Resistin-specific antibodies and uses thereof

Abstract

The present invention relates to an antibody against resistin and uses thereof, more specifically to a resistin antibody or an antigen-binding fragment thereof that inhibits resistin activity by blocking resistin / CAP1 binding, a nucleic acid encoding the same, a vector containing the nucleic acid, a cell transformed with the vector, a method for producing the antibody or its antigen-binding fragment, an antibody-drug conjugate containing the antibody or its antigen-binding fragment, a bi- or multispecific antibody, a chimeric antigen receptor, an immune cell containing the same, and a composition containing the same for preventing or treating a disease treatable by inhibiting resistin activity. The novel antibody or antigen-binding fragment thereof that binds to resistin of the present invention can bind to resistin and inhibit its activity through blocking resistin / CAP1 binding, and is therefore useful for developing therapeutic agents for various resistin-related diseases.

Description

[Technical Field]

[0001] The present application relates to an antibody against resistin and uses thereof, and more specifically to an anti-resistin antibody or antigen-binding fragment thereof that inhibits resistin activity by blocking resistin / CAP1 binding, a nucleic acid encoding the same, a vector containing the nucleic acid, a cell transformed with the vector, a method for producing the antibody or its antigen-binding fragment, an antibody-drug conjugate comprising the antibody or its antigen-binding fragment, a bispecific or multispecific antibody, a chimeric antigen receptor, an immune cell comprising the same, and a composition comprising the same for preventing or treating a disease treatable by inhibiting resistin activity. [Background technology]

[0002] Resistin, a cytokine first identified as a mediator of insulin resistance in obese mice, belongs to the cysteine-rich protein family, also known as resistin-like molecules (RELMs), and is involved in regulating inflammatory processes. Rat murine resistin has also been implicated in obesity-mediated insulin resistance and the development of type 2 diabetes (Li et al., Endocrine 35:243-251, 2009; Nakata et al., Biochem Biophys Res Commun. 353:1046-1051, 2007; Steppan et al., Nature 409:307-312, 2001).

[0003] In fact, the protein base sequences of rat and human resistin are only about 60% identical, and rodent resistin is primarily expressed and secreted in mature adipocytes, whereas human resistin is primarily secreted by peripheral blood mononuclear cells (PBMCs), such as leukocytes, and macrophages. Numerous studies have revealed that the roles of resistin differ between humans and rodents.

[0004] Human resistin has been reported to promote the influx of immune cells and induce the secretion of pro-inflammatory factors, and there is evidence that it induces inflammatory diseases and atherosclerosis, in addition to promoting insulin resistance (Bokarewa et al, J Immunol. 174:5789-5795, 2005; Silswal et al, Biochem Biophys Res Commun. 334:1092-1101, 2005; Burnett et al, Atherosclerosis 182:241-248, 2005; Jung et al, Cardiovasc. Res 69:76-85, 2006; Reilly et al, Circulation 111:932-939, 2005). Resistin, which is present in both rat and human atherosclerotic lesions, is known as an inflammatory marker of atherosclerosis in humans and is known to promote atherosclerosis by activating monocytes (Cho et al., J Am Coll Cardiol 57:99-109, 2011). Therefore, human resistin is thought to be a key element in stimulating monocytes that leads to atherosclerosis.

[0005] The mechanism by which human resistin induces inflammation appears to be through activation of the NF-κB (nuclear factor kappa B) transcription factor; however, the signaling pathway underlying resistin's pro-inflammatory effects remains unclear, and the role of resistin in cancer remains unknown.

[0006] The present inventors were the first to discover CAP1 (adenylyl cyclase associated protein 1), a receptor that directly interacts with human resistin (Lee S et al., Cell Metabolism, 19(3), 2014):484-97, 2014). Therefore, it is now possible to develop therapeutic agents for diseases caused by resistin using CAP1, a receptor that mediates the action of human resistin.

[0007] Therefore, the inventors have made extensive efforts to treat resistin-related diseases such as cancer by blocking resistin / CAP1 binding, and as a result, they have produced resistin-specific antibodies that bind to resistin with high affinity.They have confirmed that such anti-resistin antibodies exhibit resistin / CAP1 binding inhibitory activity and high therapeutic effects on diseases that can be treated through inhibition of resistin activity, thereby completing the present invention.

[0008] The information provided in this Background section is intended solely to provide a better understanding of the background of the present invention and may therefore not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a novel antibody against resistin or an antigen-binding fragment thereof. Another object of the present invention is to provide a nucleic acid encoding the antibody or antigen-binding fragment thereof.

[0010] Another object of the present invention is to provide a vector containing the nucleic acid, a cell transformed with the vector, and a method for producing the antibody or antigen-binding fragment thereof using the same. Another object of the present invention is to provide an antibody-drug conjugate or a bi- or multispecific antibody comprising said antibody or antigen-binding fragment thereof. Another object of the present invention is to provide a chimeric antigen receptor comprising the antibody or its antigen-binding fragment, and an immune cell comprising the chimeric antigen receptor.

[0011] Other objects of the present invention are to provide a pharmaceutical composition for preventing or treating a disease treatable by inhibition of resistin activity, comprising the antibody or antigen-binding fragment thereof, the antibody-drug conjugate, the bispecific or multispecific antibody, or the chimeric antigen receptor, and a method for preventing or treating a disease treatable by inhibition of resistin activity; use of the antibody or antigen-binding fragment thereof, antibody-drug conjugate, bispecific or multispecific antibody, or chimeric antigen receptor for preventing or treating a disease treatable by inhibition of resistin activity; and use of the antibody or antigen-binding fragment thereof, antibody-drug conjugate, bispecific or multispecific antibody, or chimeric antigen receptor for the manufacture of a medicament for preventing or treating a disease treatable by inhibition of resistin activity. [Means for solving the problem]

[0012] To achieve the above object, the present invention provides a heavy chain variable region comprising CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; and

[0013] Provided is an antibody or antigen-binding fragment thereof that specifically binds to resistin, comprising a light chain variable region including CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 11, CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 19, and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 27.

[0014] The present invention also provides a nucleic acid encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof. The present invention also provides a nucleic acid encoding the light chain variable region of the antibody or antigen-binding fragment thereof. The present invention also provides a vector comprising the nucleic acid. The present invention also provides a cell transformed with the vector.

[0015] The present invention also provides a method for producing the antibody or antigen-binding fragment thereof, comprising the steps of: (a) culturing the cells; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

[0016] The present invention also provides an antibody-drug conjugate or a bi- or multispecific antibody comprising said antibody or antigen-binding fragment thereof. The present invention also provides a chimeric antigen receptor comprising the antibody or antigen-binding fragment thereof, or an immune cell comprising the chimeric antigen receptor.

[0017] The present invention also provides a pharmaceutical composition for preventing or treating a disease treatable by inhibiting the activity of resistin, comprising the antibody or antigen-binding fragment thereof, antibody-drug conjugate, bispecific or multispecific antibody, or chimeric antigen receptor.

[0018] The present invention also provides a method for preventing or treating a disease treatable by inhibition of resistin activity using the antibody or antigen-binding fragment thereof, antibody-drug conjugate, bispecific or multispecific antibody, or chimeric antigen receptor; use of the antibody or antigen-binding fragment thereof, antibody-drug conjugate, bispecific or multispecific antibody, or chimeric antigen receptor for the prevention or treatment of a disease treatable by inhibition of resistin activity; and use of the antibody or antigen-binding fragment thereof, antibody-drug conjugate, bispecific or multispecific antibody, or chimeric antigen receptor for the manufacture of a medicament for the prevention or treatment of a disease treatable by inhibition of resistin activity. [Brief explanation of the drawings]

[0019] [Figure 1] After primary purification, the RETN-C-Fc antigen and RETN-flag-His antigen were analyzed by SDS-PAGE to confirm their purity. [Figure 2a] This shows the results of polyphage ELISA of the scFv phage pool using RETN-flag-His as the antigen in the first round of biopanning. [Figure 2b] This shows the results of polyphage ELISA of the scFv phage pool using RETN-C-Fc as the antigen in the first round of biopanning. [Figure 3a] This shows the results of analyzing the antigen-specific binding of monoclonal antibodies obtained in the first round of biopanning using RETN-flag-His as the antigen. [Figure 3b] This shows the results of analyzing the antigen-specific binding of monoclonal antibodies obtained in the first round of biopanning using RETN-C-Fc as the antigen. [Figure 4] This shows the results of an analysis of the antigen receptor binding inhibitory ability of 16 proteins obtained in the first screening. [Figure 5] The results are from an analysis of the antigen affinity of four clones using OCTET. [Figure 6] This shows the results of an analysis of the antigen-specific binding of 11 monoclonal antibodies obtained from the second screening. [Figure 7a] This shows the results of an analysis of antigen-specific binding of 10 monoclonal antibodies obtained from the first biopanning of the third screening. [Figure 7b] This shows the results of analyzing the antigen-specific binding of nine monoclonal antibodies obtained from the second biopanning of the third screening. [Figure 8] Five antibodies (11G01, 27A04, 32B05, 32E06, and 32G09) with excellent antigen receptor binding inhibitory effects are shown. [Figure 9a] This shows the results of ELISA analysis of the specific binding of 20 clones to the RETN-flag-His antigen. [Figure 9b] ELISA analysis results of non-specific binding of 20 clones. [Figure 10] The results are from an analysis of the antigen affinity of six clones using OCTET. [Figure 11] FIG. 1 is a schematic diagram for the construction of a light chain shuffling library (LC shuffling library). [Figure 12] This shows the results of polyphage ELISA of the scFv phage pool obtained by biopanning with the light chain shuffling library. [Figure 13] This shows the change in productivity of 18 antibody-optimized clones relative to the parent antibody RETN-5A2 clone. [Figure 14] This shows the results of SDS-PAGE analysis of 18 antibody-optimized clones. [Figure 15a] The binding ability of 18 optimized antibodies to the RETN-flag-His antigen was analyzed by ELISA. [Figure 15b] The non-specific binding of 18 optimized antibodies was analyzed by ELISA. [Figure 15c] The binding ability of 13 optimized antibodies to the intact form resistin antigen was analyzed by ELISA. [Figure 16] The cross-species cross-binding activity of eight optimized antibodies against mouse resistin antigen was examined by ELISA. [Figure 17a] FIG. 1 is a schematic diagram of an antigen receptor competitive enzyme immunoassay. [Figure 17b] The ability of 18 optimized antibodies to inhibit antigen receptor binding was evaluated using an antigen receptor competitive enzyme immunoassay. [Figure 18] The ability to inhibit antigen receptor binding was analyzed by pull-down assay. [Figure 19] The results show the antigen affinity of seven optimized antibody clones analyzed using OCTET. [Figure 20]After treating breast cancer cells with resistin, the activation of NF-κB by resistin was measured by the phosphorylation of p65. [Figure 21] The 5A2 antibody and hIgG control were administered to mice, and the pharmacokinetics was measured. [Figure 22] FIG. 1 shows the inhibition of breast cancer cell migration in vitro by the 5A2 antibody. [Figure 23] FIG. 1 shows the in vivo suppression of breast cancer cell metastasis by the 5A2 antibody. [Figure 24] FIG. 1 is a schematic diagram of an experiment in a mouse NASH model to confirm the effect of the 5A2 antibody. [Figure 25] This shows the results of H&E analysis confirming that administration of 5A2 antibody to mice suppressed fatty liver. [Figure 26] This shows the results of oil-red-o analysis, confirming that administration of 5A2 antibody to mice suppressed fatty liver. [Figure 27] 1 is a graph showing insulin sensitivity when 5A2 antibody was administered to mice. [Figure 28] 1 is a graph confirming the reduction in fasting blood glucose levels when 5A2 antibody was administered to mice. [Figure 29] 10 is a graph confirming the reduction in LDL cholesterol when 5A2 antibody was administered to mice. [Figure 30] 1 is a graph confirming the reduction in liver triglycerides when 5A2 antibody was administered to mice. [Figure 31] FIG. 1 is a schematic diagram of an experiment in a mouse IBD model to confirm the effect of the RETN-5A2_LS_2F11 antibody. [Figure 32] FIG. 1 shows the effect of administering the RETN-5A2_LS_2F11 antibody on suppressing weight loss in an IBD mouse model. [Figure 33] FIG. 1 shows the inhibitory effect of administration of the RETN-5A2_LS_2F11 antibody on an increase in disease activity score in an IBD mouse model. [Figure 34]FIG. 1 shows the intestinal protective effect of administration of the RETN-5A2_LS_2F11 antibody in an IBD mouse model. [Figure 35] This shows the results of measuring histological activity scores when the RETN-5A2_LS_2F11 antibody was administered to an IBD mouse model. DETAILED DESCRIPTION OF THE INVENTION

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the art.

[0021] The inventors of the present application used a biopanning method to screen for novel human resistin antibodies that have high affinity for human resistin and excellent ability to inhibit binding between resistin antigen receptors, and confirmed that the screened human resistin antibodies exhibit anti-cancer effects that suppress the migration and metastasis of cancer cells.

[0022] Therefore, in one aspect, the present invention provides a heavy chain variable region comprising: CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1; CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2; and CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; and The present invention relates to an antibody or antigen-binding fragment thereof that specifically binds to resistin, comprising a light chain variable region including CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 11, CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12 to 19, and CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 20 to 27.

[0023] In the present invention, the light chain variable region comprises: a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:13, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21; a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:23; a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO:8, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:16, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:24; a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO:9, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25; a light chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 10, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 18, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 26; or The light chain variable region may be characterized as comprising a CDR1 comprising the amino acid sequence shown in SEQ ID NO: 11, a CDR2 comprising the amino acid sequence shown in SEQ ID NO: 19, and a CDR3 comprising the amino acid sequence shown in SEQ ID NO: 27.

[0024] Furthermore, in the present invention, the heavy chain variable region preferably comprises the amino acid sequence shown in SEQ ID NO: 28, and the light chain variable region preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, and 46. More preferably, the antibody or antigen-binding fragment thereof of the present invention comprises, but is not limited to, a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28; and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, and 46.

[0025] The heavy chain variable region CDR sequences (SEQ ID NOs: 1, 2, and 3) of the antibodies of the present invention that specifically bind to resistin are shown in Table 1 below.

[0026] The light chain variable region CDR sequences (SEQ ID NOs: 4 to 27) of the antibodies of the present invention that specifically bind to resistin are shown in Table 2. SEQ ID NOs: 11, 19, and 27 represent the sequences of CDR1, 2, and 3 of the light chain variable region of the resistin 5A2 parent antibody.

[0027] The amino acid sequence of the heavy chain variable region (SEQ ID NO: 28) and the amino acid sequence of the heavy chain constant region (SEQ ID NO: 29) of the antibody of the present invention that specifically binds to resistin are shown in Table 3 below.

[0028] The amino acid sequences of the light chain variable regions (SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, and 46) and the light chain constant regions (SEQ ID NOs: 33, 35, 37, 39, 41, 43, 45, and 47) of the antibodies of the present invention that specifically bind to resistin are shown in Table 5. SEQ ID NO: 46 is the amino acid sequence of the light chain variable region of the resistin 5A2 parent antibody, and SEQ ID NO: 47 is the amino acid sequence of the light chain constant region of the resistin 5A2 parent antibody.

[0029] [Table 1]

[0030] [Table 2]

[0031] [Table 3]

[0032] [Table 4] TIFF0007763948000005.tif72170

[0033] [Table 5] TIFF0007763948000007.tif211170

[0034] [Table 6] TIFF0007763948000009.tif244170TIFF0007763948000010.tif244170TIFF0007763948000011.tif243170TIFF0007763948000012.tif97170

[0035] As used herein, the term "antibody" refers to an anti-resistin antibody that specifically binds to resistin, particularly human resistin. The scope of the present invention includes not only intact antibody forms that specifically bind to resistin, but also antigen-binding fragments of the antibody molecules.

[0036] Intact antibodies have two full-length light chains and two full-length heavy chains, each connected to a heavy chain by a disulfide bond. The heavy chain constant regions are of gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, with subclasses of gamma 1 (γ1), gamma 2 (γ2), gamma 3 (γ3), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The light chain constant regions are of kappa (κ) and lambda (λ) types.

[0037] Antigen-binding fragments of antibodies or antibody fragments refer to fragments that retain antigen-binding function and include Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure comprising light-chain and heavy-chain variable regions, a light-chain constant region, and the first constant region of the heavy chain (CH1), and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy-chain CH1 domain. F(ab')2 antibodies are generated by disulfide bond formation between cysteine ​​residues in the hinge region of Fab'. Fv is the smallest antibody fragment containing only the heavy-chain variable region and the light-chain variable region, and recombinant techniques for generating Fv fragments are disclosed in PCT International Publication Nos. WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. In two-chain Fvs, the heavy chain variable region and the light chain variable region are linked non-covalently, while in single-chain Fvs (scFvs), the heavy chain variable region and the light chain variable region are generally linked covalently via a peptide linker or directly at the C-terminus, and can form a dimeric structure like two-chain Fvs. Such antibody fragments can be obtained using protease hydrolases (for example, Fab can be obtained by restrictive cleavage of a whole antibody with papain, or F(ab')2 fragments can be obtained by cleavage with pepsin), or can be produced through genetic recombination techniques.

[0038] In one embodiment, the antibody of the present invention is in Fv format (e.g., scFv) or in whole antibody format. The heavy chain constant region may be selected from the gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) isotype. For example, the constant region may be gamma 1 (IgG1), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region may be kappa or lambda type.

[0039] As used herein, the term "heavy chain" refers to both a full-length heavy chain and fragments thereof that includes a variable region domain VH and three constant region domains CH1, CH2, and CH3, each of which contains an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen. Additionally, the term "light chain" refers to both a full-length light chain and fragments thereof that includes a variable region domain VL and a constant region domain CL, each of which contains an amino acid sequence with sufficient variable region sequence to confer specificity to an antigen.

[0040] Antibodies of the present invention include, but are not limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single-chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv) and anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of the above antibodies.

[0041] The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies constituting the population are identical except for minor naturally occurring mutations that may be present. Monoclonal antibodies are highly specific and directed against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0042] For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma method or may be prepared using recombinant DNA methods from bacterial, eukaryotic, or plant cells (see U.S. Patent No. 4,816,567). The monoclonal antibodies may also be isolated from phage antibody libraries.

[0043] "Epitope" refers to a determinant on a protein to which an antibody can specifically bind. Epitopes are usually composed of chemically active surface groupings of molecules, such as amino acids or sugar side chains, and generally possess specific three-dimensional structural characteristics as well as specific charge characteristics. Conformational and nonconformational epitopes are distinguished in that the binding to the former, but not the latter, is lost in the presence of denaturing solvents.

[0044] The "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies) in which residues from a hypervariable region of a receptor have been substituted with residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and capacity.

[0045] The term "human antibody" refers to a molecule derived from human immunoglobulin, in which all amino acid sequences constituting the antibody, including complementarity-determining regions and structural regions, are entirely composed of human immunoglobulin.

[0046] These include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) is / are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0047] As used herein, "antibody variable domain" refers to the light and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and framework regions (FRs). VH refers to the variable domain of the heavy chain. VL refers to the variable domain of the light chain.

[0048] "Complementarity-determining region" (CDR; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues of an antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3.

[0049] An "Fv" fragment is an antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in substantially tight covalent association, e.g., as an scFv.

[0050] The "Fab" fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1) of the heavy chain. F(ab')2 antibody fragments typically contain a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them.

[0051] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0052] The antibody according to the present invention has increased affinity for an antigen. The term "affinity" refers to the ability to specifically recognize and bind to a specific site on an antigen. High affinity, along with the specificity of an antibody for an antigen, is an important factor in an immune response. Affinity can be determined using any of a variety of assays known in the art, such as radioimmunoassay (RIA) and ELISA, and can be expressed as various quantitative values. The affinity of an antibody for an antigen can generally be expressed by the dissociation constant (Kd) of a specific antibody-antigen interaction. A lower Kd value indicates a higher affinity of the antibody for the antigen.

[0053] The antibodies or antibody fragments of the present invention may include not only the sequences of the anti-resistin antibodies of the present invention described herein, but also their biological equivalents, as long as they can specifically recognize resistin. For example, additional modifications may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletion, insertion, and / or substitution of residues in the amino acid sequence of the antibody. Such amino acid modifications are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine are considered to be biologically functionally equivalent.

[0054] Considering the above-mentioned biologically equivalent mutations, the antibodies of the present invention or nucleic acid molecules encoding them are also understood to include sequences that exhibit substantial identity to the sequences set forth in SEQ ID NOs. The term "substantial identity" refers to a sequence that exhibits at least 90% homology, most preferably at least 95%, 96% or more, 97% or more, 98% or more, or 99% or more when the sequences are aligned as closely as possible and analyzed using algorithms commonly used in the art. Alignment methods for sequence comparison are well known in the art. The NCBI Basic Local Alignment Search Tool (BLAST) is available at NBCI and elsewhere 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 https: / / blast.ncbi.nlm.nih.gov / Blast.cgi. The sequence homology comparison method using this program can be found at https: / / blast.ncbi.nlm.nih.gov / doc / blast-help.

[0055] Based on this, antibodies or antigen-binding fragments thereof of the present invention may have 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology to the sequences described herein or to the entirety. Such homology may be determined by sequence comparison and / or alignment using methods known to those of skill in the art. For example, percent sequence homology of nucleic acids or proteins of the present invention may be determined using sequence comparison algorithms (i.e., BLAST or BLAST 2.0), manual alignment, and visual inspection.

[0056] In another aspect, the present invention relates to a nucleic acid encoding the heavy chain variable region of the antibody or antigen-binding fragment thereof.

[0057] In the present invention, the nucleic acid is preferably a nucleic acid encoding a heavy chain variable region of an antibody that specifically binds to resistin, comprising the amino acid sequence shown in SEQ ID NO: 28, and more preferably, the nucleic acid may be characterized as having the base sequence shown in SEQ ID NO: 30, the sequence of which is shown in Table 4 above.

[0058] In another aspect, the present invention relates to a nucleic acid encoding the light chain variable region of the antibody or antigen-binding fragment thereof.

[0059] In the present invention, the nucleic acid is preferably a nucleic acid encoding a light chain variable region of an antibody that specifically binds to resistin, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44, and 46, and more preferably a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60, and 62, the sequences of which are shown in Table 6 above.

[0060] The term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic building blocks of nucleic acids, include not only naturally occurring nucleotides but also analogues in which the sugar or base moiety has been modified. The nucleic acid sequences encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include addition, deletion, or non-conservative or conservative substitution of nucleotides.

[0061] DNA encoding the antibody is readily isolated or synthesized using conventional processes (e.g., by using oligonucleotide probes capable of specifically binding to DNA encoding the antibody heavy and light chains). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0062] The antibody or antigen-binding fragment thereof may be produced recombinantly by isolating nucleic acid encoding the antibody or antigen-binding fragment thereof of the invention, isolating the nucleic acid, and inserting it into a replicable vector for further cloning (amplification of the DNA) or for further expression.

[0063] Based on this, another aspect of the present invention relates to a vector containing the nucleic acid. The term "vector" as used herein includes, as a means for expressing a gene of interest in a host cell, plasmid vectors, cosmid vectors, and viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. In the vector, the nucleic acid encoding the antibody is operably linked to a promoter.

[0064] "Operably linked" refers to a functional connection between a nucleic acid expression control sequence (e.g., a promoter, a signal sequence, or an array of transcriptional regulator binding sites) and another nucleic acid sequence, whereby the control sequence controls the transcription and / or detoxification of the other nucleic acid sequence.

[0065] When a prokaryotic cell is used as the host, it generally contains a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for initiating detoxification, and a transcription / detoxification termination sequence. Furthermore, for example, when eukaryotic cells are used as hosts, promoters derived from mammalian cell genomes (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse mammary tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) may be used, and typically have a polyadenylation sequence as a transcription termination sequence.

[0066] In some cases, the vector may be fused to other sequences to facilitate purification of the antibody expressed therefrom, such as glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), and 6x His (hexahistidine; Quiagen, USA).

[0067] The vectors contain as selectable markers antibiotic resistance genes commonly used in the art, such as resistance genes to ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.

[0068] Meanwhile, the vector expressing the resistin-specific antibody or its antigen-binding fragment can be either a vector system in which nucleic acids encoding the heavy chain variable region and the light chain variable region are simultaneously expressed in a single vector, or a system in which the light chain and the heavy chain are each expressed in separate vectors. In the latter case, the two vectors can be introduced into host cells by co-transformation and targeted transformation. Alternatively, cells transformed with a vector containing the light chain (or heavy chain) can be screened, and the screened cells can be further transformed with a vector containing the heavy chain (or light chain), followed by final screening for cells expressing both the light chain and the heavy chain.

[0069] Although not limited thereto, a preferred vector system is one in which the nucleic acid encoding the heavy chain variable region and the nucleic acid encoding the light chain variable region of a resistin-specific antibody or its antigen-binding fragment are simultaneously expressed in a single vector.

[0070] From another aspect, the present invention relates to a cell transformed with the vector. In the present invention, the cells may be characterized by being selected from the group consisting of animal cells, plant cells, yeast, E. coli, and insect cells, but are not limited thereto. The cells used to produce the antibodies of the present invention may be, but are not limited to, prokaryotic, yeast, or higher eukaryotic cells.

[0071] Prokaryotic host cells such as strains of the genus Bacillus, including Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) may be used.

[0072] However, of most interest to animal cells, examples of useful host cell lines may be, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, COS-7, HEK293, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0073] In another aspect, the present invention relates to a method for producing the antibody or antigen-binding fragment thereof, comprising: (a) culturing the cells; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

[0074] The cells may be cultured in various media. The culture medium may be any commercially available medium without limitation. Any other necessary supplements known to those skilled in the art may be included at appropriate concentrations. The culture conditions, such as temperature, pH, etc., are already used with the host cells selected for expression and will be apparent to those skilled in the art.

[0075] The antibody or antigen-binding fragment thereof may be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and the resulting product may be purified, for example, by affinity chromatography. Additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxylapatite chromatography, etc., may also be used.

[0076] From another aspect, the present invention relates to an antibody-drug conjugate (ADC) in which a drug is bound to an antibody or antigen-binding fragment thereof that specifically binds to resistin.

[0077] In antibody-drug conjugates, the drug must remain stably bound to the antibody until it is delivered to the target cell. Once delivered, the drug, e.g., an anticancer drug, must be released from the antibody and induce target cell death. To achieve this, the drug must be stably bound to the antibody and, at the same time, have sufficient cytotoxicity to induce target cell death upon release from the target cell.

[0078] In the present invention, the antibody or its antigen-binding fragment and the cytotoxic substance, including a drug such as an anticancer drug, may be linked to each other (e.g., by a covalent bond, a peptide bond, etc.) and used in the form of a conjugate or a fusion protein (when the cytotoxic substance and / or the marker substance is a protein). The cytotoxic substance may be any substance that is toxic to cancer cells, particularly solid cancer cells, and may be one or more selected from the group consisting of a radioisotope, a cytotoxic small molecule, a cytotoxic protein, an anticancer drug, etc., but is not limited to this. The cytotoxic protein may be one or more selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, pseudomonas toxin, etc., but is not limited to this. The radioisotope may be one or more selected from the group consisting of 131I, 188Rh, 90Y, etc., but is not limited to this. The cytotoxic compound may be one or more selected from the group consisting of duocarmycin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)maytansine (DM1), PBD (pyrrolobenzodiazepine) dimer, and the like, but is not limited to these.

[0079] In the present invention, the antibody-drug conjugate may be produced by a technique well known in the technical field to which the present invention pertains. In the present invention, the antibody-drug conjugate may be characterized in that the antibody or antigen-binding fragment thereof is bound to a drug via a linker. In the present invention, the linker may be characterized as being a cleavable linker or a non-cleavable linker.

[0080] The linker is a moiety that connects the anti-resistin antibody and the drug, and for example, the linker is cleavable under intracellular conditions, i.e., allows the drug to be released from the antibody by cleavage of the linker in the intracellular environment.

[0081] The linker may be a peptide linker that can be cleaved by a cleaving agent present in the intracellular environment, such as a lysosome or endosome, or by an intracellular peptidase or protease enzyme, such as a lysosomal or endosomal protease. Generally, the peptide linker is at least two amino acids long. The cleaving agent may include cathepsin B, cathepsin D, or plasmin, which hydrolyzes the peptide and allows the drug to be released into the target cell. The peptide linker may be cleaved by the thiol-dependent protease cathepsin B, which is highly expressed in cancer tissues. For example, a Phe-Leu or Gly-Phe-Leu-Gly linker may be used. The peptide linker may also be, for example, a Val-Cit linker or a Phe-Lys linker, which can be cleaved by an intracellular protease.

[0082] In the present invention, the cleavable linker may be pH-sensitive and sensitive to hydrolysis at a specific pH value. Generally, a pH-sensitive linker indicates that it can be hydrolyzed under acidic conditions. For example, an acid-labile linker that can be hydrolyzed in the lysosome may be, for example, a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, etc.

[0083] The linker may be cleaved under reducing conditions, for example, a disulfide linker. Various disulfide bonds may be formed using SATA (N-succinimidyl-S-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene).

[0084] In the present invention, the drug and / or drug linker may be randomly bound via lysines on the antibody or via cysteines exposed upon reduction of disulfide-bonded chains. In some cases, the linker drug may be bound via cysteines present in a genetically engineered tag, e.g., a peptide or protein. The genetically engineered tag, for example, a peptide or protein, may contain an amino acid motif that can be recognized by isoprenoid transferase. The peptide or protein may have a deletion at the carboxy terminus of the peptide or protein or a spacer unit covalently attached to the carboxy (C) terminus of the peptide or protein. The peptide or protein may be directly covalently bound to the amino acid motif or may be linked to the amino acid motif via a spacer unit. The amino acid spacer unit is composed of 1 to 20 amino acids, with a glycine unit being preferred.

[0085] The linker may include a β-glucuronide linker that is recognized and hydrolyzed by β-glucuronidase, which is abundantly present in lysosomes or overexpressed in some tumor cells. Unlike peptide linkers, β-glucuronide linkers have a high hydrophilicity, which has the advantage of increasing the solubility of antibody-drug conjugates when conjugated with highly hydrophobic drugs.

[0086] In this regard, the present invention may use a β-glucuronide linker disclosed in Korean Patent Publication No. 2015-0137015, for example, a β-glucuronide linker containing a self-immolative group.

[0087] Alternatively, the linker may be, for example, a non-cleavable linker, which allows the drug to be released in a single step by antibody hydrolysis, for example, to form an amino acid linker-drug conjugate. This type of linker may be a thioether group or a maleimidocaproyl group, and can maintain stability in the blood.

[0088] In the present invention, the drug may be a chemotherapeutic agent, a toxin, a microRNA (miRNA), a siRNA, an shRNA, or a radioisotope. The drug may be bound to an antibody as a pharmaceutical agent that exhibits a pharmacological effect.

[0089] The chemotherapeutic agent may be a cytotoxic agent or an immunosuppressant. Specifically, it may include a chemotherapeutic agent that can function as a microtubulin inhibitor, a mitotic inhibitor, a topoisomerase inhibitor, or a DNA intercalator. It may also include an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an antiparasitic agent, or a combination thereof.

[0090] Examples of such drugs include maytansinoids, orlistatin, aminopterin, actinomycin, bleomycin, thalidomide, camptothecin, N8-acetylspermidine, 1-(2-chloroethyl)-1,2-dimethylsulfonylhydrazide, esperamicin, etoposide, 6-mercaptopurine, drostatin, trichothecenes, calicheamicin, taxol, taxanes, paclitaxel, docetaxel, methotrexate, vincristine, vinblastine, doxorubicin, melphalan, chlorambucil, duocarmycin, L-asparaginase, and the like. ginase, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosourea, cisplatin, carboplatin, mitomycin (mitomycin A, mitomycin C), dacarbazine, procarbazine, topotecan, nitrogen mustardmustard, cytoxan, 5-fluorouracil, CNU (bischloroethylnitrosourea), irinotecan, camptothecin, idarubicin, daunorubicin, dactinomycin, plicamycin, asparaginase, vinorelbine, chlorambucil chlorambucil, melphalan, carmustine, lomustine, busulfan, treosulfan, dacarbazine, teniposide, topotecan, 9-aminocamptothecin, crisnatol, trimetrexate, mycophenolic acid acid, tiazofurin, ribavirin, EICAR (5-ethynyl-1-beta-dribofuranosylimidazole-4-carboxamide), hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed, cytarabine (ara C), cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, leuprolide acetateacetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, gemcitabine, velcade, Revlimid, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, actinomycin D The antiviral agent may be, but is not limited to, one or more selected from the group consisting of benzodiazepines, benzocaine, benzoyl peroxidase, benzocaine, benzoyl peroxidase, benzoyl peroxidase D), dactinomycin, bleomycin A2, bleomycin B2, peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone, verapamil, and thapsigargin, nucleases, and toxins derived from bacteria, animals, or plants.

[0091] In the present invention, the drug may contain one or more nucleophilic groups selected from the group consisting of amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react to form covalent bonds with electrophilic groups on linkers and linker reagents.

[0092] From another aspect, the present invention relates to a bisspecific or multi-specific antibody comprising the antibody or antigen-binding fragment thereof that specifically binds to resistin.

[0093] A bispecific antibody refers to an antibody that has the ability to bind to or compete with more than one target, and refers to a form in which antibodies that have the ability to bind to or compete with two different targets are bound together, or an antibody in which an antibody that has the ability to bind to one target is bound to a substance that has the ability to compete with another target.

[0094] A multispecific antibody refers to an antibody that has binding specificities for at least three or more different antigens. Multispecific antibodies may include trispecific or higher-specific antibodies, such as trispecific antibodies, tetraspecific antibodies, or antibodies that target more than two targets.

[0095] Methods for producing bispecific or multispecific antibodies are widely known. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two or more immunoglobulin heavy / light chain pairs, under conditions where the two or more heavy chains have different specificities.

[0096] The antigen to which the antibody other than the anti-resistin antibody contained in the bispecific or multispecific antibody binds is preferably a cancer-associated antigen or an immune checkpoint protein antigen, such as HGF, EGFR, EGFRvIII, Her2, Her3, IGF-1R, VEGF, VEGFR-1, VEGFR-2, VEGFR-3, Ang2, Dll4, NRP1, FGFR, FGFR2, FGFR3, c-Kit, MUC1, MUC16, CD20, CD22, CD27, CD30, CD33, CD40, CD52, CD70, CD79, DDL3, Folate R1, or Nectin. 4, Trop2, gpNMB, Axl, BCMA, PD-1, PD-L1, PD-L2, CTLA4, BTLA, 4-1BB, ICOS, GITR, OX40, VISTA, TIM-3, LAG-3, KIR, B7.1, B7.2, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7, EphA2, EphA4, EphB2, E-selectin, EpCam, CEA, PSMA, PSA, c-MET, etc., and immune effector cell-associated antigens may include, but are not limited to, TCR / CD3, CD16 (FcγRIIIa), CD44, CD56, CD69, CD64 (FcγRI), CD89, CD11b / CD18 (CR3), etc.

[0097] From another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof that specifically binds to resistin.

[0098] The CAR may comprise an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, and the antigen binding domain may be linked to the transmembrane domain via a linker. The extracellular domain comprising the antigen binding domain may comprise a signal peptide.

[0099] From another aspect, the present invention relates to an immune cell containing the chimeric antigen receptor. The immune cells may be characterized as comprising cells genetically modified to express the chimeric antigen receptor, and preferably being T cells or NK cells.

[0100] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating a disease treatable by inhibiting the activity of resistin, comprising the antibody or antigen-binding fragment thereof, the antibody-drug conjugate, the bispecific or multispecific antibody, or the chimeric antigen receptor.

[0101] In the present invention, the activity of resistin may be characterized in that the resistin / CAP1 binding is blocked by a resistin antibody, thereby inhibiting the activity. In the present invention, the diseases treatable by inhibiting the activity of resistin are preferably, but not limited to, cancer, cardiometabolic diseases, autoimmune diseases, or inflammatory diseases.

[0102] The present invention may be, for example, a pharmaceutical composition for preventing or treating cancer, cardiometabolic disease, autoimmune disease, or inflammatory disease, comprising: (a) a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof that specifically binds to resistin according to the present invention; and (b) a pharmaceutically acceptable carrier. The present invention also relates to a method for preventing or treating cancer, cardiometabolic disease, autoimmune disease, or inflammatory disease, comprising the step of administering to a patient an effective amount of an antibody or antigen-binding fragment thereof that specifically binds to resistin according to the present invention.

[0103] Therefore, from another aspect, the present invention relates to a method for preventing or treating a disease that can be treated by inhibiting the activity of resistin, the method comprising the step of administering to a subject a composition comprising the antibody or antigen-binding fragment thereof, the antibody-drug conjugate, the bispecific or multispecific antibody, or the chimeric antigen receptor.

[0104] In another aspect, the present invention relates to use of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate, the bispecific or multispecific antibody, or the chimeric antigen receptor, or a composition comprising them, for the prevention or treatment of a disease treatable by inhibiting the activity of resistin.

[0105] In another aspect, the present invention relates to use of the antibody or antigen-binding fragment thereof, the antibody-drug conjugate, the bispecific or multispecific antibody, or the chimeric antigen receptor, or a composition comprising any of them, for the manufacture of a medicament for the prevention or treatment of a disease treatable by inhibiting the activity of resistin.

[0106] The compositions, methods, uses, and applications use the anti-resistin antibody or antigen-binding fragment thereof of the present invention as an active ingredient, and therefore overlapping descriptions will be omitted.

[0107] "Prevention" means any action of administering a composition according to the present invention to suppress or slow the progression of a disease treatable by inhibiting the activity of resistin, and "treatment" means suppressing the progression, alleviating or eliminating a disease treatable by inhibiting the activity of resistin.

[0108] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals in which a population of cells is characterized by unregulated cell growth.

[0109] "Tumor" refers to any mass of tissue caused by excessive cell growth or proliferation, which may be benign (non-cancerous) or malignant (cancerous), including pre-cancerous lesions.

[0110] "Cancer cells," "tumor cells," and grammatical equivalents refer to the total population of cells derived from a tumor or precancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the tumor cell population, and tumorigenic stem cells (cancer stem cells).

[0111] The term "cancer" as used herein includes any cancer in which resistin is expressed, without limitation. The cancer in which resistin is expressed is preferably selected from the group consisting of breast cancer, metastatic cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, lung cancer, liver cancer, glioma, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, gastric cancer, pancreatic cancer, and recurrent cancer, but is not limited thereto.

[0112] In particular, the antibody or antigen-binding fragment thereof of the present invention, or the pharmaceutical composition comprising the same may be characterized by inhibiting cancer metastasis. In the present invention, the "cardiometabolic disease, autoimmune disease, or inflammatory disease" includes, without limitation, any disease in which resistin is expressed.

[0113] In the present invention, the cardiometabolic disease is preferably selected from the group consisting of obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, insulin-resistant diabetes, type 2 diabetes, liver disease, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH), but is not limited thereto.

[0114] In the present invention, the autoimmune disease is preferably selected from the group consisting of chronic heart disease, rheumatoid arthritis, systemic erythematous cyst, digestive system diabetes, atopic dermatitis, autoimmune encephalomyelitis, asthma, and Crohn's disease, but is not limited thereto.

[0115] In the present invention, the inflammatory disease is preferably selected from the group consisting of inflammatory skin diseases including asthma, eczema, psoriasis, acne, allergies, rheumatoid arthritis, psoriatic arthritis, atopic dermatitis, atopic rhinitis, allergic dermatitis, chronic sinusitis or seborrheic dermatitis; inflammatory bowel diseases (IBD) including Crohn's disease or ulcerative colitis, ankylosing spondylitis, sepsis, septic shock, vasculitis and bursitis, but is not limited to these.

[0116] The pharmaceutical composition of the present invention may contain an antibody or a fragment thereof specific to resistin, and the components may further include a pharmaceutically acceptable carrier for administering the pharmaceutical composition of the present invention.

[0117] In the present invention, the term "pharmaceutically acceptable carrier" refers to a carrier or diluent that does not stimulate the body and does not inhibit the biological activity and properties of the administered compound. For compositions formulated into liquid solutions, pharmaceutically acceptable carriers include those that are sterile and biocompatible, such as saline, sterile water, buffered saline, albumin injection, dextrose solution, maltodextrin solution, glycerol, and mixtures of one or more of these components. Other common additives, such as antioxidants, buffers, and bacteriostatic agents, may also be added as needed. Furthermore, diluents, dispersants, surfactants, binders, and lubricants may be added to formulate the compositions into injectable solutions, such as aqueous solutions, suspensions, and emulsions, as well as pills, capsules, granules, or tablets.

[0118] The pharmaceutical compositions of the present invention may be in various oral or parenteral dosage forms. When formulated, they are prepared using commonly used diluents or excipients, such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid formulations for oral administration include tablets, pills, powders, granules, capsules, and the like. These solid formulations are prepared by mixing one or more compounds with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid formulations for oral administration include suspensions, liquids, emulsions, syrups, and the like. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as wetting agents, sweeteners, flavorings, and preservatives, may also be used. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, and suppositories. Non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases that can be used include witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin.

[0119] In another aspect, the present invention provides a method for inhibiting the growth, migration, or metastasis of resistin-expressing tumor cells, comprising contacting the cells with the antibody or antigen-binding fragment thereof.

[0120] The tumor cells may be any type of tumor cells that express resistin, and preferably include, but are not limited to, breast cancer, metastatic cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, lung cancer, liver cancer, glioma, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, gastric cancer, pancreatic cancer, and recurrent cancer cells.

[0121] The present invention relates to a method for treating cancer, a cardiometabolic disease, an autoimmune disease or an inflammatory disease comprising the step of administering to an individual a pharmaceutically effective amount of an antibody or antigen-binding fragment thereof.

[0122] Therapeutic methods using the antibodies or antigen-binding fragments thereof of the present invention include administering a pharmaceutically effective amount of the antibody or antigen-binding fragment. It will be apparent to those skilled in the art that the appropriate total daily dose can be determined by a treating physician within the scope of sound medical judgment. The antibody or antigen-binding fragment may be administered in a single dose or multiple doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will vary depending on various factors, including the type and degree of response to be achieved, the specific composition, including whether other formulations are used, the patient's age, weight, general health, sex, and diet, the time, route of administration, and 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.

[0123] Subjects to which the compositions of the present invention are administered include, without limitation, mammals, including humans. The term "administration" in the present invention means introducing the pharmaceutical composition of the present invention into a patient by any appropriate method, and the administration route of the composition of the present invention may be via various routes, such as oral or parenteral, as long as it can reach the target tissue. [Example]

[0124] The present invention will be described in more detail below with reference to examples. It will be obvious to those skilled in the art that these examples are merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the present invention.

[0125] Example 1: Expression and purification of resistin antigen To express the RETN-C-Fc protein, which is a resistin sequence fused with a human antibody Fc sequence at its C-terminus, and the RETN-flag-His protein, which is a resistin sequence fused with a flag sequence and a histidine sequence at its C-terminus, in animal cells, HEK-293F cells were transfected with RETN-flag-His DNA and cultured for a certain period of time, after which the culture medium was harvested. For transfection, DNA was added to FreeStyle 293 expression medium, and 200 μg of PEI (polyethylenimine) was added to create a polyplex reaction solution. The polyplex reaction solution was left to react at room temperature for 20 minutes, and then 1x10 cells cultured in HEK293 culture medium were used. 6 The cells were placed in an 80 ml HEK293F cell culture flask and cultured at 37°C, 8% CO2, and 85 rpm for 7 days.

[0126] After 7 days, the culture medium was collected and centrifuged at 5,000 rpm for 10 minutes to precipitate and remove cells and cell debris. To purify the antigen protein in the resulting supernatant, the supernatant from the RETN-C-Fc antigen DNA transfection culture was loaded onto a column packed with recombinant protein A agarose resin for 16 hours at 4°C to allow the antigen protein to adsorb to the resin, followed by elution with 0.1 M pH 3.3 glycine solution. The eluted protein was neutralized to pH 7-7.4 with 1 M Tris-HCl solution and the solvent was exchanged with DPBS using a Maxi GeBAflex tube (12,000-14,000 MWCO, D050-100). To purify the RETN-flag-His antigen, the culture supernatant was loaded onto a column packed with Ni Sepharose 6 Fast Flow (GE Healthcare, 17531803) resin at 4°C for 16 hours to allow the antigen protein to adsorb to the resin, followed by elution with 500 mM imidazole. The eluted protein was solvent exchanged with DPBS and concentrated using a Maxi GeBAflex-tube (12000-14000 MWCO, D050-100). The purity of the purified protein was confirmed using SDS-PAGE gel (Figure 1).

[0127] Example 2: Primary screening of resistin human antibodies Example 2-1: Biopanning Biopanning was performed to obtain a phage pool that binds to the resistin antigen. The RETN-C-Fc or RETN-flag-His antigen prepared in Example 1 was coated onto an immunosorbent tube, followed by blocking. Human antibody library phages were prepared by infecting E. coli with the human scFv library, culturing the cells, and then recovering and concentrating the culture medium. The human antibody library phages were then introduced into the antigen-coated immunotube for reaction, after which the antigen-bound scFv phages were eluted. The phages obtained in the first round of panning were recovered and amplified by reinfecting E. coli. Then, second and third rounds of biopanning were performed using the RETN-C-Fc or RETN-flag-His antigen. A polyphage pool that bound to the antigen was obtained in each round.

[0128] Example 2-2: Polyphage ELISA Polyphage ELISA was performed to confirm the specific binding of the scFv phage pools obtained in each round to their antigens. RETN-flag-His and RETN-C-Fc antigens were coated at 100 ng / well on a 96-well immunoplate (NUNC, 439454) as specific antigens and blocked with skim milk. After incubating with the scFv phage obtained in each round, the wells were washed with PBS-T and incubated with anti-M13-HRP (Amersham, 27-9421-01) at a dilution of 1:2000. After washing with PBS-T, 100 μl of OPD (Sigma, 8787-TAB) solution was added to each well and allowed to react with the substrate for 10 minutes. After 10 minutes, each well was treated with 50 μl of 1N H 2 SO 4 to stop the substrate reaction, and then the activity was measured at 490 nm using a spectrophotometer (Thermo Fisher Scientific, 51119200).

[0129] As a result, it was confirmed that the antigen binding ability of the polyphage pool obtained in each round increased (enrichment) as the rounds progressed (Fig. 2a and Fig. 2b).

[0130] Example 2-3: Screening for positive phages (monophage ELISA) Colonies obtained from the third-round polyphage pool with high antigen binding ability were inoculated into 1 ml of 2xYTCM (2% glucose, 5 mM MgCl2) medium, placed in a 96-well deep-well plate (Bionia, 90030), and cultured at 37°C for 16 hours. A 100-200 μl aliquot of the cultured cell broth was inoculated into 1 ml of 2xYTCM (2% glucose, 5 mM MgCl2) medium to achieve an OD600 of 0.1 and cultured at 37°C for 2-3 hours. After incubation, M1 helper phage was added at an M ratio of 1:20, inoculated into 2xYTCMK (5 mM MgCl2, 1 mM IPTG), and incubated at 30°C for 16 hours.

[0131] Resistin antigen was coated at 100 ng / well on a 96-well immunoplate (NUNC, 439454) and blocked with skim milk. Monoclonal scFv phage cultured for 16 hours was added to each well (100 μl per well) for 2 hours. After washing with PBS-T, the plate was incubated with anti-M13-HRP (Amersham, 27-9421-01) secondary antibody (1:2000). After washing with PBS-T, each well was treated with 100 μl of OPD (Sigma, 8787-TAB) solution for 10 minutes. After 10 minutes, each well was treated with 50 μl of 1N H2SO4 to stop the substrate reaction, and the intensity was measured at 490 nm using a spectrophotometer (Thermo Fisher Scientific, 51119200). A total of 20 antigen-specific clones were obtained (FIGS. 3a and 3b).

[0132] Example 3: Production of primary screening resistin human antibodies Example 3-1: Conversion to IgG type The 20 anti-resistin monoclonal phage antibodies selected in Examples 2-3 were converted from scFv to IgG structures. To insert the DNA of each clone into the entire vector, PCR was performed to add restriction enzyme sequences before and after each heavy and light chain. The resulting heavy and light chain DNA and the entire vector were digested with restriction enzymes, then treated with ligase (T4 DNA ligase, Thermofisher Scientific, #EL0011) and ligated at 22°C for 10 minutes to insert the heavy and light chain sequences into the vector. The resulting vectors were transformed into competent cells (XLI-blue) by heat shock at 42°C, spread on LB ampicillin plates, and cultured at 37°C for at least 16 hours to obtain colonies. The obtained colonies were inoculated into LB ampicillin plates and cultured at 37°C for 16 hours. After incubation, the colony culture was collected and centrifuged at 3000 rpm to obtain the supernatant. DNA was extracted from the supernatant using a DNA prep kit (Nuclogen). DNA sequencing confirmed successful cloning.

[0133] Example 3-2: Production of human antibody protein The cloned heavy and light chain vector DNAs were co-transfected into HEK293F cells at a ratio of 6:4 (light chain: heavy chain). Polyethylenimine (PEI) and vector DNA were mixed to form a polyplex reaction mixture, which was then transfected into cells. The culture medium was harvested on day 7 post-transfection, and the supernatant protein was purified using recombinant protein A agarose resin.

[0134] Example 4: Primary Screening Resistin Human Monoclonal Antibody Characteristics Example 4-1 Analysis of antigen receptor binding inhibitory activity Antibody candidates were selected based on their inhibitory effect on the binding of CAP1 to resistin using an in vitro pull-down assay. THP-1 cell lysate was mixed with Fc-fused recombinant resistin protein and each antibody, and then precipitated with Fc beads. Bound CAP1 was measured by Western blotting, and the binding of resistin to CAP1 and the inhibitory effect of each antibody were compared.

[0135] Specifically, THP-1 (ATCC TIP-202, Homo sapiens, Human) cells were cultured using the cell line culture method provided by ATCC (American Type Culture Collection), Inc. They were cultured in RPMI-1640 medium (pH 7.4) containing 100 units / ml penicillin and 100 μg / ml streptomycin at 37°C in an incubator maintained at 5% CO2. THP-1 cells were lysed in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail) to extract proteins. Extracted THP-1 proteins (500 μg) were added to mFc-hResistin (0.5 μg) and 100 ng of antibody and incubated overnight at 4°C. mFc beads (20 μl) were added and the mixture was pulled down at 4°C for 3 hours. After washing three times with lysis buffer, the mixture was added to SDS-PAGE sample buffer containing β-mercaptoethanol, boiled at 100°C for 5 minutes, and centrifuged to obtain the supernatant. Western blotting was performed using hCAP1 antibody and mFc-HRP antibody to confirm whether the binding of hCAP1 to mFc-hResistin was inhibited by the VS peptide.

[0136] As a result of analyzing the antigen receptor binding inhibitory ability of the 16 proteins obtained in the primary screening, five proteins (1A2, 2E12, 5A2, 3B5, and 4D12) that showed excellent effects were identified (Figure 4).

[0137] Example 4-2: Analysis of antigen affinity To investigate the affinity between the antibody and the resistin antigen, we used the Octet (Forte bio) system, which can investigate ligand-receptor binding in real time using a biosensor. This system binds the resistin antigen protein to the biosensor, and optically analyzes the degree of antibody binding to the antigen bound to the sensor by flowing the antibody. The level of antibody binding to the antigen is measured with high sensitivity at the optical wavelength level. The affinity for the resistin antigen is determined by the K on (association constants, binding rate) and K dis The dissociation constants (dissociation rate) and equilibrium dissociation constant (KD) for the binding and dissociation reactions were expressed as the dissociation rate.

[0138] As a result, of the five candidate antibodies 1A2, 2E12, 5A2, 3B5, and 4D12 that showed excellent effects in the antigen receptor binding inhibitory assay, four clones except for 2E12 showed antigen affinity, with a KD of 1 nM (Figure 5).

[0139] Example 5: Secondary screening of resistin human antibodies Example 5-1: Biopanning In addition to the primary screening, a secondary screening was performed to obtain candidate antibodies with high affinity for the antigen. Using the RETN-C-Fc or RETN-flag-His antigen prepared in Example 1, an immunosorb tube was coated with the antigen and reacted with human antibody library phages, as in Example 2, and the scFV phages that bound to the antigen were eluted. A total of three rounds of biopanning were performed, and a polyphage pool that bound to the antigen was obtained in each round.

[0140] Example 5-2: Analysis of antigen-specific binding ability of scFv phage antibodies The scFv polyphage pool obtained from biopanning was cloned by monophage ELISA, yielding 84 scFv monoclonals. To analyze their antigen-specific binding, 96-well immunoplates (NUNC, 439454) were coated with 100 ng / well of resistin antigen and nonspecific antigen, and the monoclonal scFv phages were incubated. After incubation with the secondary antibody anti-M13-HRP (Amersham, 27-9421-01), 100 μl of OPD (Sigma, 8787-TAB) solution was added to each well for 10 minutes. The substrate reaction was stopped with 1N H2SO4, and then the intensity was measured at 490 nm using a spectrophotometer (Thermo Fisher Scientific, 51119200). As a result, 11 new clones with novel sequences and antigen-specific binding were identified (Fig. 6).

[0141] Example 6: Tertiary screening of resistin human antibodies Example 6-1: Biopanning In addition to the primary and secondary screenings, a tertiary screening was performed to obtain candidate antibodies with high affinity for the antigen. Using the RETN-C-Fc or RETN-flag-His antigen prepared in Example 1, an immunosorbent tube was coated with the antigen and reacted with human antibody library phages, as in Example 2, and the scFV phages that bound to the antigen were eluted. A total of three rounds of biopanning were performed, and a polyphage pool that bound to the antigen was obtained in each round.

[0142] Example 6-2: Analysis of antigen-specific binding ability of scFv phage antibodies The scFv polyphage pool obtained from biopanning was cloned by monophage ELISA, yielding 88 scFv monoclonals. Their antigen-specific binding ability was analyzed by enzyme-linked immunosorbent assay (ELISA), yielding 10 novel clones with novel sequences and antigen-specific binding (Figures 7a and 7b).

[0143] Example 7: Production of human resistin antibodies for secondary and tertiary screening Example 7-1: Conversion to IgG type A total of 20 monoclonal phage antibodies obtained from the secondary and tertiary screenings were converted from scFv to IgG structures. As in Example 3, the DNA of each clone was ligated into a production vector and then transformed into competent cells (XLI-blue). The transformed cells were cultured in selective medium, and DNA was extracted from the cells and sequenced for identification.

[0144] Example 7-2: Production of human antibody protein The cloned heavy and light chain vector DNAs were co-transfected into HEK293F cells at a ratio of 6:4 (light chain: heavy chain). Polyethylenimine (PEI) and vector DNA were mixed to form a polyplex reaction mixture, which was then transfected into cells. Soytone (BD, USA) was used as an enhancer. The culture medium was harvested on day 7 post-transfection, and the supernatant protein was purified using recombinant protein A agarose resin.

[0145] Example 8: Secondary and tertiary screening characteristics of resistin human monoclonal antibodies Example 8-1: Analysis of antigen receptor binding inhibitory ability Antibody candidates were selected based on their inhibitory effect on the binding of CAP1 to resistin using an in vitro pull-down assay. THP-1 cell lysate was mixed with Fc-fused recombinant resistin protein and each antibody, and then precipitated with Fc beads. Bound CAP1 was measured by Western blotting, and the binding of resistin to CAP1 and the inhibitory effect of each antibody were compared.

[0146] Specifically, THP-1 (ATCC TIP-202, Homo sapiens, Human) cells were cultured using the cell line culture method provided by ATCC (American Type Culture Collection), Inc. They were cultured in RPMI-1640 medium (pH 7.4) containing 100 units / ml penicillin and 100 μg / ml streptomycin at 37°C in an incubator maintained at 5% CO2. THP-1 cells were lysed in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail) to extract proteins. Extracted THP-1 proteins (500 μg) were added to mFc-hResistin (0.5 μg) and 100 ng of antibody and incubated overnight at 4°C. mFc beads (20 μl) were added and the mixture was pulled down at 4°C for 3 hours. After washing three times with lysis buffer, the mixture was added to SDS-PAGE sample buffer containing β-mercaptoethanol, boiled at 100°C for 5 minutes, and centrifuged to obtain the supernatant. Western blotting was performed using hCAP1 antibody and mFc-HRP antibody to confirm whether the binding of hCAP1 to mFc-hResistin by the VS peptide was inhibited. As a result, five species (11G01, 27A04, 32B05, 32E06, and 32G09) that showed excellent effects were identified (Figure 8).

[0147] Example 8-2: Analysis of antigen-specific binding ability Enzyme-linked immunosorbent assay (ELISA) was performed to examine the specific binding of the screened clones to human resistin protein and to confirm the presence of nonspecific binding to other antigenic proteins. The RETN-flag-His antigen prepared in Example 1 and a nonspecific antigen were diluted to 10 nM in DPBS and added at 100 μl per well to a 96-well immunoplate (NUNC, 439454). The plates were then coated at 4°C for 16 hours. The plates were then blocked with 4% skim milk in PBS at 37°C for 1 hour. The antibody proteins were diluted to 1, 10, and 100 nM in 1% skim milk in PBS, and 100 μl of each was added to each well and incubated at 37°C for 2 hours. After washing with PBS-T, the secondary antibody, anti-human Fc-HRP (Pierce® Peroxidase Conjugated Goat Anti-Human IgG FC (Thermo Fisher Scientific, 31413) was diluted 1:10,000 and incubated at 37°C for 1 hour. After washing with PBS-T, 100 μl of TMB (Sigma, T0440) solution was added to each well, and the substrate reaction was carried out for 10 minutes. After 10 minutes, each well was treated with 50 μl of 1N H2SO4 to stop the substrate reaction, and the fluorescence was measured at 490 nm using a spectrophotometer (Thermo Fisher Scientific, 51119200).

[0148] As a result, all five antibodies, 11G01, 27A04, 32B05, 32E06, and 32G09, which showed excellent effects in the antigen receptor binding inhibitory assay, exhibited high specific binding to the RETN-flag-His antigen protein (Fig. 9a).Furthermore, all five antibodies that showed excellent effects in the antigen receptor binding inhibitory assay against five nonspecific antigens did not bind nonspecifically (Fig. 9b).

[0149] Example 8-3: Analysis of antigen affinity To investigate the affinity between the antibody and resistin antigen, a ligand-receptor assay was performed using the Octet (Forte bio) system biosensor. As a result, four of the five candidate antibodies that showed efficacy in the antigen receptor binding inhibitory assay showed antigen affinity at a KD of 1 nM (FIG. 10).

[0150] Example 8-4: Anti-resistin primary antibody screening

[0151] From the sequence analysis results of 40 monoclonal antibodies obtained in the first to third screenings, the CDR regions of VH and VL of each clone were investigated, and the similarity to the germline antibody group was analyzed using the Ig BLAST program on the NCBI webpage http: / / www.ncbi.nlm.nih.gov / igblast / . The results are shown in Table 7 below.

[0152] [Table 7] TIFF0007763948000014.tif134170

[0153] Example 9: Antibody optimization for the resistin antibody RETN-5A2 clone For antibody optimization, the heavy chain of the RETN-5A2 clone was immobilized and diluted to 1x10 mAb from Ybiologics. 6 A light chain (LC) pool with a diversity of 1x10 was used to create a light chain shuffling library with new combinations of existing heavy chains and light chains. The light chain site of the RETN-5A2 gene was removed by restriction enzyme digestion, and then 1x10 6 The diverse light chain pool was ligated to RETN-5A2 DNA, which had been digested with the same restriction enzyme to remove the light chain. The cloned DNA was transformed into competent cells (XLI-blue), and the cells were then pooled to generate a library (Figure 11).

[0154] Example 10: Screening for resistin human antibodies Example 10-1: Biopanning Biopanning was performed to obtain an optimized phage pool using the 5A2 antibody, which binds to the resistin antigen, as the parent antibody. The RETN-C-Fc or RETN-flag-His antigen prepared in Example 1 was coated onto an immunosorbent tube, followed by blocking. The 5A2 antibody-optimized library phages were placed in the antigen-coated immunotube and reacted, after which the antigen-bound scFv phages were eluted. The obtained phages were collected and amplified by reinfecting E. coli.

[0155] Example 10-2: Screening of positive phages and obtaining monoclonal antibody sequences Phage ELISA was performed to identify specific binding scFv pools for the antigen from the scFv phage pools obtained. RETN-flag-His antigen was coated at 100 ng / well on a 96-well immunoplate (NUNC, 439454) and blocked with skim milk. Each scFv phage obtained in each round was applied to each well for reaction, then washed with PBS-T. Anti-M13-HRP (Amersham, 27-9421-01) was added as a secondary antibody at a dilution of 1:2000. After washing with PBS-T, 100 μl of OPD (Sigma, 8787-TAB) solution was added to each well for 10 minutes. After 10 minutes, each well was treated with 50 μl of 1N H2SO4 to stop the substrate reaction, and the activity was measured at 490 nm using a spectrophotometer (Thermo Fisher Scientific, 51119200). As a result, it was confirmed that the antigen binding ability of the phage pool was slightly enriched after biopanning (Figure 12).

[0156] Colonies obtained from the first-round polyphage pool, whose antigen-binding ability was confirmed, were inoculated into 1 ml of 2xYTCM (2% glucose, 5 mM MgCl2) medium and placed in a 96-well deep-well plate (Bionia, 90030) and cultured for 16 hours at 37°C in an incubator. A 10-200 ml of the cultured cell broth was inoculated into 1 ml of 2xYTCM (2% glucose, 5 mM MgCl2) medium to achieve an OD600 of 0.1 and cultured for 2-3 hours at 37°C. After incubation, M1 helper phage was added at an M ratio of 1:20, and the mixture was inoculated into 2xYTCMK (5 mM MgCl2, 1 mM IPTG) and incubated for 16 hours at 30°C.

[0157] Resistin antigen was coated at 100 ng / well on a 96-well immunoplate (NUNC, 439454) and blocked with skim milk. Monoclonal scFv phage cultured for 16 hours was added to each well (100 μl per well) for 2 hours. After washing with PBS-T, the plate was incubated with anti-M13-HRP (Amersham, 27-9421-01) secondary antibody (1:2000). After washing with PBS-T, each well was treated with 100 μl of OPD (Sigma, 8787-TAB) solution for 10 minutes. After 10 minutes, each well was treated with 50 μl of 1N H2SO4 to stop the substrate reaction, and the intensity was measured at 490 nm using a spectrophotometer (Thermo Fisher Scientific, 51119200).

[0158] As a result, a total of 36 antigen-specific clones were obtained, and the sequences of the obtained 36 clones were analyzed to identify 18 clones with unique sequences different from existing sequences (Table 8).

[0159] [Table 8]

[0160] Example 11: Production of resistin human antibody Example 11-1: Conversion to IgG type The 18 selected anti-resistin monoclonal phage antibodies were converted from scFv to IgG. To insert the DNA of each clone into the entire vector, PCR was performed to add restriction enzyme sequences before and after each heavy and light chain. The resulting heavy and light chain DNA and the entire vector were digested with restriction enzymes and then ligated with T4 DNA ligase (Thermofisher Scientific, #EL0011) at room temperature for 10 minutes to insert the heavy and light chain sequences into the vector. The resulting vectors were transformed into competent cells (XLI-Blue) by heat shock at 42°C, spread on LB ampicillin plates, and cultured at 37°C for at least 16 hours to obtain colonies. The colonies were then inoculated onto LB ampicillin plates and cultured at 37°C for 16 hours. After incubation, the colony culture medium was harvested and centrifuged at 3000 rpm to obtain the supernatant. DNA was extracted from the supernatant using a DNA prep kit (Nuclogen).

[0161] Example 11-2: Production of human antibody protein The cloned heavy chain vector and light chain vector DNA were co-transfected into HEK293F cells at a ratio of 6:4 (light chain: heavy chain). Polyethylenimine (PEI) and vector DNA were mixed to form a polyplex reaction mixture, which was then transfected into cells. The culture medium was harvested on day 7 post-transfection, and the supernatant protein was purified using recombinant protein A agarose resin. The concentration of the purified protein was quantified by measuring absorbance at 280 nm using a nano-drop, and the productivity of each clone was evaluated based on this (Table 9). As a result, the productivity was improved by an average of 92% compared to the parent antibody 5A2 clone (FIG. 13).

[0162] [Table 9]

[0163] In addition, protein expression was evaluated by injecting reduced and non-reduced proteins into SDS-PAGE and analyzing the band pattern, thereby analyzing the purity and size of the produced proteins.

[0164] As a result, all 18 screened antibodies showed a size of 150 kDa under non-reducing conditions, and under reducing conditions, two proteins of predicted sizes were detected: a heavy chain of ~50 kDa and a light chain of ~25 kDa (Figure 14).

[0165] Example 12: Characterization of resistin human monoclonal antibodies Example 12-1: Antigen-specific and non-specific binding (including intact human binding) Enzyme-linked immunosorbent assay (ELISA) was performed to confirm the specific binding ability of the resistin-optimized antibody protein to the resistin antigen. The RETN-flag-His antigen prepared in Example 1 and a nonspecific antigen were diluted to 10 nM in DPBS and added at 100 μl per well to a 96-well immunoplate (NUNC, 439454). The plates were then coated at 4°C for 16 hours. The plates were then blocked with 4% skim milk in PBS at 37°C for 1 hour. The antibody proteins were diluted to 1, 10, and 100 nM in 1% skim milk in PBS, and 100 μl of each was added to each well and incubated at 37°C for 2 hours. After washing with PBS-T, the secondary antibody, anti-human Fc-HRP (Pierce® Peroxidase Conjugated Goat Anti-Human IgG FC (Thermo Fisher Scientific, 31413) was diluted 1:10,000 and incubated at 37°C for 1 hour. After washing with PBS-T, 100 μl of TMB (Sigma, T0440) solution was added to each well, and the substrate reaction was carried out for 10 minutes. After 10 minutes, each well was treated with 50 μl of 1N H2SO4 to stop the substrate reaction, and the fluorescence was measured at 490 nm using a spectrophotometer (Thermo Fisher Scientific, 51119200).

[0166] As a result, most clones, except for a few clones (LS_1F09, LS_1G11), showed antigen-binding reactions similar to those of 5A2 (Fig. 15a).Furthermore, none of the 18 optimized antibodies bound nonspecifically to the five nonspecific antigens (Fig. 15b).

[0167] The antigens prepared in Example 1 had artificially added human antibody Fc sequences or histidine sequences to the C-terminus of the antigen for easier purification and analysis. To confirm that the effects of the antibodies on the natural antigen were the same as those observed with the artificial antigen, enzyme-linked immunosorbent assays (ELISAs) were performed using recombinant human resistin (Recombinant Human Resistin, Peprotech, #450-19) with the same natural structure. Thirteen optimized antibodies were evaluated, excluding five antibodies with significantly lower production and resistin antigen binding affinity than the parent antibody 5A2 clone: ​​LS_1E09, LS_2B10, LS_2B12, LS_2E11, and LS_2D09. Recombinant human antigen (Peprotech, #450-19) was diluted to 10 nM in DPBS and added at 100 μl per well to coat the wells at 4°C for 16 hours. Then, the sections were blocked with 4% skim milk diluted in PBS at 37°C for 1 hour. The antibody proteins were diluted to 1, 10, and 100 nM in 1% skim milk in PBS, and 100 μl of each was added to each well and incubated at 37°C for 2 hours. After washing with PBS-T, the secondary antibody, anti-human Fc-HRP (Pierce® Peroxidase Conjugated Goat Anti-Human IgG FC (Thermo Fisher Scientific, 31413) was diluted 1:10,000 and incubated at 37°C for 1 hour. After washing with PBS-T, 100 μl of TMB (Sigma, T0440) solution was added to each well, and the substrate reaction was carried out for 10 minutes. After 10 minutes, each well was treated with 50 μl of 1N H2SO4 to stop the substrate reaction, and the fluorescence was measured at 490 nm using a spectrophotometer (Thermo Fisher Scientific, 51119200).

[0168] As a result, all clones, including the parent antibody 5A2, showed binding activity to intact form resistin that was very similar to that of existing his-tagged resistin, which is expected to enable prediction of the efficacy of candidate clones against the natural antigen (Fig. 15c).

[0169] Example 12-2: Interspecies cross-linking reaction against mouse antigens Enzyme-linked immunosorbent assays (ELISAs) were performed to examine the interspecies cross-binding activity of eight optimized antibodies against mouse resistin antigen, excluding five antibodies with significantly lower production and binding affinity to the resistin antigen than the parent antibody clone 5A2: LS_1E09, LS_2B10, LS_2B12, LS_2E11, and LS_2D09. The mouse antigen used was murine resistin (#450-28) from Peprotech. Mouse antigen was diluted to 10 nM in DPBS and added at 100 μl per well to a 96-well immunoplate (NUNC, 439454). The plate was then coated at 4°C for 16 hours. The plate was then blocked with 4% skim milk in PBS at 37°C for 1 hour. The antibody proteins were diluted to 1, 10, or 100 nM in 1% skim milk in PBS, and 100 μl of each was added to each well and incubated at 37°C for 2 hours. After washing with PBS-T, anti-human Fc-HRP (Pierce® Peroxidase Conjugated Goat Anti-Human IgG FC (Thermo Fisher Scientific, 31413) was added as a secondary antibody at a 1:10,000 dilution and incubated at 37°C for 1 hour. After washing with PBS-T, 100 μl of TMB (Sigma, T0440) solution was added to each well, and the substrate reaction was carried out for 10 minutes. After 10 minutes, each well was treated with 50 μl of 1N H2SO4 to stop the substrate reaction, and the fluorescence was measured at 490 nm using a spectrophotometer (Thermo Fisher Scientific, 51119200).

[0170] In contrast to their binding ability to the RETN-flag-His antigen, the parent antibody 5A2 clone and the eight optimized antibodies did not bind to the mouse antigen, confirming the absence of interspecies cross-reactivity with mouse resistin (Figure 16).

[0171] Example 12-3 Analysis of antigen receptor binding inhibitory activity using antigen receptor competitive enzyme immunoassay (resistin-CAP1 competitive ELISA) To evaluate the antibody's ability to bind to resistin and inhibit antigen receptor binding, we performed an antigen-receptor competitive enzyme-linked immunosorbent assay (ELISA). A 96-well plate was coated with antigen protein, treated with various concentrations of antibody, and then treated with the receptor to detect the receptor protein bound to the antigen. A schematic diagram of the assay is shown in Figure 17a. The coated antigen protein was the RETN-C-Fc protein prepared in Example 1, and the receptor protein was purchased from LSbio, containing a histidine-linked CAP1-His (LS-G13682-100).

[0172] RETN-C-Fc protein was diluted to 100 nM in DPBS and added at 100 μl per well to a 96-well immunoplate (NUNC, 439454) for 16 hours at room temperature. It was then blocked with 4% skim milk in PBS at 37°C for 1 hour. Antibody proteins were serially diluted 5-fold from a maximum concentration of 500 nM to 0.0064 nM and added at 100 μl per well for 2 hours at room temperature. After washing with PBS-T, CAP1-His was diluted to 100 nM in 1% skim milk in PBS and added at 100 μl per well for 2 hours at room temperature. After washing with PBS-T, anti-histidine-biotin (Invitrogen, MA1-21315-BTIN) was diluted 1:2000 in 1% skim milk and added at 100 μl per well. The reaction was allowed to proceed at room temperature for 1 hour. Streptavidin-HRP (Thermo Fisher Scientific, 21140) was then added at 1:1000 and allowed to react for 1 hour. After washing with PBS-T, 100 μl of TMB (Sigma, T0440) solution was added to each well for 10 minutes. After 10 minutes, the reaction was stopped by adding 50 μl of 1N H2SO4 to each well, and the activity was measured at 490 nm using a spectrophotometer (Thermo Fisher Scientific, 51119200).

[0173] As a result, in Examples 11 and 12, among the clones with productivity and antigen binding ability superior to that of the parent antibody 5A2, seven top clones (5A2_LS_1F11, 5A2_LS_2B02, 5A2_LS_2D07, 5A2_LS_2F11, 5A2_LS_2G03, 5A2_LS_2E10, and 5A2_LS_2H10) with superior ability to inhibit antigen receptor binding to 5A2 were selected (Figure 17b).

[0174] Example 12-4: Analysis of antigen receptor binding inhibitory ability Antibody candidates were selected based on their inhibitory effect on the binding of CAP1 to resistin using an in vitro pull-down assay. THP-1 cell lysate was mixed with Fc-fused recombinant resistin protein and each antibody, and then precipitated with Fc beads. Bound CAP1 was measured by Western blotting, and the binding of resistin to CAP1 and the inhibitory effect of each antibody were compared.

[0175] Specifically, THP-1 (ATCC TIP-202, Homo sapiens, Human) cells were cultured using the cell line culture method provided by ATCC (American Type Culture Collection), Inc. They were cultured in RPMI-1640 medium (pH 7.4) containing 100 units / ml penicillin and 100 μg / ml streptomycin at 37°C in an incubator maintained at 5% CO2. THP-1 cells were lysed in lysis buffer (20 mM Tris pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM NaF, 1 mM Na3VO4, and protease inhibitor cocktail) to extract proteins. Extracted THP-1 protein (500 μg) was added to mFc-hResistin (0.5 μg) and 100 ng of antibody and incubated overnight at 4°C. mFc beads (20 μl) were added and the mixture was pulled down at 4°C for 3 hours. After washing three times with lysis buffer, the mixture was added to SDS-PAGE sample buffer containing β-mercaptoethanol, boiled at 100°C for 5 minutes, and centrifuged to obtain the supernatant. Western blotting was performed using hCAP1 antibody and mFc-HRP antibody to confirm whether the binding of hCAP1 to mFc-hResistin was inhibited by the VS peptide. As a result, an antibody was identified that showed a superior effect to the parent antibody 5A2 (FIG. 18).

[0176] Example 12-5: Investigation of affinity between antibody and resistin antigen using Octet (Forte bio) system To investigate the affinity between the antibody and the resistin antigen, we used the Octet (Forte bio) system, which can investigate ligand-receptor binding in real time using a biosensor. This system binds the resistin antigen protein to the biosensor, and optically analyzes the degree of antibody binding to the antigen bound to the sensor by flowing the antibody. The level of antibody binding to the antigen is measured with high sensitivity at the optical wavelength level. The affinity for the resistin antigen is determined by the K on (association constants, binding rate) and K dis The dissociation constants (dissociation rate) and equilibrium dissociation constant (KD) for the binding and dissociation reactions were expressed as the dissociation rate.

[0177] As a result, the seven antibodies that showed superior effects to the parent antibody in the antigen receptor competitive enzyme immunoassay showed antigen affinity of KD 1 nM or less, which is equal to or higher than that of the parent antibody 5A2 (Figure 19).

[0178] Example 13: Analysis of the anti-cancer effect of resistin antibodies Example 13-1: NF-κB activation by resistin To confirm the effect of the antibody on NF-κB activity, we activated NF-κB in MB231 cells with resistin, and then measured the NF-κB activity by measuring p65 phosphorylation when 5A2 and IgG were added (Fig. 20).

[0179] Specifically, the MDA-MB-231 (ATCC HTB-26, Homo sapiens, Human) 293F cell line was cultured in DMEM medium (pH 7.4) containing 100 units / ml penicillin and 100 μg / ml streptomycin, using the cell line culture method of the American Type Culture Collection (ATCC), the distributor, in an incubator maintained at 37°C and 5% CO2 partial pressure. 5.0 x 10 cells per well were cultured. 5MB-231 cells were seeded into four 6-well plates and treated with 100 ng / ml antiresistin 5A2 (Y-BIOLOGICS / ANRT) in 1% FBS-containing DMEM (Invitrogen Life Technologies) medium for 6 h, followed by sequential treatment with 50 ng / ml recombinant human resistin (Y-BIOLOGICS / ANRT, Korea) for 10, 30, and 60 min.

[0180] For Western blotting analysis, equal amounts of cell lysates were collected and lysed in lysis buffer containing protease inhibitors (Roche, Cat. 11836153001). Total protein (10–30 μg) was immunoblotted with specific primary antibodies: Phospho-Src (Tyr416) ​​(Cell Signaling Technology, #6943), p-p65 (Ser276) (Cell Signaling Technology, #3037), total p65 (Santa Cruz Biotechnology, sc-372), p-CREB (Ser133) (Santa Cruz Biotechnology, sc-101663), total CREB (Cell Signaling Technology, #9197), and α-Tubulin (Calbiochem, Cat. CP06). Anti-mouse IgG HRP and anti-rabbit IgG HRP were purchased from Promega as secondary antibodies, and detection was performed with ECL and ECL-PLUS (Amersham).

[0181] Example 13-2: Analysis of cancer cell migration in vitro To confirm the effect of antibodies on resistin-induced cancer cell migration, we treated MB231 breast cancer cells with 5A2 antibody and IgG and performed cell migration experiments.

[0182] 5x10 5MDA-MB-231 breast cancer cells were cultured in 6-well plates. At 90% confluency, the cells were starvated for 24 hours in RPMI 1640 medium supplemented with 1% serum. A scratch was then induced using a 200-p tip, followed by the addition of 100 ng / ml recombinant resistin, 100 ng / ml human normal IgG, and 100 ng / ml 5A2 antibody. After 6 hours, migrated cells were observed using a Leica microscope (DMI 3000 B) and quantified using Image J software based on the area of ​​cell migration.

[0183] As a result, 100ng / ml resistin increased the migration of MDA-MB-231 breast cancer cells by approximately 137%. Human normal IgG, used as a negative control, induced breast cancer cell migration at a level similar to that of the resistin-only treatment group, but when treated with the anti-resistin antibody 5A2, cell migration was inhibited by 55% compared to the resistin-only treatment group and by 76% compared to the vehicle treatment group (Figure 22).

[0184] Example 13-3: In vivo metastasis of cancer cells To determine pharmacokinetics, a pharmacokinetic experiment was performed in mice administered the 5A2 antibody and a hIgG control group (FIG. 21).

[0185] To confirm the in vivo metastasis suppression ability of the 5A2 antibody against breast cancer cells, we first induced hyper-resistinemia in NOD / SCID mice using adenovirus. We confirmed through luminescence imaging and tissue extraction that hyper-resistinemia promotes metastasis of breast cancer cells to the liver and lungs. Specifically, 1x10 7 pfu of Adv.GFP and Adv.hResistin were intraperitoneally injected (IP) into NOD / SCID mice, and 1 week later, 6x10 luciferase-overexpressing MDA-MB-231 cells were transfected.5 The tumor cells were intravenously injected (IV) with 10 mg / kg human normal IgG and 10 mg / kg 5A2 antibody, which were then administered continuously three times a week. At the fourth week, 150 mg / kg Luciferin was administered to each experimental group, and luminescence images were obtained. Images were captured using a KODAK FX-PRO instrument.

[0186] Administration of 10 mg / kg of the 5A2 antibody for 4 weeks (3 times a week) significantly reduced metastasis to the relevant organ. Furthermore, when the tissues were excised and the number of metastatic colons visible to the naked eye was compared (indicated by arrows in Figure 23), a significant reduction in the number of metastatic colons was observed in the 5A2 antibody-treated group, consistent with the results of the luminescence imaging (Figure 23).

[0187] Example 14: Analysis of the effect of resistin antibody on NASH (non-alcoholic steatohepatitis) Example 14-1: Confirmation of the effect of resistin antibody on improving high-fat diet-induced NASH To confirm whether the antibody is effective in ameliorating high-fat diet-induced NASH, fatty liver was induced with a high-fat diet for 2 months, followed by a one-month high-fat diet, and 5A2 was administered intravenously (IV) (Figure 24).

[0188] Mouse liver tissue was fixed in 4% PFA for 5 days, then paraffin-blocked and stained with H&E. The results were measured using the Nonalcoholic Fatty Liver Disease Activity Score (NAS). The NAS score was significantly reduced in the 5A2 antibody-treated group (Figure 25).

[0189] Mouse liver tissue was prepared as an OCT block and stained with Oil-Red-O. As a result, it was confirmed that neutral triglycerides and lipids were significantly reduced in the 5A2 antibody administration group (FIG. 26). Example 14-2: Confirmation of the insulin sensitivity improving effect of resistin antibody

[0190] Four weeks after 5A2 administration, an insulin tolerance test was performed. Mice were kept fasting for 12 hours prior to the test, with water provided. Before insulin administration, the tip of the mouse's tail was clipped, and fasting blood glucose levels were measured using a Roche Accu-Chek blood glucose meter. Then, 0.75 IU of insulin was administered intraperitoneally (IP), and blood glucose levels were measured 15, 30, 60, and 120 minutes later.

[0191] The results showed that the 5A2 antibody-administered group had greater insulin sensitivity. In particular, the fasting blood glucose level reduction rate in the 5A2 antibody-administered group 30 minutes after insulin administration was 56%, which was more than 20% lower than that in the control group (Figure 27).

[0192] Example 14-3: Confirmation of the fasting glucose level lowering effect of resistin antibody Four weeks after 5A2 administration, fasting blood glucose tests were performed. Mice were kept fasting for 12 hours, but water was provided. Blood samples were obtained by cutting the tip of the mouse's tail with scissors and used with a Roche AccuTec blood glucose meter.

[0193] As a result, it was found that fasting blood glucose level in the humanized resistin mice overexpressing human resistin was 105.2 mg / dL, which was even lower than that of the control group (125.5 mg / dL) (Figure 28).

[0194] Example 14-4: Confirmation of the LDL cholesterol lowering effect of resistin antibody Blood was collected from mice and separated into serum using a capillary blood collection tube. An equal volume of serum was mixed with the precipitation buffer provided in the cholesterol assay kit (ab65390) and spun down at 13,000 rpm to separate the supernatant (high-density lipoprotein) and pellet (low-density lipoprotein). The pellet was dissolved in PBS to isolate LDL. Results of two independent experiments showed that LDL was significantly reduced in the 5A2 antibody-treated group compared to the control group (Figure 29).

[0195] Example 14-5: Confirmation of the triglyceride accumulation-reducing effect of resistin antibody 50 mg of mouse liver was washed several times with cold PBS and then homogenized in 1 ml of 5% NP-40 / ddH2O. Triglycerides were then dissolved by heating to 100°C. Triglycerides were measured using a triglyceride assay kit (ab65336), and the 5A2 antibody-treated group showed a reduction of more than 30% compared to the control group (Figure 30).

[0196] Example 15: Analysis of the effect of resistin antibody on IBD (inflammatory bowel disease) To confirm whether the antibody was effective in ameliorating acute intestinal disease, mice were given 5% dextran sodium sulfate (DSS) in their drinking water for 5 days to induce intestinal disease, and saline, Remsima (1 mg / kg), and RETN-5A2_LS_2F11 antibody (10 mg / kg) were administered intravenously (IV) on days 1 to 3 (Figure 31).

[0197] The body weight of the mice was measured daily from the start of DSS administration, and weight loss began on day 3. Both Remsima and RETN-5A2_LS_2F11 caused less weight loss than the saline group, with RETN-5A2_LS_2F11 being approximately 3.6% more effective than Remsima (Figure 32).

[0198] The Disease Activity Score (DAI) was calculated by observing the amount of weight loss (%), stool condition, and bloody stool, and then calculating a score from 0 to 4. The DAI was measured daily from the start of DSS administration, and began to increase from the third day. Both Remsima and RETN-5A2_LS_2F11 showed a smaller increase in DAI than the saline group, with RETN-5A2_LS_2F11 being approximately 1.4 times more effective than Remsima (Figure 33).

[0199] The length of the mouse colon was measured from the cecum to the anus. In the normal group, the intestinal length was 7.1 cm, which was reduced to 4.9 cm by DSS. Both Remsima and RETN-5A2_LS_2F11 reduced the intestinal length of the mice less than the saline group, and RETN-5A2_LS_2F11 had a greater protective effect on intestinal length than Remsima, approximately 0.5 cm (Figure 34).

[0200] Mouse colon tissue was fixed in 4% PFA for 5 days, then paraffin-blocked and stained with H&E. The results were evaluated using a histological activity score. Both Remsima and RETN-5A2_LS_2F11 showed a smaller increase in histological activity score compared to the saline group, with RETN-5A2_LS_2F11 being approximately 0.8 times more effective than Remsima (Figure 35). [Industrial Applicability]

[0201] The novel antibody or antigen-binding fragment thereof that binds to resistin of the present invention can bind to resistin and inhibit its activity by blocking resistin / CAP1 binding, and is therefore useful for developing treatments for various resistin-related diseases.

[0202] Although certain parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the true scope of the present invention is to be defined by the appended claims and their equivalents.

Claims

1. a heavy chain variable region comprising a CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, a CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, and a CDR3 comprising the amino acid sequence set forth in SEQ ID NO:3; and a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 4, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 20; a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO:5, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:13, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:21; a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 14, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 22; a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO:7, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:15, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:23; a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 8, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 16, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 24; a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO:9, CDR2 comprising the amino acid sequence set forth in SEQ ID NO:17, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO:25; a light chain variable region comprising CDR1 comprising the amino acid sequence represented by SEQ ID NO: 10, CDR2 comprising the amino acid sequence represented by SEQ ID NO: 18, and CDR3 comprising the amino acid sequence represented by SEQ ID NO: 26; or a light chain variable region comprising CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 19, and CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 27; An antibody or antigen-binding fragment thereof that specifically binds to resistin, comprising:

2. The antibody or antigen-binding fragment thereof that specifically binds to resistin according to claim 1, characterized in that the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO:

28.

3. The antibody or antigen-binding fragment thereof that specifically binds to resistin according to claim 2, characterized in that the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44 and 46.

4. A heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 28; and An antibody or antigen-binding fragment thereof that specifically binds to resistin according to claim 1, characterized in that it comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 32, 34, 36, 38, 40, 42, 44 and 46.

5. A nucleic acid encoding the antibody or antigen-binding fragment thereof of claim 1.

6. The nucleic acid described in claim 5, characterized in that the nucleic acid encoding the heavy chain variable region comprises the base sequence shown in SEQ ID NO:

30.

7. The nucleic acid described in claim 5, characterized in that the nucleic acid encoding the light chain variable region comprises a base sequence selected from the group consisting of SEQ ID NOs: 48, 50, 52, 54, 56, 58, 60 and 62.

8. An expression vector comprising a nucleic acid encoding the antibody or antigen-binding fragment thereof described in claim 5.

9. A cell transformed with the expression vector of claim 8.

10. 10. The cell according to claim 9, characterized in that it is selected from the group consisting of animal cells, plant cells, yeast, E. coli and insect cells.

11. A method for producing an antibody or antigen-binding fragment thereof that specifically binds to resistin, comprising the steps of: (a) culturing the cells of claim 9; and (b) recovering the antibody or antigen-binding fragment thereof from the cultured cells.

12. An antibody-drug conjugate (ADC) in which a drug is bound to the antibody or antigen-binding fragment thereof described in claim 1.

13. The antibody-drug conjugate of claim 12, wherein the antibody or antigen-binding fragment thereof is conjugated to the drug via a linker.

14. The antibody-drug conjugate of claim 13, wherein the linker is a cleavable linker or a non-cleavable linker.

15. 13. The antibody-drug conjugate of claim 12, wherein the drug is a chemotherapeutic agent, a toxin, a microRNA (miRNA), an siRNA, an shRNA, or a radioisotope.

16. A bispecific or multi-specific antibody comprising the antibody or antigen-binding fragment thereof described in claim 1.

17. A chimeric antigen receptor (CAR) comprising the antibody or antigen-binding fragment thereof described in claim 1.

18. An immune cell comprising the chimeric antigen receptor of claim 17.

19. The immune cell according to claim 18, characterized in that the immune cell is a T cell or an NK cell.

20. A pharmaceutical composition for preventing or treating a disease treatable by inhibiting the activity of resistin, comprising the antibody or antigen-binding fragment thereof described in claim 1, the antibody-drug conjugate described in claim 12, the bispecific or multispecific antibody described in claim 16, or the chimeric antigen receptor described in claim 17.

21. The pharmaceutical composition of claim 20, wherein the activity of resistin is inhibited by blocking resistin / CAP1 binding with a resistin antibody.

22. The pharmaceutical composition according to claim 20, wherein the disease treatable by inhibiting the activity of resistin is cancer, a cardiometabolic disease, an autoimmune disease, or an inflammatory disease.

23. 23. The pharmaceutical composition of claim 22, wherein the cancer is selected from the group consisting of breast cancer, metastatic cancer, uterine cancer, ovarian cancer, prostate cancer, melanoma, lung cancer, liver cancer, glioma, colorectal cancer, head and neck cancer, bladder cancer, renal cell carcinoma, gastric cancer, pancreatic cancer, and recurrent cancer.

24. 23. The pharmaceutical composition of claim 22, wherein the cardiometabolic disease is selected from the group consisting of obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, insulin-resistant diabetes, type 2 diabetes, liver disease, non-alcoholic fatty liver disease, and non-alcoholic steatohepatitis.

25. 23. The pharmaceutical composition according to claim 22, wherein the autoimmune disease is selected from the group consisting of chronic heart disease, rheumatoid arthritis, generalized erythematous cyst, digestive system diabetes, atopic dermatitis, autoimmune encephalomyelitis, asthma, and Crohn's disease.

26. 23. The pharmaceutical composition according to claim 22, wherein the inflammatory disease is selected from the group consisting of inflammatory skin diseases including asthma, eczema, psoriasis, acne, allergies, rheumatoid arthritis, psoriatic arthritis, atopic dermatitis, atopic rhinitis, allergic dermatitis, chronic sinusitis or seborrheic dermatitis; inflammatory bowel diseases including Crohn's disease or ulcerative colitis, ankylosing spondylitis, sepsis, septic shock, vasculitis and bursitis, and acute or chronic inflammatory diseases.

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