctgtc cttggaagtg ctggatctga gttccaacaa catcacggaa 360 attcggagct cctgtttccc gaacggcctg cgtataaggg aactcaactt ggcgagcaac 420 cgcatcagca tcctggagtc tggagcattt gatggtctgt cgcggtcact gctgactctc 480 cgtctgagca aaaacaggat cacccagctt cctgtgaaag cgttcaagct acccaggctg 540 acacaactag acctgaatcg gaatcggatt cggctgattg aaggcctcac gttccagggg 600 ctcgacagct tagaggtgct gaggcttcag aggaacaaca tcagcaggct gacggacggg 660 gccttctggg ggctgtctaa gatgcacgtg ctgcacctgg agtacaacag tctggtggaa 720 gtgaacagtg gctccctcta tggcctcaca gccctgcacc agctgcacct cagcaacaac 780 tccatctctc gaattcagcg tgatggctgg agcttctgcc aaaagctgca tgagttgatt 840 ctgtccttca acaacctcac gcggctggat gaggagagtc tagcggagtt gagcagcctc 900 agtatcctgc gcctcagtca caacgccatc agtcacattg ctgaaggcgc cttcaaggga 960 ctcaagagtc tgcgggtctt ggacctggac cataacgaga tctcgggtac aatcgaggat 1020 accagtggtg cctttacggg gcttgacaac ctcagcaagc tgactctgtt tggaaacaag 1080 atcaaatctg tggctaagag agccttctcg ggcctggaaa gcctggaaca cctgaacctt 1140 ggagagaatg caatcaggtc tgtccagttt gatgcctttg caaagatgaa gaaccttaaa 1200 gagctctaca tcagcagtga gagcttcctg tgtgactgcc agctcaagtg gctgccccca 1260 tggctaatgg gtaggatgct gcaggccttt gtgacagcca cctgtgccca tccagagtcg 1320 ctgaagggcc agagcatttt ctcagtgctg ccagacagct ttgtgtgtga tgactttcca 1380 aagccacaga tcatcaccca gcctgagacg accatggctg tggtgggcaa ggacatccgt 1440 ttcacatgct ccgcagccag cagcagcagc tcaccaatga ccttcgcctg gaagaaggac 1500 aatgaggtcc tggccaatgc agacatggag aactttgccc acgtccgtgc acaggacggc 1560 gaagtgatgg agtataccac tatcctgcac ctccgtcacg tcacctttgg gcacgagggc 1620 cgctaccagt gtatcatcac aaaccacttt ggctccacat actcccacaa agccaggctc 1680 actgtgaatg tgttgccatc attcactaaa ataccccatg acattgccat ccggactggc 1740 accacagccc gcctcgagtg tgctgccacg ggccacccta accctcagat tgcctggcag 1800 aaggatggag gcaccgattt cccggcagct cgtgagcgac gcatgcatgt tatgccagac 1860 gatgatgtgt tcttcatcac tgatgtgaaa atagacgaca tgggggtcta cagctgcact gcccagaact cggcaggctc ggtttcagcc aacgctaccc tcacagtctt agaaactcca tccttggcag tgcctctgga agaccgtgtg gtaactgtgg gagaaacagt ggccttccag tgcaaagcaa ccggggagccc cacaccacgc atcacctggc ttaaggggagg tcgcccattg agcctcacag agcgccacca tttcactcca ggcaaccagc tgctggttgt tcagaatgtg atgatagcg atgcagggcg gtatacctgt gagatgtcta atcccctggg cactgagcga gcacatagcc agctgagcat tttacctacc cctggctgcc ggaaggatgg gaccaccgta ggc 2283 <210> 5 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR <400> 5 Asn Tyr With Ser 1 5 <210> 6 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR 2 <400> 6 Val Ile Ser His Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly <210> 7 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain CDR 3 <400> 7 Val Ile Ser Asn Cys His Leu Gly Val Cys Tyr Tyr Ser Asn Gly Met 1 5 10 15 Asp Val <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR 1 <400> 8 Ser Gly Ser Ser Ser Asn Ile Gly Asp Asn Tyr Val Ser 1 5 10 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR 2 <400> 9 Asp Asn Asn Lys Arg Pro Ser 1 5 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Light chain CDR 3 <400> 10 Trp Asp Gly Ser Leu Ser Ala Gly Arg Val 1 5 10 <210> 11 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain variable region <400> 11 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser His Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Ile Ser Asn Cys His Leu Gly Val Cys Tyr Tyr Ser Asn 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 12 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Light chain variable region <400> 12 Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Arg Val Ser Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asp Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Gly Ser Leu 85 90 95 Ser Ala Gly Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 13 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain CDR 1 <400> 13 aactacgcca tgagc 15 <210> 14 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain CDR 2 <400> 14 gtgatctctc acggcggagg cagcacctac tacgccgatt ctgtgaaggg c 51 <210> 15 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain CDR 3 <400> 15 gtgatcagca actgccacct gggcgtgtgc tactacagca acggcatgga tgtg 54 <210> 16 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Light chain CDR 1 <400> 16 agcggcagca gcagcaacat cggcgacaac tacgtgtcc 39 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Light chain CDR 2 <400> 17 gacaacaaca agcggcccag c 21 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Light chain CDR 3 <400> 18 tgggatggct ccctgagcgc cggaagagtt 30 <210> 19 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain variable region <400> 19 gaagttcagc tgcttgagtc tggcggagga ctggttcaac ctggcggaag cctgagactg 60 tcttgtgccg ccagcggctt caccttcagc aactacgcca tgagctgggt ccgacaggcc 120 cctggaaaag gccttgaatg ggtgtccgtg atctctcacg gcggaggcag cacctactac 180 gccgattctg tgaagggcag attcaccatc agccgggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actattgcgc cagagtgatc 300 agcaactgcc acctgggcgt gtgctactac agcaacggca tggatgtgtg gggccagggc 360 acactggtta ccgttagttc t 381 <210> 20 <211> 333 <212> DNA <213> Artificial Sequence <220> <223> Light chain variable region <400> 20 caactggttc tgacacagcc tccaagcgtg tcagctgccc ctggacagag agtgtccatc 60 <h2 style=";text-align:left;direction:ltr">agctgtagcg gcagcagcag caacatcggc gacaactacg tgtcctggta tcagcaggtc 120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccaggctctg cccctaagct gctgatctac gacaacaaca agcggcccag cggcatcccc 180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatagatttt ctgccagcaa gagcggcacc agcgccacac tgggaattac aggactgcag 240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acaggcgacg aggccgacta ctattgtgcc acatgggatg gctccctgag cgccggaaga 300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gtttttggcg gaggcaccaa gctgaccgtg ctt 333<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 21<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 328<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> Mus musculus<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 21<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser<h2 style=";text-align:left;direction:ltr"> 50 55 60 Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys 65 70 75 80 Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu 85 90 95 Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala 100 105 110 Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile 115 120 125 Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val 130 135 140 Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val 145 150 155 160 Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp 165 170 175 Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln 180 185 190 Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp 195 200 205 Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val 210 215 220 Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr 225 230 235 240 Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu 245 250 255 Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr 260 265 270 Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr 275 280 285 Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr 290 295 300 Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys 305 310 315 320 Ser Phe Ser Arg Thr Pro Gly Lys 325 <210> 22 <211> 107 <212> PRT <213> Mus musculus <400> 22 Arg Thr Val Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 <210> 23 <211> 330 <212> PRT <213> Homo sapiens <400> 23 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 24 <211> 330 <212> PRT <213> Homo sapiens <400> 24 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 25 <211> 107 <212> PRT <213> Homo sapiens <400> 25 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 26 <211> 107 <212> PRT <213> Homo sapiens <400> 26 Arg Ser Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 27 <211> 326 <212> PRT <213> Homo sapiens <400> 27 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ser Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Lys 325 <210> 28 <211> 377 <212> PRT <213> Homo sapiens <400> 28 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180 185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300 Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305 310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 <210> 29 <211> 327 <212> PRT <213> Homo sapiens <400> 29 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 30 <211> 332 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain constant region <400> 30 Ala Ser Ala Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val 1 5 10 15 Cys Gly Asp Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys 20 25 30 Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu 35 40 45 Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr 50 55 60 Thr Leu Ser Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln 65 70 75 80 Ser Ile Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp 85 90 95 Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys 100 105 110 Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe 115 120 125 Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val 130 135 140 Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile 145 150 155 160 Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr 165 170 175 His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro 180 185 190 Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val 195 200 205 Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro 210 215 220 Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu 225 230 235 240 Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp 245 250 255 Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr 260 265 270 Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser 275 280 285 Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu 290 295 300 Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His 305 310 315 320 His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys 325 330 <210> 31 <211> 107 <212> PRT <213> Artificial Sequence <220> <223> Light chain constant region <400> 31 Arg Thr Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu 1 5 10 15 Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe 20 25 30 Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg 35 40 45 Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu 65 70 75 80 Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser 85 90 95 Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 100 105 <210> 32 <211> 245 <212> PRT <213> Artificial Sequence <220> <223> Hybrid Fc Heavy region <400> 32 Arg Asn Thr Gly Arg Gly Gly Glu Glu Lys Lys Lys Glu Lys Glu Lys 1 5 10 15 Glu Glu Gln Glu Glu Arg Glu Thr Lys Thr Pro Glu Cys Pro Ser His 20 25 30 Thr Gln Pro Leu Gly Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 35 40 45 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 50 55 60 Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 65 70 75 80 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser 85 90 95 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 100 105 110 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser 115 120 125 Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 130 135 140 Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln 145 150 155 160 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 165 170 175 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 180 185 190 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 195 200 205 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 210 215 220 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 225 230 235 240 Leu Ser Leu Gly Lys 245 <210> 33 <211> 990 <212> DNA <213> Homo sapiens <400> 33 gctagcacca agggcccatc ggtcttcccc ctggcgccct cctccaagag cacctctggg 60 ggcacagcgg cctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120 tggaactcag gggccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcctggg cacccagacc 240 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggaggag 720 atgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960 cagaagagcc tctccctgtc tccgggtaaa 990 <210> 34 <211> 321 <212> DNA <213> Homo sapiens <400> 34 agatctgtgg ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct 60 ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc caaagtacag 120 tggaaggtgg ataacgccct ccaatcgggt aactcccagg agagtgtcac agagcaggac 180 agcaaggaca gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag 240 aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag 300 agcttcaaca ggggagagtg t 321 <210> 35 <211> 999 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain constant region <400> 35 gctagcgcca aaacaacagc cccctccgta taccccctag cccccgtatg cggcgacacc 60 accggctcct ccgtaaccct aggctgccta gtaaaaggct acttccccga acccgtaacc 120 ctaacctgga actccggctc cctatcctcc ggcgtacaca ccttccccgc cgtactacaa 180 tccgacctat acaccctatc ctcctccgta accgtaacct cctccacctg gccctcccaa 240 tccatcacct gcaacgtagc ccaccccgcc tcctccacca aagtagacaa aaaaatcgag 300 ccaaggggcc ccaccatcaa gccttgccct ccctgcaagt gccctgcccc caacctgctg 360 ggcggcccct ccgtgttcat cttccctcca aagatcaagg acgtgctgat gatctccctg 420 agccccatcg tgacctgcgt ggtggtggac gtgtccgagg acgaccctga cgtgcagatc 480 agctggttcg tgaacaacgt ggaggtgcac accgcccaga cccagaccca cagagaggac 540 tacaactcca ccctgagggt ggtgagcgcc ctgccaatcc agcaccagga ctggatgtcc 600 ggcaaggagt tcaagtgcaa ggtgaacaac aaggacctgc cagccccaat cgagaggacc 660 atcagcaagc ctaagggctc cgtgagggcc ccccaggtgt acgtgctgcc ccctcctgag 720 gaggagatga ccaagaagca ggtgaccctg acctgcatgg tgaccgactt catgcctgag 780 gacatctacg tggagtggac caacaacggc aagaccgagc tgaactacaa gaacaccgag 840 cctgtgctgg acagcgacgg cagctacttc atgtacagca agctgagggt ggagaagaag 900 aactgggtgg agagaaactc ctactcctgc tccgtggtgc acgagggcct gcacaaccac 960 cacaccacca agagcttctc cagaaccccc gggaagtga 999 <210> 36 <211> 321 <212> DNA <213> Artificial Sequence <220> <223> Light chain constant region <400> 36 cgtacggatg ctgcaccaac tgtatccatc ttcccaccat ccagtgagca gttaacatct 60 ggaggtgcct cagtcgtgtg cttcttgaac aacttctacc ccaaagacat caatgtcaag 120 tggaagattg atggcagtga acgacaaaat ggcgtcctga acagttggac tgatcaggac 180 agcaaagaca gcacctacag catgagcagc accctcacgt tgaccaagga cgagtatgaa 240 cgacataaca gctatacctg tgaggccact cacaagacat caacttcacc cattgtcaag 300 agcttcaaca ggaatgagtg t 321 <210> 37 <211> 457 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 37 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser His Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Ile Ser Asn Cys His Leu Gly Val Cys Tyr Tyr Ser Asn 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 125 Lys Thr Thr Ala Pro Ser Val Tyr Pro Leu Ala Pro Val Cys Gly Asp 130 135 140 Thr Thr Gly Ser Ser Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Leu Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser 180 185 190 Ser Ser Val Thr Val Thr Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr 195 200 205 Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile 210 215 220 Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro 225 230 235 240 Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys 245 250 255 Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val 260 265 270 Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe 275 280 285 Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu 290 295 300 Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His 305 310 315 320 Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys 325 330 335 Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser 340 345 350 Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met 355 360 365 Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro 370 375 380 Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn 385 390 395 400 Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met 405 410 415 Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser 420 425 430 Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr 435 440 445 Lys Ser Phe Ser Arg Thr Pro Gly Lys 450 455 <210> 38 <211> 457 <212> PRT <213> Artificial Sequence <220> <223> Heavy chain <400> 38 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser His Gly Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Ile Ser Asn Cys His Leu Gly Val Cys Tyr Tyr Ser Asn 100 105 110 Gly Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 125 Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser 130 135 140 Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe 145 150 155 160 Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly 165 170 175 Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu 180 185 190 Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr 195 200 205 Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys 210 215 220 Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro 225 230 235 240 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 245 250 255 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 260 265 270 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 275 280 285 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 290 295 300 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 305 310 315 320 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 325 330 335 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 340 345 350 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 355 360 365 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 370 375 380 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 385 390 395 400 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 405 410 415 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 420 425 430 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 435 440 445 Lys Ser Leu Ser Leu Ser Pro Gly Lys 450 455 <210> 39 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 39 Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Arg Val Ser Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asp Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Gly Ser Leu 85 90 95 Ser Ala Gly Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg 100 105 110 Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln 115 120 125 Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr 130 135 140 Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln 145 150 155 160 Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg 180 185 190 His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro 195 200 205 Ile Val Lys Ser Phe Asn Arg Asn Glu Cys 210 215 <210> 40 <211> 218 <212> PRT <213> Artificial Sequence <220> <223> Light chain <400> 40 Gln Leu Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Arg Val Ser Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asp Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Val Pro Gly Ser Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Gly Ser Leu 85 90 95 Ser Ala Gly Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 41 <211> 1380 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain <400> 41 gaagttcagc tgcttgagtc tggcggagga ctggttcaac ctggcggaag cctgagactg 60 tcttgtgccg ccagcggctt caccttcagc aactacgcca tgagctgggt ccgacaggcc 120 cctggaaaag gccttgaatg ggtgtccgtg atctctcacg gcggaggcag cacctactac 180 gccgattctg tgaagggcag attcaccatc agccgggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actattgcgc cagagtgatc 300 agcaactgcc acctgggcgt gtgctactac agcaacggca tggatgtgtg gggccagggc 360 acactggtta ccgttagttc tgctagcgcc aaaacaacag ccccctccgt atacccccta 420 gcccccgtat gcggcgacac caccggctcc tccgtaaccc taggctgcct agtaaaaggc 480 tacttccccg aacccgtaac cctaacctgg aactccggct ccctatcctc cggcgtacac 540 accttccccg ccgtactaca atccgaccta tacaccta cctcctccgt aaccgtacc 600 tcctccacct ggccctccca atccatcacc tgcaacgtag cccaccccgc ctcctccacc 660 aaagtagaca aaaaaatcga gccaaggc cccaccatca agccttgccc tccctgcaag 720 tgccctgccc ccaacctgct gggcggcccc tccgtgttca tctccctcc aagatcaag 780 gacgtgctga tgatctccct gagccccatc gtgacctgcg tggtggtgga cgtgtccgag 840 gacgaccctg acgtgcagat cagctggttc gtgacaacg tggaggtgca caccgcccag 900 acccagaccc acagagagga ctacaactcc accctgaggg tggtgagcgc cctgccaatc 960 cagcaccagg actggactgtc cggcaggag ttcagtgca aggtgacaa caggacctg 1020 ccagccccaa tcgaggaggac catcagcaag cctaagggct ccgtgaggc cccccaggtg 1080 tacgtgctgc cccctcctga gaggagatg accagaagc aggtgaccct gacctgcatg 1140 gtgaccgact tcatgcctga ggacatctac gtggagtgga ccaacaacgg caagaccgag 1200 ctgaactaca agaacaccga gcctgtgctg gacagcgacg gcagctactt catgtacagc 1260 aagctgaggg tggagaagaa gaactgggtg gagagaaact cctactcctg ctccgtggtg 1320 cacgagggcc tgcacaacca ccacaccacc aagagcttct ccagaacccc cgggaagtga 1380 1380 <210> 42 <211> 1371 <212> DNA <213> Artificial Sequence <220> <223> Heavy chain <400> 42 gaagttcagc tgcttgagtc tggcggagga ctggttcaac ctggcggaag cctgagactg 60 tcttgtgccg ccagcggctt caccttcagc aactacgcca tgagctgggt ccgacaggcc 120 cctggaaaag gccttgaatg ggtgtccgtg atctctcacg gcggaggcag cacctactac 180 gccgattctg tgaagggcag attcaccatc agccgggaca acagcaagaa caccctgtac 240 ctgcagatga acagcctgag agccgaggac accgccgtgt actattgcgc cagagtgatc 300 agcaactgcc acctgggcgt gtgctactac agcaacggca tggatgtgtg gggccagggc 360 acactggtta ccgttagttc tgctagcacc aagggcccat cggtcttccc cctggcgccc 420 tcctccaaga gcacctctgg gggcacagcg gccctgggct gcctggtcaa ggactacttc 480 cccgaaccgg tgacggtgtc gtggaactca ggggccctga ccagcggcgt gcacaccttc 540 ccggctgtcc tacagtcctc aggactctac tccctcagca gcgtggtgac cgtgccctcc 600 agcagcctgg gcacccagac ctacatctgc aacgtgaatc acaagcccag caacaccaag 660 gtggacaaga aagttgagcc caaatcttgt gacaaaactc acacatgccc accgtgccca 720 gcacctgaac tcctgggggg accgtcagtc ttcctcttcc ccccaaaacc caaggacacc 780 ctcatgatct cccggacccc tgaggtcaca tgcgtggtgg tggacgtgag ccacgaagac 840 cctgaggtca agttcaactg gtacgtggac ggcgtggagg tgcataatgc caagacaaag 900 ccgcgggagg agcagtacaa cagcacgtac cgtgtggtca gcgtcctcac cgtcctgcac 960 caggactggc tgaatggcaa ggagtacaag tgcaaggtct ccaacaaagc cctcccagcc 1020 cccatcgaga aaaccatctc caaagccaaa gggcagcccc gagaaccaca ggtgtacacc 1080 ctgcccccat cccgggagga gatgaccaag aaccaggtca gcctgacctg cctggtcaaa 1140 ggcttctatc ccagcgacat cgccgtggag tgggagagca atgggcagcc ggagaacaac 1200 tacaagacca cgcctcccgt gctggactcc gacggctcct tcttcctcta cagcaagctc 1260 accgtggaca agagcaggtg gcagcagggg aacgtcttct catgctccgt gatgcatgag 1320 gctctgcaca accactacac gcagaagagc ctctccctgt ctccgggtaa a 1371 <210> 43 <211> 654 <212> DNA <213> Artificial Sequence <220> <223> Light chain <400> 43 caactggttc tgacacagcc tccaagcgtg tcagctgccc ctggacagag agtgtccatc 60 agctgtagcg gcagcagcag caacatcggc gacaactacg tgtcctggta tcagcaggtc 120 ccaggctctg cccctaagct gctgatctac gacaacaaca agcggcccag cggcatcccc 180 gatagatttt ctgccagcaa gagcggcacc agcgccacac tgggaattac aggactgcag 240 acaggcgacg aggccgacta ctattgtgcc acatgggatg gctccctgag cgccggaaga 300 gtttttggcg gaggcaccaa gctgaccgtg cttcgtacgg atgctgcacc aactgtatcc 360 atcttcccac catccagtga gcagttaaca tctggaggtg cctcagtcgt gtgcttcttg 420 aacaacttct accccaaaga catcaatgtc aagtggaaga ttgatggcag tgaacgacaa 480 aatggcgtcc tgaacagttg gactgatcag gacagcaaag acagcaccta cagcatgagc 540 agcaccctca cgttgaccaa ggacgagtat gaacgacata acagctatac ctgtgaggcc 600 actcacaaga catcaacttc acccattgtc aagagcttca acaggaatga gtgt 654 <210> 44 <211> 654 <212> DNA <213> Artificial Sequence <220> <223> Light chain <400> 44 caactggttc tgacacagcc tccaagcgtg tcagctgccc ctggacagag agtgtccatc 60 agctgtagcg gcagcagcag caacatcggc gacaactacg tgtcctggta tcagcaggtc 120 ccaggctctg cccctaagct gctgatctac gacaacaaca agcggcccag cggcatcccc 180 gatagatttt ctgccagcaa gagcggcacc agcgccacac tgggaattac aggactgcag 240 acaggcgacg aggccgacta ctattgtgcc acatgggatg gctccctgag cgccggaaga 300 gtttttggcg gaggcaccaa gctgaccgtg cttagatctg tggctgcacc atctgtcttc 360 atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 420 aataacttct atcccagaga ggccaaagta cagtggaagg tggataacgc cctccaatcg ggtaactccc aggagagtgt cacagagcag cacagcaagg acagcaccta cagcctcagc 540 agcaccctga cgctgagcaa agcacctc gagaaacaca aagtctacgc ctgcgaagtc acccatcagg gcctgagctc gcccgtcaca aagagcttca acaggggaga gtgt 654 <210> 45 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mouse Lrig-1 forward primer <400> 45 gacggaattc rich acct <210> 46 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mouse Lrig-1 reverse primer <400> 46 caactggtag tggcagcttg tag <210> 47 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mouse Lrig-2 forward primer <400> 47 tcacaaggaa cattgtctga acca 24 <210> 48 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mouse Lrig-2 reverse primer <400> 48 gcctgatcta acacatcctc ctca 24 <210> 49 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mouse Lrig-3 forward primer <400> 49 cagcaccttg agctgaacag aaac 24 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mouse Lrig-3 reverse primer <400> 50 ccagcctttg gtaatctcgg ttag 24 <210> 51 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mouse FOXP3 forward primer <400> 51 ctttcaccta tcccaccctt atcc 24 <210> 52 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> mouse FOXP3 reverse primer <400> 52 attcatctac ggtccacact gctc 24 <210> 53 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACTG1 forward primer <400> 53 ggcgtcatgg tgggcatggg 20 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ACTG1 reverse primer <400> 54 atggcgtggg gaagggcgta 20

Claims

1. A heavy chain variable region comprising: a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and A binding molecule comprising a light chain variable region including: a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:8; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:9; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:

10.

2. The binding molecule comprises a heavy chain variable region including a heavy chain CDR1 encoded by the base sequence represented by SEQ ID NO: 13; a heavy chain CDR2 encoded by the base sequence represented by SEQ ID NO: 14; a heavy chain CDR3 encoded by the base sequence represented by SEQ ID NO: 15; and The binding molecule of claim 1, comprising a light chain variable region comprising: a light chain CDR1 encoded by the base sequence represented by SEQ ID NO: 16; a light chain CDR2 encoded by the base sequence represented by SEQ ID NO: 17; and a light chain CDR3 encoded by the base sequence represented by SEQ ID NO:

18.

3. The binding molecule comprises a heavy chain variable region consisting of the amino acid sequence represented by SEQ ID NO: 11; and The binding molecule of claim 1 , comprising a light chain variable region consisting of the amino acid sequence represented by SEQ ID NO:

12.

4. The binding molecule comprises a heavy chain variable region encoded by the base sequence represented by SEQ ID NO: 19; and The binding molecule of claim 1 , comprising a light chain variable region encoded by the base sequence represented by SEQ ID NO:

20.

5. The binding molecule of claim 1 , further comprising an Fc region (Fragment crystallization region) or a constant region.

6. 6. The binding molecule of claim 5, wherein the Fc region is an Fc region of an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 antibody, or a hybrid Fc region.

7. The binding molecule of claim 5, further comprising a heavy chain constant region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 23, 24, 27, 28, 29, 30 and 32.

8. The binding molecule of claim 5, further comprising a light chain constant region consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 25, 26 and 31.

9. The binding molecule comprises: A heavy chain constant region encoded by the base sequence represented by SEQ ID NO: 33 or 35; and The binding molecule of claim 5 , further comprising a light chain constant region encoded by the base sequence represented by SEQ ID NO: 34 or 36.

10. The binding molecule comprises: A heavy chain consisting of the amino acid sequence represented by SEQ ID NO: 37 or 38; and The binding molecule of claim 1 , comprising a light chain consisting of the amino acid sequence represented by SEQ ID NO: 39 or 40.

11. The binding molecule of claim 10, comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO:37; and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

39.

12. The binding molecule of claim 10, comprising a heavy chain consisting of the amino acid sequence represented by SEQ ID NO:38; and a light chain consisting of the amino acid sequence represented by SEQ ID NO:

40.

13. The binding molecule comprises: A heavy chain encoded by the base sequence represented by SEQ ID NO: 41 or 43; and The binding molecule of claim 10, comprising a light chain encoded by the base sequence represented by SEQ ID NO: 42 or 44.

14. The binding molecule of claim 1 , comprising a heavy chain encoded by the base sequence represented by SEQ ID NO: 41; and a light chain encoded by the base sequence represented by SEQ ID NO:

42.

15. The binding molecule of claim 1 , comprising a heavy chain encoded by the base sequence represented by SEQ ID NO: 43; and a light chain encoded by the base sequence represented by SEQ ID NO:

44.

16. The binding molecule of claim 1 , wherein the binding molecule is an antibody or a fragment thereof.

17. 17. The binding molecule of claim 16, wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody, a tetrabody or a fragment thereof.

18. A nucleic acid molecule encoding a binding molecule according to any one of claims 1 to 17.

19. An expression vector into which the nucleic acid molecule of claim 18 has been inserted.

20. 20. A host cell line transfected with the expression vector of claim 19.

21. A heavy chain variable region comprising: a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and An antibody comprising a light chain variable region including: a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 8; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 9; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO: 10; and an antibody-drug conjugate (ADC) comprising a drug.

22. A pharmaceutical composition for preventing or treating a brain and nervous system disease, comprising the binding molecule according to any one of claims 1 to 17 or 21 as an active ingredient.

23. The pharmaceutical composition according to claim 22, wherein the cranial nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

24. The neurodegenerative or neuroinflammatory disease may be stroke, cognitive impairment, Alzheimer's disease (AD), or other neurodegenerative or neuroinflammatory diseases. Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, dementia with Lewy bodies, amyotrophic lateral sclerosis (AlzAmyotrophic lateral sclerosis), paraneoplastic syndrome (Parathyropathy), 24. The pharmaceutical composition of claim 23, wherein the therapeutic agent is selected from the group consisting of: chronic myocardial infarction syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain disorders, cerebrovascular diseases, spinal cord injury and tauopathy.

25. In a chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain and a CD3 zeta (ζ) signaling domain, The antigen-specific binding domain comprises a heavy chain variable region including a heavy chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO:5; a heavy chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO:6; and a heavy chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:7; and A chimeric antigen receptor which is a light chain variable region comprising: a light chain CDR1 consisting of the amino acid sequence represented by SEQ ID NO: 8; a light chain CDR2 consisting of the amino acid sequence represented by SEQ ID NO: 9; and a light chain CDR3 consisting of the amino acid sequence represented by SEQ ID NO:

10.

26. A pharmaceutical composition for preventing or treating a brain and nervous system disease, comprising the chimeric antigen receptor according to claim 25 as an active ingredient.

27. A composition for diagnosing a brain and nervous system disease, comprising the binding molecule of claim 1.

28. A diagnostic kit for a central nervous system disease comprising the composition according to claim 27.

Images