Novel antibodies affinity matured

Abstract

The present application relates to a binding molecule capable of specifically binding to Lrig-1 protein, which is a protein expressed on the surface of regulatory T cells. The binding molecule provided by the present application can inhibit the function of regulatory T cells, thereby effectively preventing, improving or treating cancer, especially solid tumors. In addition, compared with commercially available antibodies against Lrig-1, the binding molecule provided by the present application can more effectively target Lrig-1 protein and show excellent binding affinity.

Description

Technical Field

[0001] This invention relates to a novel binding molecule that exhibits superior binding affinity for the Lrig-1 antigen compared to existing antibodies; it also relates to the application of this binding molecule in the prevention, improvement, or treatment of cancer, immune-related diseases, and neurological disorders. Background Technology

[0002] One of the most important characteristics of all normal individuals is the ability to recognize and eliminate non-self antigens without reacting harmfully to antigens that constitute the self. Therefore, the lack of response to self antigens in an organism is called immune non-responsiveness or immune tolerance. Self-tolerance arises either by clearing lymphocytes that may have specific receptors for self antigens or by the inactivation of the organism's ability to react upon contact with self antigens. If the induction or maintenance of self-tolerance is impaired, an immune response against self antigens will occur, resulting in diseases known as autoimmune diseases.

[0003] To treat autoimmune diseases, Gershon first proposed and demonstrated the concept of suppressor T cells in the early 1970s, suggesting the possible existence of T cells capable of controlling and suppressing the effector functions of regular T cells (RK Gershon and K. Kondo, Immunology, 1970, 18: 723-37). Since then, extensive research has been conducted to elucidate the biological characteristics and functions of regulatory T cells in numerous areas of immunology. Studies have shown that regulatory T cells (Treg cells) play a crucial role in the natural prevention of excessive inflammation and immune responses; however, the function and number of regulatory T cells are significantly reduced in autoimmune and chronic inflammatory diseases. Therefore, for patients with immune and inflammatory diseases, generating regulatory T cells at normal levels is crucial and could be one of the treatment options for these diseases. Summary of the Invention

[0004] Technical issues

[0005] One object of the present invention is to provide a binding molecule that, compared with previously known antibodies, has a superior binding capacity to the Lrig-1 protein expressed on the surface of regulatory T cells (Treg cells) and exhibits greater efficacy in the prevention, improvement or treatment of immune-related diseases.

[0006] Another object of the present invention is to provide a composition for the prevention, improvement or treatment of nervous system diseases, said composition comprising the binding molecules of the present invention.

[0007] Another object of the present invention is to provide a composition for the prevention, improvement or treatment of cancer, said composition comprising the binding molecules of the present invention.

[0008] Another object of the present invention is to provide: a composition for diagnosing cancer, immune-related diseases, or neurological diseases; a kit comprising said composition; and a method for providing information for diagnosing cancer, immune-related diseases, or neurological diseases.

[0009] However, the technical problem to be solved by the present invention is not limited to the above-mentioned problem. Other technical problems not mentioned can be clearly understood by those skilled in the art through the following description. Detailed Implementation

[0010] Technical solution

[0011] The various embodiments described herein will be described below with reference to the accompanying drawings. Numerous specific details, such as specific constructions, compositions, and methods, are set forth in the following description to provide a thorough understanding of the invention. However, in practice, some embodiments may omit more than one specific detail or be combined with other known methods and constructions. In other instances, known methods and preparation techniques are not described in particular detail to avoid unnecessarily obscuring the invention. Throughout this specification, the phrase "an embodiment" or "one embodiment" refers to a specific feature, construction, composition, or characteristic described in association with that embodiment, which is included in at least one embodiment of the invention. Therefore, the expressions "in an embodiment" or "an embodiment" appearing in different places throughout this specification do not necessarily refer to the same embodiment of the invention. Furthermore, these specific features, constructions, compositions, or characteristics can be combined in any suitable manner in one or more embodiments.

[0012] As used herein, the term "binding molecule" refers to a variable region comprising either an intact immunoglobulin (including monoclonal antibodies, such as chimeric antibodies, humanized antibodies, or human monoclonal antibodies) or an antigen-binding immunoglobulin (e.g., an immunoglobulin fragment that competes with the intact immunoglobulin for binding to influenza A virus monomeric HA or trimeric HA). Regardless of structure, the antigen-binding fragment binds to the same antigen recognized by the intact immunoglobulin. The antigen-binding fragment may include a peptide or polypeptide, said peptide or polypeptide comprising the following amino acid sequences in the binding molecule: two or more consecutive amino acid residues, 20 or more consecutive amino acid residues, 25 or more consecutive amino acid residues, 30 or more consecutive amino acid residues, 35 or more consecutive amino acid residues, 40 or more consecutive amino acid residues, 50 or more consecutive amino acid residues, 60 or more consecutive amino acid residues, 70 or more consecutive amino acid residues, 80 or more consecutive amino acid residues, 90 or more consecutive amino acid residues, 100 or more consecutive amino acid residues, 125 or more consecutive amino acid residues, 150 or more consecutive amino acid residues, 175 or more consecutive amino acid residues, 200 or more consecutive amino acid residues, or 250 or more consecutive amino acid residues. The term "antigen-binding fragment" specifically includes 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, diabody, triabody, tetrabody, polypeptides containing one or more immunoglobulin fragments (said to be sufficient to bind a specific antigen to the polypeptide), and so on. This fragment can be produced by synthetic methods or by enzymatic or chemical digestion of intact immunoglobulins, or by genetic engineering methods using recombinant DNA technology. Production methods are well known in the art.

[0013] The term "Lrig-1 (leucine-rich and immunoglobulin-like domain 1) protein" as used in this article refers to a 1091-amino acid transmembrane protein present on the surface of regulatory T cells, consisting of a leucine-rich repeat sequence (LRR) on the extracellular (or luminal) side, three immunoglobulin-like domains, a transmembrane sequence, and a cytoplasmic tail. The LRIG gene family includes LRIG1, LRIG2, and LRIG3, whose amino acid sequences are highly conserved. The LRIG1 gene is highly expressed in normal skin and can be expressed in basal cells and hair follicle cells to regulate the proliferation of epithelial stem cells. Therefore, the LRIG1 gene plays an important role in maintaining epidermal homeostasis, and its deletion may lead to psoriasis or skin cancer. It has been reported that if the portion of chromosome 3p14.3 containing LRIG1 is cleaved, cancer cells may develop. In fact, significantly reduced expression of LRIG1 has been identified in renal cell carcinoma and squamous cell carcinoma of the skin. Recent studies have also found that Lrig-1 is expressed in only about 20% to 30% of cancers. On the other hand, for the purposes of this invention, the Lrig-1 protein may be, but is not limited to, a protein present in humans or mice.

[0014] In this invention, the binding molecule may also include a crystalline fragment (Fc) region or a constant region. Here, the Fc region may be the Fc region of an IgG1, IgG2, IgG3, or IgG4 antibody, or derived from these Fc regions. Alternatively, the Fc region may be a hybrid Fc region.

[0015] In this invention, the Fc region can be the Fc region of mammalian-derived IgG1, IgG2, IgG3, or IgG4 antibodies, preferably the Fc region of human-derived IgG1, IgG2, IgG3, or IgG4 antibodies. However, the Fc region is not limited to this.

[0016] As an example of the present invention, the Fc region may be the λ constant region of human-derived immunoglobulins, but is not limited thereto.

[0017] In this invention, the "hybrid Fc" can be derived from a combination of human IgG subclasses or a combination of human IgD and IgG. When the hybrid Fc binds to bioactive molecules, peptides, etc., the role of the hybrid Fc is not only to prolong the serum half-life of the bioactive molecules, but also to increase the expression level of the peptide when the nucleotide sequence encoding the Fc-peptide fusion protein is expressed.

[0018] In the binding molecule of the present invention, the Fc or constant region can be connected to the variable region via a connector. Here, the connector can be connected to the C-terminus of the Fc, and the N-terminus of the binding molecule of the present invention can be connected to the connector. However, the present invention is not limited thereto.

[0019] In this invention, the "connector" may comprise a sequence that can be cleaved by an enzyme, wherein the enzyme is overexpressed in tissues or cells with the target disease. When the connector can be cleaved by the overexpressed enzyme (as described above), the reduction in peptide activity due to the Fc moiety can be effectively prevented. In this invention, an example of a connector is preferably a peptide connector consisting of 33 amino acids located at positions 282 to 314 of human albumin (the most abundant protein in blood), more preferably a peptide connector consisting of 13 amino acids located at positions 292 to 304 of human albumin. These portions are the parts primarily exposed externally in the three-dimensional structure, thus minimizing the likelihood of inducing an immune response in vivo. However, the connector is not limited to this.

[0020] The binding molecule of the present invention is characterized in that it is an antibody, but is not limited thereto. The antibody includes all monoclonal antibodies, full-length antibodies, or antibody fragments (parts of an antibody) capable of binding to the Lrig-1 protein and competing with the binding molecule of the present invention for binding to epitopes on Lrig-1.

[0021] As used herein, the term "antibody" refers to a protein molecule that acts as a receptor for specifically recognizing an antigen, including immunoglobulin molecules that react with a specific antigen. For the purposes of this 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.

[0022] In this invention, "immunoglobulin" has heavy and light chains, each containing constant and variable regions. The variable regions of each light and heavy chain contain three hypervariable regions called complementarity-determining regions (hereinafter referred to as "CDRs") and four framework regions. The primary function of CDRs is to bind to epitopes on antigens. The CDRs on each chain are typically named CDR1, CDR2, and CDR3 sequentially, starting from the N-terminus, and are further distinguished by the chain in which the specific CDR is located.

[0023] Furthermore, as used herein, the term "monoclonal antibody" refers to an antibody molecule obtained from a substantially identical population of antibodies that has a single molecular composition and exhibits a single binding specificity and affinity for a particular epitope.

[0024] In this invention, the structure of the "full-length antibody" has two full-length light chains and two full-length heavy chains, wherein each light chain is linked to the heavy chain by a disulfide bond. The "full-length antibody" includes IgA, IgD, IgE, IgM, and IgG. IgG includes IgG1, IgG2, IgG3, and IgG4 as its subtypes.

[0025] Furthermore, as used herein, the term "antigen fragment" refers to a fragment that retains antigen-binding function, including Fab, Fab', F(ab')2, Fv, etc. Fab has a structure with variable regions in the light and heavy chains, a constant region in the light chain, and a first constant region (CH1 domain) in the heavy chain. Fab has one antigen-binding site. Furthermore, Fab' differs from Fab in that Fab' has a hinge region containing at least one cysteine ​​residue at the C-terminus of the CH1 domain in the heavy chain. F(ab')2 antibodies are generated by forming disulfide bonds through the cysteine ​​residues in the Fab' hinge region. Fv (variable fragment) refers to the smallest antibody fragment containing only the variable regions in the heavy and light chains. Double-chain Fv (dsFv) is constructed with the variable regions in the heavy and light chains linked together by disulfide bonds, while single-chain Fv (scFv) is constructed with the variable regions in the heavy and light chains covalently linked together, usually through peptide linkers. Antibody fragments can be obtained in the form of Fab or F(ab')2 fragments (when using proteolytic enzymes such as papain or pepsin)) or produced by recombinant genetic technology.

[0026] Furthermore, in this invention, the antibody may be, but is not limited to, chimeric antibodies, humanized antibodies, bivalent bispecific molecules, minibody, domain antibody, bispecific antibody, antibody mimic, biantibody, triantibody or tetraantibody, or fragments thereof.

[0027] In this invention, the "chimeric antibody" is an antibody obtained by recombination of the variable region of a mouse antibody with the constant region of a human antibody, which significantly enhances the immune response compared to the mouse antibody.

[0028] Furthermore, as used herein, the term "humanized antibody" refers to an antibody obtained by modifying the protein sequence of a non-human species-derived antibody so that the protein sequence is similar to a naturally occurring human antibody variant. For example, humanized antibodies can be prepared by recombining a mouse-derived CDR with a human antibody-derived FR to prepare a humanized variable region, which can then be recombined with a preferred constant region of a human antibody to prepare the humanized antibody. In this invention, the binding molecule can be provided in the form of a bispecific antibody or a bispecific antigen-binding fragment capable of binding to the Lrig-1 protein and also capable of binding to another protein.

[0029] In this invention, the binding molecule can bind to the Lrig-1 protein, or it can be provided in the form of a bispecific antibody or bispecific antigen binding fragment capable of binding to Lrig-1 and at least one other protein.

[0030] In this invention, the bispecific antibody and the bispecific antigen-binding fragment may comprise the binding molecule of this invention. As an example of this invention, the bispecific antibody and the bispecific antigen-binding fragment comprise an antigen-binding domain capable of binding to the Lrig-1 protein, wherein the antigen-binding domain capable of binding to the Lrig-1 protein may comprise the binding molecule of this invention or be composed of the binding molecule of this invention.

[0031] The bispecific antibody and bispecific antigen-binding fragment provided by this invention comprise an antigen-binding domain (which is the binding molecule of this invention capable of binding to the Lrig-1 protein) and an antigen-binding domain capable of binding to another target protein. Here, the antigen-binding domain capable of binding to the other target protein can be an antigen-binding domain capable of binding to proteins other than the Lrig-1 protein (e.g., but not limited to PD-1 or cell surface receptors). However, the antigen-binding domain is not limited to this.

[0032] The bispecific antibody and bispecific antigen binding fragment of the present invention may be provided in any suitable form, such as the form described in “Kontermann MAbs 2012, 4(2): 182-197”, the full text of which is incorporated herein by reference. For example, bispecific antibodies or bispecific antigen-binding fragments can be bispecific antibody conjugates (e.g., IgG2, F(ab')2, or CovX-body), bispecific IgG or IgG-like molecules (e.g., IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, or dual IgG, mAb2, or tandem ab universal LC), asymmetric bispecific IgG or IgG-like molecules (e.g., kih IgG, kih IgG universal LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair, or SEED-body), small bispecific antibody molecules (e.g., bispecific antibody (Db), dsDb, DART, scDb, tandAb, tandem scFv (taFv), tandem dAb / VHH, triple body, triple body). (e.g., head), Fab-scFv or F(ab')2-scFv2), bispecific Fc and CH3 fusion proteins (e.g., taFv-Fc, di-biantibody, 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-cytokine 2). The bispecific antibodies and bispecific antigen-binding fragments of the present invention can be designed and prepared by those skilled in the art.

[0033] The method for preparing the bispecific antibody of the present invention includes forming a reduced disulfide bond or a non-reduced thioether bond, and chemically cross-linking the antibody or antibody fragment. For example, N-succinimide-3-(2-pyridyldithio)propionate (SPDP) can be used to chemically cross-link the Fab fragment through the SH- group in the hinge region, thereby generating a disulfide-linked bispecific F(ab)2 heterodimer.

[0034] In addition, an alternative method for preparing the bispecific antibody of the present invention includes using, for example, polyethylene glycol to fuse antibody-producing hybridoma cells to produce quadroma cells capable of secreting bispecific antibodies.

[0035] The bispecific antibody and bispecific antigen-binding fragment of the present invention can also be prepared, for example, by recombination of a nucleic acid construct expressing an antigen-binding polypeptide.

[0036] For example, a DNA construct can be prepared using molecular cloning techniques. This construct comprises sequences encoding light chain variable domains and heavy chain variable domains encoding two antigen-binding domains (i.e., light chain variable domains and heavy chain variable domains capable of binding the antigen-binding domain of PD-1, and light chain variable domains and heavy chain variable domains capable of binding the antigen-binding domain of another target protein), and sequences encoding suitable linker or dimerizing domains between the antigen-binding domains. Subsequently, a recombinant bispecific antibody can be prepared by expressing the construct in a suitable host cell (e.g., mammalian host cells) (e.g., in vitro expression), and optionally, the expressed recombinant bispecific antibody can then be purified.

[0037] Antibodies can be prepared via an affinity maturation process, in which modified antibodies are produced that exhibit enhanced antigen affinity compared to unmodified parent antibodies. Affinity-matured antibodies can be prepared using methods known in the art.

[0038] Furthermore, the binding molecules provided by this invention may include variants of the amino acid sequence, as long as the variant can specifically bind to the Lrig-1 protein. For example, the amino acid sequence of the antibody may be modified to improve its binding affinity and / or other biological properties. Such modifications include, for example, the deletion, insertion, and / or substitution of amino acid sequence residues in the antibody.

[0039] Such amino acid variations are based on the relative similarity (e.g., hydrophobicity, hydrophilicity, charge, and size) of amino acid side chain substituents. Analysis of the size, shape, and type of the amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine are similar in size; and phenylalanine, tryptophan, and tyrosine are similar in shape. Therefore, based on these considerations, arginine, lysine, and histidine, alanine, glycine, and serine, and phenylalanine, tryptophan, and tyrosine can be considered biologically equivalent.

[0040] When introducing variations, the hydropathic index of amino acids can be considered. Each amino acid is assigned a hydropathic index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); arginine (-4.5). The hydropathic index of amino acids is crucial for conferring biological functions to protein interactions. It is known that replacing proteins with amino acids with similar hydropathic indices can preserve similar biological activities. When a variation is introduced in the reference hydrophilicity index, substitutions are made between amino acids whose hydrophilicity index difference is preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0041] Furthermore, it is well known that substitutions between amino acids with similar hydrophilicity values ​​can produce proteins with equivalent biological activities. As disclosed in U.S. Patent No. 4,554,101, the amino acid residues are assigned the following hydrophilicity values: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+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 a variation is introduced in the reference hydrophilicity value, substitutions can be made between amino acids whose hydrophilicity value difference is preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0042] It is known in the art that amino acid substitutions in proteins do not completely alter the activity of the molecule. The most common substitutions are between the following amino acid residues: Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, and Gln / Glu.

[0043] In view of the above-mentioned variations with bioequivalent activity, it can be understood that the binding molecules of the present invention also include sequences that are substantially identical to the sequences listed in the sequence listing.

[0044] As used herein, the term "substantial identity" refers to a sequence that, when aligned with any other sequence to correspond to it to the greatest extent possible, and when analyzed using algorithms commonly used in the art, exhibits at least 61% homology, more preferably 70%, more preferably 80%, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various alignment methods and algorithms are available from NCBI's Basic Local Alignment Search Tool (BLAST), the National Center for Biotechnology Information (NCBI), and can be used in conjunction with sequence analysis programs available on the Internet (e.g., blastp, blasm, blastx, tblastn, and tblastx). BLAST is accessible at ncbi.nlm.nih.gov / BLAST / . Sequence homology comparison methods using this program can be found online (ncbi.nlm.nih.gov / BLAST / blast_help.html).

[0045] In this invention, the binding molecule (preferably an antibody) can be prepared by conventional methods for preparing antibodies, or by affinity maturation.

[0046] As used herein, the terms “H6 antibody” and “GTC110-04 antibody” refer to the same known monoclonal antibody that is effective in preventing and treating cancer and neurological diseases.

[0047] As used herein, the terms “GTC310-01 antibody” and “1C07 antibody” refer to the same known monoclonal antibody that is effective in preventing and treating neurological diseases.

[0048] As used herein, the term "affinity maturation" refers to the process by which activated B cells produce antibodies with higher affinity for antigens during an immune response. For the purposes of this invention, affinity maturation enables the production of antibodies or antibody fragments through a process identical to the spontaneously occurring process, based on the principles of mutation and selection.

[0049] The ten affinity-matured antibody clones (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01) provided by this invention are all derived from the previously described H6 antibody or GTC310-01 antibody. In some clones (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, and MH6), only one of the six complementarity-determining regions (CDRs) of the parental GTC310-01 antibody is partially modified. In clone 6F01, three CDRs contained in the light chain are modified. Compared with the parental H6 or GTC310-01 antibody, the affinity-matured clones exhibit increased binding affinity to Lrig-1. Because only a portion of the six total CDRs are modified, the affinity-matured antibodies retain the pharmacological activity of the parental H6 or GTC310-01 antibody while exhibiting higher binding affinity to the Lrig-1 antigen.

[0050] Generally, the biological or pharmacological effects of an antibody or antigen-binding fragment are influenced by the antigen to which it is bound. Therefore, it is generally understood in the art that an antibody with a higher binding affinity to the same antigen is expected to exhibit pharmacological effects comparable to or improved upon those of a previously known antibody with established pharmacological activity.

[0051] In particular, the ten affinity-matured antibody clones (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01) presented herein exhibit significantly enhanced binding affinity compared to previously characterized GTC310-01 or H6 antibodies with established pharmacological activity. Specifically, the affinity-matured clones exhibit equilibrium dissociation constant (KD) values ​​approximately 2 to 737 times lower than the KD values ​​of GTC310-01 or H6, where a lower KD value indicates higher binding affinity. Furthermore, when administered at a dose of 5 µg, the affinity-matured clones showed improved binding affinity to CD4+. + FoxP3 + Lrig1 + The binding rate of regulatory T cells is at least approximately 90%.

[0052] Therefore, those skilled in the art can reasonably expect that the antibody provided by this invention (whose KD value is about 2 to 737 times lower than the KD value of the previously characterized GTC310-01 antibody, wherein a lower KD value indicates higher binding affinity; and / or, after administration of 5 µg, with CD4 + FoxP 3+ Lrig1 + (A binding rate of at least about 90% to regulatory T cells) will exhibit pharmacological activity comparable to or improved upon that of previously established H6 or GTC310-01 antibodies.

[0053] The binding molecules (preferably antibodies) provided by this invention can be used to prevent, improve or treat immune-related diseases, nervous system diseases and cancer.

[0054] According to another embodiment of the present invention, a nucleic acid molecule encoding the binding molecule provided by the present invention is provided.

[0055] The nucleic acid molecules of this invention include all nucleic acid molecules obtained by translating the amino acid sequence of the binding molecule provided by this invention into a polynucleotide sequence, as is known to those skilled in the art. Therefore, various polynucleotide sequences can be prepared using open reading frames (ORFs), all of which are also included in the nucleic acid molecules of this invention.

[0056] According to another embodiment of the present invention, an expression vector is provided in which the isolated nucleic acid molecule provided by the present invention is inserted.

[0057] In this invention, a "vector" is a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which is a circular double-stranded DNA in which an additional DNA fragment can be linked. Another type of vector is a bacteriophage vector. Yet another type of vector is a viral vector, in which an additional DNA fragment can be linked to a viral genome. Some vectors are capable of autonomous replication after introduction into a host cell (e.g., bacterial vectors with a bacterial origin of replication are free mammalian vectors). Other vectors (e.g., non-free mammalian vectors) can integrate into the host cell's genome after introduction into a host cell, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Generally, expression vectors used for recombinant DNA technology are typically in plasmid form. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector.

[0058] Specific examples of expression vectors in this invention may be selected from, but are not limited to: commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; granules; bacteriophages, such as λ phage, lambdoid phage, M13 phage, Mu phage, p1 P22 phage, Qμμ phage, T-even phage, T2 phage, T3 phage, T7 phage; and plant viruses. Any expression vector known to those skilled in the art can be used as the expression vector in this invention, and the expression vector may be selected according to the properties of the target host cell. Vector introduction into host cells can be performed by: calcium phosphate transfection, viral infection, DEAE-glucan-mediated transfection, liposome transfection, or electroporation. However, this invention is not limited to these methods, and those skilled in the art can adopt and use appropriate introduction methods for the expression vector and host cells. The vector preferably contains at least one selection marker. However, this invention is not limited to these methods, and vectors without selection markers may also be used and screened based on whether a product is produced. The selection markers are chosen based on the target host cell, which is done by methods known to those skilled in the art, and therefore the present invention is not limited thereto.

[0059] To facilitate the purification of the nucleic acid molecules of this invention, a tag sequence can be inserted and fused into the expression vector. Tags include, but are not limited to, hexahistine tags, hemagglutinin tags, Myc tags, or Flag tags; any tag known to those skilled in the art that facilitates purification can be used in this invention.

[0060] According to another embodiment of the present invention, a host cell line is provided, which is transfected with the expression vector provided by the present invention.

[0061] In this invention, "host cell" includes individual cells or cell cultures that may be, or have been, recipients of a vector for introducing a polypeptide insert. Host cells include progeny of a single host cell, which may not necessarily be identical to the original parent cell (in terms of morphology or genomic DNA complementarity) due to natural, accidental, or intentional mutations. Host cells include cells transfected in vivo with the polynucleotides described herein.

[0062] In this invention, the host cell may include: mammalian, plant, insect, fungal, or cellular origin cells; for example, the host cell may be: bacterial cells, such as Escherichia coli, Streptomyces, and Salmonella typhimurium; fungal cells, such as yeast cells and Pichia pastoris; insect cells, such as fruit fly and S. beet armyworm Sf9 cells; animal cells, such as Chinese hamster ovary (CHO) cells, SP2 / 0 (mouse myeloma cells), human lymphoblasts, COS, NSO (mouse myeloma cells), 293T, Bowes melanoma cells, HT-1080, young hamster kidney (BHK) cells, human embryonic kidney (HEK) cells, or PERC.6 (human retinal cells); or plant cells. However, the host cell is not limited thereto, and any cell known to those skilled in the art as a host cell line may be used.

[0063] According to another embodiment of the present invention, an antibody-drug conjugate (ADC) is provided, the antibody-drug conjugate (ADC) comprising the antibody and drug provided by the present invention.

[0064] As used herein, the term "antibody-drug conjugate (ADC)" refers to a form in which a drug and an antibody are chemically linked together without diminishing the biological activity of the antibody and the drug. In this invention, an antibody-drug conjugate refers to a form in which a drug binds 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 binds to an α-amino group at the N-terminus of the heavy chain and / or light chain of an antibody.

[0065] As used herein, the term "drug" can refer to any substance that has specific biological activity against cells, including DNA, RNA, or peptides. Drugs can be in forms containing reactive groups capable of reacting and cross-linking with α-amino groups, and also in forms containing reactive groups capable of reacting and cross-linking with α-amino groups and having a linker attached.

[0066] In this invention, there is no particular limitation on the type of reactive group capable of reacting and crosslinking with α-amino groups, as long as the reactive group can react and crosslink with the α-amino group at the N-terminus of the heavy or light chain of the antibody. The reactive group includes all types of groups known in the art capable of reacting with amino groups. For example, the reactive group can be any one of, but is not limited to, isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, ethylene oxides, carbonates, aryl halides, imine esters, carbodiimides, acid anhydrides, and fluorophenyl esters.

[0067] As used herein, the term "drug" is not specifically limited and includes any therapeutic agent that can treat immune-related diseases, neurological disorders, or cancer.

[0068] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of neurological diseases is provided, the composition comprising a chimeric antigen receptor (CAR) as an active ingredient, the CAR comprising an antigen-specific binding domain, a hinge domain and a CD3ζ signaling domain.

[0069] As used herein, the term "chimeric antigen receptor" or "CAR" refers to an engineered receptor comprising an extracellular antigen-binding domain and an intracellular signaling domain. While the most common type of CAR comprises a single-chain variable fragment (scFv) derived from a monoclonal antibody fused to the transmembrane domain and intracellular domain (e.g., the CD3ζ chain) of a T-cell co-receptor, this invention 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 any extracellular antigen-binding domain fused to or linked thereto. In this invention, the binding domain may comprise a single-chain variable fragment (scFv) capable of specifically recognizing the Lrig-1 protein. In this invention, the term "single-chain variable fragment" or "scFv" refers to a fusion protein formed by fusing the variable heavy chain (VH) and variable light chain (VL) of an antibody via a peptide linker between VL and VH.

[0070] Furthermore, in this invention, the VH and VL domains can be connected via a flexible linker. In this invention, the flexible linker can be a glycine / serine linker of approximately 10 to 30 amino acids (e.g., 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids), preferably 15 amino acids in length. In this invention, the linker length can be a crucial determinant of chimeric antigen receptors; therefore, linkers shorter than the aforementioned range can increase affinity but may also lead to the formation of intracellular multimers, thereby impairing CAR expression; while linkers longer than the aforementioned range may reduce antigen affinity by increasing the distance between the CDRs of adjacent VL and VH domains.

[0071] The chimeric antigen receptor of the present invention may further include at least one of a hinge region (or spacer region) and / or a signaling domain. In the present invention, the hinge region is the region connecting the antigen-binding domain and the transmembrane domain, also referred to as the "spacer region," and its purpose is to extend the antigen-binding domain from the T cell membrane or NK cell membrane. In the present invention, the hinge region may be obtained from any suitable sequence of any genus (including, for example, human or parts thereof), or may include all or part of CD8, CD28, 4-1BB, OX40, CD3ζ chains commonly used in the art, T cell receptor α or β chains, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, their functional derivatives, or combinations thereof, but is not limited thereto. Furthermore, the hinge region may include, but is not limited to, one selected from, immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD), but is not limited thereto.

[0072] In this invention, the signal domain refers to a portion of the chimeric antigen receptor present within T cells or engineered to exist within T cells. The signal domain may or may not include a transmembrane region for anchoring the chimeric antigen receptor to the plasma membrane of the T cell. The transmembrane domain and the signal domain may originate from the same protein (e.g., the CD3ζ molecule), or they may originate from different proteins (e.g., the transmembrane domain of CD28 and the intracellular signal domain of the CD3ζ molecule, or vice versa).

[0073] In this invention, the transmembrane domain includes, for example, all or part of the T cell receptor α or β chain, CD3ζ chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, their functional derivatives, or combinations thereof, but is not limited thereto. Co-stimulatory domains may include, but are not limited to, 4-1BB (CD137), OX40, CD27, CD28, CD30, CD40, PD-1, CD2, CD7, CD258, Natural Killer Family 2 Member C (NKG2C), Natural Killer Family 2 Member (NKG2D), B7-H3, CD83, ICAM-1, ligands or ICOS that bind to LFA-1 (CD11a / CD18), their active fragments, their functional derivatives, or functional signaling domains derived from combinations of these domains of polypeptides.

[0074] In this invention, the signal domain may include a functional signal domain derived from a polypeptide comprising all or part of CD3ζ, universal FcRγ (FcER1G), FcγRIIIa, FcRβ (Fcεlip), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DNAX activator 10 (DAP10), DNAX activator 12 (DAP12), their active fragments, their functional derivatives, or combinations thereof, but not limited thereto. Such signal domains are known in the art.

[0075] According to another embodiment of the present invention, a composition for preventing, improving or treating nervous system diseases is provided, the composition comprising, as an active ingredient, a binding molecule of the present invention, a nucleic acid molecule encoding the binding molecule, an expression vector containing the nucleic acid molecule, a host cell expressing the binding molecule, or an antibody-drug conjugate (ADC) containing the binding molecule.

[0076] In this invention, "immune-related diseases" can be diseases induced by the overactivation and expression of various immune cells and inflammatory cells. Immune-related diseases can include, but are not limited to, autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopy, or acute or chronic inflammatory diseases.

[0077] Furthermore, in this invention, "autoimmune disease" can be, but is not limited to, one or more of the following: rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjögren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibrositis, and polyarteritis nodosa.

[0078] As used herein, the term "nervous system disease" for which the compositions provided by the present invention prevent, improve or treat may include neurodegenerative diseases or neuroinflammatory diseases.

[0079] In this invention, "neurodegenerative disease" can refer to a disease caused by the decline or loss of neuronal function, and "neuroinflammatory disease" can refer to a disease caused by an excessive inflammatory response of the nervous system. As specific examples, the neurodegenerative disease or neuroinflammatory disease in this invention can be selected from, but is not limited to: stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prions, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy body dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, cortical degeneration syndrome, multiple system atrophy, progressive supranuclear palsy, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular diseases, spinal cord injury, and tau proteinosis.

[0080] Neuroinflammatory responses are increasingly recognized as a major contributing factor to neurodegenerative diseases. Immune cells, such as glial cells, present in the central nervous system can be activated by various exogenous and endogenous stimuli. Upon activation, glial cells produce and release inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), nitric oxide, prostaglandins, and superoxide (Gao et al., J. Neurochem., 81:1285–1297, 2002; Nelson et al., Ann. Med., 34:491–500, 2002; Griffin et al., J. Neuroinflammation, 3:5, 2006). While the production of these mediators may initially contribute to host defense and immune regulation, excessive or persistent production has been reported to promote neuronal damage and cell death, leading to the progression of neurodegenerative processes.

[0081] Given the recognized importance of neuroinflammatory responses in neurodegenerative diseases, reducing the expression levels of pro-inflammatory mediators in activated glial cells is expected to alleviate neuroinflammation, which may thus help treat related neurodegenerative diseases.

[0082] It has been reported that activated glial cells induce or upregulate the production of nitric oxide (NO), reactive oxygen species (ROS), inflammatory enzymes (such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2)), and pro-inflammatory mediators (including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α)). Persistent or excessive activation of these inflammatory pathways is associated with the pathogenesis of various central nervous system diseases, including multiple sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease (Gao et al., 2002; Nelson et al., 2002; Eikelenboomh and Van Gool, 2004).

[0083] It has been reported that reducing pro-inflammatory mediators within glial cells can suppress or alleviate symptoms of such neurological disorders (Matsumoto et al., 1992; Liu and Hong, 2003). In cell-based models, lipopolysaccharide (LPS)-induced glial cell activation has been widely used to study inflammatory mechanisms, and LPS stimulation has been shown to induce the production of various pro-inflammatory factors and neurotoxic mediators that lead to neuronal damage during inflammation (Kim et al., 2004a, b). Furthermore, numerous studies have reported that LPS-activated glial cells enhance neurotoxicity by releasing pro-inflammatory mediators and cytotoxic substances (Dawson et al., 1994). Given the recognized importance of inflammatory responses in neuroinflammation, inhibiting the production of such inflammatory factors in activated glial cells represents a promising therapeutic approach for diseases associated with neuroinflammatory conditions.

[0084] As used herein, the term "cancer" refers to a physiological condition in mammals characterized by uncontrolled or abnormal cell growth. In some embodiments, the cancer to be prevented, improved, or treated according to the present invention is a solid tumor formed by abnormal cell proliferation within a solid organ. Depending on the primary site, the solid tumor may include, but is not limited to: gastric cancer, liver cancer, glioma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer. In some embodiments, the cancer is melanoma or colorectal cancer, but the scope of the invention is not limited thereto.

[0085] Furthermore, the compositions provided in this invention can be formulated into pharmaceutical compositions or food compositions, but the scope of this invention is not limited thereto.

[0086] Meanwhile, in this invention, "prevention" may include, but is not limited to, any behavior that uses the pharmaceutical composition of this invention to block disease symptoms, or to suppress or delay disease symptoms.

[0087] As used herein, “treatment,” “curing,” and “improvement” refer to any act of applying the compositions of the present invention to a subject in need to improve, alleviate, reduce, or otherwise benefit one or more symptoms caused by a disease. These terms are not limited to a complete cure of the disease, but also include partial improvement or relief of one or more disease-related symptoms.

[0088] In this invention, the pharmaceutical composition is characterized in that it may be in the form of capsules, tablets, granules, injections, ointments, powders or beverages; and the pharmaceutical composition is characterized in that it may be intended for human use.

[0089] In this invention, the pharmaceutical composition can be formulated into oral formulations (e.g., powders, granules, capsules, tablets, and aqueous suspensions), topical formulations (e.g., suppositories), and sterile injections using conventional methods, and used accordingly. However, the pharmaceutical composition is not limited thereto. The pharmaceutical composition of this invention may also contain a pharmaceutically acceptable carrier. As a pharmaceutically acceptable carrier, binders, flow aids, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc., can be used for oral administration; buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc., can be used for injectable mixtures; matrices, excipients, lubricants, preservatives, etc., can be used for topical administration. The formulation of the pharmaceutical composition of this invention can be prepared by mixing with the above-mentioned pharmaceutically acceptable carriers in various ways. For example, for oral administration, the pharmaceutical composition can be formulated into tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, etc. For injection, the pharmaceutical composition can be formulated into single-dose ampoules or multi-dose dosage forms. Alternatively, the pharmaceutical composition can be formulated into solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

[0090] Examples of suitable carriers, diluents, or excipients for preparing formulations include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. Furthermore, fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives may also be included.

[0091] The routes of administration for the pharmaceutical compositions of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, percutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal routes. Oral or parenteral administration is preferred.

[0092] In this invention, "parenteral administration" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrabursal, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical compositions of this invention can also be administered in suppository form for rectal administration.

[0093] The pharmaceutical compositions of the present invention can vary depending on a variety of factors, including: the activity of the specific compound used, the patient's age, weight, overall health status, sex, diet, time of administration, route of administration, excretion rate, drug combination, and the severity of the disease to be prevented or treated. The dosage of the pharmaceutical composition can vary depending on the patient's condition, weight, disease severity, dosage form, route of administration, and duration, and can be appropriately selected by those skilled in the art. The pharmaceutical compositions can be administered at doses of 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg daily. They can be administered once daily or multiple times daily. The dosage is not intended to limit the scope of the invention in any way. The pharmaceutical compositions of the present invention can be formulated into dosage forms such as pills, sugar-coated tablets, capsules, liquids, gels, syrups, pastes, or suspensions.

[0094] Food compositions containing the compositions of the present invention as active ingredients can be formulated into various food forms, including but not limited to beverages, chewing gum, tea, vitamin complexes, powders, granules, tablets, capsules, candies, rice cakes, and bread. In some embodiments, the food compositions contain plant extracts that are generally considered to have low toxicity and minimal adverse reactions. Therefore, after appropriate safety assessments, the compositions may be suitable for long-term preventative use.

[0095] When the compositions of the present invention are added to a food composition, the content of the compositions of the present invention may be from about 0.1% to about 50% by weight of the total food composition. When the food composition is formulated as a beverage, there are no particular limitations other than including the compositions of the present invention in the indicated proportions. The beverage may also contain various flavorings or natural carbohydrates, which is conventional in beverage formulations. Examples of natural carbohydrates include monosaccharides (e.g., glucose), disaccharides (e.g., fructose and sucrose), and polysaccharides (e.g., dextrin and cyclodextrin). Sugar alcohols may also be included, including xylitol, sorbitol, and erythritol. Exemplary flavorings include natural flavorings (e.g., semathymidine, stevia extract (e.g., rebaudioside A and glycyrrhizic acid)) and synthetic sweeteners (e.g., saccharin and aspartame).

[0096] Furthermore, the food compositions of the present invention may also contain various additives commonly used in food formulations. Such additives include, but are not limited to, nutrients, vitamins, minerals (electrolytes), synthetic and natural flavorings, colorings, pectic acids and their salts, alginic acids and their salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, and carbonating agents used in carbonated beverages.

[0097] These ingredients can be used alone or in combination with each other. While there is no particular limitation on the specific amount of each additive, it is generally selected to be in the range of about 0.1 to about 50 parts by weight relative to 100 parts by weight of the composition of the present invention.

[0098] According to one embodiment of the present invention, a binding molecule is provided, the binding molecule comprising: (a) a heavy chain variable region comprising a heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23 and a heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24; and (b) a light chain variable region comprising a light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26 and a light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27.

[0099] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule comprising: (a) a heavy chain variable region comprising a heavy chain CDR3 composed of an amino acid sequence selected from SEQ ID NO: 25 and 43; and (b) a light chain variable region comprising: (i) a light chain CDR1 composed of an amino acid sequence selected from SEQ ID NO: 26 and 79; (ii) a light chain CDR2 composed of an amino acid sequence selected from SEQ ID NO: 27 and 80; and (iii) a light chain CDR3 composed of an amino acid sequence selected from SEQ ID NO: 28, 47, 51, 55, 59, 63, 67, 71, 75 and 81.

[0100] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule comprising: (a) a heavy chain variable region comprising: (i) a heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31; and (ii) a heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32; and (b) a light chain variable region comprising: (i) a light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34; and (ii) a light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35.

[0101] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule comprising: (a) a heavy chain variable region comprising: (i) a heavy chain CDR1 encoded by a nucleotide sequence of SEQ ID NO: 31; (ii) a heavy chain CDR2 encoded by a nucleotide sequence of SEQ ID NO: 32; (iii) a heavy chain CDR3 encoded by a nucleotide sequence selected from SEQ ID NO: 33 and 45; and (b) a light chain variable region comprising: (i) a light chain CDR1 encoded by a nucleotide sequence selected from SEQ ID NO: 34 and 84; (ii) a light chain CDR2 encoded by a nucleotide sequence selected from SEQ ID NO: 35 and 85; and (iii) a light chain CDR3 encoded by a nucleotide sequence selected from SEQ ID NO: 36, 49, 53, 57, 61, 65, 69, 73, 77 and 86.

[0102] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule being selected from:

[0103] (1) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 28;

[0104] (2) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24 and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 43, and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27 and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 28;

[0105] (3) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 47;

[0106] (4) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 51;

[0107] (5) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 55;

[0108] (6) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 59;

[0109] (7) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 63;

[0110] (8) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 67;

[0111] (9) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 71;

[0112] (10) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 27, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 75;

[0113] (11) A conjugated molecule comprising the following heavy chain variable regions and light chain variable regions: (i) a heavy chain variable region comprising: a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 23, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 24, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 25; and (ii) a light chain variable region comprising: a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 79, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 80, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 81.

[0114] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule being selected from:

[0115] (1) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 33; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 36;

[0116] (2) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 45; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 36;

[0117] (3) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 33; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 49;

[0118] (4) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 33; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 53;

[0119] (5) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 33; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 57;

[0120] (6) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 33; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 61;

[0121] (7) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 33; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 65;

[0122] (8) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 33; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 69;

[0123] (9) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 33; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 73;

[0124] (10) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 33; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 35 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 77;

[0125] (11) A binding molecule comprising the following heavy chain variable region and light chain variable region: (i) a heavy chain variable region comprising: heavy chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 32 and heavy chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 33; and (ii) a light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence of SEQ ID NO: 84, light chain CDR2 encoded by the nucleotide sequence of SEQ ID NO: 85 and light chain CDR3 encoded by the nucleotide sequence of SEQ ID NO: 86.

[0126] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule comprising: (a) a heavy chain variable region comprising an amino acid sequence selected from SEQ ID NO: 29, 44 and 82; and (b) a light chain variable region comprising an amino acid sequence selected from SEQ ID NO: 30, 48, 52, 56, 60, 64, 68, 72, 76 and 83.

[0127] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule comprising: (a) a heavy chain variable region encoded by a nucleotide sequence selected from SEQ ID NO: 37, 46 and 87; and (b) a light chain variable region encoded by a nucleotide sequence selected from SEQ ID NO: 38, 50, 54, 58, 62, 66, 70, 74, 78 and 88.

[0128] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule further comprising an Fc region or a constant region.

[0129] According to another embodiment of the present invention, a binding molecule is provided, wherein the Fc region is the Fc region of an IgG1, IgG2, IgG3 or IgG4 antibody, or a hybrid Fc region.

[0130] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule further comprising a heavy chain constant region composed of an amino acid sequence selected from SEQ ID NO: 1, 3, 4, 7, 8, 9, 10 and 12.

[0131] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule further comprising a light chain constant region composed of an amino acid sequence selected from SEQ ID NO: 2, 5, 6 and 11.

[0132] According to another embodiment of the present invention, a binding molecule is provided, the binding molecule further comprising a heavy chain constant region and a light chain constant region, the heavy chain constant region being encoded by a nucleotide sequence selected from SEQ ID NO: 13, 15 and 17, and the light chain constant region being encoded by a nucleotide sequence selected from SEQ ID NO: 14, 16 and 18.

[0133] According to another embodiment of the present invention, a binding molecule is provided, said binding molecule being an antibody or a fragment thereof.

[0134] According to another embodiment of the present invention, a binding molecule is provided, wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent molecule, a bispecific molecule, a microantibody, a domain antibody, a bispecific antibody, an antibody mimic, a biantibody, a triantibody, a tetraantibody, or fragments thereof.

[0135] According to another embodiment of the present invention, a nucleic acid molecule encoding the binding molecule described herein is provided.

[0136] According to another embodiment of the present invention, an expression vector comprising the nucleic acid molecules described herein is provided.

[0137] According to another embodiment of the present invention, a host cell transformed with the expression vector described herein is provided.

[0138] According to another embodiment of the present invention, an antibody-drug conjugate comprising the binding molecule and the drug described herein is provided.

[0139] According to another embodiment of the present invention, a chimeric antigen receptor (CAR) is provided, the chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linker domain, and a CD3ζ signaling domain, wherein the antigen-specific binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a heavy chain CDR1 composed of the amino acid sequence of SEQ ID NO: 23 and a heavy chain CDR2 composed of the amino acid sequence of SEQ ID NO: 24, and the light chain variable region comprising a light chain CDR1 composed of the amino acid sequence of SEQ ID NO: 26 and a light chain CDR2 composed of the amino acid sequence of SEQ ID NO: 27; the antigen-specific binding domain is selected from the antigen-specific binding domains described herein, and the CAR further comprises an immunoglobulin Fc region to form a fusion protein.

[0140] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of cancer is provided, the pharmaceutical composition comprising the binding molecules described herein as active ingredients.

[0141] According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the cancer is a solid tumor.

[0142] According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the cancer is selected from: gastric cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, and lung cancer.

[0143] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of central nervous system diseases is provided, the pharmaceutical composition comprising the binding molecules described herein as active ingredients.

[0144] According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the central nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

[0145] According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the neurodegenerative disease or neuroinflammatory disease is selected from: stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prions, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy body dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, neuroautoimmune diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular diseases, spinal cord injury, and tau proteinosis.

[0146] According to another embodiment of the present invention, a pharmaceutical composition for the prevention or treatment of immune-related diseases is provided, the pharmaceutical composition comprising the binding molecules described herein as active ingredients.

[0147] The composition wherein the immune-related disease is selected from autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopic disease, and acute or chronic inflammatory diseases.

[0148] According to another embodiment of the present invention, a pharmaceutical composition is provided, wherein the autoimmune disease is selected from: rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjögren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa.

[0149] Advantages and effects

[0150] The binding molecule provided by this invention has enhanced binding affinity for the Lrig-1 antigen, and therefore can specifically prevent, improve or treat various immune-related diseases, neurodegenerative diseases or neuroinflammatory diseases and cancer. Attached Figure Description

[0151] Figure 1 Linear graphs showing the ELISA results of 11 affinity-matured antibodies with mLrig1-His.

[0152] Figure 2 Line graph showing the ELISA results of another group of 11 affinity-matured antibodies with mLrig1-His.

[0153] Figure 3 The BLI results for the selected 10 TregL1-mFc molecules are shown, primarily focusing on their KD values.

[0154] Figure 4 A graph comparing the binding affinity of 10 mature antibodies with control H6 antibody and GTC310-01 antibody.

[0155] Figure 5 An experimental design for evaluating anticancer efficacy using an Lrig-1 protein-specific monoclonal antibody (H6) according to one embodiment of the present invention is illustrated.

[0156] Figure 6 The results of analyzing anticancer efficacy using an Lrig-1 protein-specific monoclonal antibody (H6) according to one embodiment of the present invention are shown.

[0157] Figure 7This illustration schematically demonstrates an experimental design for evaluating anticancer efficacy using a monoclonal antibody (ML2) with Lrig-1 protein-specific affinity maturation according to one embodiment of the present invention.

[0158] Figure 8 The results of analyzing anticancer efficacy using a monoclonal antibody (ML2) with Lrig-1 protein-specific affinity maturation according to one embodiment of the present invention are presented.

[0159] Figure 9 A schematic diagram of an experiment for evaluating the therapeutic effect of a monoclonal antibody on Alzheimer's disease is shown according to one embodiment of the present invention.

[0160] Figure 10 A graph showing the percentage (%) of thioflavone S (ThS) positive regions in the cortex and hippocampus of Alzheimer's disease-induced mice after various treatments (including monoclonal antibodies according to one embodiment of the present invention) and staining with thioflavone S (ThS) to show the proportion of thioflavone S (ThS) positive regions in normal mice after various treatments (including monoclonal antibodies according to one embodiment of the present invention).

[0161] Figure 11 A graph showing the change in the rate (%) of spontaneous alternation in the Y-maze test in Alzheimer's disease-induced mice after various treatments (including monoclonal antibodies according to one embodiment of the present invention).

[0162] Figure 12 A graph showing the changes in preference values ​​in a new object recognition test in mice induced by Alzheimer's disease after various treatments, including monoclonal antibodies according to one embodiment of the invention.

[0163] Figure 13 This is a schematic diagram illustrating an experiment using Alzheimer's disease-induced mice (specifically 5xFAD mice and 6xTg mice) to evaluate the therapeutic effect of a monoclonal antibody according to one embodiment of the present invention.

[0164] Figure 14 The graph shows the change in the rate of spontaneous alternation (%) in a Y-maze test performed on Alzheimer's disease-induced 5xFAD mice and 6xTg mice after treatment with a monoclonal antibody according to an embodiment of the present invention.

[0165] Figure 15 A graph showing the change in preference values ​​in a novel object recognition test performed on Alzheimer's disease-induced 6xTg mice after treatment with a monoclonal antibody according to an embodiment of the present invention.

[0166] Figure 16The graph shows the changes in dwell time during a passive avoidance test in Alzheimer's disease-induced 5xFAD mice and 6xTg mice after treatment with a monoclonal antibody according to an embodiment of the present invention.

[0167] Figure 17 This is a schematic diagram of an experimental design used to evaluate the therapeutic effects of a monoclonal antibody according to one embodiment of the present invention on neurodegenerative diseases and autoimmune diseases.

[0168] Figure 18 The therapeutic effects of MH6 and 6F01 antibodies on neurodegenerative diseases and autoimmune diseases were demonstrated.

[0169] Figure 19 An experimental design is presented for evaluating the therapeutic effects of a monoclonal antibody according to one embodiment of the present invention on neurodegenerative diseases and autoimmune diseases.

[0170] Figure 20 The therapeutic effects of ML4 antibodies on neurodegenerative diseases and autoimmune diseases were demonstrated.

[0171] Invention patterns

[0172] The present invention will now be described in more detail through embodiments. These embodiments are merely for illustrating the invention in more detail, and it will be apparent to those skilled in the art that the scope of the invention according to its subject matter is not limited to these embodiments. Example

[0173]

Preparation Example 1

[0174] An Lrig-1 protein-specific antibody according to the present invention was prepared. This antibody does not target a specific epitope, thus enabling the preparation of an antibody capable of binding to any region of the Lrig-1 protein.

[0175] To prepare the aforementioned antibody, cells expressing the Lrig-1 protein were constructed. More specifically, the DNA fragment corresponding to SEQ ID NO: 20 and the pcDNA (hygro) vector were digested with a restriction endonuclease and ligated at 37°C to generate a pcDNA vector containing the inserted Lrig-1 protein DNA sequence. The resulting pcDNA containing SEQ ID NO: 20 was transfected into T cells, thereby enabling the expression of the Lrig-1 protein on the surface of T cells.

[0176] Light and heavy chain sequences capable of binding to Lrig-1 expressed on the cell surface were screened from a human scFv library. The selected heavy and light chain amino acid sequences were fused with the heavy chain constant region of SEQ ID NO: 1 or 4 and the light chain constant region of SEQ ID NO: 2 or 6, respectively, to prepare monoclonal antibodies. The sequences of the monoclonal antibodies are shown in Table 1.

[0177] [Table 1]

[0178]

[0179] [Preparation Example 2] Preparation of affinity-matured GTC310-01 antibody

[0180] To prepare antibodies that could bind specifically to Lrig-1 with higher specificity than the H6 and GTC310-01 antibodies prepared in Table 1, ten affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, ML9, and 6F01) were generated. The ML (modified light chain) clone underwent affinity maturation by modifying a portion of the light chain CDR3 sequence to enhance specific binding to Lrig-1; while the MH (modified heavy chain) clone underwent affinity maturation by modifying a portion of the heavy chain CDR3 sequence to enhance specific binding to Lrig-1. The 6F01 clone retained the same heavy chain but modified only the light chain CDR to enhance specific binding to Lrig-1.

[0181] [Example 1] Analyzing the binding affinity of ten novel antibodies with mature affinity using ELISA

[0182] After screening with magnetic beads, the monoclonal antibodies grown on the square plates were inoculated into each well of a 96-well plate and cultured until OD (October Expiratory Time). 600 The concentration reached 0.5. Subsequently, IPTG induction was performed overnight. Recombinant His-tagged mouse Lrig-1 protein (antigen) was immobilized on immunotubes and incubated overnight at 4°C. The antigen-coated plates were washed three times with TBST and blocked with 3% skim milk. Then, 50 μL of the cell supernatant from overnight IPTG induction was added to each well of the antigen-coated plate. Next, 50 μL of a mixture of 3% skim milk and HA-HRP (secondary antibody) (1000:1 ratio) was added to each well, followed by 50 μL of substrate solution. After approximately 10 minutes of reaction, 50 μL of sulfuric acid or stop buffer was added to terminate the reaction. The absorbance at 450 nm was then measured using a microplate reader.

[0183] like Figures 1 to 2 As shown in Table 2, the Kd value of the previously prepared 6F01 antibody is about half that of the H6 antibody, indicating that the 6F01 antibody binds to the His-tagged mouse Lrig-1 antigen about twice as efficiently as the H6 antibody.

[0184] [Table 2]

[0185]

[0186] [Example 2] Analysis of the binding affinity of ten novel affinity maturation antibodies using Octet®

[0187]

Example 2-1

[0188] Count the number of peripheral blood mononuclear cells (PBMCs), dividing the cells into groups of 10-1. 7 Each cell was resuspended in 40 µL of buffer. Subsequently, every 10 cells... 7 Add 10µL of initial CD4 to each cell. + T-cell biotin antibody mixture II was incubated at 4°C for 10 minutes, during which the cells were gently tapped once per minute. The cells were then incubated at 10... 7 Resuspend each cell in 30 µL of buffer, every 10 cells 7 Add 20µL of initial CD4 to each cell. + T cell microbead mixture II was incubated at 4°C for 15 minutes, with gentle tapping once per minute during this period. LS columns were prepared, and cells containing initial CD4+ were collected. + The effluent from T cells was collected at a rate of 2 × 10⁶ cells per well. 6 10 cells / 2 mL were seeded into 24-well plates at a density of 2 × 10⁶ cells / well. 5 Cells were stimulated with Dynabead® CD3 / CD28 at a ratio of 1 cell / 4 µL. Cells were treated with 5 ng / mL TGF-β and 20 ng / mL IL-2 and cultured for 4 days. For surface protein staining, 200 µL of differentiated cells were transferred to round-bottom wells and centrifuged at 2500 rpm for 2 minutes. The supernatant was removed, and 50 µL of PBS was mixed with the antibody per well, which was then pipetted to stain the cell surface proteins. The plate was wrapped in aluminum foil and incubated at 4°C for 30 minutes. Afterward, 150 µL of PBS was added to each well, and the plate was centrifuged at 2500 rpm for 2 minutes. For intracellular protein staining, the supernatant was removed, and 4× fixation / permeabilization buffer was diluted to 1×. 80 µL of the solution was added to each well, and the plate was wrapped in aluminum foil and incubated at 4°C for 30 minutes. Subsequently, 120 µL of 1× permeabilization buffer (prepared by diluting 10× permeabilization buffer with distilled water) was added to each well, and the cells were centrifuged at 2500 rpm for 2 minutes. Add 50 µL of 1× permeation buffer mixed with antibody to each well and incubate at 4 °C for 30 minutes. Then, add 150 µL of 1× permeation buffer to each well and centrifuge at 2500 rpm for 2 minutes. Wash each well with 200 µL of PBS and centrifuge at 2500 rpm for 2 minutes.

[0189] [Example 2-2] Analysis of binding affinity using Octet®

[0190] First, the analyte was equilibrated in water for 60 seconds. Then, it was activated with EDC / s-NHS buffer for 300 seconds, followed by immobilization of the ligand (20 μg / mL anti-L1 antibody) in 10 mM acetate buffer at pH 6.0 for 10 minutes. Next, the reaction was quenched with ethanolamine for 5 minutes, and the analyte was treated with 1X Kinetics buffer for 2 minutes. Then, it was treated with regeneration buffer and 1X Kinetics buffer for 20 seconds, including 5 seconds of regeneration and 5 seconds of neutralization, repeated twice. Afterward, the analyte was treated again with 1X Kinetics buffer for 2 minutes, and the analyte was allowed to bind to the immobilized ligand in 1X Kinetics buffer for 5 minutes. Finally, the analyte was washed with 1X Kinetics buffer for 5 minutes, followed by another 2 minutes of treatment with 1X Kinetics buffer.

[0191] Results of Examples 2-3

[0192] The results are as follows Figure 3 and Figure 4 As shown. Figure 3 As shown, the KD value of the affinity-matured novel antibody (the KD value decreases as the affinity for the antigen increases) is at least 2 times lower and at most approximately 737 times lower than that of the GTC310-01 antibody. Therefore, it is confirmed that the affinity of the affinity-matured novel antibody to the Lrig-1 antigen is at least 2 times higher and at most approximately 737 times higher than that of the conventional GTC310-01 antibody.

[0193] Finally, as Figure 4 As shown, this confirms that the novel antibody with maturing affinity interacts with CD4. + FoxP3 + Lrig1 + The cell binding affinity is higher than that of conventional H6 and GTC310-01 antibodies.

[0194] [Example 3] Anticancer efficacy of the antibody of the present invention

[0195]

Example 3-1

[0196] To evaluate the therapeutic efficacy of the monoclonal antibody (H6) prepared in Table 1 against solid tumors, such as Figure 5 As shown, B16F10 melanoma cells were divided into two groups at a rate of 3 × 10⁶ cells per mouse. 5 A dose of 100 μg of melanoma cells was subcutaneously injected into the back of mice. On days 4, 8, and 12 post-inoculation, antibodies were administered intraperitoneally at a dose of 200 μg. Tumor volume was measured over time after melanoma cell implantation, and the results are as follows: Figure 8 As shown.

[0197] like Figure 6As shown, treatment with conventional Lrig-1-specific monoclonal antibody (H6) significantly reduced tumor size compared to the untreated negative control group.

[0198] These results demonstrate that conventional Lrig-1-specific monoclonal antibodies effectively inhibit the growth of various solid tumor cells, proving their potential in the prevention, improvement, or treatment of such cancers.

[0199] [Example 3-2] Anticancer efficacy of affinity maturation antibodies

[0200] based on Figure 4 The results shown indicate that among the affinity-matured antibodies prepared in Example 2, the ML2 antibody was selected (compared to the conventional H6 antibody, the ML2 antibody showed better affinity for CD4 at a concentration of 5 μg). + FoxP3 + Lrig1 + The anticancer efficacy was assessed by using a cell-specific binding rate that was more than 9 times higher (i.e., more than 90%).

[0201] like Figure 7 As shown, a CT26 mouse model was established. 3×10 5 Two CT26 cancer cells (200 μL) were subcutaneously injected into the back of 7-week-old female mice, and tumor size was measured 7 days later. Subsequently, ML2 antibody was administered intraperitoneally every two days at doses of 100 μg, 200 μg, and 500 μg, and tumor size was measured. Results are as follows: Figure 8 As shown.

[0202] The results showed that, compared with the control group, the novel affinity-matured ML2 antibody provided by this invention reduced tumor size, demonstrating its therapeutic potential for cancer. Therefore, at a concentration of 5 μg, it showed efficacy against CD4+. + FoxP3 + Lrig1 + Other mature antibodies with at least nine times (or more than 90%) higher affinity than conventional H6 antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01) can specifically bind to Lrig-1 on regulatory T cells. These antibodies activate effector T cells by inhibiting the function of regulatory T cells, thereby achieving cancer therapy. The mechanism will be clearly understood by those skilled in the art.

[0203] Ultimately, as observed in Example 3-1, even the H6 antibody, which has a relatively low affinity for the Lrig-1 protein, exhibited anticancer activity. This is because the ML2 antibody (at a concentration of 5 μg, binds to CD4) has been shown to have anticancer activity. + FoxP3 + Lrig1 +The anticancer effects of other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01) that have a cell-specific binding efficiency at least nine times (or more than 90%) higher than that of conventional H6 antibodies will be readily understood by those skilled in the art. These other affinity-matured antibodies, which similarly bind specifically to Lrig-1 on regulatory T cells, can inhibit the function of regulatory T cells, thereby activating effector T cells to treat cancer.

[0204] [Example 4] Evaluation of the therapeutic effect of the antibody of the present invention on central nervous system diseases

[0205] [Example 4-1] Evaluation of the therapeutic effect of conventional GTC310-01 antibody on central nervous system diseases – 5xFAD mice

[0206] To evaluate the therapeutic effect of the antibody of this invention on central nervous system diseases, such as Figure 9 The experimental group was designed as shown. Specifically, the GTC310-01 mouse antibody of this invention was administered intravenously at a dose of 10 mg / kg to male 5-6 month old 5xFAD mice with Alzheimer's disease for 3 weeks. For comparison, the positive control group was administered glatiramer acetate (GA, Copaxone®) subcutaneously at a dose of 100 µg for 3 weeks, or the H6 antibody (GTC110-04 antibody) of this embodiment was administered intravenously at a dose of 10 mg / kg for 4 weeks.

[0207] 1. Reduction of β-amyloid plaques

[0208] For immunohistochemical analysis, each treated mouse was euthanized, and cortical and hippocampal brain tissue was collected and fixed in 4% paraformaldehyde (pH 7.4) for 16 hours. The fixed cortical and hippocampal tissues were then cryoprotected in 30% sucrose solution and sectioned to a thickness of 35 μm using a cryostat microtome (Thermo Scientific Microm HM 525, Waltham, MA). To observe Aβ plaques, brain sections were stained with thioflavone S (ThS) for 7 minutes. Thioflavone S was purchased from Sigma-Aldrich (catalog number T-1892) and dissolved in 50% ethanol at 500 μM. Sections were washed sequentially with 100%, 95%, and 70% ethanol and then transferred to PBS buffer. For immunofluorescence staining, relatively thick, free-floating brain sections were incubated with anti-GFAP goat antibody at 4°C for 7 days. Subsequently, the sections were incubated overnight at 4°C with Alexa Fluor 594-labeled donkey anti-goat IgG (1:200, Abcam, ab150132). Nuclear counterstaining was performed using DAPI (4',6-diamidinyl-2-phenylindole dihydrochloride, 1 mg / mL, 1:2000, Sigma). Images were acquired using a Leica DM2500 fluorescence microscope, and the results are shown below. Figure 12 As shown. Furthermore, the percentage (%) of the ThS+ region relative to normal mice was calculated using the ImageJ software program, and the results are also shown in... Figure 12 middle.

[0209] like Figure 10 As shown, the ThS+ region was significantly increased in Alzheimer's disease model mice; however, after administration of the previously described GTC310-01 and H6 (GTC110-04) antibodies, the ThS+ region was significantly reduced, especially in the cortex. This indicates that these antibodies have a significant effect in reducing β-amyloid plaques.

[0210] 2. Y-maze test

[0211] The Y-maze test uses a maze consisting of three identical arms (each 40 cm long and 15 cm high), arranged at 120° angles. This behavioral test utilizes the innate exploratory tendency of rodents, based on the principle that rodents are more inclined to explore new areas. When a rodent avoids re-entering an arm it has just explored, it indicates good memory performance. Each animal has 8 minutes to explore, and the final result is expressed as a spontaneous alternation rate (%). Figure 13 As shown. The spontaneous alternation rate (%) was calculated according to Formula 1. Behavioral patterns were analyzed using SMART VIDEO TRACKING software (Panlab, USA).

[0212] [Formula 1]

[0213] Spontaneous alternation rate (%) = Number of three-arm attempts / (Total number of arm advances - 2)

[0214] like Figure 11 As shown, in this experiment, the relative frequency of the test animals entering the maze arms sequentially based on environmental cues was measured. The spontaneous alternation rate values ​​obtained by administering conventional GTC310-01 or H6 (GTC110-04) antibodies according to this embodiment to Alzheimer's disease-induced mice were comparable to those of the normal control group.

[0215] 3. New Object Recognition Test

[0216] The Novel Object Recognition (NOR) test uses a 40cm x 40cm acrylic cage and two different objects to assess memory function. After the animal becomes familiar with the cage, the two objects are placed in fixed positions, and the time spent exploring each object is recorded. After a 24-hour delay, one of the objects is replaced with the new object placed in the same position. A longer exploration time for the new object is interpreted as better memory performance. If the animal fails to remember the object from 24 hours ago, it will be unable to distinguish between the new and familiar objects, resulting in roughly equal exploration times for both. The animal is allowed 10 minutes of free exploration. Results are expressed as a preference index (new object exploration time / total exploration time), such as... Figure 14 As shown. The analysis was performed using SMART VIDEO TRACKING software (Panlab, USA).

[0217] like Figure 12 As shown, the preference index for novel objects was lower in the Alzheimer's disease-induced group compared to the normal control group. However, after administration of the previously prepared GTC310-01 antibody or H6 (GTC110-04) antibody, the preference index increased to levels comparable to or higher than those in the normal control group.

[0218] These results indicate that the previously prepared GTC310-01 antibody or H6 (GTC110-04) antibody has preventive, ameliorative, or therapeutic effects on neurodegenerative diseases.

[0219] [Example 4-2] Evaluation of the therapeutic efficacy of existing GTC310-01 antibodies against neurodegenerative diseases – 6xTg small mouse

[0220] To evaluate the therapeutic efficacy of existing GTC310-01 antibodies against Alzheimer's disease, such as Figure 13The study group was designed and experiments were conducted as shown. Specifically, female 5xFAD mice and female 6xTg mice that had undergone a 4.5-month adaptation period were administered the GTC310-01 antibody from Example 1 at a dose of 10 mpk, intravenously every other week for 2 months. As a control, the positive control group received 100 μg glatiramer acetate (GA, Copaxone®) intravenously every other week for 2 months. After 2 months, the mice were subjected to the Y-maze test, the novel object recognition test, and the passive avoidance test.

[0221] 1. Characteristics of 6xTg mice

[0222] While 5xFAD mice are widely used as a model of Alzheimer's disease (AD), the 6xTg mice used in this experiment carry mutations not only in β-amyloid protein (Aβ42) but also in tau protein (MAPT), leading to their accumulation. This makes the 6xTg model more closely resemble the pathology of human diseases than the 5xFAD mouse model, making it an ideal animal model for Alzheimer's disease and other neurodegenerative diseases.

[0223] 2. Y-maze test

[0224] The Y-maze test uses a maze consisting of three identical arms (each 40 cm long and 15 cm high), arranged at 120° angles. This behavioral test utilizes the innate exploratory tendency of rodents, based on the principle that animals are more inclined to explore new arms. When an animal avoids re-entering an arm it has just explored, it indicates better memory performance. Each animal had 8 minutes to explore the maze, and the final results are expressed as the rate of spontaneous alternation (%). Figure 14 As shown. The spontaneous alternation rate (%) was calculated according to Formula 1. Behavioral patterns were analyzed using SMART VIDEO TRACKING software (Panlab, USA).

[0225] [Formula 2]

[0226] Spontaneous alternation rate (%) = Number of three-arm attempts / (Total number of arm advances - 2)

[0227] like Figure 14 As shown, in this experiment, the relative frequency of animals sequentially entering the arm based on environmental cues was measured. Administration of the GTC310-01 antibody according to the present invention to Alzheimer's disease-induced mice resulted in a spontaneous alternation rate comparable to that of the normal control group. Furthermore, in the 6xTg mouse model (which more closely mimics human disease than the 5xFAD model), a spontaneous alternation rate even closer to that of the normal control group was observed.

[0228] 3. New Object Recognition Test

[0229] To assess memory, a Novel Object Recognition (NOR) test was conducted using two different objects placed in a 40cm x 40cm acrylic cage. After the animal became familiar with the cage, the two objects were placed in a fixed location, and the time spent exploring each object was measured. After a 24-hour delay, one of the objects was replaced with the new object placed in the same location. The longer the exploration time for the new object, the better the memory retention. If the animal failed to remember the object from 24 hours prior, it explored both the familiar and new objects equally. The animal was allowed 10 minutes of free exploration. Results are expressed as a preference index (new object exploration time ÷ total exploration time), as shown in Figure 21. Behavioral analysis was performed using SMART VIDEO TRACKING software (Panlab, USA).

[0230] like Figure 15 As shown, the Alzheimer's disease-induced group had a lower preference index for novel objects compared to the normal control group. However, when the GTC310-01 antibody according to the present invention was administered, the preference index of the 6xTg model, which is closer to the human disease model, was observed to increase to a level comparable to or higher than that of the normal control group.

[0231] 4. Passive Avoidance Test

[0232] The passive avoidance testing device consisted of two chambers: a bright chamber and a dark chamber, with a grid bottom. On day one, mice were allowed to freely explore the device to acclimatize. On day two, each 5xFAD and 6xTg mouse was placed in the dark chamber for 1 minute for initial acclimatization, followed by 2 minutes of illumination. Immediately after entering the dark chamber, mice were given a mild plantar electric shock (0.5mA, 1 second) for a training test. On day two, a retention test was performed to assess memory. Each mouse was placed in the illuminated chamber, and the latency (the time required for all four limbs to enter the dark chamber) was recorded, with a maximum cutoff time of 300 seconds. Figure 16 As shown, administration of GTC310-01 antibody to 5xFAD mice and 6xTg mice resulted in a latency period close to that of the normal control group, indicating significant recovery.

[0233]

Example 4-3

[0234] Among the affinity-matured antibodies prepared in Example 2, the antibodies with the lowest and highest binding affinity were selected to evaluate their therapeutic effects on autoimmune diseases and neurodegenerative diseases. If the therapeutic effects of these antibodies are confirmed, it will be easily understood by those skilled in the art without extensive experimentation or specialized knowledge that antibodies with moderate binding affinity also have similar therapeutic effects.

[0235] Therefore, according to Figure 3The MH6 antibody was selected. Among the other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01), the MH6 antibody had the lowest binding affinity to Lrig-1, but its KD value was approximately twice that of the original GTC310-01 antibody, indicating a moderate enhancement in its binding affinity to Lrig-1. Conversely, the ML4 antibody was also selected. The ML4 antibody had the highest binding affinity to Lrig-1 among all affinity-matured antibodies, with a KD value more than 737 times lower than that of GTC310-01. Furthermore, according to Table 2, the therapeutic effect of the 6F01 antibody on neurodegenerative diseases was evaluated; the 6F01 antibody had a KD value of 6.1 × 10⁻⁶. 11 The KD value of the original H6 antibody was 1.2 × 10⁻⁶. 10 This also indicates that the KD value is about twice as low.

[0236] 1. Establish a mouse model of EAE (experimental autoimmune encephalomyelitis).

[0237] To assess the reduction in inflammatory response, a representative animal model—the experimental autoimmune encephalomyelitis (EAE) mouse model—was used. Because neuroinflammatory responses are widely considered a major factor leading to neurodegeneration, this model is also used as a representative mouse model for neurodegenerative diseases.

[0238] To construct an EAE mouse model, according to... Figure 17 The experiment was conducted according to the experimental design shown. On day 0, 9-week-old female C57Bl / 6J mice were subcutaneously injected with MOG peptide and intraperitoneally injected with 260 ng PTX. On day 1, another 260 ng PTX was administered to further stimulate the mice's immune system. On day 10, mice were administered 50 µg of the 6F01 and MH6 antibodies prepared in the above examples, along with 50 µg of anti-IL-17a antibody as a positive control.

[0239] In addition, to evaluate the therapeutic effect of ML4 antibodies, according to Figure 19 The experiment was conducted according to the experimental design shown. On day 0, 11-week-old female C57L / 6N mice were subcutaneously injected with MOG peptide and intraperitoneally injected with 200 ng PTX. On day 1, another 200 ng PTX was administered to further stimulate the mice's immune system. As shown in Table 3, mice were treated three times with the ML4 antibody prepared in the above examples at 2.5 mg / kg on days 12, 14, and 16, or once at 10 mg / kg on day 12. As a positive control, 200 µg of anti-IL-17a antibody was administered.

[0240] [Table 3]

[0241]

[0242] 2. Evaluate the therapeutic effects of 6F01 and MH6 antibodies on multiple sclerosis.

[0243] Starting from day 7, clinical scores were recorded according to the following evaluation criteria to assess the therapeutic effect of the antibody on multiple sclerosis in mice. Results are as follows: Figure 18 As shown.

[0244] <Evaluation Criteria>

[0245] 0: Asymptomatic, normal activity.

[0246] 1: The tip of the tail is paralyzed, and the tail droops.

[0247] 2: The tail is completely paralyzed, and the entire tail hangs down.

[0248] 3: Partial paralysis of one hind limb, but still responsive to stimuli.

[0249] 4: Complete paralysis of one hind limb, no response to stimuli, and dragging of the leg when moving.

[0250] 5: Bilateral hind limb paralysis, the mouse drags its body with its forelimbs to move.

[0251] 6: Difficulty moving the body with the forelimbs, but the forelimbs still respond to stimuli.

[0252] 7: The mouse was unable to move, but one forelimb responded to stimulation.

[0253] 8: The mouse was unable to move, and its forelimbs did not respond to stimuli.

[0254] 9: The mouse was unable to move and its breathing was irregular.

[0255] 10: Death.

[0256] The results showed that among other antibodies with mature affinity, even the 6F01 and MH6 antibodies, which had the lowest binding affinity to Lrig-1, exhibited significant therapeutic effects in a mouse model induced by multiple sclerosis.

[0257] 3. Evaluate the therapeutic effect of ML4 on multiple sclerosis.

[0258] Starting from day 12, clinical scores were assessed according to the following evaluation criteria to evaluate the therapeutic effect on multiple sclerosis in mice. Results are as follows: Figure 20 As shown.

[0259] <Evaluation Criteria>

[0260] 0: Asymptomatic, normal activity.

[0261] 1: The tip of the tail is paralyzed, and the tail droops.

[0262] 2: The tail is completely paralyzed, and the entire tail hangs down.

[0263] 3: Partial paralysis of one hind limb, but still responsive to stimuli.

[0264] 4: Complete paralysis of one hind limb, no response to stimuli, and dragging of the leg when moving.

[0265] 5: Bilateral hind limb paralysis, the mouse drags its body with its forelimbs to move.

[0266] 6: Difficulty moving the body with the forelimbs, but the forelimbs still respond to stimuli.

[0267] 7: The mouse was unable to move, but one forelimb responded to stimulation.

[0268] 8: The mouse was unable to move, and its forelimbs did not respond to stimuli.

[0269] 9: The mouse was unable to move and its breathing was irregular.

[0270] 10: Death.

[0271] The results showed that, in a multiple sclerosis-induced model, ML4 (the antibody with the highest binding affinity to Lrig-1 among all other affinity-matured antibodies) could also significantly improve multiple sclerosis.

[0272] 4. Conclusion

[0273] As described in Examples 4-1 and 4-2 above, H6 antibody and GTC310-01 antibody (both with relatively low binding affinity to Lrig-1 protein) have been shown to be effective in treating neurological diseases.

[0274] Furthermore, as described in Examples 4-3 above, in the affinity-matured antibodies prepared in Preparation Example 2, the antibody against MH6 (based on...) Figure 4 Among the affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01), ML4 showed the lowest binding affinity to Lrig-1, with a KD value approximately twice that of the conventional GTC310-01 antibody; ML4 showed the highest binding affinity to Lrig-1 among the affinity-matured antibodies, with a KD value at least 737 times lower than that of the conventional GTC310-01 antibody; and 6F01 (as shown in Table 2, 6F01 has a KD value of 6.1 × 10⁻⁶). 11 The KD value of conventional H6 antibodies is 1.2 × 10⁻⁶. 10These antibodies (equivalent to approximately twice the KD value) were evaluated in a representative animal model (i.e., an experimental autoimmune encephalomyelitis (EAE) mouse disease model) to assess the reduction in inflammatory response. The results confirmed that these antibodies reduced the inflammatory response in the EAE model, thus demonstrating their therapeutic efficacy in treating autoimmune diseases and neurological disorders.

[0275] Therefore, those skilled in the art will understand that other mature antibodies with higher binding affinity to Lrig-1 protein (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01) can also bind to Lrig-1 on the surface of regulatory T cells more efficiently and reduce inflammatory responses by modulating the function of regulatory T cells, thereby playing a therapeutic role in the treatment of autoimmune diseases and neurological disorders.

[0276] Although certain specific embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that these embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention. Therefore, the scope of the invention should be defined by the appended claims and their equivalents.

Claims

1. A binding molecule, said binding molecule comprising: (a) A heavy chain variable region, the heavy chain variable region comprising: Heavy chain CDR1, wherein the heavy chain CDR1 consists of the amino acid sequence shown in SEQ ID NO: 23; and Heavy chain CDR2, wherein the heavy chain CDR2 consists of the amino acid sequence shown in SEQ ID NO: 24; and (b) A light chain variable region, said light chain variable region comprising: Light chain CDR1, wherein the light chain CDR1 consists of the amino acid sequence shown in SEQ ID NO: 26; and The light chain CDR2, which consists of the amino acid sequence shown in SEQ ID NO:

27.

2. The binding molecule according to claim 1, wherein, The heavy chain variable region further comprises heavy chain CDR3, which is composed of an amino acid sequence selected from SEQ ID NO: 25 and 43; and The light chain variable region includes: Light chain CDR1, wherein the light chain CDR1 is composed of an amino acid sequence selected from SEQ ID NO: 26 and 79; Light chain CDR2, wherein the light chain CDR2 is composed of an amino acid sequence selected from SEQ ID NO: 27 and 80; and The light chain CDR3 is composed of an amino acid sequence selected from SEQ ID NO: 28, 47, 51, 55, 59, 63, 67, 71, 75 and 81.

3. The binding molecule according to claim 1, wherein, The heavy chain variable region includes: Heavy chain CDR1, which is encoded by the nucleotide sequence shown in SEQ ID NO: 31; and Heavy chain CDR2, wherein the heavy chain CDR2 is encoded by the nucleotide sequence shown in SEQ ID NO: 32; as well as The light chain variable region includes: Light chain CDR1, wherein the light chain CDR1 is encoded by the nucleotide sequence shown in SEQ ID NO: 34; and The light chain CDR2 is encoded by the nucleotide sequence shown in SEQ ID NO:

35.

4. The binding molecule according to claim 1, wherein, The heavy chain variable region includes: Heavy chain CDR1, wherein the heavy chain CDR1 is encoded by the nucleotide sequence shown in SEQ ID NO: 31; Heavy chain CDR2, wherein the heavy chain CDR2 is encoded by the nucleotide sequence shown in SEQ ID NO: 32; and Heavy chain CDR3, wherein the heavy chain CDR3 is encoded by a nucleotide sequence selected from SEQ ID NO: 33 and 45; as well as The light chain variable region includes: Light chain CDR1, wherein the light chain CDR1 is encoded by a nucleotide sequence selected from SEQ ID NO: 34 and 84; Light chain CDR2, wherein the light chain CDR2 is encoded by a nucleotide sequence selected from SEQ ID NO: 35 and 85; and The light chain CDR3 is encoded by a nucleotide sequence selected from SEQ ID NO: 36, 49, 53, 57, 61, 65, 69, 73, 77 and 86.

5. The binding molecule according to claim 1, wherein, The binding molecules are selected from: (a) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 25; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 28; (b) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 43; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 28; (c) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 25; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 47; (d) Combined molecules containing the following heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 25; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 51; (e) A molecule containing both a heavy chain variable region and a light chain variable region: The heavy chain variable region comprises: heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 25; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 55; (f) Combined molecules containing the following heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 25; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 59; (g) Combined molecules containing the following heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 25; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 63; (h) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 25; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 67; (i) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 25; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 71; (j) A molecule containing the following heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 25; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 75; as well as (k) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 23, heavy chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 24, and heavy chain CDR3 composed of the amino acid sequence shown in SEQ ID NO: 25; and The light chain variable region comprises: light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 79, light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 80, and light chain CDR3 composed of the amino acid sequence shown in SEQ ID NO:

81.

6. The binding molecule according to claim 1, wherein, The binding molecules are selected from: (a) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 33; and The light chain variable region comprises: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 35, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 36; (b) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 45; and The light chain variable region comprises: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 35, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 36; (c) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 33; and The light chain variable region comprises: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 35, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 49; (d) Combined molecules containing the following heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 33; and The light chain variable region comprises: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 35, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 53; (e) A molecule containing both a heavy chain variable region and a light chain variable region: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 33; and The light chain variable region comprises: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 35, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 57; (f) Combined molecules containing the following heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 33; and The light chain variable region comprises: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 35, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 61; (g) Combined molecules containing the following heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 33; and The light chain variable region comprises: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 35, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 65; (h) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 33; and The light chain variable region comprises: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 35, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 69; (i) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 33; and The light chain variable region comprises: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 35, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 73; (j) A molecule containing the following heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 33; and A light chain variable region comprising: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 34, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 35, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 77; and (k) A molecule containing the following combined heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: heavy chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 31, heavy chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 32, and heavy chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO: 33; and The light chain variable region comprises: light chain CDR1 encoded by the nucleotide sequence shown in SEQ ID NO: 84, light chain CDR2 encoded by the nucleotide sequence shown in SEQ ID NO: 85, and light chain CDR3 encoded by the nucleotide sequence shown in SEQ ID NO:

86.

7. The binding molecule according to claim 1, wherein, The binding molecule comprises: The heavy chain variable region is composed of an amino acid sequence selected from SEQ ID NO: 29, 44 and 82; and The light chain variable region is composed of an amino acid sequence selected from SEQ ID NO: 30, 48, 52, 56, 60, 64, 68, 72, 76 and 83.

8. The binding molecule according to claim 1, wherein, The binding molecule comprises: Heavy chain variable region, wherein the heavy chain variable region is encoded by a nucleotide sequence selected from SEQ ID NO: 37, 46 and 87; and The light chain variable region is encoded by a nucleotide sequence selected from SEQ ID NO: 38, 50, 54, 58, 62, 66, 70, 74, 78 and 88.

9. The binding molecule according to claim 1, wherein, The binding molecule also includes an Fc region or a constant region.

10. The binding molecule according to claim 9, wherein, The Fc region is the Fc region of IgG1, IgG2, IgG3 or IgG4 antibodies, or a hybrid Fc region.

11. The binding molecule according to claim 1, wherein, The binding molecule further comprises a heavy chain constant region consisting of an amino acid sequence selected from SEQ ID NO: 1, 3, 4, 7, 8, 9, 10 and 12.

12. The binding molecule according to claim 1, wherein, The binding molecule further comprises a light chain constant region consisting of an amino acid sequence selected from SEQ ID NO: 2, 5, 6 and 11.

13. The binding molecule according to claim 1, wherein, The binding molecule further comprises a heavy chain constant region and a light chain constant region, the heavy chain constant region being encoded by nucleotide sequences selected from SEQ ID NO: 13, 15 and 17, and the light chain constant region being encoded by nucleotide sequences selected from SEQ ID NO: 14, 16 and 18.

14. The binding molecule according to claim 1, wherein, The binding molecule is an antibody or its antigen-binding fragment.

15. The binding molecule according to claim 14, wherein, The antibody is a chimeric antibody, humanized antibody, bivalent antibody, bispecific molecule, microantibody, domain antibody, bispecific antibody, antibody mimic, biantibody, triantibody, tetraantibody, or fragments thereof.

16. A nucleic acid molecule, said nucleic acid molecule encoding a binding molecule as described in any one of claims 1 to 15.

17. An expression vector comprising the nucleic acid molecule of claim 16.

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

19. An antibody-drug conjugate comprising the binding molecule and drug as described in claim 5.

20. A chimeric antigen receptor (CAR), said chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a hinge domain, and a CD3ζ signaling domain, wherein, The antigen-specific binding domain is selected from antigen-specific binding domains that include the following heavy chain variable regions and light chain variable regions: The heavy chain variable region comprises: a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 23 and a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 24; and The light chain variable region comprises: a light chain CDR1 composed of the amino acid sequence shown in SEQ ID NO: 26 and a light chain CDR2 composed of the amino acid sequence shown in SEQ ID NO: 27; The CAR is a fusion protein that also contains the Fc region of an immunoglobulin.

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

22. The pharmaceutical composition according to claim 21, wherein, The cancer in question is a solid tumor.

23. The pharmaceutical composition according to claim 21, wherein, The cancers mentioned are stomach cancer, liver cancer, glioblastoma, ovarian cancer, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer.

24. A pharmaceutical composition for the prevention or treatment of nervous system diseases, said pharmaceutical composition comprising the binding molecule of any one of claims 1 to 20 as an active ingredient.

25. The pharmaceutical composition according to claim 24, wherein, The neurological diseases mentioned are neurodegenerative diseases or neuroinflammatory diseases.

26. The pharmaceutical composition according to claim 25, wherein, The neurodegenerative or neuroinflammatory diseases mentioned are selected from: stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prions, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy body dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, autoimmune neurological diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular diseases, spinal cord injury, and tau proteinosis.

27. A pharmaceutical composition for the prevention or treatment of immune-related diseases, said pharmaceutical composition comprising the binding molecule of any one of claims 1 to 20 as an active ingredient.

28. The pharmaceutical composition according to claim 27, wherein, The immune-related diseases mentioned are autoimmune diseases, graft-versus-host disease, organ transplant rejection, asthma, atopic disease, or acute or chronic inflammatory diseases.

29. The pharmaceutical composition according to claim 28, wherein, The autoimmune diseases mentioned are selected from: rheumatoid arthritis, systemic sclerosis, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjögren's syndrome, Guillain-Barré syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa.

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

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

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

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

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

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

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