Surface antigens of regulatory T cells and antibodies that specifically bind to them

Abstract

The present invention relates to an epitope of the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein, an antigen present on the surface of regulatory T cells or adipocytes, and an antibody or antigen-binding fragment that specifically binds to the epitope. The present invention also provides a diagnostic composition, diagnostic kit, method for providing diagnostic information, or diagnostic device for metabolic diseases, which comprises, as an active ingredient, an antibody or antigen-binding fragment capable of specifically binding to Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present on the surface of regulatory T cells or adipocytes.

Description

[Technical Field]

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

[0002] The most important characteristic of all normal individuals is the ability to recognize and eliminate non-self antigens while avoiding harmful reactions to antigenic substances that constitute the self. This lack of response by the body to self-antigens is called immunological unresponsiveness or tolerance. Self-tolerance develops by eliminating lymphocytes that may have specific receptors for self-antigens or by inactivating the body's own response after exposure to self-antigens. Problems in inducing or maintaining self-tolerance can lead to immune responses to self-antigens, resulting in autoimmune diseases.

[0003] For the treatment of autoimmune diseases, Gershon first proposed the concept of regulatory T cells in the early 1970s, which suggested the existence of T cells that could control and suppress the effector function of conventional T cells. Since then, many areas of epidemiology have been conducting research to clarify the biological characteristics and functions of regulatory T cells.

[0004] Regulatory T cells (Tregs) play an important role in naturally preventing excessive inflammation and immune responses, but it has been reported that the function and number of regulatory T cells are significantly reduced in autoimmune diseases and late-onset inflammatory diseases. Therefore, it is important for patients with immune and inflammatory diseases to produce regulatory T cells at normal levels, which could be one of the treatments for these diseases. To date, research has been conducted on genes and proteins that are specifically present in regulatory T cells, and substances such as CD25, CTLA4, CD62L, CD38, CD103, GITR, and CD45RB have been suggested as potential markers; however, there are currently no genes or proteins that can independently target regulatory T cells.

[0005] Each chain contains three variable regions called complementarity determining regions (CDRs) and four framework regions. The CDRs primarily function to bind to the epitope of an antigen. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, starting from the N-terminus, and are identified by the chain in which the particular CDR is located. Summary of the Invention [Problem to be solved by the invention]

[0006] One object of the present invention is to provide a pharmaceutical composition for preventing or treating metabolic diseases, which comprises, as an active ingredient, an antibody or antigen-binding fragment thereof capable of specifically binding to Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present on the surface of regulatory T cells (Treg cells).

[0007] It is yet another object of the present invention to provide an antibody-drug conjugate (ADC) in which the antibody according to the present invention is conjugated to a drug for preventing or treating a metabolic disease.

[0008] It is yet another object of the present invention to provide a pharmaceutical composition for preventing or treating various diseases, such as metabolic diseases, which comprises an antibody-drug conjugate as an active ingredient.

[0009] One object of the present invention is to provide a diagnostic composition, diagnostic kit, method for providing diagnostic information, or diagnostic device for metabolic diseases, which comprises, as an active ingredient, an antibody or antigen-binding fragment capable of specifically binding to Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present on the surface of regulatory T cells (Treg cells).

[0010] However, the technical problems that the present invention aims to achieve are not limited to those mentioned above, and other problems not mentioned above will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0011] The present invention relates to an epitope of the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein, or an antibody or antigen-binding fragment that specifically binds to said epitope.

[0012] As used herein, "epitope" refers to a sequence of amino acids within an antigen, the amino acids within which (or a subset thereof) are specifically recognized by an antibody or binding fragment, such as an antibody or binding fragment described herein. An epitope may comprise one or more antigenic determinants. For example, an antibody raised against an isolated peptide corresponding to a topographical epitope may recognize part or all of the epitope sequence.

[0013] In the present invention, the "Lrig-1 protein" is a transmembrane protein present on the surface of regulatory T cells. It consists of an extracellular or lumenal leucine-rich repeat (LRR), three immunoglobulin-like domains, a transmembrane sequence, and a cytoplasmic tail. The LRIG gene family consists of LRIG1, LRIG2, and LRIG3, and the amino acid structure of each family is highly conserved. The LRIG1 gene is highly expressed in normal skin, expressed in basal and hair follicle cells, and can regulate the proliferation of epidermal stem cells. Therefore, it plays an important role in maintaining epidermal homeostasis, and its absence can lead to the development of psoriasis and skin cancer. It has been reported that disruption of chromosome 3p14.3, where LRIG1 is located, can lead to the development of cancer cells. In fact, it has been confirmed that LRIG1 expression is significantly reduced in renal cell carcinoma and cutaneous squamous cell carcinoma. However, it has recently been revealed that only about 20-30% of cancers express the Lrig-1 protein. Meanwhile, for the purposes of the present invention, the Lrig-1 protein may be derived from mammals or mice, but is not limited thereto.

[0014] In one embodiment of the present invention, the Lrig-1 protein may be derived from mammals, for example, primates such as humans and monkeys, and rodents such as mice and rats.

[0015] In one embodiment of the present invention, the Lrig-1 protein may be a human-derived Lrig-1 protein represented by SEQ ID NO: 1, which may be encoded by the nucleic acid sequence represented by SEQ ID NO: 2, but is not limited thereto.

[0016] In another embodiment of the present invention, the Lrig-1 protein may be a mouse-derived Lrig-1 protein represented by SEQ ID NO: 3, which may be encoded by the nucleic acid sequence represented by SEQ ID NO: 4, but is not limited thereto.

[0017] In another embodiment of the present invention, the Lrig-1 protein may be, but is not limited to, the extracellular domain of the Lrig-1 protein.

[0018] The Lrig-1 extracellular domain of the present invention may be an extracellular domain of an Lrig-1 protein derived from a mammal, for example, a primate such as a human or a monkey, or a rodent such as a mouse or a rat. For purposes of the present invention, the extracellular domain of an Lrig-1 protein may be, but is not limited to, an extracellular domain of an Lrig-1 protein derived from a human or a mouse.

[0019] In one example of the present invention, the extracellular domain of the Lrig-1 protein may be represented by SEQ ID NO: 5, which corresponds to the amino acid sequence from positions 35 to 794 of the human Lrig-1 protein, but is not limited thereto.

[0020] In another embodiment of the present invention, the extracellular domain of the Lrig-1 protein may be represented by SEQ ID NO: 6, which corresponds to the amino acid sequence from amino acid 35 to amino acid 794 of the mouse-derived Lrig-1 protein, but is not limited thereto.

[0021] The present invention will be described in more detail below.

[0022] According to one embodiment of the present invention, the epitope of the Lrig-1 protein includes at least one polypeptide selected from the group consisting of polypeptides having the amino acid sequences represented by SEQ ID NOs: 35 to 45.

[0023] In one example of the present invention, the epitope of the Lirg-1 protein may be an epitope comprising at least one polypeptide selected from the group consisting of polypeptides having the amino acid sequences represented by SEQ ID NOs: 35 to 40, but is not limited thereto.

[0024] As another example of the present invention, the epitope of the Lrig-1 protein may be an epitope comprising at least one polypeptide selected from the group consisting of polypeptides having the amino acid sequences represented by SEQ ID NO: 41 to SEQ ID NO: 45, but is not limited thereto.

[0025] According to one embodiment of the present invention, the epitope of the Lrig-1 protein may be a site corresponding to the base sequence from bases 62 to 101, or from bases 88 to 92, or from bases 90 to 101, or from bases 111 to 134, or from bases 452 to 476, or from bases 472 to 480, or from bases 542 to 553 of the Lrig-1 protein.

[0026] According to another embodiment of the present invention, the epitope of the Lrig-1 protein may be a site corresponding to the 95th to 101st or 472nd to 476th bases of the nucleotide sequence of the Lrig-1 protein.

[0027] The epitope of the present invention may be a conformational epitope.

[0028] The "conformational epitope" of the present invention is composed of a discontinuous amino acid sequence, unlike a one-dimensional linear epitope composed of a continuous sequence. Such a conformational epitope reacts with a three-dimensional structure such as an antibody-antigen binding site.

[0029] According to another embodiment of the present invention, there is provided a binding molecule that specifically binds to an epitope of the present invention.

[0030] The "binding molecule" of the present invention may be, but is not limited to, an antibody or an antigen-binding fragment.

[0031] In the present invention, the "antibody" includes both a full-length antibody and an antibody fragment, which is a portion of an antibody and has the ability to bind to the Lrig-1 protein and binds to the Lrig-1 antigen crystal site competitively with the binding molecule of the present invention.

[0032] In the present invention, the term "antibody" refers to a protein molecule that acts as a receptor to specifically recognize an antigen, including an immunoglobulin molecule immunologically reactive with a specific antigen. For purposes of the present invention, the antigen may be the Lrig-1 protein present on the surface of regulatory T cells. Preferably, the antibody may specifically recognize the leucine-rich region or immunoglobulin-like domain of the Lrig-1 protein, but is not limited thereto.

[0033] In the present invention, the "immunoglobulin" has a heavy chain and a light chain, each of which contains a constant region and a variable region. The variable regions of the light and heavy chains contain three variable regions called complementarity determining regions (hereinafter referred to as "CDRs") and four framework regions. The CDRs primarily function to bind to epitopes of antigens. The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, sequentially, starting from the N-terminus, and are identified by the chain in which the particular CDR is located.

[0034] In the present invention, the antibodies or antigen-binding fragments thereof provided by the present invention are chimeric antibodies or fragments comprising heavy chain CDRs and light chain CDRs selected from the CDRs provided by the present invention, or comprising conservative variants of the CDRs provided by the present invention.

[0035] For purposes of the present invention, "variants" of an amino acid sequence include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. Amino acid deletion variants, including deletions at the N-terminus and / or C-terminus of a protein, are also referred to as N-terminal and / or C-terminal truncation variants.

[0036] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertions with appropriate screening of the resulting products are also possible.

[0037] Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids.

[0038] Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be at any position in the protein.

[0039] Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Preferably, the alterations occur at positions in the amino acid sequence that are not conserved among homologous proteins or peptides and / or involve the substitution of an amino acid with another amino acid with similar properties. Preferably, the amino acid changes in the protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve the substitution of a member of a family of amino acids that are related in sequence. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes also classified as aromatic amino acids.

[0040] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence may be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given over at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given over the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, may be performed using tools known in the art, preferably the best sequence alignment, for example, Align using standard settings, preferably EMBOSS::Needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0041] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of identical amino acids between the sequences.

[0042] The term "percent identity" is intended to indicate the percentage of identical amino acid residues between the two sequences being compared after optimal alignment; this percentage is purely statistical, with the differences between the two sequences being distributed randomly and throughout their entire length. Sequence comparison between two amino acid sequences is usually performed by optimally aligning these sequences and then comparing them, which identifies local regions of sequence similarity and is performed by segments or "comparison windows" for comparison. Optimal alignment of sequences for comparison can be generated not only passively but also by local homology algorithms or similarity search methods, or by computer programs using these algorithms (GAP, BESTFIT, FASTA, BLASTP, BLASTN, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Medison, Wisconsin, USA).

[0043] Percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

[0044] Homologous amino acid sequences according to the present invention exhibit at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98 or at least 99% identity of the amino acid residues.

[0045] The amino acid sequence variants described herein can be readily prepared by one of skill in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to produce proteins and peptides with substitutions, additions, insertions, or deletions is well known. The peptides and amino acid variants described herein can also be readily prepared through known peptide synthesis techniques, such as, for example, solid phase synthesis and similar methods.

[0046] In the present invention, the antibody or antigen-binding fragment thereof provided by the present invention is a humanized antibody or fragment comprising a heavy chain CDR and a light chain CDR selected from the CDRs provided by the present invention, or comprising conservative variants of the CDRs provided by the present invention.

[0047] The antibodies or antigen-binding fragments thereof provided herein comprise a light chain and / or a heavy chain comprising a sequence provided herein or a conservative variant thereof. In one embodiment, the conservative variant has a sequence that is 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical to the reference sequence provided herein. In one embodiment, the conservative variant contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 amino acid substitutions, insertions, or deletions.

[0048] In the present invention, antibodies that specifically bind to the Lrig-1 protein provided herein comprise the sequences provided herein or conservative variants thereof. As used herein, "conservative variants" include conservative amino acid substitutions, insertions, or deletions. Those skilled in the art will recognize that a conservative amino acid substitution is the substitution of one amino acid with another amino acid that has similar structural or chemical properties, such as a similar amino acid sequence, while maintaining the biological activity of the reference sequence. Exemplary conservative substitutions are well known in the art.

[0049] In the present invention, the "full-length antibody" has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond, and includes IgA, IgD, IgE, IgM, and IgG. The IgG subtypes include IgG1, IgG2, IgG3, and IgG4.

[0050] In addition, in the present invention, the term "antigen-binding fragment" refers to a fragment that retains antigen-binding function, and examples of antigen-binding fragments include: (1) a Fab fragment consisting of a light chain variable region (VL), a heavy chain variable region (VH), a light chain constant region (CL), and the first heavy chain constant region (CH1); (2) an Fd fragment consisting of a VH and CH1 domain; (3) an Fv fragment consisting of the VL and VH domains of a single antibody; (4) a dAb fragment consisting of a VH domain; (5) separated CDR regions; (6) an F(ab')2 fragment, which is a bivalent fragment comprising two linked Fab fragments; (7) a single-chain Fv molecule (scFv) in which the VH and VL domains are linked by a peptide linker to form an antigen-binding site; (8) a bispecific single-chain Fv dimer; and (9) a diabody, which is a multivalent or multispecific fragment produced by gene fusion. The antigen-binding fragment can be produced by using proteolytic enzymes such as papain or pepsin to obtain Fab or F(ab')2 fragments, or by genetic recombination techniques.

[0051] Furthermore, in the present invention, the antibody and fragment thereof may be, but is not limited to, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human antibody, a bivalent antibody, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody, a tetrabody, or a fragment thereof.

[0052] In the present invention, the "chimeric antibody" is an antibody obtained by recombining the variable region of a mouse antibody and the constant region of a human antibody, and exhibits significantly improved immune responses compared to mouse antibodies.

[0053] In the present invention, the term "humanized antibody" refers to an antibody in which the protein sequence of an antibody derived from a non-human species has been modified to resemble a naturally occurring human antibody variant. For example, the humanized antibody can be prepared by combining mouse-derived CDRs with human-derived FRs to prepare humanized variable regions, which can then be combined with a preferred human antibody constant region to prepare a humanized antibody.

[0054] In the present invention, the binding molecule may also be a bispecific antibody or bispecific antigen-binding fragment that can bind to the Lrig-1 protein and also to other proteins.

[0055] In the present invention, the bispecific antibodies and bispecific antigen-binding fragments may comprise a binding molecule of the present invention. In one embodiment, the bispecific antibodies and bispecific antigen-binding fragments comprise an antigen-binding domain capable of binding to Lrig-1 protein, and the antigen-binding domain capable of binding to Lrig-1 protein may comprise or consist of a binding molecule of the present invention.

[0056] The bispecific antibodies and bispecific antigen-binding fragments provided herein comprise an antigen-binding domain that is a binding molecule capable of binding to the Lrig-1 protein of the present invention, and an antigen-binding domain capable of binding to another target protein. Here, the antigen-binding domain capable of binding to another target protein is a protein other than the Lrig-1 protein, but is not limited to this, and may be, for example, an antigen-binding domain capable of binding to PD-1 or a cell surface receptor, but is not limited to this.

[0057] The bispecific antibodies and bispecific antigen-binding fragments of the invention can be provided in any suitable format, such as those described in the documents incorporated herein by reference in their entirety. For example, the bispecific antibodies or bispecific antigen-binding fragments can be provided as bispecific antibody conjugates (e.g., IgG2, F(ab')2, or CovX-bodies), bispecific IgG or IgG-type molecules (e.g., IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, or 2in1-IgG, mAb2, or Tandemab common LC), asymmetric bispecific IgG or IgG-type molecules (e.g., kih IgG, kih IgG common LC, CrossMab, kih The antibody may be an IgG-scFab, mAb-Fv, charge pair or SEED-body), a small bispecific antibody molecule (e.g., diabody (Db), dsDb, DART, scDb, tandAbs, tandem scFv (taFv), tandem dAb / VHH, triple body, triple head, Fab-scFv, or F(ab')2-scFv2), a bispecific Fc and CH3 fusion protein (e.g., taFv-Fc, di-diabody, scDb-CH3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-CH3), or a bispecific fusion protein (e.g., scFv2-albumin, scDb-albumin, taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine2). One skilled in the art can design and produce bispecific antibodies and bispecific antigen-binding fragments according to the invention.

[0058] In the present invention, methods for producing the bispecific antibodies include chemical cross-linking of antibodies or antibody fragments with reducible disulfide or non-reducible thioether bonds. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) may be used to chemically cross-link Fab fragments, for example, through hinge region SH groups, to generate disulfide-linked bispecific F(ab)2 heterodimers.

[0059] Another method for producing the bispecific antibody of the present invention involves fusing an antibody-producing hybridoma with, for example, polyethylene glycol, to generate a quadroma cell capable of secreting the bispecific antibody.

[0060] The bispecific antibodies and bispecific antigen-binding fragments of the present invention may be produced recombinantly, for example, by expression from nucleic acid constructs encoding the polypeptides for the antigen-binding molecules.

[0061] For example, a DNA construct encoding light and heavy chain variable domains for two antigen-binding domains (i.e., light and heavy chain variable domains for an antigen-binding domain capable of binding, e.g., PD-1, and light and heavy chain variable domains for an antigen-binding domain capable of binding another target protein), along with sequences encoding a suitable linker or dimerization domain between the antigen-binding domains, can be produced by molecular cloning techniques. Recombinant bispecific antibodies may then be produced by expression (e.g., in vitro) of the construct in a suitable host cell (e.g., a mammalian host cell), and the expressed recombinant bispecific antibody may then optionally be purified.

[0062] Antibodies may also be produced by an affinity maturation process that produces modified antibodies that have improved affinity for the antigen compared to the unmodified parent antibody. Affinity matured antibodies may be produced by procedures known in the art.

[0063] Furthermore, the binding molecules provided by the present invention may contain variants of the amino acid sequences, so long as they are capable of specifically binding to the Lrig-1 protein. For example, the amino acid sequence of the antibody may be altered to improve the antibody's binding affinity and / or other biological properties. Such modifications include, for example, deletion, insertion, and / or substitution of residues in the amino acid sequence of the antibody.

[0064] Such amino acid mutations are performed based on the relative similarity of the amino acid side chain substitutions, such as hydrophobicity, hydrophobicity, charge, size, etc. Analysis of the size, pattern, and type of amino acid side chain substitutions reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tylosin have similar patterns. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tylosin can be considered biologically functional equivalents.

[0065] When introducing mutations, the hydropathic index of amino acids may be taken into consideration. Each amino acid is assigned a hydrophobic index based on its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tylosin (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The hydrophobic amino acid index is very important in imparting interactive biological functions to proteins. It is well known that similar biological activity can be maintained only by substituting amino acids with similar hydrophobic indexes. When introducing mutations based on the hydrophobic index, substitutions are made between amino acids showing a difference in hydrophobic index within ±2, preferably within ±1, and even more preferably within ±0.5.

[0066] On the other hand, it is also well known that substitutions between amino acids with similar hydrophilicity values ​​result in proteins with equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tylosin (-2.3); phenylalanine (-2.5); tryptophan (-3.4). When introducing mutations with reference to the new water value, substitutions can be made between amino acids that show a difference in new water value within ±2, more preferably within ±1, and even more preferably within ±0.5.

[0067] Amino acid exchanges in proteins that do not overall alter the activity of the molecule are known in the art. The most commonly occurring exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, and Gln / Glu.

[0068] In view of the above-mentioned bioequivalent variants, the binding molecules of the present invention are also understood to include sequences that show substantial identity to the sequences set forth in the sequence listing.

[0069] As used herein, the term "substantial identity" refers to a sequence that exhibits at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology, when the sequence of the present invention is aligned with any other sequence for maximum correspondence and the aligned sequences are analyzed using algorithms commonly used in the art. Alignment methods for sequence comparison are well known in the art. Various alignment methods and algorithms are available from the NCBI Basic Local Alignment Search Tool (BLAST), the National Center for Biological Information (NBCI), and the like, and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet. BLAST can be accessed at the following address (www.ncbi.nlm.nih.gov / BLAST / ). Sequence homology comparison methods using this program can be viewed online (www.ncbi.nlm.nih.gov / BLAST / blast_help.html).

[0070] In the present invention, the binding molecule, preferably the antibody, may be produced by a conventional method for producing an antibody, but may also be produced by affinity maturation.

[0071] In the present invention, the term "affinity maturation" refers to the process by which activated B cells produce antibodies with increased affinity for an antigen during an immune response. For the purposes of the present invention, affinity maturation can be performed based on the principles of mutation and selection, similar to the process that occurs in nature, to produce antibodies or antibody fragments produced by affinity maturation.

[0072] In addition, in the present invention, the term "monoclonal antibody" refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, and exhibits a single binding specificity and affinity for a specific antigenic determinant (epitope).

[0073] In the present invention, the term "binding" or "specific binding" refers to the affinity of the present antibody or antibody composition for an antigen. In antigen-antibody binding, "specific binding" typically refers to a dissociation constant (Kd) of 1x10 -5 Less than M or 1x10 -6 Less than M or 1x10 -7 If the binding is less than M, it can be distinguished from nonspecific background binding. Specific binding may be detected by methods known in the art, such as ELISA, surface plasmon resonance (SPR), immunoprecipitation, coprecipitation, etc., and includes appropriate controls that can distinguish specific binding from nonspecific binding.

[0074] The antibodies or antigen-binding fragments of the present invention can exist in multimers, such as dimers, trimers, tetramers, and pentamers, that contain at least a portion of the antigen-binding ability of the monomers. Such multimers also include homomultimers and heteromultimers. Antibody multimers contain multiple antigen-binding sites and therefore have superior antigen-binding ability compared to monomers. Antibody multimers also facilitate the production of multifunctional (bifunctional, trifunctional, and tetrafunctional) antibodies.

[0075] In the present invention, the term "multifunctional" refers to an antibody or antigen-binding fragment having two or more activities or functions (e.g., antigen-binding ability, enzymatic activity, ligand- or receptor-binding ability). For example, the antibody of the present invention may be conjugated to a polypeptide having enzymatic activity, such as luciferase, acetyltransferase, or galactosidase. Multifunctional antibodies also include multivalent or multispecific (bispecific, trispecific, etc.) antibodies.

[0076] According to another embodiment of the present invention, there is provided a nucleic acid molecule encoding the binding molecule provided herein.

[0077] The nucleic acid molecules of the present invention include any nucleic acid molecule in which the amino acid sequence of the binding molecule provided by the present invention has been translated into a polynucleotide sequence as known to those skilled in the art. Therefore, various polynucleotide sequences based on ORF (open reading frame) can be produced, and all of these are also included in the nucleic acid molecules of the present invention.

[0078] According to another embodiment of the present invention, there is provided an expression vector into which the isolated nucleic acid molecule provided by the present invention is inserted.

[0079] As used herein, a "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid or a nucleic acid to which it has been ligated. One type of vector is a "plasmid," which refers to a circular double-stranded DNA to which additional DNA segments can be ligated. Another type of vector is a phage vector. Yet another type of vector is a viral vector, to which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in host cells into which they are introduced (e.g., bacterial vectors are episomal mammalian vectors with a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction, thereby replicating along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors." Expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector.

[0080] Specific examples of the expression vector used in the present invention include, but are not limited to, commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1P22, Qμμ, T-even, T2, T3, and T7; and plant viruses. All expression vectors known to those skilled in the art can be used in the present invention, and the selection of an expression vector depends on the properties of the target host cell. Introduction of the vector into the host cell can be performed by calcium phosphate transfection, viral infection, DEAE-dextran-mediated transfection, lipofectamine transfection, or electroporation, but is not limited thereto. Those skilled in the art can select an appropriate introduction method for the expression vector and host cell used. Preferably, the vector contains one or more selectable markers, but is not limited thereto. Using a vector without a selectable marker allows for selection based on the production of a product. The selection of the selectable marker is determined by the desired host cell, and this is done using methods already known to those skilled in the art, and the present invention is not limited thereto.

[0081] To facilitate purification of the nucleic acid molecule of the present invention, a tag sequence can be inserted into an expression vector to fuse the nucleic acid molecule. Examples of such tags include, but are not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag. Any tag known to those skilled in the art that facilitates purification can be used in the present invention.

[0082] According to another embodiment of the present invention, there is provided a host cell line transformed with the expression vector provided by the present invention.

[0083] As used herein, the term "host cell" includes an individual cell or culture of cells that may be or has been the recipient of a vector for incorporation of a polypeptide insert. Host cells include the progeny of a single host cell, which may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Host cells include cells transfected with the polypeptides of the present application.

[0084] In the present invention, the host cells may include cells of mammalian, plant, insect, fungal or cellular origin, such as bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, etc.; fungal cells such as yeast cells and Pichia pastoris, etc.; insect cells such as Drosophila and Spodoptera Sf9 cells, etc.; CHO (Chinese hamster ovary cells), SP2 / 0 (mouse myeloma), human lymphoblastoid, COS, NSO (mouse myeloma), 293T, bow melanoma cells, HT-1080, BHK (Baby Hamster Kidney cells), HEK (Human Embryonic Kidney cells), etc. The host cell line may be, but is not limited to, animal cells such as PERC.6 (human retinal cells) or PERC.6 (human retinal cells); or plant cells, and any cells that can be used as a host cell line known to those skilled in the art can be used.

[0085] As used herein, the term "combination" or "pharmaceutical combination" refers to a product formed by mixing or combining more than one active ingredient, including both fixed and non-fixed combinations of the active ingredients. The term "fixed combination" means that the active ingredients, e.g., a compound of the present invention, and one or more additional therapeutic agents are administered to a patient simultaneously in the form of a single substance or dosage. The term "non-fixed combination" means that the active ingredients, e.g., a compound of the present invention, and one or more additional therapeutic agents are administered to a patient simultaneously, jointly, or sequentially without specific time limitations as separate substances, where such administration provides a therapeutically effective level of the active ingredients in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0086] The term "composition" or "pharmaceutical composition" as used herein refers to a mixture of at least one compound of the present invention and, optionally, one or more different pharmaceutically acceptable chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, and / or excipients.

[0087] As used herein, the terms "optical isomer" or "stereoisomer" refer to any of the various stereoisomeric configurations that can exist for a given compound of the present invention, including geometric isomers. It is understood that substituents can be attached to chiral carbon atoms at chiral centers. The term "chiral" refers to a molecule that possesses the property of non-superimposability on its mirror image partner, while the term "non-chiral" refers to a molecule that is superimposable on its mirror image partner. Thus, the present invention includes enantiomers, partial stereoisomers, or racemates of a compound. "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A 1:1 mixture of a pair of enantiomers is a "racemic" mixture. The terms are used to designate racemic mixtures where appropriate. "Partial stereoisomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other. Absolute stereochemistry is specified by the Cahn-Ingold-Prelog RS system. When a compound is a pure enantiomer, the stereochemistry at each chiral carbon may be designated by R or S. Resolved compounds of unknown absolute configuration may be designated (+) or (-) according to the direction (right- or left-handed) they rotate plane-polarized light at the wavelength of the sodium D line. Certain compounds described herein may contain one or more asymmetric centers or axes and thus produce enantiomers, partial stereoisomers, and other stereoisomeric forms that can be defined in terms of absolute stereochemistry as (R)- or (S)-.

[0088] As used herein, the term "efficacy agent" or "agonist" refers to a compound or antigen-binding fragment that is capable of activating a Toll-like receptor.

[0089] The terms "treating" or "treatment" as used herein refer to a method of alleviating, alleviating or reversing the symptoms of a disease or condition, preventing additional symptoms, reversing or preventing the underlying metabolic cause of the symptoms, inhibiting the disease or condition, halting the development of the disease or condition, relieving the disease or condition, inducing regression of the disease or condition, alleviating conditions caused by the disease or condition, or prophylactically and / or therapeutically halting the symptoms of a disease or condition.

[0090] The salt of the compound of the present invention having at least one salt-forming group can be prepared by a method known to those skilled in the art.For example, the salt of the compound of the present invention having an acid group can be prepared by treating the compound with a metal compound, for example, the alkali metal salt of a suitable organic carboxylic acid, for example, sodium 2-ethylhexanoate, with an organic alkali metal or alkaline earth metal compound, for example, the corresponding hydroxide, carbonate or hydrogen carbonate, for example, sodium hydroxide or potassium hydroxide, carbonate or hydrogen carbonate, with the corresponding calcium compound, or with ammonia or a suitable organic amine, preferably using a stoichiometric amount or a small excess of the salt-forming agent.The acid addition salt of the compound of the present invention can be obtained by a conventional method, for example, by treating the compound with an acid or a suitable anion exchange reagent. Internal salts of compounds of the present invention containing acid and base salt-forming groups, such as carboxy and amino groups, may be formed, for example, by neutralizing salts, e.g., acid addition salts, at the isoelectric point, for example, with a weak base or by treatment with an ion exchanger.

[0091] The salts may be converted to glass compounds by methods known to those of ordinary skill in the relevant art: metal and ammonium salts may be converted, for example, by treatment with a suitable acid, and acid addition salts may be converted, for example, by treatment with a suitable basic agent.

[0092] All of the process steps referred to above may be carried out under reaction conditions known to those of ordinary skill in the relevant art, including those specifically mentioned, in the absence or usually presence of solvents or diluents (including, for example, solvents or diluents that are inert to and dissolve the reagents used), in the absence or presence of catalysts, condensing agents or neutralizing agents, e.g., ion exchangers, e.g., cation exchangers (e.g., in H+ form), depending on the nature of the reaction and / or reactants, at reduced, normal, or elevated temperatures, e.g., in the temperature range of about −100° C. to about 190° C. (e.g., about −80° C. to about 150° C., e.g., −80 to −60° C., room temperature, −20 to 40° C., or reflux temperature), under atmospheric pressure or in a sealed vessel, under elevated pressure where appropriate, and / or in an inert atmosphere, e.g., under an argon or nitrogen atmosphere.

[0093] In one embodiment, the present invention provides a compound selected from the group consisting of acetate, adipate, ascorbate, aspartate, benzoate, besylate, benzenesulfonate, bicarbonate / carbonate, bisulfate / sulfate, bromide / hydrobromide, camphorsulfonate, camsylate, caprate, chloride / hydrochloride, chlortheophyllonate, citrate, Edisylate, ethanedisulfonate, fumarate, gluceptate, glucoheptanoate, gluconate, glucuronate, glutamate, glutarate, glycolate, hippurate, hydroiodide / iodide, isethionate, lactate, lactobionate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, mucate, naphthoate, napsylate, 2-napsylate, naphthalenesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, octadecanoate, It may be provided as an oleate, oxalate, palmitate, pamoate, phosphate / hydrogenphosphate / dihydrogenphosphate, polygalacturonate, propionate, sebacate, stearate, succinate, sulfosalicylate, sulfate, tartrate, tosylate, p-toluenesulfonate, trifluoroacetate, triphenate, triphenylacetate or xinafoate salt form.

[0094] The compounds of the present invention can inherently or by design form solvates with pharmaceutically acceptable solvents, including water; therefore, the present invention is intended to encompass both solvated and unsolvated forms. The term "solvate" refers to a molecular complex of a compound of the present invention (including pharmaceutically acceptable salts thereof) with one or more solvent molecules. Such solvent molecules are those commonly used in the pharmaceutical arts that are known to be harmless to the recipient, e.g., water, ethanol, etc. The term "hydrate" refers to a complex in which the solvent molecule is water.

[0095] Any asymmetric atom (e.g., carbon, etc.) of the compounds of the present invention can be present in racemic or enantiomerically enriched form, for example, in the (R)-, (S)-, or (R,S)-configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R)- or (S)-configuration. Substituents at atoms having unsaturated double bonds can exist in cis-(Z)- or trans-(E)-forms, where possible.

[0096] Thus, as used herein, the compounds of the present invention may be in the form of one of the possible isomers, rotational isomers, rotationally hindered isomers, tautomers or mixtures thereof, such as substantially pure geometric (cis or trans) isomers, partial stereoisomers, optical isomers (enantiomers), racemates or mixtures thereof.

[0097] Any resulting mixture of isomers can be separated into pure or substantially pure geometric or optical isomers, partial stereoisomers, racemates, based on the physicochemical differences of the constituent components, for example, by chromatography and / or fractional crystallization.

[0098] Any resulting racemic forms of the final products or intermediates can be resolved into their optical antipodes by known methods, for example, by separating the resulting partial stereoisomeric salts with optically active acids or bases to liberate the optically active acidic or basic compounds. In particular, basic moieties can be used to resolve the compounds of the present invention into their optical antipodes by fractional crystallization of salts formed with optically active acids, such as tartaric acid, dibenzoyltartaric acid, diacetyltartaric acid, di-O,O'-p-toluoyltartaric acid, mandelic acid, malic acid, or camphor-10-sulfonic acid. Racemic products can also be resolved by chiral chromatography, for example, high-pressure liquid chromatography (HPLC) using a chiral adsorbent.

[0099] In certain embodiments, compounds are prepared as their individual stereoisomers. In other embodiments, compounds are prepared as their individual stereoisomers by reacting a racemic mixture of the compound with an optically active resolving agent to form a pair of partial stereoisomer compounds, separating the partial stereoisomers, and recovering the optically pure enantiomers. In certain embodiments, this is done using covalent partial stereoisomer derivatives of the enantiomeric compounds or using dissociable complexes (e.g., crystalline partial stereoisomer salts). The partial stereoisomers have distinct physical properties (e.g., melting points, boiling points, solubilities, reactivity, etc.) and are easily separated by exploiting these differences. In certain embodiments, the partial stereoisomers are separated by chromatography or by separation / resolution techniques based on solubility differences. The optically pure enantiomers are subsequently recovered along with the resolving agent by any means that does not induce racemization. A more detailed description of techniques applicable to the resolution of stereoisomers of a compound from its racemic mixture can be found in the literature [Jean Jacques, Andre Collet, Samuel H. Wilen, "Enantiomers, Racemates and Resolutions," John Wiley and Sons, Inc., 1981].

[0100] The mixture of isomers obtained according to the present invention can be separated into individual isomers in a manner known to those of ordinary skill in the relevant art; partial stereoisomers can be separated, for example, by partition between a multiphase solvent mixture, recrystallization and / or chromatographic separation, for example, on silica gel, or by pressure liquid chromatography, for example, on a reversed-phase column, and racemates can be separated, for example, by salt formation with an optically pure salt-forming reagent and separation of the mixture of partial stereoisomers thus obtained, for example, by means of fractional crystallization or by optically active column chromatography.

[0101] Depending on the selection of starting materials and procedures, certain embodiments of the compounds of the present invention exist in the form of one of the possible isomers or as a mixture thereof, depending on the number of asymmetric carbon atoms, e.g., as pure optical isomers or as isomeric mixtures, e.g., racemic and partial stereoisomeric mixtures. The present invention is intended to include all such possible isomers, including racemic mixtures, partial stereoisomeric mixtures, and optically pure forms. Optically active (R)- and (S)-isomers can be prepared using chiral synthesis units or chiral reagents, or resolved using conventional techniques. When compounds contain double bonds, the substituents may be in the E- or Z-configuration. When compounds contain disubstituted cycloalkyl, the cycloalkyl substituents can have cis- or trans-configuration. All tautomeric forms are further intended to be included.

[0102] Intermediates and final products may be worked up and / or purified by standard methods, for example using chromatographic methods, distribution methods, (re)crystallization, etc. The present invention also relates to processes in which compounds obtained as intermediates at any step of the process are used as starting materials to carry out the remaining process steps, or in which starting materials are formed under the reaction conditions or used in the form of derivatives, e.g., protected forms or salt forms, or in which compounds obtained by the process according to the invention are prepared under the process conditions and further processed in situ. All starting materials, building blocks, reagents, acids, bases, dehydrating agents, solvents and catalysts used to synthesize the compounds of the present invention are commercially available or can be prepared by organic synthesis methods known to those of ordinary skill in the relevant art.

[0103] The terms "alkyl" and "alk" refer to a straight or branched chain alkane (hydrocarbon) radical containing 1 to 12 carbon atoms, preferably 1 to 6 carbon atoms. Exemplary "alkyl" groups include methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutylpentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like. The term "(C1-C4) alkyl" refers to a straight or branched chain alkane (hydrocarbon) radical containing 1 to 4 carbon atoms, e.g., methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, and isobutyl. "Substituted alkyl" refers to an alkyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.

[0104] The term "alkenyl" refers to a linear or branched chain hydrocarbon radical containing 2 to 12 carbon atoms and at least one carbon-carbon double bond. Exemplary such groups include ethenyl or allyl. The term "C2-C6 alkenyl" refers to a linear or branched chain hydrocarbon radical containing 2 to 6 carbon atoms and at least one carbon-carbon double bond, such as ethylenyl, propenyl, 2-propenyl, (E)-but-2-enyl, (Z)-but-2-enyl, 2-methy(E)-but-2-enyl, 2-methy(Z)-but-2-enyl, 2,3-dimethyl ... "Alkenyl" refers to alkenyl groups substituted with one or more substituents, preferably from 1 to 4 substituents, at any available point of attachment.

[0105] The term "alkynyl" refers to a linear or branched chain hydrocarbon radical containing 2 to 12 carbon atoms and at least one carbon-carbon triple bond. Exemplary such groups include ethynyl. The term "C2-C6 alkynyl" refers to a linear or branched chain hydrocarbon radical containing 2 to 6 carbon atoms and at least one carbon-carbon triple bond, such as ethynyl, prop-1-ynyl, prop-2-ynyl, but-1-ynyl, but-2-ynyl, pent-1-ynyl, pent-2-ynyl, hex-1-ynyl, hex-2-ynyl, and hex-3-ynyl. "Substituted alkynyl" refers to an alkynyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.

[0106] The term "cycloalkyl" refers to a fully saturated cyclic hydrocarbon group containing from 1 to 4 rings and from 3 to 8 carbons per ring. "C3-C7 cycloalkyl" refers to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or cycloheptyl. "Substituted cycloalkyl" refers to a cycloalkyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.

[0107] The term "cycloalkenyl" refers to a partially unsaturated cyclic hydrocarbon group containing 1 to 4 rings and 3 to 8 carbons per ring. Exemplary groups include cyclobutenyl, cyclopentenyl, cyclohexenyl, and the like. "Substituted cycloalkenyl" refers to a cycloalkenyl group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.

[0108] The term "aryl" refers to a cyclic, aromatic hydrocarbon group having one to five aromatic rings, particularly a monocyclic or bicyclic group, such as phenyl, biphenyl, or naphthyl. When containing two or more aromatic rings (e.g., bicyclic), the aromatic rings of the aryl group may be joined at a single point (e.g., biphenyl) or fused (e.g., naphthyl, phenanthrenyl, etc.). "Substituted aryl" refers to an aryl group substituted with one or more substituents, preferably one to three substituents, at any available point of attachment.

[0109] The term "carbocycle" refers to a fully saturated or partially saturated cyclic hydrocarbon group containing one to four rings and three to eight carbons per ring, or a cyclic, aromatic hydrocarbon group having one to five aromatic rings, particularly a monocyclic or bicyclic group, such as phenyl, biphenyl, or naphthyl. The term "carbocycle" encompasses cycloalkyl, cycloalkenyl, cycloalkynyl, and aryl as defined above. The term "substituted carbocycle" refers to a carbocycle or carbocyclic group substituted at any available attachment point with one or more substituents, preferably one to four substituents.

[0110] The terms "heterocycle" and "heterocyclic" refer to fully saturated or partially or fully unsaturated, including aromatic (i.e., "heteroaryl") cyclic groups (e.g., 4- to 7-membered monocyclic, 7- to 11-membered bicyclic, or 8- to 16-membered tricyclic ring systems) having at least one heteroatom within a ring containing at least one carbon atom. Each ring of a heteroatom-containing heterocyclic group can have 1, 2, 3, or 4 heteroatoms selected from nitrogen, oxygen, and / or sulfur atoms, where the nitrogen and sulfur heteroatoms can be optionally oxidized and the nitrogen heteroatom can be optionally quaternized. (The term "heteroarylium" refers to a heteroaryl group containing a quaternary nitrogen atom and is positively charged.) The heterocyclic group can be attached to any heteroatom or carbon atom within the ring or ring system remaining in the molecule. Exemplary monocyclic heterocyclic groups include azetidinyl, pyrrolidinyl, pyrroline, pyrazolyl, oxetanyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, oxazolyl, oxazolidinyl, isoxazolinyl, isoxazolyl, thiazolyl, thiadiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, furyl, tetrahydrofuryl, thienyl, oxadiazolyl, piperidinyl, piperazinyl, 2-oxo piperazinyl, 2-oxopiperidinyl, 2-oxopyrrolodinyl, 2-oxoazepinyl, azepinyl, hexahydrodiazepinyl, 4-piperidonyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl, triazolyl, tetrazolyl, tetrahydropyranyl, thiamolporinyl, thiamolporinyl sulfoxide, thiamolporinyl sulfone, 1,3-deoxolane, and tetrahydro-1,1-dioxothienyl.

[0111] "Substituted heterocycle" and "substituted heterocyclic" (e.g., "substituted heteroaryl") refer to a heterocycle or heterocyclic group substituted with one or more substituents, preferably 1 to 4 substituents, at any available point of attachment.

[0112] The term "alkylamino" refers to a group having the structure --NHR', where R' is hydrogen, alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, as defined above.

[0113] The term "dialkylamino" refers to a group having the structure -NRR', where R and R' are each independently alkyl or substituted alkyl, cycloalkyl or substituted cycloalkyl, cycloalkenyl or substituted cycloalkenyl, aryl or substituted aryl, or heterocyclic or substituted heterocyclic, as defined above. R and R' may be the same or different dialkyamino moieties. Examples of dialkylamino groups include, but are not limited to, dimethylamino, methylethylamino, diethylamino, methylpropylamino, di(n-propyl)amino, di(isopropyl)amino, di(cyclopropyl)amino, di(n-butyl)amino, di(tert-butyl)amino, di(neopentyl)amino, di(n-pentyl)amino, di(hexyl)amino, di(cyclohexyl)amino, and the like. In certain embodiments, R and R' are linked to form a cyclic structure. The resulting cyclic structure may be aromatic or non-aromatic. Examples of cyclic diaminoalkyl groups include, but are not limited to, aziridinyl, pyrrolidinyl, piperidinyl, molybdenum polynyl, pyrroline, imidazolyl, 1,3,4-trianolyl, and tetrazolyl.

[0114] The terms "halogen" or "halo" refer to chlorine, bromine, fluorine or iodine.

[0115] Unless otherwise specified, any heteroatom with unsatisfied valences is assumed to have enough hydrogen atoms to satisfy the valences.

[0116] The compounds of the present invention can form salts, which are also within the scope of the present invention. Reference to a compound of the present invention is understood to include reference to its salts, unless otherwise specified. As used herein, the term "salt" refers to acidic and / or basic salts formed with inorganic and / or organic acids and bases. Additionally, when a compound of the present invention contains both a basic moiety, such as, but not limited to, pyridine or imidazole, and an acidic moiety, such as, but not limited to, a carboxylic acid, zwitterions ("internal salts") can be formed and are included within the term "salt" as used herein. Although other salts may be useful, for example, in isolation or purification steps that may be used during formulation, pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred. Salts of the compounds of the present invention can be formed, for example, by reacting Compound I with a predetermined amount, e.g., an equivalent amount, of acid or base in the same medium in which the salt is precipitated, or in an aqueous medium, followed by lyophilization.

[0117] Compounds of the present invention that contain a basic moiety, such as, without limitation, an amine or pyridine or imidazoline, can form salts with a variety of organic and inorganic acids. Exemplary acid addition salts include acetate (e.g., formed with acetic acid or a trihaloacetic acid, such as trifluoroacetic acid), adipate, alginate, ascorbate, aspartate, benzoate, benzenesulfonate, bisulfate, borate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, hydroxybenzoate, and the like. Examples of suitable amines include dimethylsulfonates (e.g., 2-hydroxyethanesulfonate), lactate, malate, methanesulfonate, naphthalenesulfonates (e.g., 2-naphthalenesulfonate), nicotinate, nitrate, oxalate, pectinate, persulfate, phenylpropionate (e.g., 3-phenylpropionate), phosphate, picrate, pivalate, propionate, salicylate, succinate, sulfate (e.g., formed with sulfuric acid), sulfonate, tartrate, thiocyanate, toluenesulfonates, e.g., tosylate, undecanoate, and the like.

[0118] Compounds of the present invention that contain an acidic moiety, such as, without limitation, a carboxylic acid, can form salts with a variety of organic and inorganic bases. Exemplary base salts include ammonium salts, alkali metal salts, such as sodium, lithium, and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases (e.g., organic amines), such as benzathine, dicyclohexylamine, hydrabamine (formed with N,N-bis(dehydroabietyl)ethylenediamine), N-methyl-D-glucamine, N-methyl-D-glycamide, t-butylamine, and salts with amino acids, such as arginine and lysine. Basic nitrogen-containing groups may be quaternized with materials such as lower alkyl halides (e.g., methyl, ethyl, propyl, and butyl chlorides, bromides, and iodides), dialkyl sulfates (e.g., dimethyl, diethyl, dibutyl, and diamyl sulfate), long chain halides (e.g., decyl, lauryl, myristyl, and stearyl chlorides, bromides, and iodides), aralkyl halides (e.g., benzyl and phenethyl bromide), and the like.

[0119] Prodrugs and solvates of the compounds of the present invention are also contemplated herein. As used herein, the term "prodrug" refers to a compound that, upon administration to a subject, undergoes chemical conversion by metabolic or chemical processes to yield the compounds of the present invention, or salts and / or solvates thereof. Solvates of the compounds of the present invention include, for example, hydrates.

[0120] Compounds of the present invention, and salts or solvates thereof, may exist in their tautomeric form (for example, as an amide or imino ether), and all such tautomeric forms are contemplated herein as part of the present invention.

[0121] All stereoisomers of the present compounds, including enantiomeric and partial stereoisomeric forms (e.g., those that may exist due to the presence of asymmetric carbons on various substituents), are contemplated within the scope of the present invention. Individual stereoisomers of the compounds of the present invention may, for example, be substantially free of other isomers (e.g., as pure or substantially pure optical isomers having the specified activity), or may be mixed, for example, as racemates or with all different, or differently selected, stereoisomers. The chiral centers of the present invention may have the S or R configuration as defined by the 1974 Recommendations of the International Union of Pure and Applied Chemistry (IUPAC). Racemic forms can be resolved by physical methods, such as fractional crystallization, separation or crystallization of partial stereoisomeric derivatives, or separation by chiral column chromatography. Individual optical isomers can be obtained from the racemates by any suitable method, including, but not limited to, conventional methods, such as salt formation with an optically active acid followed by crystallization.

[0122] Definitions of specific functional groups and chemical terms are described in more detail below. For purposes of the present invention, chemical elements are identified by their internal designations in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., and specific functional groups are generally defined as described in said book. Additionally, specific functional moieties and reactivities, as well as general principles of organic chemistry, are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito:1999, the entire contents of which are incorporated herein by reference.

[0123] Isomeric mixtures containing any of a variety of isomer ratios can be used according to the present invention. For example, when only two isomers are combined, mixtures containing 50:50, 60:40, 70:30, 80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios are all contemplated by the present invention. Those skilled in the art will readily appreciate that similar ratios are contemplated for more complex isomer mixtures.

[0124] The present invention also includes isotopically labeled compounds identical to the compounds disclosed herein, but in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature. Examples of isotopes that may be contained in compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, and chlorine, e.g., individually 2 H, 3 H, 13 C. 11 C. 14 C. 15 N, 18 O. 17 O. 31 P, 32 P, 35 S, 18 F, and 36 Compounds of the present invention that contain the aforementioned isotopes and / or other isotopes of other atoms, or their enantiomers, partial stereoisomers, tautomers, or pharmaceutically acceptable salts or solvates, are within the scope of the present invention. Certain isotopically labeled compounds of the present invention, e.g., 3 H, and 14 Those incorporating radioisotopes such as 1C are useful in drug and / or substrate tissue distribution assays. Tritiated, i.e. 3 H, and carbon-14, i.e. 14 C isotopes are particularly preferred for their ease of preparation and detectability. Heavier isotopes, such as deuterium, i.e. 2Substitution with H can provide certain therapeutic advantages resulting from additional metabolic safety, e.g., increased in vivo half-life or reduced dosage requirements, and thus may be preferred in some cases. Isotopically labeled compounds can generally be prepared by following the procedures disclosed in the following schemes and / or examples, by substituting a readily available isotopically labeled reagent for a non-isotopically labeled reagent.

[0125] Furthermore, the antibody or antigen-binding fragment of the present invention specifically binds to an epitope containing a polypeptide represented by any one of the amino acid sequences of SEQ ID NOs: 35 to 45 present on regulatory T cells, thereby suppressing the function of the regulatory T cells and regulating the activity of effector T cells, thereby preventing or treating various diseases, such as metabolic diseases.

[0126] In the present invention, "obesity" refers not only to being overweight but also to a state in which body fat is excessively accumulated. Even if a person appears to be of normal weight, a high percentage of body fat can be considered obese. Obesity is typically assessed using the body mass index (BMI), with a BMI of 23-24.9 being considered overweight, 25-29.9 being considered severely obese, 30-34.9 being considered moderately obese, and 35 or higher being considered severely obese. Obesity is caused by a combination of multiple factors rather than a single one, including Westernized eating habits, poor eating habits, decreased activity, emotional factors, and genetic factors. Obesity resulting from these factors ultimately increases the risk of developing late-onset diseases such as hyperlipidemia, diabetes, and hypertension.

[0127] In the present invention, the term "metabolic disease" refers to a condition or disease that is closely related to or caused by obesity, and specifically may be one or more selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, and arteriosclerosis.

[0128] In the present invention, fatty liver refers to a condition or disease in which excessive amounts of fat accumulate in hepatocytes due to a disorder in hepatic fat metabolism.

[0129] In the present invention, hyperlipidemia refers to a condition or disease in which the concentrations of lipid components in the blood, especially cholesterol and triglycerides, are higher than normal, and is used in a broad sense to include all conditions requiring a reduction in blood lipid concentrations.

[0130] As used herein, arteriosclerosis refers to a condition or disease in which arterial walls thicken and lose elasticity, resulting in reduced blood circulation to internal organs and tissues, and includes the term "atherosclerosis," which refers to a condition or disease in which fat, cholesterol, and other substances are deposited on the inner walls of arteries to form plaque, narrowing the lumen and reducing blood circulation. Arteriosclerosis can occur anywhere in the body; when it occurs in the blood vessels within the heart, it can lead to coronary artery diseases such as angina pectoris and myocardial infarction, and cerebral infarction, and when it occurs in the kidneys, it can lead to renal failure.

[0131] Meanwhile, Treg Foxp3, T cell receptor (TCR) strength, binding and expression, ability to produce anti-inflammatory cytokines such as interleukin-10 (IL-10) and IL-35, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), programmed cell death protein 1 (PD-1), CD39 and CD73 are known to play important roles in immune regulation.

[0132] Recent research has shown that the expression levels of these proteins are related to metabolism. Specifically, after Treg activation, mTOR signaling is upregulated to induce lipid synthesis, mevalonate metabolism, and mitochondrial function. CD39, which converts adenosine triphosphate (ATP) and adenosine diphosphate (ADP) to adenosine monophosphate (AMP), and CD73, which converts AMP to adenosine, eliminate ATP effects such as P2 receptor-mediated cytotoxicity and ATP-induced maturation of dendritic cells.

[0133] Alterations in Treg numbers and function have been widely documented in human autoimmune, infectious, and allergic diseases, as well as cancer. Furthermore, decreased Treg numbers have been reported in patients with type 2 diabetes, contributing to both elevated blood glucose and high-density lipoprotein concentrations. Circulating and visceral adipose Tregs are reduced in obese individuals and correlate inversely with measures of adiposity, inflammation, and glucose tolerance, potentially identifying subjects at increased metabolic and cardiovascular risk. PPARγ signaling for Treg energy homeostasis maintains the inflammatory state of adipose tissue and insulin sensitivity in lean adipose tissue, whereas dysfunction attenuates insulin production. Interestingly, attenuation of acute graft-to-host disease and multiple sclerosis has been observed in animal models, yet Treg frequency and suppressive capacity remain unchanged after silencing of INSR.

[0134] Therefore, changes in the number and function of Tregs are closely related not only to autoimmune and cancer diseases, but also to metabolic diseases including type 2 diabetes, obesity, and cardiovascular disease. Binding molecules that specifically bind to the Lrig-1 protein or its epitope present in Tregs and can alter the number and function of Tregs can prevent or treat metabolic diseases including type 2 diabetes, obesity, and cardiovascular disease.

[0135] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating various diseases, such as metabolic diseases, comprising the antibody or antigen-binding fragment thereof or antibody-drug conjugate (ADC) provided by the present invention as an active ingredient.

[0136] In the present invention, the binding molecule, antibody, or antigen-binding fragment contained as an active ingredient in the pharmaceutical composition; or an antibody-drug conjugate having a drug bound thereto; specifically binds to an epitope containing a polypeptide represented by any one of the amino acid sequences of SEQ ID NOs: 35 to 45, thereby regulating the function of the regulatory T cells and the activity of effector T cells, thereby enabling highly effective treatment of various diseases.

[0137] In the present invention, the term "antibody-drug conjugate (ADC)" refers to a form in which a drug and an antibody are chemically linked without reducing the biological activities of the antibody and the drug. In the present invention, the antibody-drug conjugate refers to a form in which a drug is bound to the N-terminal amino acid residue of the heavy chain and / or light chain of the antibody, specifically, a form in which a drug is bound to the α-amine group at the N-terminus of the heavy chain and / or light chain of the antibody.

[0138] In the present invention, the term "drug" refers to any substance having a specific biological activity in cells, including DNA, RNA, and peptides. The drug may be in a form containing a reactive group capable of reacting with an α-amine group to crosslink, or may be linked to a linker containing a reactive group capable of reacting with an α-amine group to crosslink.

[0139] In the present invention, the reactive group capable of reacting with the α-amine group to crosslink is not particularly limited as long as it can react with the α-amine group at the N-terminus of the heavy or light chain of an antibody to crosslink, and includes any reactive group known in the art that reacts with an amine group, such as, but not limited to, isothiocyanate, isocyanate, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester.

[0140] In the present invention, the antibody-drug conjugate comprises an antibody or antigen-binding fragment that specifically binds to an epitope of the present invention, i.e., the Lrig-1 protein; or an epitope comprising a polypeptide consisting of a partial amino acid sequence of the extracellular domain of the Lrig-1 protein; or an epitope comprising a polypeptide represented by any one of the amino acid sequences of SEQ ID NOs: 7 to 17, and in this case, the drug may include any drug that can treat a disease targeted by an antibody that specifically binds to the Lrig-1 protein, for example, a drug that can treat a metabolic disease, but is not limited to this.

[0141] In the present invention, the term "metabolic disease" refers to a condition or disease that is closely related to obesity or that is caused by obesity, and specifically may be one or more selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, and arteriosclerosis.

[0142] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating a metabolic disease, comprising, as an active ingredient, a chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain.

[0143] 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 monoclonal antibody-derived scFv (scFv) fused to the transmembrane and intracellular domains of a T cell receptor, such as the CD3 zeta (ζ) chain, the invention described herein is not limited to these domains. Rather, as used herein, "chimeric antigen receptor" or "CAR" refers to any receptor engineered to express any intracellular signaling molecule and its extracellular antigen-binding domain fused or linked thereto. In the present invention, the binding domain may comprise a scFv capable of specifically recognizing the Lrig-1 protein. In the present invention, the "scFv" refers to a fusion protein of the variable heavy chain (VH) and variable light chain (VL) of an antibody via a peptide linker between the VL and VH.

[0144] In the present invention, the VH domain and VL domain may be linked via a flexible linker. The flexible linker may be a glycine / serine linker of about 10 to 30 amino acids (e.g., 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 amino acids), preferably 15 amino acids in length. The linker length may act as an important crystallization site for the chimeric antigen receptor. Linkers shorter than this range may increase affinity but may also cause intracellular aggregation and impair CAR expression. Linkers longer than this range may reduce antigen affinity by moving the VL and VH CDRs farther apart in space.

[0145] The chimeric antigen receptor of the present invention may further comprise at least one of a hinge region (or spacer) and a signaling domain. In the present invention, the hinge region, which is also called a "spacer" and connects the antigen-binding domain to the transmembrane domain, serves to extend the antigen-binding domain from the T cell membrane or NK cell membrane. In the present invention, the hinge region may be derived from any suitable sequence, including, for example, a human or a portion thereof, or may include, but is not limited to, hinge regions of human proteins commonly used in the art, including CD8, CD28, 4-1BB, OX40, all or a portion of the CD3 zeta (ζ) chain, T cell receptor α or β chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof. Furthermore, in the present invention, the hinge region may include, but is not limited to, one selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD).

[0146] In the present invention, the signaling domain refers to a portion of a chimeric antigen receptor that is found or engineered to be found inside T cells. In the present invention, the signaling domain may or may not include a transmembrane domain that serves to anchor the chimeric antigen receptor in the plasma membrane of a T cell. In the present invention, the transmembrane domain and the signaling domain may be derived from the same protein (e.g., a CD3 zeta (ζ) molecule), or the transmembrane domain and the signaling domain may be derived from different proteins (e.g., the transmembrane domain of CD28 and the intracellular signaling domain of a CD3 zeta (ζ) molecule, or vice versa).

[0147] In the present invention, the transmembrane domain may include, but is not limited to, the T cell receptor α or β chain, all or part of the CD3 zeta (ζ) chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, functional derivatives thereof, or combinations thereof. In the present invention, the costimulatory domain may include, but is not limited to, a functional signaling domain derived from a polypeptide containing 4-1BB (CD137); OX40; CD27; CD28; CD30; CD40; PD-1; CD2; CD7; CD258; natural killer group 2 member C (NKG2C); natural killer group 2 member (NKG2D); B7-H3; CD83; ICAM-1; LFA-1 (CD11a / CD18) or a ligand that binds to ICOS; active fragments thereof; functional derivatives thereof; or a combination thereof.

[0148] In the present invention, the signaling domain may include, but is not limited to, a functional signaling domain derived from a polypeptide including all or a portion of CD3 zeta (ζ), common FcR gamma (FcER1G), Fc gamma RIIIa, FcR beta (Fc epsilon RIP), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX-activating protein 10 (DAP10), DNAX-activating protein 12 (DAP12), an active fragment thereof, a functional derivative thereof, or a combination thereof, and such signaling domains are known in the art.

[0149] In the present invention, the term "metabolic disease" refers to a condition or disease that is closely related to obesity or that is caused by obesity, and specifically may be one or more selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, and arteriosclerosis.

[0150] Meanwhile, in the present invention, "prevention" may include, without limitation, any action of blocking, suppressing, or delaying the symptoms of a disease using the pharmaceutical composition of the present invention.

[0151] Furthermore, in the present invention, "treatment" may include, without limitation, any action that improves or is beneficial to the symptoms of a disease using the pharmaceutical composition of the present invention.

[0152] In the present invention, the pharmaceutical composition may be in the form of a capsule, tablet, granule, injection, ointment, powder or drink, and may be intended for humans.

[0153] In the present invention, the pharmaceutical composition can be formulated into oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, as well as topical preparations, suppositories, and sterile injectable solutions, using conventional methods, but is not limited thereto. The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, dyes, flavorings, etc.; for injections, the pharmaceutically acceptable carrier may include buffers, preservatives, soothing agents, solubilizers, isotonicity agents, stabilizers, etc.; and for topical administration, the pharmaceutically acceptable carrier may include bases, excipients, lubricants, preservatives, etc. The pharmaceutical composition of the present invention can be formulated into various dosage forms by mixing with the pharmaceutically acceptable carriers described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injection, it can be prepared in unit dose ampoules or multiple dose forms. It can also be formulated into solutions, suspensions, tablets, capsules, sustained release preparations, etc.

[0154] On the other hand, examples of carriers, excipients, and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, etc. Furthermore, fillers, anti-agglomerating agents, lubricants, wetting agents, flavorings, emulsifiers, preservatives, etc. may also be included.

[0155] The routes of administration of the pharmaceutical composition of the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, cutaneous, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, or rectal, with oral or parenteral administration being preferred.

[0156] In the present invention, the term "parenteral" includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intradural, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of a suppository for office administration.

[0157] The dosage of the pharmaceutical composition of the present invention may vary depending on many factors, including the activity of the specific compound used, age, body weight, general health, sex, dietary intake, administration time, administration route, excretion rate, drug composition, and the severity of the specific disease being prevented or treated. The dosage of the pharmaceutical composition varies depending on the patient's condition, body weight, severity of disease, drug form, administration route, and duration, but can be appropriately selected by those skilled in the art and may be administered at 0.0001 to 50 mg / kg or 0.001 to 50 mg / kg per day. Administration may be once a day or in several divided doses. The dosage does not limit the scope of the present invention in any respect. The pharmaceutical composition of the present invention may be formulated as a tablet, sugar tablet, capsule, liquid, gel, syrup, slurry, or suspension.

[0158] Another embodiment of the present invention relates to a method for preventing or treating, for example, a metabolic disease, comprising administering a pharmaceutically effective amount of a binding molecule, or an antibody-drug conjugate (ADC), or a chimeric antigen receptor (CAR) according to the present invention to an individual.

[0159] In the present invention, the term "metabolic disease" refers to a condition or disease that is closely related to obesity or that is caused by obesity, and specifically may be one or more selected from the group consisting of fatty liver, type 2 diabetes, hyperlipidemia, cardiovascular disease, and arteriosclerosis.

[0160] The antibodies or antigen-binding fragments and antibody-drug conjugates of the present invention specifically bind to epitopes containing polypeptides represented by any one of the amino acid sequences of SEQ ID NOs: 35 to 45, thereby regulating the function of regulatory T cells and the activity of effector T cells, thereby enabling highly effective treatment of various diseases.

[0161] In the present invention, the "individual" refers to an individual suspected of developing a metabolic disease, and the individual suspected of developing a metabolic disease refers to mammals, including rodents, including humans, and livestock, that have developed or may develop the disease, but includes, without limitation, individuals that can be treated with the antibody, antigen-binding fragment, or antibody-drug conjugate of the present invention.

[0162] The methods of the present invention may involve administering a pharmaceutically effective amount of an antibody or antibody-drug conjugate. The appropriate total daily amount can be determined by the treating physician within the scope of sound medical judgment and may be administered in single or divided doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will vary depending on a variety of factors, including the type and degree of response to be achieved, the specific composition, including whether or not other drugs are used, the patient's age, weight, general health, sex, and diet, the time of administration, the route of administration, and the excretion rate of the composition, the duration of treatment, and drugs used in conjunction with or concurrently with the specific composition, as well as similar factors well known in the pharmaceutical arts.

[0163] Meanwhile, the method for preventing or treating the disease may be, but is not limited to, a combination therapy that further comprises administering a compound or substance having therapeutic activity against one or more diseases.

[0164] In the present invention, the term "combination" should be understood to refer to simultaneous, separate or sequential administration. If the administration is sequential or separate, the interval between administrations of the two components should be such that the beneficial effect of the combination is not lost.

[0165] In the present invention, the administration dose of the antibody or antibody-drug conjugate may be about 0.0001 μg to 500 mg per kg of patient body weight, but is not limited thereto.

[0166] According to another embodiment of the present invention, there is provided a composition for diagnosing metabolic diseases, comprising an agent for measuring the expression level of Lrig-1 protein or its extracellular domain present on the surface of T cells, or the gene encoding it, using the binding molecule.

[0167] The T cells of the present invention may be regulatory T cells.

[0168] The diagnostic composition of the present invention may include, but is not limited to, an agent for measuring the expression level of the Lrig-1 protein or its extracellular domain present on the surface of activated regulatory T cells, i.e., regulatory T cells whose suppressive ability on effector T cells has been activated; or a gene encoding the same.

[0169] The diagnostic composition of the present invention may further comprise an agent for measuring the expression level of one or more proteins present on the surface of T cells selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and Lrig-1 protein; or genes encoding the same. In this way, when the expression levels of various proteins present on the surface of T cells or genes encoding them are further measured, a significant synergistic effect can be achieved in diagnosing a target disease compared to measuring the Lrig-1 protein or its extracellular domain; or genes encoding it alone.

[0170] The agent for measuring the expression level of the Lrig-1 protein or its extracellular domain of the present invention and the expression level of one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and Lrig-1 protein is not particularly limited, and may include, for example, one or more proteins selected from the group consisting of antibodies, oligopeptides, ligands, peptide nucleic acids (PNAs), and aptamers that specifically bind to the proteins.

[0171] In the present invention, "PNA (Peptide Nucleic Acid)" refers to an artificially synthesized polymer similar to DNA or RNA. It was first introduced in 1991 by Professors Nielsen, Egholm, Berg, and Buchardt of the University of Copenhagen, Denmark. While DNA has a phosphate-ribose backbone, PNA has repeated N-(2-aminoethyl)-glycine backbones linked by peptide bonds. This significantly increases its binding strength and safety to DNA or RNA, making it suitable for use in molecular biology, diagnostic analysis, and antisense therapy. PNA is described in detail in the literature [Nielsen PE, Egholm M, Berg RH, Buchardt O (December 1991). "Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide." Science 254 (5037): 1497-1500].

[0172] In the present invention, the "aptamer" is an oligonucleic acid or peptide molecule, and the general content of aptamers is disclosed in the literature [Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine", J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial approach" in detail].

[0173] The agent for measuring the expression level of the gene encoding the Lrig-1 protein (i.e., LAIR1) or the gene encoding the extracellular domain of the Lrig-1 protein of the present invention, and the gene encoding one or more proteins selected from the group consisting of CCD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and Lrig-1 protein, may include one or more selected from the group consisting of primers, probes, LNAs, and antisense nucleotides that specifically bind to the genes.

[0174] The "primer" of the present invention is a fragment that recognizes a target gene sequence and includes a forward and reverse primer pair, preferably a primer pair that provides analytical results with specificity and sensitivity. Since the nucleic acid sequence of the primer is a sequence that does not match non-target sequences present in the sample, high specificity can be achieved when the primer is a primer that amplifies only the target gene sequence containing the complementary primer binding site and does not induce non-specific amplification.

[0175] The term "probe" as used herein refers to a substance capable of specifically binding to a target substance to be detected in a sample and capable of specifically confirming the presence of the target substance in the sample through this binding. The type of probe may be any substance commonly used in the art, but is preferably a peptide nucleic acid (PNA), locked nucleic acid (LNA), peptide, polypeptide, protein, RNA, or DNA, and is most preferably a PNA. More specifically, the probe may be a biological substance, including those derived from or similar to living organisms or those produced in vitro, such as enzymes, proteins, antibodies, microorganisms, animal and plant cells and tissues, nerve cells, DNA, and RNA. DNA includes cDNA, genomic DNA, and oligonucleotides; RNA includes genomic RNA, mRNA, and oligonucleotides; and proteins include antibodies, antigens, enzymes, and peptides.

[0176] In the present invention, the term "LNA (Locked nucleic acids)" refers to a nucleic acid analog containing a 2'-O, 4'-C methylene bridge [J. Weiler, J. Hunziker and J. Hall, Gene Therapy (2006) 13, 496, 502]. LNA nucleosides contain the common nucleic acid bases of DNA and RNA and can form base pairs according to the Watson-Crick base pairing rules. However, due to the "locking" of the molecule by the methylene bridge, LNA cannot form the ideal shape for Watson-Crick binding. When LNA is included in a DNA or RNA oligonucleotide, it can pair more quickly with the complementary nucleotide strand, increasing the security of the double helix.

[0177] In the present invention, the term "antisense" refers to an oligomer having a sequence of nucleotide bases and an intersubunit backbone that allows the antisense oligomer to hybridize to a target sequence in RNA through Watson-Crick base pairing, typically with mRNA, and form an RNA:oligomer heteroduplex within the target sequence. The oligomer can have exact sequence complementarity or approximate complementarity to the target sequence.

[0178] Since information about the Lrig1 protein of the present invention and the gene (LRIG1) that encodes it is known, a person skilled in the art can easily design primers, probes, or antisense nucleotides that specifically bind to the gene encoding the protein based on this information.

[0179] Another embodiment of the present invention relates to a diagnostic kit for metabolic diseases, comprising the antibody or antigen-binding fragment provided herein.

[0180] The kit of the present invention may be, but is not limited to, an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0181] The kit of the present invention may further comprise one or more other component compositions, solutions or devices compatible with the analytical method.

[0182] The kit of the present invention may further include essential components necessary for performing a reverse transcription polymerase reaction, for example. The reverse transcription polymerase reaction kit includes a primer pair specific to a gene encoding a marker protein. The primer pair is a nucleotide having a sequence specific to the nucleic acid sequence of the gene and may be, for example, about 7 bp to 50 bp in length, or about 10 bp to 30 bp in length. A primer pair specific to the nucleic acid sequence of a control gene may also be included. The reverse transcription polymerase reaction kit may further include test tubes or other suitable containers, a reaction buffer (with various pH and magnesium concentrations), deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor DEPC-water, sterile water, etc.

[0183] The kit of the present invention may include essential elements required for performing a DNA chip. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to genes or their fragments are attached, and reagents, drugs, enzymes, etc. for producing fluorescently labeled probes. The substrate may also include cDNA or oligonucleotides corresponding to control genes or their fragments.

[0184] The kit of the present invention may contain essential elements necessary for performing ELISA. The ELISA kit includes an antibody specific to the protein. The antibody has high specificity and affinity for the marker protein and little cross-reactivity with other proteins, and may be a monoclonal antibody, polyclonal antibody, or recombinant antibody. The ELISA kit may also include an antibody specific to a control protein. In addition, the ELISA kit may include reagents capable of detecting the bound antibody, such as a labeled secondary antibody, a chromophore, an enzyme (e.g., conjugated to the antibody) and its substrate, or other substances capable of binding to the antibody.

[0185] According to another embodiment of the present invention, there is provided a method for providing information for diagnosing metabolic diseases, comprising measuring the expression level of Lrig-1 protein or its extracellular domain present on the surface of T cells in a biological sample isolated from a target individual using the binding molecule; or a gene encoding the same.

[0186] The T cells of the present invention may be regulatory T cells.

[0187] In the present invention, the step of measuring the expression level may further measure the expression level of one or more proteins present on the surface of T cells selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and Lrig-1 protein; or genes encoding the same. In this way, when the expression levels of various proteins present on the surface of T cells or genes encoding them are further measured, a significant synergistic effect can be achieved in diagnosing a target disease compared to measuring the Lrig-1 protein or genes encoding it alone.

[0188] The "target individual" of the present invention refers to an individual whose presence or absence of the disease in question is uncertain, but who has a high probability of developing the disease.

[0189] The "biological sample" of the present invention means any substance, biological fluid, tissue, or cell obtained from or derived from an individual, such as whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, pelvic fluids, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, and pancreatic fluid. The fluid may include, but is not limited to, fluid, lymph fluid, pleural fluid, nipple aspirate, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, or cerebrospinal fluid.

[0190] The Lrig-1 protein of the present invention; or its extracellular domain, may be expressed on the cell surface of T cells, particularly regulatory T cells, for example, activated regulatory T cells, i.e., regulatory T cells whose suppressive ability on effector T cells has been activated.

[0191] The method for measuring or comparing the expression levels of the Lrig-1 protein or its extracellular domain and one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and Lrig-1 protein of the present invention may include protein chip analysis, immunoassay, ligand binding assay, MALDI-TOF (Matrix Assisted Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, SELDI-TOF (Surface Enhanced Laser Desorption / Ionization Time of Flight Mass Spectrometry) analysis, radioimmunoassay, radial immunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, complement fixation analysis, two-dimensional electrophoresis, liquid chromatography-mass spectrometry (LC-MS), LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry), and the like. Examples of such methods include, but are not limited to, immunoblotting (ELISA), Western blotting (Western blotting), and enzyme-linked immunosorbent assay (ELISA).

[0192] In order to confirm the presence and expression level of the gene encoding the Lrig-1 protein or its extracellular domain of the present invention and the gene encoding one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16, and Lrig-1 protein, analytical methods for measuring the expression level of the gene include, but are not limited to, reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase reaction (Competitive RT-PCR), real-time reverse transcription polymerase reaction (Real-time RT-PCR), RNase protection assay (RPA), Northern blotting, or DNA chip.

[0193] The present invention may include a step of predicting that there is a high possibility of developing a bone disease if the expression level of the Lrig-1 protein or its extracellular domain; or the gene encoding it, measured in a biological sample of the target individual is changed compared to a normal control group.

[0194] In the present invention, the "control group" may be a normal control group, more specifically, a group obtained from serum samples of patients confirmed to be free of bone-related diseases, or an average or median TregL1 expression level of patients finally confirmed to be free of bone-related diseases by bone density testing, ultrasound, and CT. The expression level of the marker protein or the gene encoding it in the control group can be compared with the expression level of the marker protein or the gene encoding it in a biological sample from a patient with a bone-related disease to be analyzed, and the presence or absence of a significant change in the expression level can be determined to diagnose the bone-related disease.

[0195] In the present invention, "high level" means that the expression is significantly higher than that of a normal control group to a measurable extent, and specifically means, for example, that the expression is 20% or more than that of a normal control group, more specifically, 30%, even more specifically, 40%, and most specifically, 50% or more, but is not limited thereto.

[0196] The method of the present invention may further comprise predicting that there is a high possibility of developing a bone disease when the expression level of one or more proteins selected from the group consisting of CD25, TIGIT, LAG3, CTLA-4, GITR, OX40, ICOS, PD-1, TIM-3, CCR4, FR4, CD15s, PI-16 and Lrig-1 proteins present on the surface of the T cells or genes encoding the same is altered (increased or decreased) compared to a normal control group.

[0197] In another embodiment of the present invention, the method may include predicting that there is a high possibility of developing a bone disease if the expression level of the Lrig-1 protein or the gene encoding it is reduced compared to a normal control group.

[0198] Furthermore, in the present invention, when the expression level of the Lrig-1 protein or its extracellular domain; or the gene encoding it, measured in a biological sample of a target individual as described above is measured to predict or diagnose a high possibility of developing a bone disease, the method may further include a step of administering a drug for the disease to the target individual.

[0199] In the present invention, the term "diagnosis" means to confirm the existence or characteristics of a pathological state, and more specifically to determine a metabolic disease.

[0200] Another embodiment of the present invention relates to a diagnostic device for diagnosing metabolic diseases.

[0201] The measurement unit of the diagnostic device of the present invention can measure the expression level of TregL1 in a biological sample obtained from an individual of interest using an agent for measuring the expression level of TregL1.

[0202] In the present invention, the target individual may be selected from the group consisting of humans, rats, mice, guinea pigs, hamsters, rabbits, monkeys, dogs, cats, cows, horses, pigs, sheep, and goats, and specifically may be, but is not limited to, humans.

[0203] In the present invention, the biological sample may be whole blood, leukocytes, peripheral blood mononuclear cells, buffy coat, plasma, serum, sputum, tears, mucus, nasal washes, nasal aspirate, breath, urine, semen, saliva, peritoneal washings, ascites, cystic fluid, meningeal fluid, amniotic fluid, glandular fluid, pancreatic fluid, lymph fluid, pleural fluid, nipple aspirate, or the like. The material may be one or more selected from the group consisting of, but not limited to, bronchial aspirate, synovial fluid, joint aspirate, organ secretions, cells, cell extracts, and tissues.

[0204] The reagent used in the measurement unit of the diagnostic device of the present invention may be a reagent for measuring the expression level of TregL1, and more specifically, may include at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to the gene.

[0205] The presence or absence of a metabolic disease can be predicted by confirming the expression level of the gene using the drug in the measuring unit of the diagnostic device of the present invention.

[0206] The metabolic disease diagnostic device of the present invention may further include a detection unit that predicts and outputs the presence or absence of a metabolic disease in the target individual based on the expression level of the gene obtained by the measurement unit.

[0207] In the present invention, when the expression level of the gene obtained by the measurement unit is higher than the expression level of TregL1 measured in a control group, the detection unit generates and classifies information indicating that a metabolic disease has occurred or is likely to occur, thereby diagnosing a metabolic disease.

[0208] In addition, in the present invention, when the expression level of the gene obtained by the measurement unit is higher than the expression level of TregL1 measured in a control group, the detection unit generates and classifies information indicating that a metabolic disease has occurred or is likely to occur, thereby diagnosing a metabolic disease.

[0209] According to one embodiment of the present invention, there is provided a composition for diagnosing metabolic diseases, comprising an agent for measuring the expression level of Lrig-1 protein.

[0210] According to one embodiment of the present invention, there is provided a diagnostic composition, wherein the Lrig-1 protein is present on the surface of adipocytes.

[0211] According to one embodiment of the present invention, a diagnostic composition is provided in which an agent for measuring the expression level of the Lrig-1 protein comprises one or more selected from the group consisting of primers, probes, and antisense nucleotides that specifically bind to the gene.

[0212] According to one embodiment of the present invention, there is provided a diagnostic composition, wherein the agent for measuring the expression level of the Lrig-1 protein is an antibody or antigen-binding fragment that specifically binds to Lrig-1.

[0213] According to one embodiment of the present invention, there is provided a diagnostic composition, wherein the antibody or antigen-binding fragment specifically binds to at least one epitope selected from the group consisting of the amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO: 45.

[0214] According to one embodiment of the present invention, there is provided a diagnostic composition, wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent antibody, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, a tetrabody, or a fragment thereof.

[0215] According to one embodiment of the present invention, there is provided a diagnostic composition, wherein the metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension.

[0216] According to one embodiment of the present invention, there is provided a kit for diagnosing metabolic diseases, comprising the composition.

[0217] According to another embodiment of the present invention, there is provided a diagnostic kit, wherein the metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension.

[0218] According to one embodiment of the present invention, there is provided a method for providing information for diagnosing metabolic diseases, comprising measuring the expression level of Lrig-1 protein in a biological sample isolated from a target individual.

[0219] According to another embodiment of the present invention, there is provided a method for providing information, wherein the Lrig-1 protein is present on the surface of adipocytes.

[0220] According to another embodiment of the present invention, there is provided an information providing method, which predicts that the target individual has a metabolic disease if the expression level of Lrig-1 protein measured in the biological sample is higher or lower than that of a normal control group.

[0221] According to another embodiment of the present invention, there is provided an information providing method, wherein the metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension.

[0222] According to another embodiment of the present invention, a diagnostic device for metabolic diseases is provided, which includes: (a) a measuring unit that measures the expression level of Lrig-1 in a biological sample obtained from a target individual; and (b) a detecting unit that outputs, based on the expression level of Lrig-1 measured by the measuring unit, whether or not the target individual has developed or is likely to develop a bone disease.

[0223] According to another embodiment of the present invention, there is provided a diagnostic device wherein the metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension.

[0224] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating metabolic diseases, comprising an antibody or antigen-binding fragment that specifically binds to Lrig-1 as an active ingredient.

[0225] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating a metabolic disease, comprising, as an active ingredient, a binding molecule that specifically binds to at least one epitope selected from the group consisting of amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO: 45.

[0226] According to another embodiment of the present invention, there is provided a pharmaceutical composition wherein the binding molecule is an antibody or an antigen-binding fragment thereof.

[0227] According to another embodiment of the present invention, there is provided a pharmaceutical composition wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent antibody, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, a tetrabody, or a fragment thereof.

[0228] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating a metabolic disease, comprising, as an active ingredient, a chimeric antigen receptor (CAR) that is a fusion protein comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain, wherein the antigen-specific binding domain specifically binds to at least one epitope selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 35 to 45; and an immunoglobulin Fc region.

[0229] According to another embodiment of the present invention, there is provided a pharmaceutical composition for preventing or treating metabolic diseases, comprising as an active ingredient an antibody-drug conjugate comprising a drug and a binding molecule that specifically binds to at least one epitope selected from the group consisting of amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO: 45.

[0230] According to another embodiment of the present invention, there is provided a pharmaceutical composition wherein the binding molecule is an antibody or an antigen-binding fragment thereof.

[0231] According to another embodiment of the present invention, there is provided a pharmaceutical composition wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent antibody, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, a tetrabody, or a fragment thereof.

[0232] According to another embodiment of the present invention, there is provided a pharmaceutical composition, wherein the metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension. [Effects of the Invention]

[0233] The Lrig-1 protein of the present invention and antibodies or antigen-binding fragments thereof that specifically bind to its epitopes specifically bind to the epitopes of the present invention present on regulatory T cells, thereby regulating the function of the regulatory T cells and, as a result, regulating the activity of effector T cells, can be used very efficiently in the prevention, amelioration, or treatment of metabolic diseases. [Brief explanation of the drawings]

[0234] [Figure 1] FIG. 1 shows the structure of the Lrig-1 protein according to one embodiment of the present invention. [Figure 2] FIG. 2 shows the structure of the Lrig-1 protein according to one embodiment of the present invention. [Figure 3] FIG. 3 shows the expression level of Lrig-1 mRNA according to one embodiment of the present invention. [Figure 4] FIG. 4 shows the expression level of Lrig-1 mRNA according to one embodiment of the present invention. [Figure 5]FIG. 5 shows the expression level of Lrig-1 mRNA according to one embodiment of the present invention.

[0235] [Figure 6] FIG. 6 shows the expression levels of Lrig-1, Lrig-2, and Lrig-3 mRNA according to one embodiment of the present invention. [Figure 7] FIG. 7 shows the results of comparing the expression levels of Lrig-1 protein in regulatory T cells and non-regulatory T cells according to one embodiment of the present invention. [Figure 8] FIG. 8 shows the expression of Lrig-1 protein on the surface of regulatory T cells according to one embodiment of the present invention. [Figure 9] FIG. 9 shows the results of analyzing the binding affinity of antibodies (GTC210-01, GTC210-02, GTC210-03, GTC210-04, GTC110-01, GTC110-02, GTC110-03, and GTC110-04) to Lrig-1 protein in one example of the present invention. [Figure 10] Figure 10 shows the results of an analysis of the mechanism of Lrig-1 protein-induced Stat3 phosphorylation regulation in regulatory T cells using monoclonal antibodies specific to Lrig-1 protein (GTC210-01, GTC210-02, GTC210-03, GTC210-04, GTC110-01, GTC110-02, GTC110-03 and GTC110-04) in one embodiment of the present invention.

[0236] [Figure 11] FIG. 11 shows the results of covalent labeling analysis of peptides digested by treatment with proteases according to one embodiment of the present invention (chymotrypsin (FIG. 11), trypsin (FIG. 12), and Asp-N / Lys-C (FIG. 13)). [Figure 12] FIG. 12 shows the results of covalent labeling analysis of peptides digested by treatment with proteases according to one embodiment of the present invention (chymotrypsin (FIG. 11), trypsin (FIG. 12), and Asp-N / Lys-C (FIG. 13)). [Figure 13] FIG. 13 shows the results of covalent labeling analysis of peptides digested by treatment with proteases according to one embodiment of the present invention (chymotrypsin (FIG. 11), trypsin (FIG. 12), and Asp-N / Lys-C (FIG. 13)). [Figure 14] FIG. 14 shows the results of aligning and comparing peptides digested by each protease according to one embodiment of the present invention based on the sequences confirmed by covalent labeling analysis. [Figure 15] FIG. 15 shows the results of aligning and comparing peptides digested by each protease according to one embodiment of the present invention based on the sequences confirmed by covalent labeling analysis.

[0237] [Figure 16] FIG. 16 shows the results of cross-linking analysis of peptides digested by treating with proteases (chymotrypsin (FIG. 16) and trypsin (FIG. 17)) according to an embodiment of the present invention. [Figure 17] FIG. 17 shows the results of cross-linking analysis of peptides digested by treating with proteases (chymotrypsin (FIG. 16) and trypsin (FIG. 17)) according to an embodiment of the present invention. [Figure 18] FIG. 18 shows the results of aligning and comparing peptides digested by each protease according to one embodiment of the present invention based on the sequences confirmed by cross-linking analysis. [Figure 19] FIG. 19 shows the results of aligning and comparing peptides digested by each protease according to one embodiment of the present invention based on the sequences confirmed by cross-linking analysis. [Figure 20] FIG. 20 shows the results of aligning and analyzing epitopes identified by covalent labeling and cross-linking analysis according to one embodiment of the present invention.

[0238] [Figure 21] FIG. 21 shows the results of examining changes in body weight in female and male Lrig-1 knockout mice. [Figure 22] FIG. 22 shows the results of the weight change in the first Lrig-1 knockout male mice, divided into heterozygous and homozygous genotypes. [Figure 23] Figure 23 shows the results of the first observation of changes in adipocytes in Lrig-1 knockout male mice, divided into visceral and subcutaneous fat. In the figure, PGF stands for eWAT, i.e., visceral fat, and SCF stands for iWAT, i.e., subcutaneous fat. [Figure 24] FIG. 24 shows the results of the weight change in the first Lrig-1 knockout female mice, divided into heterozygous and homozygous genotypes. [Figure 25] Figure 25 shows the results of the first observation of changes in adipocytes in Lrig-1 knockout female mice, divided into visceral and subcutaneous fat. In the figure, PGF stands for eWAT, i.e., visceral fat, and SCF stands for iWAT, i.e., subcutaneous fat.

[0239] [Figure 26] FIG. 26 shows the results of the weight change in the second confirmed Lrig-1 knockout male mice, divided into heterozygous and homozygous genotypes. [Figure 27] Figure 27 shows the results of the second study, examining changes in adipocytes in Lrig-1 knockout male mice, divided into visceral and subcutaneous fat. In the figure, PGF stands for eWAT (visceral fat), SCF stands for iWAT (subcutaneous fat), WAT stands for white adipose tissue (white adipocytes), and BAT stands for brown adipose tissue (brown adipocytes). [Figure 28] FIG. 28 shows the results of the weight change in the second confirmed Lrig-1 knockout female mice, divided into heterozygous and homozygous genotypes. [Figure 29]Figure 29 shows the results of the second study, examining changes in adipocytes in Lrig-1 knockout female mice, divided into visceral and subcutaneous fat. In the figure, PGF represents eWAT, i.e., visceral fat, and SCF represents iWAT, i.e., subcutaneous fat. [Figure 30] FIG. 30 shows the experimental process for differentiating the 3T3 cell line to express Lrig-1.

[0240] [Figure 31] FIG. 31 shows the expression level of Lrig-1 confirmed through breast cell analysis. [Figure 32] FIG. 32 shows the level of Lrig-1 expression over time. DETAILED DESCRIPTION OF THE INVENTION

[0241] The present invention will be described in more detail with reference to the following examples. It will be obvious to those skilled in the art that these examples are provided solely to illustrate the present invention in more detail, and that the scope of the present invention is not limited by these examples, according to the gist of the present invention.

[0242] [Example] [Preparation Example 1] Cultivation of T cell subtypes To confirm whether Lrig-1 protein is expressed only in regulatory T cells (Tregs), we prepared T cell subtypes Th0, Th1, Th2, Th17, and iTregs. iTregs are distinct from naturally isolated nTregs, and refer to cells artificially induced to differentiate in a medium containing the following components:

[0243] To differentiate T cell subtypes, naive T cells were isolated from mouse spleens and then cultured in RPMI1640 (Invitrogen Gibco, Grand Island, NY) nutrient medium containing 10% fetal bovine serum (FBS; Hyclone, Logan, UT) and the additional components listed in Table 1 below for 72 hours at 37°C in 5% CO2.

[0244] [Table 1]

[0245] Example 1: Analysis of Lrig-1 structure In order to produce antibodies specific to Lrig-1 protein, a surface protein of regulatory T cells, the three-dimensional structure of the extracellular domain of Lrig-1 protein was predicted.

[0246] First, to predict the epitope base sequence, we confirmed the structure of the extracellular domain (ECD) of the Lrig-1 protein and predicted its three-dimensional structure using Uniprot (uniprot.org) and RCSB Protein Data Bank (rcsb.org / pdb) tools. The results are shown in Figures 1 and 2.

[0247] As can be seen from Figure 1, the Lrig-LRR domain (amino acid sequence 41 to 494) of the extracellular domain of the Lrig-1 protein contains a total of 15 leucine-rich regions, LRR1 to LRR15. Each of the LRR domains consists of 23 to 27 amino acids and contains 3 to 5 leucines.

[0248] Furthermore, as can be seen from FIG. 2, the amino acid sequence from 494 to 781 of the Lrig-1 protein contained three immunoglobulin-like domains in the extracellular domain of the Lrig-1 protein.

[0249] [Example 2] Confirmation of specific expression of Lrig-1 mRNA in regulatory T cells We investigated whether Lrig-1 protein could act as a specific biomarker for regulatory T cells.

[0250] For this verification, CD4 was isolated from the spleen of the mice using a magnet-activated cell sorting (MACS) with CD4 beads. + T cells were isolated. Thereafter, regulatory T cells (CD4 + CD25 + T) cells and non-regulated T (CD4 + CD25 - T) cells were isolated. mRNA was extracted from each cell and the cells differentiated in Preparation Example 1 using Trizol. Genomic RNA was then extracted using a gDNA extraction kit (Qiagen) and gDNA was removed according to the manufacturer's protocol. The gDNA-removed mRNA was synthesized into cDNA using the BDsprint cDNA synthesis kit (Clonetech).

[0251] To quantitatively confirm the expression level of Lrig-1 mRNA using the cDNA, real-time polymerase chain reaction (real-time PCR) was performed.

[0252] The real-time polymerase chain reaction was performed using SYBR Green (Molecular Probes) according to the protocol provided by the manufacturer, with 40 cycles of 95°C for 3 minutes, 61°C for 15 seconds, and 72°C for 30 seconds, using the primers listed in Table 2 below. Relative gene expression levels were calculated using the △CT method and normalized using HPRT. The results are shown in Figures 3 to 6.

[0253] [Table 2]

[0254] As can be seen in Figure 3, non-regulatory T (CD4 + CD25 - T) cells, compared with regulatory T (CD4 + CD25 + It can be seen that Lrig-1 expression was 18.1-fold higher in regulatory T cells. This was approximately 10-fold higher than the previously known regulatory T cell markers, Lag3 and Ikzf4. Furthermore, as can be seen from Figures 4 and 5, Lrig-1 mRNA expression was significantly higher in regulatory T cells compared to other types of immune cells, particularly in naturally isolated regulatory T cells (nTreg) compared to induced regulatory T cells (iTreg).

[0255] Furthermore, as can be seen from FIG. 6, among Lrig-1, Lrig-2, and Lrig-3, which belong to the Lrig family, Lrig-1 expression was the highest.

[0256] These results demonstrate that the Lrig-1 protein of the present invention is specifically expressed in regulatory T cells, particularly naturally occurring regulatory T cells.

[0257] [Example 3] Confirmation of specific expression of Lrig-1 protein in regulatory T cells We confirmed whether Lrig-1 protein expressed from Lrig-1 mRNA was specifically expressed only in regulatory T cells.

[0258] Using FOXP3-RFP knock-in mice, in which RFP (Red Fluorescence Protein) is linked to the FOXP3 promoter, a regulatory T cell-specific transcription factor, CD4 beads were sorted from the spleen of the mice using a magnet-activated cell sorting (MACS) system. + T cells were isolated and then sorted using RFP protein using a fluorescence activated cell sorter (FACS). + RFP + T) cells and non-regulated T (CD4 + RFP -T) cells were isolated and obtained. Each of the cells was stained with a purchased Lrig-1 antibody and a negative control isotype, and the expression level of Lrig-1 was measured using a fluorescence activated cell sorter. The results are shown in Figure 7.

[0259] As can be seen from Figure 7, the non-regulatory T cells, represented by the dotted line, showed almost the same Lrig-1 expression level as the negative control group, but the regulatory T cells contained many cells with high Lrig-1 expression levels.

[0260] These results demonstrate that the Lrig-1 protein of the present invention is specifically expressed in regulatory T cells.

[0261] [Example 4] Confirmation of specific expression of Lrig-1 protein on the surface of regulatory T cells For Lrig-1 protein to be a target for cell therapy, it must be expressed on the surface of regulatory T cells for more effective targeted therapy, so we checked whether Lrig-1 protein was expressed on the surface.

[0262] Each differentiated T cell subtype from Preparatory Example 1 was stained with anti-CD4-APC and anti-Lrig-1-PE antibodies, and the expression level of Lrig-1 on the surface of each cell was measured using a fluorescence-activated cell sorter (FACS). The results are shown in Figure 8.

[0263] As can be seen from Figure 8, Lrig-1 expression was found to be at levels of 0.77 to 15.3 in activated T cells, Th1 cells, Th2 cells, Th17 cells, and naive T cells, whereas it was highly expressed at 83.9 in differentiation-induced T cells (iTreg).

[0264] These results demonstrate that the Lrig-1 protein of the present invention is not only specifically expressed in regulatory T cells (Treg) but is particularly highly expressed on the surface of regulatory T cells.

[0265] [Preparation Examples 1 to 8] Preparation of monoclonal antibodies specific to Lrig-1 protein We have produced an antibody specific to the Lrig-1 protein according to the present invention. This antibody was produced without targeting a specific antigenic determinant, but rather was capable of binding to any site on the Lrig-1 protein.

[0266] To produce the antibody, cells expressing Lrig-1 protein were prepared. More specifically, the DNA fragment corresponding to SEQ ID NO: 2 and pcDNA (hygro) were digested with a cleavage enzyme and then cultured at 37°C for ligation to prepare pcDNA with an insert of the DNA sequence encoding Lrig-1 protein. The prepared pcDNA with SEQ ID NO: 2 inserted was introduced into L cells via transfection to allow expression of Lrig-1 protein on the surface of the L cells.

[0267] The light chain and heavy chain amino acid sequences capable of binding to Lrig-1 expressed on the cell surface were screened using a human scFv library, and a total of eight heavy and light chains were selected.

[0268] The selected heavy and light chain amino acid sequences were fused to the IgG2a Fc region or human IgG1 Fc region to produce monoclonal antibodies. The sequences of the monoclonal antibodies are shown in Table 3 below.

[0269] [Table 3-1] [Table 3-2]

[0270] [Example 5] Evaluation of the binding ability of the antibody according to the present invention to the Lrig-1 protein To confirm whether the monoclonal antibodies of the present invention, Preparations 1 to 9, can effectively recognize Lrig-1, each of the antibodies of Preparations 1 to 9 was bound to L cells stably expressing Lrig-1, and then a secondary antibody conjugated with eFlour670, which can recognize mouse antibodies, was added. Then, the binding strength of the antibodies of the Preparations to the Lrig-1 protein was analyzed using FACS, and the results are shown in Figure 9.

[0271] As can be seen from Figure 9, it was confirmed that all of the Lrig-1 protein-specific monoclonal antibodies according to the present invention effectively recognized and bound to the Lrig-1 protein present on the surface of L cells.

[0272] [Example 6] Permissible CDR changes of the antibody according to the present invention To investigate the role of each amino acid position in each CDR, the CDRs of the antibodies that specifically bind to the Lrig-1 protein of Preparation Examples 1 to 9 were aligned and analyzed using GTC210-03 as the standard.

[0273] As a result of analyzing the CDR changes of acceptable antibodies, the acceptable CDR changes of antibodies that specifically bind to Lrig-1 epitopes, regulate the signaling pathway within regulatory T cells, and are effective against bone diseases compared to the control group are summarized in Table 4, and their specific sequences are shown in Table 5.

[0274] [Table 4]

[0275] [Table 5-1] [Table 5-2]

[0276] [Example 7] Regulation of the antibody according to the present invention Regulation of signal transduction pathways within T cells To analyze whether the monoclonal antibodies of Preparations 1 to 9 according to the present invention affect the signal transduction pathway within regulatory T cells through Lrig-1 protein, regulatory T cells were treated with the monoclonal antibodies of Preparations 1 to 9 to stimulate Lrig-1 present on the surface of the regulatory T cells, and the level of tyrosine phosphorylation of Stat3 protein present in the stimulated regulatory T cells was analyzed using phosphotyrosine immunoblot, and the results are shown in Figure 10.

[0277] As can be seen from Figure 10, the Lrig-1 protein-specific monoclonal antibodies according to the present invention (GTC210-01, GTC210-02, GTC210-03, and GTC210-04) were confirmed to increase Stat3 phosphorylation to a level similar to that of Th17 cells. On the other hand, the Lrig-1 protein-specific monoclonal antibodies according to the present invention (GTC110-01, GTC110-02, GTC110-03, and GTC110-04) were confirmed to subsequently maintain and decrease Stat3 phosphorylation to a level similar to that of iTreg cells.

[0278] [Example 8] Confirmation of epitopes of Lrig-1 protein In order to identify a structural epitope in the extracellular domain of Lrig-1 protein, which is a site to which the GTC210-03 antibody of Preparation Example 3 can bind, the following experiment was carried out.

[0279] [8-1] Reagents and materials CNBr-activated Sepharose™ 4B (GE, 17-0430-01) Diethylpyrocarbonate (DEPC, Sigma, 159220) Imidazole (Acros, 301870010) Sequencing grade modified trypsin (Promega, V5117) Chymotrypsin, sequencing grade (Promega, V1062) Asp-N, Sequencing grade (Promega, V1621) rLyc-C, Mass spec grade (Promega, V1671) disuccinimidyl dibutyric urea(DSBU;Thermo, A35459) Dimethyl sulfoxide (DMSO, Sigma, D5879)

[0280] Sodium bicarbonate (NaHCO3, Sigma, 792519) Sodium chloride (NaCl, Sigma, S3014) Sodium acetate (NaOAc, Sigma, S2889) Tris (GE, 17-1321-01), Glycine (GE, 17-1323-01) ProteaseMAX(TM) Surfactant, Trypsin Enhancer (Promega, V207A) Acetone (Merck, 1.07021.2521) Dithiothreitol (DTT; GE, 17-1318-02) Iodoacetamide (IAA; Sigma, I-6125) Sodium phosphate monobasic dihydrate (NaH2PO4, Sigma, 71505) Sodium phosphate dibasic hepta-hydrate(Na2HPO4, Sigma, S9390) Syringe filter 0.2 μm (Sartorius stedim, 16534) Acetonitrile, methanol, water (HPLC grade, JT Baker) Formic acid (Sigma, 33015)

[0281] [8-2] Experimental method [8-2-1] Covalent Labeling MS (CL-MS) Antigen samples were prepared at a 2:1 ratio of antigen to antibody. DEPC was added at a concentration not exceeding 1% and incubated at 37°C for 1 minute. Imidazole was then added to terminate the reaction. Acetone was then added to precipitate the sample, and the supernatant was removed by centrifugation. The precipitate was thoroughly dissolved using 0.1% PM. The dissolved precipitate was digested with trypsin, chymotrypsin, or Asp-N / Lys-C, followed by removal of glycans attached to the protein using PNGase-F. Finally, disulfide bonds between cysteines in the protein were cleaved using DTT and IAA, followed by LC-MS and MS2 analysis.

[0282] [8-2-2] Cross-linking MS (XL-MS) Two antigen samples were prepared with an antigen to antibody ratio of 2:1. Then, the crosslinker (CSBU) was thoroughly dissolved in DMSO. To one sample, the sample and DSG were added at a ratio of 1:100, and to the other, DMSO alone was added and the reaction was carried out at 25°C for 1 hour. After the reaction was complete, the protein was digested using trypsin or chymotrypsin, and then the glycans attached to the protein were removed using PNGase-F. Finally, the disulfide bonds between the cysteines in the protein were cleaved using DTT and IAA, and LC-MS and MS2 analysis was performed.

[0283] [8-2-3] Conditions for LC-MS and MS analysis 1. Liquid chromatography (LC) conditions カラム(Column):Hypersil GOLD TM (1.9μm, 1X100mm) Flow rate: 100 μl / min Mobile phase A: DW / 0.2% Formic acid Mobile phase B: ACN / 0.2% Formic acid Temperature: 30℃ LC condition (gradient)

[0284] Table 6

[0285] 2. MS, MS2 condition Table 7

[0286] Table 8

[0287] Table 9

[0288] Table 10

[0289] Table 11

[0290] [8-3] Covalent labeling MS (CL-MS) Control huTregL1-his (Ag, Control) and test huTregL1-his / E7-mlgG2a (Ag+Ab, Test) samples were treated with DEPC followed by digestion with trypsin, chymotrypsin, or Asp-N / Lys-C. Each sample was analyzed three times (see Figures 11 to 13). The MS analysis results for the control (Control 1) and test (Test 1) shown in the analysis results were compared with the control (huTregL1-his) sequence using the BioPharma finder program to identify labeled peptides. Additionally, the presence of unlabeled and labeled peptides was confirmed in the control and test samples treated with chymotrypsin, trypsin, or Asp-N / Lys-C. The results of the repeated tests were compared (decrease ≥ 5%) between the control and test groups, and the results were summarized by amino acid sequence (Res#) and shown in Figures 11 and 12. Based on these results, the huTregL1-his sequence was substituted, and two sequences were found to match the protease test results.

[0291] Among the sequences with a labeling decrease (%) of 5% or more, the sequences E62-L101, D111-L134, and Q542-Y553 showed 13.2%, 15.3%, and 14.7%, respectively, confirming a labeling decrease (%) of 10% or more. In the E62-L101 sequence, the E62-K92 portion (AL) showed 65.9%, the L65-F87 portion (Y) showed 48.7%, and the L88-L101 portion showed 88.6% labeling. These results suggest that the L88-L101 portion of the E62-L101 sequence, with a labeling decrease (%) of 50% or more, is exposed to the outside. The D111-L134 sequence showed a labeling decrease (%) of 15.3% in the chymotrypsin-treated group, with an RSD of 62.4% compared to the control. The Q542-Y553 sequence also showed a 14.7% labeling decrease (%) in the chymotrypsin-treated group, while the labeling (%) in the control antigen group was over 50%, suggesting that it is an externally exposed portion of the protein structure.

[0292] Through CL-MS analysis of the test group (huTregL1-his / E7-mlgG2a), we found that two or more of the sequences showed a labeling decrease (%) of 5% or more in the protease-treated groups, and we ultimately determined that the E62-L101 sequence, which is considered to be exposed externally, corresponds to the epitope site.

[0293] [8-4] Cross-linking MS (XL-MS) Test group (huTregL1-his / E7-mlgG2a) mixture samples were prepared and treated with the cross-linker DSBU (disuccinimidyl dibutyric urea) and then digested with proteases (chymotrypsin or trypsin) (Figures 17 and 18). Peptides digested with chymotrypsin and trypsin were then compared with the control group to identify peptide sequences that were not detected in the test tube (including less than 2%). Furthermore, the corresponding peptides were reconfirmed by examining the form in which only the cross-linker was bound (mono) (M%), and finally, the peptide sequences not detected in the test group samples were combined and determined as the epitope site. For three sequences, L88-K92, S90-L101, and N211-W222, the percentage of identical peptide sequences (T%) detected in the test group samples compared to the control group was confirmed to be less than 2% with both chymotrypsin and trypsin. T158-F174 and G442-K476 were reduced by chymotrypsin (Y) and trypsin (T), respectively. The only sequence with an undetectable T% (0%) was S362-K365 in the trypsin (T) treatment group. Cross-linking analysis identified three sequences, L88-K92, S90-L101, and N211-W222, which were significantly reduced in both protease treatment groups, as epitope sites.

[0294] [8-5] Conclusion As shown in Figures 19 and 20, epitopes were predicted based on the above results. Specifically, two sequences, E65-L101 and G452-K476, of huTreg L1-his(Ag) were identified as identical positions in CL-MS and XL-MS analyses. The L88-K92 portion of L62-L101 showed labeling decrease (%) in two protease treatment groups (Chymotrypsin and Asp-N / Lys-C) in CL-MS analysis, and was also significantly reduced in two protease treatment groups (Chymotrypsin and Trypsin) in XL-MS analysis. The S95-L101 portion was an epitope that was overlappingly identified by chymotrypsin and trypsin in XL-MS analysis. Furthermore, the L88-L101 portion, with labeling (%) of over 50%, was exposed to the outside. In particular, in the case of L88-L101, the amino acid sequence within the sequence was composed of a continuous sequence of new aqueous amino acids (Q89-S95).

[0295] In addition, in the case of G452-K476, the A472-F480 region was confirmed to be externally exposed through CL-MS analysis, and the A472-K476 region was determined to correspond to the epitope. In the case of N211-W222, although it was selected as an epitope site in XL-MS, it did not overlap with the CL-MS analysis, and the labeling (%) of the corresponding sequence was less than 50%, so it was excluded from the epitope as it is located internally in the structure.

[0296] [Example 9] Confirmation of the therapeutic effect of antibodies that specifically bind to Lrig-1 on metabolic diseases - Analysis of experimental results in TregL1 knockout mice [9-1] Preparation of L1 wKO(Lrig-1 CreERT2) mice L1 wKO (Lrig-1 CreERT2) mice are Lrig1 whole-body knockout mice in which the creERT2 sequence is inserted into the translational initiation site of the endogenous Lrig1 locus to knock out Lrig1, allowing Lrig1 expression to be reported as creERT2.

[0297] [9-2] Observation of weight changes in L1 wKO (Lrig-1 CreERT2) mice The L1 wKO (Lrig-1 CreERT2) mice prepared in Example 9-1 were divided into homozygous and heterozygous knockout mice, as well as male and female mice, and body weight changes were observed for 6 to 8 weeks.

[0298] The genotype and body weight changes of L1 wKO (Lrig-1 CreERT2) mice are shown in Table 12 below and Figure 21.

[0299] [Table 12]

[0300] As a result, it was found that the weight of homozygous L1 wKO (Lrig-1 CreERT2) mice was reduced by 22.4%, 14.7% compared to heterozygous knockout mice.

[0301] [9-2] Confirmation of adipose tissue and body weight loss in L1 wKO (Lrig-1 CreERT2) mice The weight of adipose tissue and changes in body weight were measured twice in L1 wKO (Lrig-1 CreERT2) mice. Specifically, the weight changes in adipose tissue and body weight were measured in male and female mice over a 5-12 week period, and are shown in Figures 22 to 25.

[0302] As a result, in the homozygous knockout mice, it was found that in males, body weight decreased by 12.51% compared to the control group at 12 weeks, and visceral fat and subcutaneous fat also decreased. In females, body weight decreased by 14.82% compared to the control group at 12 weeks, and visceral fat and subcutaneous fat also decreased.

[0303] Secondly, the changes in adipose tissue and body weight of male and female mice were observed again for 6 to 12 weeks, and the results are shown in FIGS. 26 to 29.

[0304] As a result, in male homozygous knockout mice, body weight decreased by 8.98% compared to the control group at 12 weeks, and it was confirmed that visceral fat and subcutaneous fat also decreased, and in particular, brown adipocytes also decreased. In female mice, body weight decreased by 14.84% compared to the control group at 12 weeks, and it was confirmed that visceral fat and subcutaneous fat also decreased.

[0305] This indicates that suppressing TregL1 function reduces fat cells and body weight, confirming that it has therapeutic effects on metabolic diseases including obesity.

[0306] [Example 10] Diagnosis of metabolic diseases by confirming increased Lrig-1 in adipocytes [10-1] Differentiation of 3T3 cells into adipocytes 3T3-L1 cells, which are a cell line prior to differentiation into adipocytes and express Lrig-1, were cultured in a 24-well plate at 1x10 5 The cells were then cultured in DMI medium for 2 days, followed by insulin medium for 2 days and DMEM medium for 2 days.

[0307] The specific experimental protocol is shown in Table 13 below and FIG.

[0308] [Table 13]

[0309] [10-2] Analysis of Lrig-1 expression in differentiated adipocytes Subsequently, breast cell analysis was performed using mouse Lrig-1 Alexa488-conjugated antibodies to analyze the level of Lrig-1 expression in differentiated adipocytes.

[0310] The results are shown in Figures 31 and 32. As a result, it was found that the expression of Lrig1-1 was highest on day 4, which can be used to diagnose metabolic diseases including obesity.

[0311] Although the present invention has been described in detail above, it is obvious to a person having ordinary skill in the art that the scope of the invention is not limited thereto, and various modifications and variations are possible within the scope that does not deviate from the technical idea of ​​the invention described in the claims.

Claims

1. A composition for diagnosing a metabolic disease, comprising an agent for measuring the expression level of Lrig-1 protein.

2. The diagnostic composition according to claim 1, wherein the Lrig-1 protein is present on the surface of adipocytes.

3. The diagnostic composition of claim 2, wherein the agent for measuring the expression level of the Lrig-1 protein comprises at least one selected from the group consisting of a primer, a probe, and an antisense nucleotide that specifically binds to the gene.

4. The diagnostic composition of claim 2, wherein the agent for measuring the expression level of Lrig-1 protein is an antibody or an antigen-binding fragment that specifically binds to Lrig-1.

5. The diagnostic composition of claim 4, wherein the antibody or antigen-binding fragment specifically binds to at least one epitope selected from the group consisting of amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO:

45.

6. 6. The diagnostic composition of claim 5, wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent antibody, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, a tetrabody, or a fragment thereof.

7. The diagnostic composition according to any one of claims 1 to 6, wherein the metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension.

8. A kit for diagnosing metabolic diseases, comprising the composition according to any one of claims 1 to 7.

9. The diagnostic kit according to claim 8, wherein the metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension.

10. An informative method for diagnosing metabolic disease comprising measuring Lrig-1 protein expression levels in a biological sample isolated from an individual of interest.

11. The information providing method according to claim 10, wherein the Lrig-1 protein is present on the surface of adipocytes.

12. The information providing method according to claim 11, wherein if the expression level of Lrig-1 protein measured in the biological sample is higher or lower than that of a normal control group, it is predicted that the individual of interest has a metabolic disease.

13. The information providing method according to claim 12, wherein the metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension.

14. (a) a measuring unit for measuring the expression level of Lrig-1 in a biological sample obtained from a target individual; and (b) a detection unit that outputs whether or not the target individual has developed or is likely to develop a bone disease based on the expression level of Lrig-1 measured by the measurement unit.

15. The diagnostic device according to claim 14, wherein the metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension.

16. A pharmaceutical composition for preventing or treating a metabolic disease, comprising as an active ingredient an antibody or antigen-binding fragment thereof that specifically binds to Lrig-1.

17. A pharmaceutical composition for preventing or treating a metabolic disease, comprising as an active ingredient a binding molecule that specifically binds to at least one epitope selected from the group consisting of amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO:

45.

18. 18. The pharmaceutical composition of claim 17, wherein the binding molecule is an antibody or an antigen-binding fragment.

19. 19. The pharmaceutical composition of claim 18, wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent antibody, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, a tetrabody, or a fragment thereof.

20. A chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain, A pharmaceutical composition for preventing or treating a metabolic disease, comprising, as an active ingredient, an antigen-specific binding domain that specifically binds to at least one epitope selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 35 to 45; and a chimeric antigen receptor that is a fusion protein comprising an immunoglobulin Fc region.

21. A pharmaceutical composition for preventing or treating a metabolic disease, comprising as an active ingredient an antibody-drug conjugate comprising a drug and a binding molecule that specifically binds to at least one epitope selected from the group consisting of amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO:

45.

22. 22. The pharmaceutical composition of claim 21, wherein the binding molecule is an antibody or an antigen-binding fragment.

23. 23. The pharmaceutical composition of claim 22, wherein the antibody is a chimeric antibody, a humanized antibody, a bivalent antibody, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, a tetrabody, or a fragment thereof.

24. 24. The pharmaceutical composition according to any one of claims 16 to 23, wherein the metabolic disease is one or more selected from the group consisting of insulin resistance disease, obesity, diabetes, dyslipidemia, liver disease, kidney damage, arteriosclerosis, and hypertension.

25. A pharmaceutical composition for preventing or treating a metabolic disease, comprising, as an active ingredient, a compound that binds to Lrig-1 and inhibits the function of Lrig-1.

26. The pharmaceutical composition according to claim 25, wherein the compound inhibits the function of Lrig-1 through Lrig-1 antagonism.

27. The pharmaceutical composition according to claim 26, wherein the Lrig-1 protein is present on the surface of adipocytes or regulatory T cells.

28. The pharmaceutical composition of claim 27, wherein the compound is an Lrig-1 inhibitor.

29. A method for treating a metabolic disease, comprising administering to an individual as an active ingredient an antibody or antigen-binding fragment that specifically binds to Lrig-1.

30. A method for treating a metabolic disease, comprising administering to an individual as an active ingredient a binding molecule that specifically binds to at least one epitope selected from the group consisting of the amino acid sequences represented by SEQ ID NO: 35 to SEQ ID NO:

45.

31. A chimeric antigen receptor (CAR) comprising an antigen-specific binding domain, a linking domain, and a CD3 zeta (ζ) signaling domain, A method for treating a metabolic disease, comprising administering to an individual as an active ingredient a chimeric antigen receptor, which is a fusion protein comprising an antigen-specific binding domain that specifically binds to at least one epitope selected from the group consisting of amino acid sequences represented by SEQ ID NOs: 35 to 45; and an immunoglobulin Fc region.

32. A method for treating a metabolic disease, comprising administering to an individual as an active ingredient an antibody-drug conjugate comprising a drug and a binding molecule that specifically binds to at least one epitope selected from the group consisting of the amino acid sequences represented by SEQ ID NOs: 35 to 45.

33. A method for treating a metabolic disease, comprising administering to an individual as an active ingredient a compound that binds to Lrig-1 and suppresses the function of Lrig-1.

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