Anti-β2-microglobulin antibody and uses thereof

Abstract

The present invention relates to an antibody that specifically binds to β2-microglobulin (β2m) and to uses thereof, in which the antibody specifically binds to free β2m protein that has dissociated from MHCI. The antibody or an antigen-binding fragment thereof binds to both the monomer and amyloid forms of β2m, and can detect the monomer and amyloid forms of free β2m in body fluids of dementia and kidney-disease animal models, and thus is useful as an antibody for diagnosing these diseases. In addition, the antibody or an antigen-binding fragment thereof protects damage to cognitive functions induced by β2m in normal mice, and exhibits: reduced amyloid beta and inflammation in the brain after intraperitoneal administration to a dementia mouse model; reduced renal fibrosis and biomarkers in a renal fibrotic disease animal model; and reduced liver fibrosis in a liver fibrotic disease animal model, and thus is useful for preventing and / or treating these diseases.

Description

Anti-beta2-microglobulin antibodies and uses thereof

[0001] The present invention relates to an antibody that specifically binds to β2-microglobulin (β2m) and its use, and more particularly, to an anti-β2m antibody or an antigen-binding fragment thereof, a nucleic acid encoding the antibody or an antigen-binding fragment thereof, a vector and a cell comprising the nucleic acid, a method for producing an anti-β2m antibody or an antigen-binding fragment thereof using the same, an antibody-drug conjugate comprising the antibody or an antigen-binding fragment thereof, a bispecific or multispecific antibody, a chimeric antigen receptor, an immune cell into which the chimeric antigen receptor has been introduced, and a diagnostic composition and a preventive / therapeutic pharmaceutical composition comprising the same for neurodegenerative diseases.

[0002]

[0003] Beta-2 microglobulin (β2m) is a protein that primarily forms the MHC class I (MHCI) complex on the cell membrane and plays a crucial role in the immune system, including presenting antigenic peptides to cytotoxic T cells. MHCI is composed of a heterodimer formed by noncovalent association between the MHCI α chain (α1, α2, and α3 domains) and the β2m protein. Under certain pathological conditions, β2m can dissociate from the MHCI α chain, and β2m levels are known to increase in the blood and urine of patients with various diseases, including aging, Alzheimer's disease, chronic kidney disease, dialysis-related amyloidosis, multiple myeloma, prostate cancer, and pulmonary fibrosis. Thus, it is highly likely that the β2m present in body fluids is free β2m that has dissociated from MHCI, and free β2m is considered to be highly related to various diseases (Zhen-Yuan Liu, et al., Cell Mol Neurobiol. 2024 May 14;44:46).

[0004] In 2011, Tony Wyss-Coray's group observed that the amount of doublecortin (DCX), a neurodevelopment marker protein, decreased in the brains of young and aged mice through parabiosis, allowing blood exchange between the two. This resulted in a decrease in the amount of DCX in the brains of young mice, whereas an increase in the amount of DCX was observed in the brains of aged mice. They hypothesized that this phenomenon was due to factors in the blood of aged mice, and reported that several aging factors, including β2m, were detected in the blood through proteomic analysis (Saul A. Villeda, et al., Nature. 2011 Aug 31;477(7362):90-4). In a subsequent study by the same group, they observed that the amount of β2m increased in the blood and brain tissue of mice, as well as in human blood and cerebrospinal fluid, with aging, and that injecting β2m into the mouse brain and blood vessels resulted in a decline in cognitive function. Accordingly, β2m was suggested as a major factor in aging phenomena such as cognitive function and neurogenesis decline (Lucas K Smith, et al., Nat Med. 2015 Aug;21(8):932-7).

[0005] Various research groups have reported increases in β2m in the cerebrospinal fluid and blood of patients with Alzheimer's disease and other neurodegenerative diseases, as well as in aging. β2m has been suggested as a potential biomarker through blood analysis of healthy individuals and patients with Alzheimer's disease, and has been found to correlate with the amount of amyloid beta (Aβ). Furthermore, increases in β2m have been observed in the blood and urine of patients with brain injury, suggesting its potential use as an indicator for assessing patients' consciousness and cognitive function or for early diagnosis. Furthermore, it has recently been reported that β2m aggregates with Aβ in an animal model of Alzheimer's disease, contributing to neurotoxicity such as cognitive dysfunction, and that it antagonizes NMDA receptors in an animal model of Down's syndrome, contributing to synaptic damage (Yue Gao, et al., Cell. 2023 Mar 2;186(5):1026-1038.e20).

[0006] Meanwhile, blood β2m levels increase in patients with chronic kidney disease undergoing long-term hemodialysis, raising the possibility that β2m may also be involved in cognitive decline in these patients. A recent report suggested damage to the blood-brain barrier as one of the causes of brain damage in patients with chronic kidney disease (Yi Li, et al., J Nephrol. 2020; 33(4): 839-848).

[0007] In 2017, the Rist PM research team analyzed American women and found that blood β2m levels were higher in patients with cerebral infarction than in normal subjects, and when evaluated by groups classified by β2m levels, the risk of cerebral infarction was 56% higher in the highest group than in the lowest group. This elevation of blood β2m was related to systemic inflammation and explained that it has value as a marker for assessing the risk of cerebral infarction in women (Pamela M Rist, et al., Neurology. 2017 Jun 6;88(23):2176-2182). Therefore, research to investigate the form of increased β2m in brain diseases including dementia and to understand its impact on the pathophysiology and its mechanism is very important.

[0008] The association between β2m and inflammatory responses has long been known in various diseases, but the detailed mechanisms were unknown. However, a recent report by Heiko Bruns' group showed that β2m phagocytosed by macrophages in an animal model of multiple myeloma accelerated disease progression by inducing lysosomal damage and NLRP3 inflammasome activation, and that antibody inhibition of β2m could control inflammasome activity (Daniel Hofbauer, et al., Immunity. 2021 Aug 10;54(8):1772-1787.e9).

[0009] Another group's study suggested that β2m fibrosis caused by inflammasome activation is the cause of β2m deposition and inflammation in the joints in dialysis amyloidosis, and it was observed that this amyloid form of β2m promotes inflammation by interacting with inflammasomes (Naoe Kaneko, et al., Int J Immunopathol Pharmacol. 2022 Jan-Dec:36:3946320221104554). However, a recent study confirmed that removing β2m during the dialysis process was not sufficient to improve cognitive decline, which may be because high molecular weight β2m that may exist in the blood is not removed during the dialysis process and still remains. Another report showed that the mechanisms of action of different forms of β2m in dialysis amyloidosis are different, and the level of β2m in the blood does not always correspond to the risk of amyloidosis, so analysis of the form of β2m present in the blood and definition of high risk are necessary for the treatment of β2m amyloidosis and others (Ignacio Portales-Castillo, et al., Kidney360. 2020 Oct 21;1(12):1447-1455).

[0010] Therefore, there is a need to investigate the conformational changes of β2m in human and animal samples of various diseases and analyze its role as a pathogenic factor, and inhibiting β2m with antibodies, etc. may have therapeutic value. However, since β2m is a protein expressed in a form bound to MHCI in most nucleated cells, targeting β2m raises concerns about serious potential side effects by suppressing immune functions such as antigen presentation by MHCI. Therefore, the need for the development of an antibody that specifically recognizes only free β2m released from MHCI has arisen, and if only free β2m can be detected as a risk factor by specifically recognizing only free β2m in body fluids, it can be expected to be utilized not only in treatment but also in diagnostic fields.

[0011]

[0012] Against this backdrop, the present inventors produced monoclonal antibodies by immunization using several peptides containing β2m sequences that bind to the MHCI α chain, and selected clones that were highly reactive specifically to free β2m. The selected clones were antibodies that targeted sequences outside the amyloid core of β2m as epitopes, and were confirmed to be able to bind to both monomeric and amyloid forms of β2m. Using the free β2m-specific antibody clone, we aimed to clearly understand the form, role, and mechanism of free β2m, which acts as a pathological factor not only in humans but also in animals (cats, dogs, etc.), and to develop a therapeutic antibody that specifically recognizes only free β2m and prevents secondary toxic effects without interfering with the immune action of MHCI. In addition, the diagnostic validity of the discovered antibody substances was evaluated so that they can be used for diagnostic purposes, such as development of companion diagnostic biomarkers, in addition to therapeutic purposes, thereby expanding the scope of application of the value of free β2m-specific antibodies, and thus the present invention was completed.

[0013]

[0014] The above information described in this background section is solely intended to enhance understanding of the background of the present invention and may not include information that constitutes prior art already known to a person of ordinary skill in the art to which the present invention pertains.

[0015]

[0016] Summary of the invention

[0017] An object of the present invention is to provide a novel antibody or antigen-binding fragment thereof targeting beta2-microglobulin (β2m), particularly free beta2-microglobulin.

[0018] Another object of the present invention is to provide a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[0019] Another object of the present invention is to provide a recombinant expression 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.

[0020] Another object of the present invention is to provide an antibody-drug conjugate or a bispecific or multispecific antibody comprising the antibody or an antigen-binding fragment thereof.

[0021] Another object of the present invention is to provide a chimeric antigen receptor comprising the antibody or an antigen-binding fragment thereof, and an immune cell into which the chimeric antigen receptor has been introduced.

[0022] Another object of the present invention is to provide a composition and a method for diagnosing a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), kidney disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer, or inflammatory disease, comprising the antibody or an antigen-binding fragment thereof.

[0023] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), renal diseases, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer or inflammatory diseases, or a method for the prevention or treatment thereof, comprising the antibody or an antigen-binding fragment thereof, the antibody-drug conjugate, the bispecific or multispecific antibody, the chimeric antigen receptor, or the immune cell.

[0024] Another object of the present invention is to provide an isolated peptide comprising an epitope of beta2-microglobulin (β2m).

[0025]

[0026] To achieve the above object, the present invention provides an anti-beta2-microglobulin antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 17; a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 18, and SEQ ID NO: 22; and a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 19, and SEQ ID NO: 23; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 6; a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 7; and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 8.

[0027] The present invention also provides a nucleic acid encoding the antibody or an antigen-binding fragment thereof.

[0028] The present invention also provides a recombinant expression vector comprising the nucleic acid.

[0029] The present invention also provides a cell transformed with the recombinant expression vector.

[0030] The present invention also provides a method for producing an anti-beta2-microglobulin antibody or an antigen-binding fragment thereof, comprising a step of culturing the cells.

[0031] The present invention also provides an antibody-drug conjugate or a bispecific or multispecific antibody comprising the antibody or an antigen-binding fragment thereof.

[0032] The present invention also provides a chimeric antigen receptor comprising the antibody or an antigen-binding fragment thereof or an immune cell into which the chimeric antigen receptor has been introduced.

[0033] The present invention also provides a composition and a method for diagnosing a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome, renal disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer, or inflammatory disease, comprising the antibody or an antigen-binding fragment thereof.

[0034] The present invention also provides a pharmaceutical composition for preventing or treating neurodegenerative diseases, aging, Down syndrome, acquired immunodeficiency syndrome, renal diseases, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer, or inflammatory diseases, comprising the antibody or antigen-binding fragment thereof, antibody-drug conjugate, bispecific or multispecific antibody, chimeric antigen receptor, or immune cell.

[0035] The present invention also provides a method for preventing or treating a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome, kidney disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer, or inflammatory disease, comprising administering the antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, a bi- or multispecific antibody, a chimeric antigen receptor, or an immune cell; a use of the antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, a bi- or multispecific antibody, a chimeric antigen receptor, or an immune cell for preventing or treating a neurodegenerative disease, etc.; and a use of the antibody or an antigen-binding fragment thereof, an antibody-drug conjugate, a bi- or multispecific antibody, a chimeric antigen receptor, or an immune cell for the manufacture of a medicament for preventing or treating a neurodegenerative disease, etc.

[0036] The present invention also provides an isolated peptide comprising an epitope of beta2-microglobulin (β2m) comprising the amino acid sequence of SEQ ID NO: 59.

[0037]

[0038] Figure 1 is a schematic diagram of antigen binding of an anti-free β2m-specific antibody (ADEL-Y03) showing that it specifically recognizes an epitope that is not exposed when β2m is in a complex state with MHCI, but is exposed only when it is dissociated from the complex.

[0039] Figure 2 shows the results of dot blot analysis of an anti-free β2m-specific antibody clone that specifically binds only to the dissociated β2m after heat treatment of the MHCI native complex into the MHCI heavy chain and β2m. (B) IP of the MHCI native complex with anti-free β2m-specific antibody clones and BBM.1, followed by western blot analysis using anti-MHCI or anti-β2m antibodies, and the results of confirming the anti-free β2m-specific antibody clone that does not precipitate the protein. (C) To confirm whether the anti-free β2m-specific antibody clone binds to β2m present in the MHCI complex on the cell surface, the surface of HeLa cells was stained with each antibody and then checked by flow cytometry. Unlike the BBM.1 antibody, the anti-free β2m-specific antibody clones showed almost no reactivity on the cell surface. In the above experiment, among the antibody clones that specifically bind only to free β2m, an excellent antibody clone was selected through comparison of biological sample reactivity, etc., and named 'Y03'. (D, E) The reactivity of Y03 to the β2m recombinant protein and the MHCI native complex was reconfirmed through antigen-coated indirect ELISA. (F) The binding affinity of each antibody (IgG, Y03, BBBM.1) to the MHCI native complex was measured through BLI.

[0040] Figure 3 is a graph showing the results of ELISA confirming that (A) Y03 shows a concentration-dependent reactivity to amyloid form of β2m. (B) Western blot results showing that the anti-free β2m specific antibody clone binds to free β2m in monomeric and amyloid forms in the blood of patients with mild cognitive impairment and Alzheimer's disease, and that free β2m in monomeric and amyloid forms may be increased in the blood of patients with Alzheimer's disease compared to those with mild cognitive impairment.

[0041] Figure 4 shows the results of a Novel Objective Recognition (NOR) behavioral experiment demonstrating that free β2m impairs memory and cognitive function in the brain, while Y03 protects them. (A) Schematic diagram of the animal experiment, and (B, C, D) show the results of movement distance, habituation, and training in the NOR behavioral experiment. Injection of β2m into the brain decreased the exploration time for a novel object and the number of times the animals entered the area containing the novel object, whereas co-injection of Y03 resulted in recovery (*P<0.05, **P<0.01).

[0042] Figure 5 shows the results of (A) ELISA using Y03 confirming that free β2m increases in the serum of patients with probable Alzheimer's disease (pAD) compared to patients with subjective memory impairment (SMI), normal pressure hydrocephalus (NPH), and mild cognitive impairment (MCI). (B) ELISA using Y03 confirming that free β2m increases in the plasma of an animal model of dementia (5xFAD mice) (*P<0.05, **P<0.01).

[0043] Figure 6 shows the results of confirming (A) the reduction of beta-amyloid (cortex, hippocampus) in the brain and (C) the reduction of inflammatory (cortex) marker proteins after intraperitoneal administration of Y03 once a week for 8 weeks to 5XFAD mice.

[0044] Figure 7 shows the results of confirming the increase in free β2m aggregates in fibrosis animal model mouse tissues (kidney, liver, lung) through western blot using an anti-free β2m antibody clone.

[0045] Figure 8 shows the results of confirming free β2m expression in various cancer tissues by performing human tissue microarray through mIgG, BBM.1, and anti-free β2m antibody clone staining.

[0046] Figure 9 shows the results showing that (A, B) the fibrosis biomarker hydroxyproline level in the kidney was reduced and the fibrosis area in the kidney tissue was reduced after intraperitoneal administration of Y03 three times at five-day intervals in a mouse model of renal fibrosis (unilateral ureteral obstruction). (C) The results showing that the fibrosis area in the liver tissue was reduced after intraperitoneal administration of Y03 six times at five-day intervals in a mouse model of liver fibrosis induced by carbon tetrachloride (CCl4).

[0047] Figure 10 shows the results of an ELISA analysis showing that the anti-free β2m specific antibody clone also reacts with β2m proteins of animal species other than humans.

[0048]

[0049] Detailed description of the invention and preferred embodiments

[0050] 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 pertains. Generally, the nomenclature used herein is well known and commonly used in the art.

[0051]

[0052] The present invention relates to an antibody or an antigen-binding fragment thereof that specifically binds to β2-microglobulin (β2m; B2M), and in particular, a monoclonal antibody having a high reactivity specifically to free β2m has been developed. In one embodiment of the present invention, it was confirmed that the anti-β2m antibody specifically recognizes only free β2m without interfering with the immune action of MHCI and exhibits reactivity to monomeric and amyloid forms of β2m, and it was confirmed that it exhibits the effects of recovering cognitive function decline induced by free β2m in a wild-type mouse model, reducing amyloid pathology in an Alzheimer's disease animal model, reducing tissue fibrosis area in renal and liver fibrosis models, and reducing fibrosis biomarker proteins. Additionally, using the above antibody, we were able to confirm an increase in free β2m levels in Alzheimer's disease patients and mouse models, an increase in free β2m aggregates in fibrosis model mouse tissues (kidney, liver, lung), and an increase in free β2m in various tumors / cancers.

[0053] Accordingly, the anti-β2m antibody according to the present invention (named 'ADEL-Y03m', 'ADEL-Y03' 'Y03m' or 'Y03') is useful for diagnosis and prevention and / or treatment of neurodegenerative diseases including Alzheimer's disease, fibrosis, cancer, etc.

[0054]

[0055] Accordingly, the present invention relates, in one aspect, to an antibody or antigen-binding fragment thereof that specifically binds to beta2-microglobulin (β2m).

[0056] In the present invention, the antibody or antigen-binding fragment thereof may be characterized by binding to an epitope comprising an amino acid sequence of SEQ ID NO: 59, and the region comprising the epitope may be characterized by comprising an amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 52, but is not limited thereto.

[0057] In one embodiment of the present invention, a region that is structurally easy to access by antibodies and is exposed but does not correspond to a β-sheet region when β2m amyloid is formed was selected as an epitope.

[0058] In the present invention, preferably, it relates to an anti-β2m antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 17; a heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 18, and SEQ ID NO: 22; and a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 19, and SEQ ID NO: 23; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence of SEQ ID NO: 6; a light chain CDR2 comprising an amino acid sequence of SEQ ID NO: 7; and a light chain CDR3 comprising an amino acid sequence of SEQ ID NO: 8.

[0059] In the present invention, the anti-β2m antibody or antigen-binding fragment thereof may be characterized by including heavy and light chain variable regions selected from the group consisting of, but not limited to:

[0060] (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8;

[0061] (ii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8;

[0062] (iii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and

[0063] (iv) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0064] In the present invention, the heavy chain variable region may be characterized by including an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO: 40, and the light chain variable region may be characterized by including an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO: 34, but is not limited thereto.

[0065] In the present invention, the anti-β2m antibody or antigen-binding fragment thereof may be characterized by including, but is not limited to, the following variable regions:

[0066] (i) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4, SEQ ID NO: 20, or SEQ ID NO: 24; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 9;

[0067] (ii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 13; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 15;

[0068] (iii) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 26; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28, 30, 32, or 34;

[0069] (iv) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 36; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28, 30, 32 or 34;

[0070] (v) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 38; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28, 30, 32, or 34; or

[0071] (vi) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 40; and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 28, 30, 32, or 34.

[0072] The sequences of each CDR and variable region of the anti-β2m antibody according to the present invention are as described in Tables 3 and 4 below.

[0073]

[0074] In the present invention, the anti-β2m antibody or antigen-binding fragment thereof may be characterized by not binding to β2m bound to an MHCI alpha chain and specifically binding only to free β2m, but is not limited thereto, and the anti-β2m antibody or antigen-binding fragment thereof may be characterized by binding to both monomeric and amyloid forms of free β2m, but is not limited thereto.

[0075]

[0076] The antibody or antibody fragment of the present invention may include not only the sequence of the anti-β2m antibody of the present invention described herein, but also biological equivalents thereof, as long as it can specifically recognize β2m. For example, additional changes 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, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations 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 biologically functional equivalents.

[0077] Considering the mutations having the above-described biological equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence number. The substantial identity means a sequence showing at least 90% homology, most preferably at least 95% homology, 96% or more, 97% or more, 98% or more, or 99% or more homology when the sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet.

[0078] The anti-β2m antibody or antigen-binding fragment thereof according to the present invention also includes an antibody or antigen-binding fragment thereof in which a part of the amino acid sequence is substituted through conservative substitution in the anti-β2m antibody or antigen-binding fragment thereof according to the present invention.

[0079] As used herein, the term "conservative substitution" refers to a modification of a polypeptide that involves replacing one or more amino acids with amino acids having similar biochemical properties, without causing a loss of biological or biochemical function of the polypeptide. A "conservative amino acid substitution" is a substitution that replaces an amino acid residue with an amino acid residue having a similar side chain. Classes of amino acid residues having similar side chains are well known and defined in the art. These classes include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). It is anticipated that antibodies of the present invention may have conservative amino acid substitutions and still retain activity.

[0080]

[0081] In this specification, “antibody” refers to a general term for a substance produced in the immune system by antigen stimulation, and its type is not particularly limited. The antibody is an immunoglobulin molecule that is immunologically reactive with a specific antigen, and refers to a protein molecule that acts as a receptor that specifically recognizes the antigen, and may include polyclonal antibodies, monoclonal antibodies, whole antibodies, and antibody fragments. The antibody may be non-naturally produced, for example, recombinantly or synthetically produced. The antibody may be an animal antibody (e.g., mouse antibody, etc.), chimeric antibody, humanized antibody, or human antibody. The antibody may be a monoclonal antibody. In addition, unless otherwise specified, the antibody may be understood to also include an antigen-binding fragment of an antibody that possesses antigen-binding ability.

[0082] The "variable region" of an antibody used in the present invention refers to the light chain and heavy chain portions of an antibody molecule, including the amino acid sequences of the complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3) and the framework regions (FR). VH refers to the variable domain of the heavy chain, and VL refers to the variable domain of the light chain.

[0083] As used herein, the term “heavy chain” refers to a full-length heavy chain and fragments thereof, comprising a variable domain VH and three constant domains CH1, CH2 and CH3, each comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen. The term “light chain” also refers to a full-length light chain and fragments thereof, comprising a variable domain VL and a constant domain CL, each comprising an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

[0084] As used herein, the term “complementarity determining region (CDR)” refers to the amino acid sequence of the hypervariable region of immunoglobulin heavy and light chains (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., US Department of Health and Human Services, National Institutes of Health (1987)). The heavy chains (CDRH1, CDRH2, and CDRH3) and light chains (CDRL1, CDRL2, and CDRL3) each contain three CDRs. CDRs provide key contact residues for antibody binding to an antigen or epitope.

[0085] As used herein, the term “framework region (FR)” refers to variable domain residues other than CDR residues. Each variable domain typically has four FRs: FR1, FR2, FR3, and FR4.

[0086] In one embodiment of the present invention, the anti-β2m antibody may be, but is not limited to, a mouse antibody, a chimeric antibody, or a humanized antibody.

[0087] As used herein, “humanized antibody”, also referred to as a reshaped human antibody, refers to an antibody in which the complementarity determining region (CDR) of a non-human mammalian antibody, such as a mouse antibody, is grafted onto the complementarity determining region (CDR) of a human antibody. Humanized antibodies can be produced using known general genetic recombination methods (see, for example, European Patent Publication No. EP 0125023 A and WO 96 / 02576).

[0088] Specifically, a DNA sequence designed to link the CDRs of a mouse antibody and the framework regions (FRs) of a human antibody is synthesized. The framework regions of the human antibody linked to the CDRs are selected so that the CDRs form a favorable antigen-binding site. If necessary, amino acids in the framework regions of the variable region of the antibody can be substituted so that the complementarity-determining regions of the humanized antibody form a suitable antigen-binding site (see K Sato, et al., Cancer Res. 1993 Feb 15; 53(4):851-6).

[0089] The framework region of a humanized antibody uses the corresponding region of a human antibody with the highest similarity, based on information from an electroporated mouse antibody. In the present invention, sequence-based and structure-based similarity were utilized to investigate the sequence of human antibodies.

[0090] While humanizing antibodies while maintaining the efficacy or binding affinity of the original antibody is typically challenging, the present invention successfully yielded a humanized antibody with activity equivalent to that of the original mouse antibody by identifying key interaction sites through structural elucidation of the antigen-antibody complex and selecting amino acid substitutions based on this information. Because humanized antibodies exhibit reduced antigenicity in the human body, they are useful for human administration, particularly for therapeutic purposes.

[0091] In the present invention, the constant region of a chimeric antibody or a humanized antibody may be that of a human antibody, and the human antibody constant region may be modified to improve the stability of the antibody or its production. The human antibody used in the humanized antibody may include any isotype of human antibody, such as IgG, IgM, IgA, IgE, and IgD. In the present invention, IgG1 is preferred, but is not limited thereto. In addition, after producing a humanized antibody, amino acids in the constant region may be substituted with other amino acids for the purpose of enhancing antibody stability.

[0092] In this specification, the term “anti-β2m antibody” refers to an antibody that binds to β2m and causes inhibition of the biological activity of β2m, and is used interchangeably with “β2m-specific antibody.” In the present invention, the anti-β2m antibody or antigen-binding fragment thereof may be characterized by having specific binding ability to human or mouse β2m, but is not limited thereto.

[0093] In the present invention, the term “anti-β2m antibody” encompasses both polyclonal antibodies and monoclonal antibodies, preferably monoclonal antibodies, and may be in the form of a complete whole antibody. The whole antibody has a structure comprising two full-length light chains and two full-length heavy chains, and includes a constant region, with each light chain being linked to a heavy chain by a disulfide bond.

[0094] The entire antibody of the anti-β2m antibody according to the present invention is a concept including IgA, IgD, IgE, IgM and IgG forms, and IgG is a subtype, including IgG1, IgG2, IgG3 and IgG4.

[0095] A full-length antibody consists of two full-length light chains and two full-length heavy chains, each light chain linked to a heavy chain by a disulfide bond. The heavy-chain constant region is of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light-chain constant region is of the kappa (κ) and lambda (λ) types.

[0096] The “antigen-binding fragment” of the anti-β2m antibody according to the present invention refers to a fragment that has the function of binding to the antigen of the anti-β2m antibody, i.e., β2m, and is a concept that includes Fab, Fab', F(ab')2, scFv, (scFv)2, scFv-Fc, and Fv, etc., and is used interchangeably with the same meaning as “antibody fragment” in this specification.

[0097] The Fab has a structure with variable regions of the light and heavy chains, a constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, 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 produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond.

[0098] Fv (variable fragment) refers to the smallest antibody fragment containing only the heavy chain variable region and the light chain variable region. In double-chain Fv (dsFv), the heavy chain variable region and the light chain variable region are linked by a disulfide bond, and in single-chain Fv (scFv), the heavy chain variable region and the light chain variable region are covalently linked, usually through a peptide linker. These antibody fragments can be obtained using a proteolytic enzyme (for example, Fab can be obtained by restriction digestion of the whole antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), and can be produced through genetic recombination technology (for example, a DNA encoding the heavy chain or its variable region and a DNA encoding the light chain or its variable region are used as templates, and a primer pair is used to amplify by the PCR (Polymerase Chain Reaction) method, and a DNA encoding a peptide linker is combined with a primer pair so that both ends are linked to the heavy chain or its variable region and the light chain or its variable region, respectively, and amplified).

[0099] 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.

[0100] The above monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., identical except for possible naturally occurring mutations that may exist in trace amounts in individual antibodies comprising the population. Monoclonal antibodies are highly specific, being 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.

[0101] For example, monoclonal antibodies useful in the present invention can be produced by hybridoma methods, or can be produced using recombinant DNA methods in bacterial, eukaryotic, or plant cells (see U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries.

[0102] An "epitope" is a protein determinant to which an antibody can specifically bind. Epitopes typically consist of chemically active surface molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former is lost in the presence of denaturing solvents, while binding to the latter is not.

[0103]

[0104] In another aspect, the present invention relates to a nucleic acid encoding an anti-β2m antibody or an antigen-binding fragment thereof according to the present invention.

[0105] The nucleic acids used herein may be present in cells, cell lysates, or in partially purified or substantially pure form. A nucleic acid is “isolated” or “rendered substantially pure” if it is purified from other cellular components or other contaminants, such as nucleic acids or proteins of other cells, by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art. The nucleic acids of the present invention may be, for example, DNA or RNA, and may or may not contain intronic sequences.

[0106] In the present invention, the nucleic acid encoding the anti-β2m antibody or antigen-binding fragment thereof may be characterized by including, but is not limited to, a nucleic acid encoding a heavy chain variable region comprising a base sequence represented by SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, or SEQ ID NO: 48; and a nucleic acid encoding a light chain variable region comprising a base sequence represented by SEQ ID NO: 43, SEQ ID NO: 45, or SEQ ID NO: 47.

[0107] The nucleic acid encoding the antibody or antigen-binding fragment thereof can be isolated to recombinantly produce the antibody or antigen-binding fragment thereof.

[0108] As used herein, the term "nucleic acid" encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, the basic structural units of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base moieties. The sequence of the nucleic acid encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, non-conservative substitutions, or conservative substitutions of nucleotides.

[0109] The DNA encoding the antibody can be readily isolated or synthesized using conventional molecular biological techniques (e.g., by using an oligonucleotide probe that can specifically bind to the DNA encoding the antibody and its heavy and light chains), and the nucleic acid can be isolated and further cloned (amplified) or further expressed by inserting it into a replicable vector.

[0110]

[0111] In another aspect, the present invention relates to a recombinant expression vector comprising the nucleic acid.

[0112] The term "vector" as used herein refers to a means for expressing a target gene in a host cell, and includes viral vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors.

[0113] For expression of the anti-β2m antibody or antigen-binding fragment thereof according to the present invention, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biology techniques (e.g., PCR amplification or cDNA cloning using a hybridoma expressing the antibody of interest) and inserted into an expression vector “operably linked” to transcriptional and translational control sequences.

[0114] The term “operatively linked” as used herein can mean that a gene encoding an antibody is ligated into a vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or both genes can be inserted into the same expression vector. The antibody is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction enzyme sites on the antibody gene fragment and the vector, or blunt-end ligation if no restriction enzyme sites are present at all).

[0115] In some cases, the recombinant expression vector may encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene may be cloned into the vector such that the signal peptide is linked in frame to the amino terminus of the antibody chain gene. The signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide derived from a protein other than immunoglobulin). In addition, the recombinant expression vector carries a regulatory sequence that controls the expression of the antibody chain gene in the host cell. The “regulatory sequence” may include a promoter, enhancer, and other expression control elements (e.g., a polyadenylation signal) that control the transcription or translation of the antibody chain gene. Those skilled in the art will recognize that the design of the expression vector may vary by selecting different regulatory sequences depending on factors such as the choice of host cell to be transformed, the level of protein expression, etc.

[0116]

[0117] In another aspect, the present invention relates to a cell (or host cell) transformed with the recombinant expression vector.

[0118] The cells according to the present invention may be characterized by being selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells, but are not limited thereto. The cells used to produce the antibodies of the present invention may be prokaryotic, yeast, or higher eukaryotic cells, but are not limited thereto.

[0119] Specifically, the host cell according to the present invention may be a prokaryotic cell such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp. In addition, it may be a eukaryotic cell such as a fungus such as Aspergillus sp., a yeast such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa, other lower eukaryotic cells and cells of higher eukaryotes such as cells from insects.

[0120] It can also be derived from plants or mammals. Preferably, monkey kidney cells 7 (COS7) cells, NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138 cells, baby hamster kidney (BHK) cells, MDCK cells, myeloma cell lines, HuT 78 cells, and HEK293 cells are available, but are not limited thereto. HEK293 cells are particularly preferred.

[0121] The nucleic acid or the vector is transfected or transformed into a host cell. Any of a variety of techniques commonly used to introduce exogenous nucleic acids (DNA or RNA) into prokaryotic or eukaryotic host cells for “transfection” or “transfection” can be used, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection. Various expression host / vector combinations can be used to express the anti-β2m antibody according to the present invention. Suitable expression vectors for eukaryotic hosts include, but are not limited to, expression control sequences derived from SV40, bovine papillomavirus, adenovirus, adeno-associated virus, cytomegalovirus, and retrovirus. Expression vectors that can be used in bacterial hosts include bacterial plasmids obtained from Escherichia coli, such as pET, pRSET, pBluescript, pGEX2T, pUC vectors, col E1, pCR1, pBR322, pMB9 and their derivatives; plasmids with a wider host range, such as RP4; phage DNA, exemplified by the numerous phage lambda derivatives, such as λgt10 and λgt11, NM989; and other DNA phages, such as M13 and filamentous single-stranded DNA phages. Useful expression vectors for yeast cells are the 2°C plasmids and their derivatives. A useful vector for insect cells is pVL941.

[0122]

[0123] In another aspect, the present invention relates to a method for producing an anti-beta2-microglobulin (β2-microglobulin; β2m) antibody or an antigen-binding fragment thereof, comprising a step of culturing the above cells.

[0124] Preferably, the method may include a step of culturing the cells to express the anti-β2m antibody or antigen-binding fragment thereof according to the present invention.

[0125] When a recombinant expression vector capable of expressing the above-described anti-β2m antibody or antigen-binding fragment thereof is introduced into a mammalian host cell, the antibody can be produced by culturing the host cell for a period of time sufficient to cause the antibody to be expressed in the host cell, or more preferably, for a period of time sufficient to cause the antibody to be secreted into the culture medium in which the host cell is cultured.

[0126] The above cells can be cultured in various media. Any commercially available medium can be used as a culture medium. Any other essential supplements known to those skilled in the art may be included at appropriate concentrations. Culture conditions, such as temperature and pH, are already used with the host cells selected for expression and will be readily apparent to those skilled in the art.

[0127] In some cases, the expressed antibody can be isolated from the host cell and purified to a homogeneous state. The separation or purification of the antibody can be performed using separation and purification methods commonly used for proteins, such as chromatography. The chromatography can include, for example, affinity chromatography using a protein A column or a protein G column, ion exchange chromatography, or hydrophobic chromatography. In addition to the chromatography, the antibody can be separated and purified by combining filtration, ultrafiltration, salting out, dialysis, and the like.

[0128]

[0129] In another aspect, the present invention relates to an antibody-drug conjugate (ADC) in which a drug is conjugated to the anti-β2m antibody or an antigen-binding fragment thereof.

[0130] Antibody-drug conjugates require the drug to be stably bound to the antibody before delivery to the target cell. Once delivered to the target, the 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, upon release from the target cell, possess sufficient cytotoxicity to induce target cell death.

[0131] In the present invention, the anti-β2m antibody or an antigen-binding fragment thereof and a cytotoxic substance including a drug such as an anticancer agent may be combined with 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 labeling substance is a protein). The cytotoxic substance may be any substance that is toxic to cancer cells, particularly solid cancer cells, and may be at least one selected from the group consisting of radioisotopes, cytotoxic compounds (small molecules), cytotoxic proteins, anticancer agents, etc., but is not limited thereto. The cytotoxic protein may be at least one selected from the group consisting of ricin, saporin, gelonin, momordin, debouganin, diphtheria toxin, pseudomonas toxin, etc., but is not limited thereto. The radioisotope may be at least one selected from the group consisting of 131I, 188Rh, 90Y, etc., but is not limited thereto. The cytotoxic compound may be at least one 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, etc., but is not limited thereto.

[0132] In the present invention, the antibody-drug conjugate may be prepared according to a technique well known in the technical field to which the present invention belongs.

[0133] In the present invention, the antibody-drug conjugate may be characterized in that the antibody or an antigen-binding fragment thereof is bound to a drug via a linker.

[0134] In the present invention, the linker may be characterized as being a cleavable linker or a non-cleavable linker.

[0135] The above linker is a portion that connects the anti-β2m antibody and the drug, for example, the linker is in a form that is cleavable under intracellular conditions, i.e., the drug can be released from the antibody through cleavage of the linker in the intracellular environment.

[0136] The linker can be cleaved by a cleavage agent present in the intracellular environment, for example, in a lysosome or endosome, and can be a peptide linker that can be cleaved by an intracellular peptidase or protease enzyme, for example, a lysosomal or endosomal protease. Typically, the peptide linker has a length of at least two amino acids. The cleavage agent can include cathepsin B, cathepsin D, or plasmin, and hydrolyzes the peptide to release the drug into the target cell. The peptide linker can be cleaved by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissues, and for example, a Phe-Leu or Gly-Phe-Leu-Gly linker can be used. In addition, the peptide linker can be cleaved by, for example, an intracellular protease, and can be a Val-Cit linker or a Phe-Lys linker.

[0137] In the present invention, the cleavable linker is pH-sensitive and may be susceptible to hydrolysis at a specific pH value. Generally, a pH-sensitive linker indicates that it can be hydrolyzed under acidic conditions. For example, it may be an acid-labile linker that can be hydrolyzed in lysosomes, such as a hydrazone, a semicarbazone, a thiosemicarbazone, a cis-aconitic amide, an orthoester, an acetal, a ketal, etc.

[0138] The above linker may also be cleaved under reducing conditions, for example, a disulfide linker. Various disulfide bonds can 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-alpha-methyl-alpha-(2-pyridyl-dithio)toluene).

[0139] In the present invention, the drug and / or drug-linker may be randomly conjugated via lysine of the antibody, or conjugated via cysteine ​​exposed when the disulfide bond chain is reduced. In some cases, the linker-drug may be conjugated via cysteine ​​present in a genetically engineered tag, for example, a peptide or a protein. The genetically engineered tag, for example, a peptide or a protein, may include an amino acid motif that can be recognized by, for example, an isoprenoid transferase. The peptide or protein may have a deletion at the carboxyl terminus of the peptide or protein, or may have an addition via covalent bonding of a spacer unit to the carboxyl (C) terminus of the peptide or protein. The peptide or protein may be covalently bonded directly to the amino acid motif, or may be covalently bonded to the spacer unit and then linked to the amino acid motif. The above amino acid spacer unit is composed of 1 to 20 amino acids, among which a glycine unit is preferred.

[0140] The above linker may include a beta-glucuronide linker that is recognized and hydrolyzed by beta-glucuronidase, which is present in large numbers in lysosomes or is overexpressed in some tumor cells. Unlike peptide linkers, it has a high hydrophilicity, which has the advantage of increasing the solubility of the antibody-drug complex when combined with a highly hydrophobic drug.

[0141] In this regard, the present invention may use a beta-glucuronide linker disclosed in Korean Patent Publication No. 2015-0137015, for example, a beta-glucuronide linker including a self-immolative group.

[0142] Additionally, the linker may be, for example, a non-cleavable linker, which releases the drug through a single step of antibody hydrolysis, producing, for example, an amino acid-linker-drug conjugate. This type of linker may be a thioether group or a maleimidocaproyl group, and may maintain stability in blood.

[0143] In the present invention, the drug may be characterized as being a chemotherapeutic agent, a toxin, a nuclease, a microRNA (miRNA), siRNA, shRNA, an antisense oligonucleotide (ASO), or a radioactive isotope. The drug may be conjugated to an antibody as a preparation exhibiting a pharmacological effect.

[0144] The chemotherapeutic agent may be a cytotoxic agent or an immunosuppressant. Specifically, it may include a microtubulin inhibitor, a mitotic inhibitor, a topoisomerase inhibitor, or a chemotherapeutic agent that can function as a DNA intercalator. It may also include an immunomodulatory compound, an anticancer agent, an antiviral agent, an antibacterial agent, an antifungal agent, an anthelmintic agent, or a combination thereof.

[0145] In the present invention, the drug is auristatin (including MMAE and MMAF), maytansinoid, aminopterin, actinomycin, bleomycin, thalidomide, camptothecin, esperamicin, etoposide, mercaptopurine, dolastatin, trichothecene, calicheamicin, taxane (including paclitaxel and docetaxel), methotrexate, vincristine, vinblastine, doxorubicin, melphalan, Mitomycin, chlorambucil, duocarmycin, L-asparaginase, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, carmustine, cisplatin, carboplatin, dacarbazine, procarbazine, topotecan, nitrogen mustard, cytoxan, 5-fluorouracil, irinotecan, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, vinorelbine, lomustine,Busulfan, treosulfan, decarbazine, teniposide, crisnatol, trimetrexate, mycophenolic acid, tiazofurin, ribavirin, EICAR (5-ethynyl-1-beta-D-ribofuranosylimidazole-4-carboxamide), defoxamine, fluxuridine, doxifluridine, raltitrexed, cytosine arabinoside, fludarabine, tamoxifen, raloxifene, megestrol, goserelin, Leuprolide acetate, flutamide, bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine, photosensitizer Pe4, demethoxy-hypocrellin A, interferon-α, interferon-γ, tumor necrosis factor, gemcitabine, bortezomib, lenalidomide, lovastatin, 1-methyl-4-phenylpyridinium ion, staurosporine, peplomycin, epirubicin, pirarubicin, zorubicin,One or more selected from the group consisting of, but not limited to, verapamil and thapsigargin.

[0146] In the present invention, the drug may include one or more nucleophilic groups selected from the group consisting of amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide groups capable of reacting to form a covalent bond with an electrophilic group on a linker and a linker reagent.

[0147]

[0148] In another aspect, the present invention relates to a bispecific or multi-specific antibody comprising the anti-β2m antibody or an antigen-binding fragment thereof.

[0149] A bispecific antibody refers to an antibody that has binding ability or antagonism to more than one target, and refers to an antibody in which antibodies with binding ability or antagonism to two different targets are combined, or an antibody in which an antibody with binding ability to one target and a substance with antagonism to another target are combined.

[0150] A multispecific antibody is an antibody that has binding specificities for at least three different antigens. A multispecific antibody may include an antibody that targets more than three specific targets, such as a trispecific antibody, a tetraspecific antibody, or an antibody that targets more than one target.

[0151] Methods for producing bispecific or multispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies relies on the co-expression of two or more immunoglobulin heavy / light chain pairs, where the two or more heavy chains have different specificities.

[0152] The antigen to which the antibody other than the anti-β2m antibody included in the above bispecific or multispecific antibody binds is preferably a cancer-related 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, Nectin 4, Trop2, gpNMB, Axl, BCMA, PD-1, PD-L1, PD-L2, CTLA4, BTLA, 4-1BB, ICOS, GITR, OX40, VISTA, TIM-3, It can be selected from 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 as an antigen related to immune effector cells, it can be selected from TCR / CD3, CD16 (FcγRIIIa), CD44, CD56, CD69, CD64 (FcγRI), CD89, CD11b / CD18 (CR3), etc., but is not limited thereto.

[0153]

[0154] In another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising the anti-β2m antibody or an antigen-binding fragment thereof.

[0155] A CAR may comprise an antigen binding domain, a transmembrane domain, and an intracellular signaling domain, wherein the antigen binding domain may be linked to the transmembrane domain by a linker. The extracellular domain comprising the antigen binding domain may also comprise a signal peptide.

[0156] In the present invention, the antigen binding site of the antigen binding domain may be characterized as being an scFv of the antibody.

[0157]

[0158] From another perspective, the present invention relates to an immune cell into which the chimeric antigen receptor has been introduced.

[0159] The above immune cells include cells genetically modified to express the chimeric antigen receptor, and are preferably capable of inducing an effect of treatment of a desired disease or condition by inducing immunity, for example, immunotherapy, and may be selected from the group consisting of, but not limited to, T cells, NK cells, cytokine-induced killer cells (CIKs), activated cytotoxic T lymphocytes (CTLs), macrophages, tumor-infiltrating lymphocytes (TILs), and dendritic cells.

[0160]

[0161] In another aspect, the present invention relates to a diagnostic composition and a diagnostic kit for a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), kidney disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer or inflammatory disease, comprising the anti-β2m antibody or an antigen-binding fragment thereof.

[0162] In another aspect, the present invention relates to a method for diagnosing a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), kidney disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer or inflammatory disease, and a method for providing information for diagnosis, comprising a step of treating a biological sample isolated from a subject with the anti-β2m antibody or an antigen-binding fragment thereof and comparing the detected β2m level with a control group.

[0163] In the present invention, the term "diagnosis" refers to accurately determining the condition of a subject with respect to a specific disease or condition. For example, the condition of a subject with respect to a specific disease or condition is used in a broad sense, including not only susceptibility to a specific disease or condition, determination of the disease the subject is currently suffering from, but also confirmation of the characteristics of the disease, such as prognosis, identification of cancer status, determination of cancer stage, or prediction of cancer sensitivity and responsiveness to treatment, obtaining a basis for appropriate treatment according to the patient's disease and condition, such as confirming the condition of the subject to confirm the therapeutic effect of a specific drug, and further predicting and confirming the presence or absence of recurrence in a subject who has been cured of a specific disease or condition. In the present invention, the diagnosis is preferably to confirm whether or not a disease has developed or is likely to develop.

[0164] In the present invention, the term "prognosis" means an expectation of medical outcome (e.g., long-term survival possibility, disease-free survival rate, etc.), and includes a positive prognosis (positive prognosis) or a negative prognosis (negative prognosis), wherein the negative prognosis includes disease progression or mortality such as recurrence, tumor growth, metastasis, drug resistance, etc., and the positive prognosis includes disease remission such as disease-free state, improvement or stabilization such as tumor regression, etc.

[0165] In the present invention, the term "prediction" means to guess in advance about the medical outcome, and for the purpose of the present invention, it means to guess in advance the course of a patient diagnosed with bile duct cancer (progression, improvement, recurrence, tumor growth, drug resistance).

[0166] In the present invention, the antibody or antigen-binding fragment thereof may be characterized in that it is linked to a signal molecule such as a radioactive isotope or fluorescent dye for detection.

[0167]

[0168] In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), renal disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer or inflammatory disease, comprising the anti-β2m antibody or an antigen-binding fragment thereof, the antibody-drug conjugate, the bispecific or multispecific antibody, the chimeric antigen receptor or the immune cell.

[0169] In another aspect, the present invention relates to a method for preventing or treating a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), renal disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer or inflammatory disease, comprising a step of administering to a subject the anti-β2m antibody or an antigen-binding fragment thereof, the antibody-drug conjugate, the bispecific or multispecific antibody, the chimeric antigen receptor or the immune cell.

[0170] In another aspect, the present invention relates to the use of the anti-β2m antibody or an antigen-binding fragment thereof, the antibody-drug conjugate, the bispecific or multispecific antibody, the chimeric antigen receptor or the immune cell for the prevention or treatment of neurodegenerative diseases, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), kidney diseases, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer or inflammatory diseases.

[0171] In another aspect, the present invention relates to the use of the anti-β2m antibody or an antigen-binding fragment thereof, the antibody-drug conjugate, the bispecific or multispecific antibody, the chimeric antigen receptor or the immune cell for the manufacture of a medicament for the prevention or treatment of a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), kidney disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer or inflammatory disease.

[0172] In this specification, the term “prevention” means any act of inhibiting or delaying the progression of a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), kidney disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer, or inflammatory disease by administering the composition of the present invention, and “treatment” means inhibiting the development of, alleviating, or eliminating symptoms of a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), kidney disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer, or inflammatory disease.

[0173] In the present invention, the pharmaceutical composition may be characterized by comprising a therapeutically effective amount of an anti-β2m antibody or an antigen-binding fragment thereof and a pharmaceutically acceptable carrier.

[0174] The above “pharmaceutically acceptable carrier” is a substance that can be added to the active ingredient to help formulate or stabilize the preparation, and does not cause significant harmful toxic effects to the patient. Pharmaceutically acceptable carriers are those commonly used in the preparation of preparations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0175] The pharmaceutical composition may further comprise, in addition to the above ingredients, lubricants, humectants, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0176] The term "administration" of the present invention means introducing the pharmaceutical composition of the present invention to a patient by any appropriate method, and the pharmaceutical composition of the present invention can be administered orally or parenterally, and can be administered by, for example, infusion, intravenous injection, intramuscular injection, subcutaneous injection, intraperitoneal injection, intrarectal administration, topical administration, intranasal injection, etc., but is not limited thereto.

[0177] The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a generally skilled physician can easily determine and prescribe a dosage effective for the desired treatment or prevention. The term "pharmaceutically effective amount" as used herein means an amount sufficient to prevent or treat a degenerative neurological disease, aging, Down syndrome, acquired immunodeficiency syndrome (AIDS), renal disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer, or inflammatory disease.

[0178] The pharmaceutical composition according to the present invention can be used in combination with conventional therapeutic agents. That is, the anti-β2m antibody or antigen-binding fragment thereof according to the present invention and the pharmaceutical composition comprising the same can be administered simultaneously with, or sequentially or in reverse order with, conventional therapeutic agents such as anticancer agents, and can be administered in combination in an appropriate effective amount within the scope of those skilled in the art.

[0179]

[0180] The present inventors selected as an epitope a region that is structurally easily accessible to antibodies and is exposed outside the β-sheet region during β2m amyloid formation. We predicted that the region of β2m that binds to the MHCI heavy chain would be exposed in β2m dissociated from MHCI. Therefore, we selected this region as the epitope and developed an antibody that recognizes only free β2m.

[0181] Accordingly, the present invention, from another aspect, relates to an isolated peptide comprising an epitope of beta2-microglobulin (β2m), wherein the epitope comprises an amino acid sequence of SEQ ID NO: 59.

[0182] In the present invention, the isolated peptide may be characterized as being a natural, synthetic or recombinant peptide.

[0183] In one embodiment of the present invention, eight antibody clones were secured by immunizing with four peptides (SEQ ID NOs: 49 to 52) including three regions of β2m reported to bind to the MHCI heavy chain, and it was confirmed that the clones immunized with SEQ ID NOs: 50 and 51 had no binding affinity to β2m or were reactive to the MHCI complex. This suggests that not all β2m sequences that bind to the MHCI heavy chain are epitope regions of free β2m-specific antibodies, and it was confirmed that the antigen regions of the four clones (#1, #2, #7, #8) that were not reactive to the MHCI complex and only showed binding affinity to free β2m commonly included the epitope sequence of SEQ ID NO: 59.

[0184] In the present invention, the region including the epitope may be characterized by including the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO: 52, but is not limited thereto.

[0185] Since the above-described isolated peptide contains the above-described epitope, description of the overlapping content with the above-described epitope is omitted to avoid excessive complexity of the present specification.

[0186]

[0187] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0188]

[0189] Example 1: Antibody development strategy and immunization (antigen boosting)

[0190] To develop an antibody that specifically binds to free β2m protein dissociated from the MHCI complex, we commissioned the production of the antibody to GenScript, a company specializing in antibody development. In particular, we gave top priority to not inhibiting the antigen presentation mechanism by not recognizing β2m, which constitutes the MHCI complex. To this end, we identified the adjacent site where β2m and the MHCI heavy chain bind and selected an epitope targeting that site. In other words, this is a strategy to specifically recognize an epitope that is not exposed when β2m is complexed with MHCI, etc., but is exposed only when it dissociates from the complex (Fig. 1).

[0191] After boosting injections of BALB / c mice with four different β2m peptides containing the above epitope as antigens, a total of eight clones, two for each peptide, were obtained (Table 1).

[0192]

[0193] Example 2: Binding capacity of anti-free-β2m antibody

[0194] Example 2-1: Binding Strength Measurement (BLI)

[0195] After activating the AR2G biosensor (18-5095, ForteBio) with EDC / NHS solution in a BLI device (BLItz, ForteBio), the amine group of β2m protein (126-11-1, Lee-biosolutions) was immobilized on the biosensor, and the association rate and dissociation rate for the analyte (antibody) were measured to calculate the KD value.

[0196] Among the eight antibody clones produced from hybridoma, five clones (#1, #2, #4, #7, #8) showed high binding affinity to human β2m (Table 2).

[0197]

[0198] To confirm whether the antibody clones specifically bind to free β2m, Dot blot, Immunoprecipitation, Cell surface staining, and FACS analysis of cells were performed using MHCI native complex protein (FCM-H82W7, Acrobiosystems). As a result, it was confirmed that four of the eight clones (#1, #2, #7, #8) did not bind to the MHCI native complex but only to dissociated free β2m (Fig. 2).

[0199] The QVYSRH (SEQ ID NO: 59) region commonly included in SEQ ID NO: 49 and SEQ ID NO: 52 targeted by four clones (#1, #2, #7, #8) that specifically bind only to dissociated free β2m suggests that it is a region that can be developed as an epitope for a free β2m-specific antibody.

[0200]

[0201] Example 2-2: MHCI complex dissociation and dot blot

[0202] The recombinant MHCI complex was dissociated into the MHCI heavy chain and β2m protein by treating with an acidic glycine buffer, and the dissociation of the complex was confirmed using a monoclonal antibody (W6 / 32) that recognizes only the MHCI complex. 1 μg of each protein was adsorbed onto a nitrocellulose membrane, left to dry at room temperature, and blocked in a buffer containing 5% nonfat dry milk (PBS, 0.1% Tween-20). The membrane was placed in a solution containing each primary antibody (free β2m-specific antibody clone, BBM.1), incubated overnight at 4°C, and then washed with a washing buffer (PBS, 0.1% Tween-20). After reacting with HRP-conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA) solution at room temperature for 1 hour, the protein dots were reacted with enhanced chemiluminescence reagent (Thermo Fisher Scientific, Rockford, IL, USA), and observed using an iBright™ CL 1500 imaging system. In the above experiment, except for clones (#3, 5, 6) that did not bind to β2m protein, most of the remaining clones recognized dissociated free β2m more strongly than the MHCI complex. In particular, clones #1, 2, 7, and 8 recognized only dissociated free β2m (Fig. 2A). A monoclonal antibody (BBM.1) that recognizes both MHCI-bound β2m and dissociated free β2m was used as a positive control, and the same amount of dissociated or native MHCI complex was used.

[0203]

[0204] Example 2-3: Immunoprecipitation

[0205] Additionally, the binding between the recombinant MHCI complex and the free β2m-specific antibody clone was confirmed by a pull-down assay. MHCI complex protein (1 mg) was reacted with 1.5 μg of each antibody (free β2m-specific antibody clone, BBM.1) in immunoprecipitation buffer (50 mM tris-HCl, pH 7.2, 150 mM NaCl, 1% Triton X-100) overnight (4°C), and then 100 μL of Protein G-Sepharose (Cytiva, 17-0618-01) was added and reacted for 1 hour at 4°C. Each immunoprecipitate was recovered by centrifugation at 6,000 rpm for 1 minute at 4°C and washed twice with immunoprecipitation buffer. The samples were then boiled in SDS sample buffer and analyzed by western blot (Example 2-4). When the free β2m-specific antibody clones were reacted, the MHCI complex was not pulled down, whereas the complex was pulled down by the BBM.1 antibody that can recognize β2m bound to MHCI and detected by western blot using antibodies against MHCI and β2m, respectively (Fig. 2B).

[0206]

[0207] Example 2-4: Western blot

[0208] Protein samples prepared for Western blot were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, USA). The membranes were incubated with solutions containing each primary antibody overnight at 4°C and then washed with washing buffer (PBS, 0.1% Tween-20). The membranes were incubated with HRP-conjugated secondary antibodies (Vector Laboratories, Burlingame, CA, USA) for 1 hour at room temperature, followed by enhanced chemiluminescence reagent (Thermo Fisher Scientific, Rockford, IL, USA). Protein bands were observed using an iBright™ CL 1500 imaging system. The band intensities of each sample were analyzed using ImageJ software (NIH, Bethesda, MD, USA) or IBA software (iBright Analysis Software, ThermoFisher).

[0209]

[0210] Example 2-5: Flow cytometry

[0211] To investigate whether the free β2m-specific antibody clones do not recognize the MHCI complex present on the cell surface, their reactivity to the MHCI complex expressed on the surface of HeLa cells was confirmed by flow cytometry.

[0212] After harvesting, HeLa cells were washed twice with cold 1X PBS containing 1% BSA and reacted with IgG, free β2m-specific antibody, or BBM.1 antibody, respectively, at 4°C for 1 h. The cells labeled with each antibody were washed twice again with cold 1X PBS containing 1% BSA and then stained with FITC-conjugated anti-mouse IgG antibody at 4°C for 1 h. A total of 10,000 gated events from each sample were acquired using a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA) and analyzed with FACSDiva software (BD Biosciences). Free β2m-specific antibody clones showed significantly lower reactivity toward the MHCI complex on the cell surface compared to BBM.1 antibody (Fig. 2C).

[0213] In the above experiments, among the four clone antibodies that showed specific reactivity only to free β2m, one excellent clone was selected through comparison of reactivity to various biological samples (#7) and was named ADEL-Y03m (hereinafter, 'Y03m' or 'Y03'). Using Y03m, reactivity to β2m and MHCI complex proteins was confirmed through ELISA (Figs. 2D and 2E), and binding strength was measured through BLI (Fig. 2F).

[0214]

[0215] Example 2-6: ELISA

[0216] To confirm the reactivity for β2m, monomeric β2m recombinant protein was diluted to various concentrations (200, 100, 50, 25, 12.5, 6.25, 3.125, 0 ng / ml) in coating buffer (sodium bicarbonate buffer, pH 9.6), dispensed 50 ㎕ per well into a 96-well plate, and coated for one day at 4℃. After removing the coating buffer, the plate was washed four times by adding and removing 160 ㎕ of washing buffer (1xPBS with 0.05% Tween-20) per well. After that, 130 ㎕ of blocking buffer (Thermo, 37532) was dispensed into each well, and blocking was performed for 2 hours at 37℃ while shaking at 700 rpm, and then the blocking buffer was removed and washed four times in the same manner. Next, 50 ㎕ of Y03m antibody solution (diluted in blocking buffer, 1,000 ng / mL) was dispensed into each well, and the mixture was incubated at 37℃ for 1 hour while shaking at 700 rpm. The detection antibody solution was removed and washed 4 times in the same manner. After this, 50 ㎕ of secondary antibody (Vector, PI-2000-1) was diluted in blocking buffer at a ratio of 1:10,000 and dispensed into each well, and the mixture was incubated at 37℃ for 1 hour while shaking at 700 rpm, and the mixture was washed 4 times in the same manner. Next, 50 μl of TMB substrate solution (TMB substrate solution, R&D systems, DY999) was added to each well, and the degree of color development was checked while reacting at room temperature. When appropriate color development was confirmed, 25 μl of stop solution (Stop solution, R&D systems, DY994) was added to each well to stop the reaction, and the absorbance was measured at 450 nm and 620 nm within 30 minutes (Fig. 2D).

[0217] To confirm the reactivity for the MHCI complex, biotin-conjugated MHCI (FCM-H82W7, Acrobiosystems) protein was diluted to a concentration of 200 ng / ml in blocking buffer (Thermo, 37532) on a streptavidin-coated plate, and 50 μl was dispensed into each well. The reaction was performed while shaking at 700 rpm for 1 hour at 37°C, and the solution was removed. The plate was washed four times by adding and removing 150 μl of washing buffer (1xPBS with 0.05% Tween-20) per well. Next, Y03m, IgG, BBM.1, and W6 / 32 antibodies were diluted in blocking buffer at various concentrations (1,000, 333.3, 111.1, 37.04, 12.35, 4.16, 1.37, 0 ng / mL), dispensed 50 ㎕ into each well, and reacted at 37℃ for 1 hour while shaking at 700 rpm. After removing the antibody solution, the wells were washed 4 times in the same manner. After this, the secondary antibody (Vector, PI-2000-1) was diluted in blocking buffer at a ratio of 1:10,000, dispensed 50 ㎕ into each well, and reacted at 37℃ for 1 hour while shaking at 700 rpm, and washed 4 times in the same manner. Next, 50 μl of TMB substrate solution (TMB substrate solution, R&D systems, DY999) was added to each well, and the degree of color development was checked while reacting at room temperature. When appropriate color development was confirmed, 25 μl of stop solution (Stop solution, R&D systems, DY994) was added to each well to stop the reaction, and the absorbance was measured at 450 nm and 620 nm within 30 minutes (Fig. 2E).

[0218]

[0219] Example 2-7: Comparison of binding strength

[0220] The binding affinity between Y03m, BBM.1, and mouse IgG2b and MHC1 was measured using BLI equipment (BLItz, ForteBio). Biotin-conjugated MHC1 (FCM-H82W7, Acrobiosystems) was loaded onto the BLI SA biosensor, and the association and dissociation rates for the analytes (each antibody) were measured to calculate the KD value.

[0221] As a result, Y03m reacted with recombinant β2m protein in a concentration-dependent manner, whereas no reactivity or binding affinity was observed for the MHCI complex (Fig. 2F). Unlike Y03m, BBM.1 and W6 / 32 antibodies showed high reactivity and binding affinity for the MHCI complex (Fig. 2F).

[0222]

[0223] Example 2-8: Amyloid β2m production and ELISA

[0224] β2m protein was dissolved in 50 mM sodium citrate (pH 4) buffer and incubated at 37°C with shaking at 300 rpm for 100 hours to induce amyloid formation. The solution was then replaced with PBS buffer (pH 7.4) using an Amicon® Ultra Centrifugal Filter (Merck, UFC503008). Amyloid β2m was diluted in coating buffer (sodium bicarbonate buffer, pH 9.6) at various concentrations (200, 100, 50, 25, 12.5, 6.25, 3.125, 0 ng / ml), dispensed into 96-well plates at 50 μl per well, and coated at 4°C for one day. ELISA was then performed in the same manner as in Example 2-6. Y03m showed a concentration-dependent reactivity toward amyloid-like β2m protein prepared in vitro (Fig. 3A).

[0225]

[0226] Example 2-9: Confirmation of β2m in human serum (Western blot)

[0227] Serum samples from patients with MCI or pAD were mixed 1:1 with 2x Laemmli Sample Buffer (BIO-RAD, 1610737EDU), separated by SDS-PAGE on a 12% Tricine gel, and subjected to Western blotting using a free β2m-specific antibody clone in the same manner as in Example 2-4. Western blotting of the sera from patients with pAD compared to those from patients with MCI revealed an increase in both monomeric and larger forms of β2m (Fig. 3B). These results demonstrate that Y03m reacts not only with monomeric but also with amyloid forms of β2m.

[0228]

[0229] Example 3: Amino acid and nucleic acid sequences of anti-free-β2m antibodies

[0230] The amino acid sequence of the anti-free-β2m antibody is as shown in Table 3 below, and the sequences shown were defined based on the IMGT system.

[0231]

[0232]

[0233]

[0234]

[0235]

[0236] The nucleic acid sequence of the anti-free-β2m antibody is as shown in Table 4 below. Y03m No. 1 clone, Y03m No. 2 clone, and Y03m No. 8 clone are mouse clone DNA sequencing sequences, and Y03m No. 7 clone is a codon-optimized DNA sequence for expression in animal cells.

[0237]

[0238]

[0239]

[0240] The sequence of the humanized antibody based on the Y03m No.7 clone is shown in Table 5 below and was also defined based on the IMGT system.

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259] Example 4: Recovery of free β2m-induced functional impairment in wild-type mice by anti-free-β2m antibodies

[0260] We aimed to confirm that cognitive impairment induced by free β2m in wild-type mice (WT mice) was restored by anti-free-β2m antibodies.

[0261] Stereotaxic injection experiments to inject β2m and antibodies into the hippocampus were performed using 12-week-old male mice (n = 30–33), randomly divided into three groups. The negative control group received only mouse immunoglobulin G (mIgG) antibody, and the positive control group received β2m and mIgG. The experimental group received a combination of β2m and Y03m antibodies. After anesthetizing the mice, they were fixed in a stereotaxic fixture, the central skull was shaved, and an incision was made along the midline. Injections were performed bilaterally into the dentate gyrus (DG) of the hippocampus, and the coordinates used are as follows:

[0262] Anterior: -1.8 mm (from bregma)

[0263] Lateral: ±1.6 mm

[0264] Depth: -2.1 mm (based on skull surface)

[0265] The injection solution was prepared by diluting β2m and antibody in 1X PBS to concentrations of 25 ng / μL and 300 ng / μL, respectively. 2 μL was injected into each hippocampus at a rate of 0.2 μL / min for a total of 10 minutes, using a 10-μL, 26-gauge Hamilton syringe (HAMILTON, Cat. 80300). After injection, the injection needle was held in place for 5 minutes to prevent reflux and then slowly withdrawn, and the incision site was sutured. Thirteen days later, a Novel Object Recognition (NOR) assay was performed (Fig. 4A).

[0266] The Novel Object Recognition (NOR) test consists of three phases: habituation, training, and testing. Rats are allowed to explore a square space (40 cm x 40 cm x 40 cm) without objects for 10 minutes (habituation). One hour later, in the training phase, two identical objects are placed in the square space, and the rats are allowed to explore it for 5 minutes. Four hours later, in the testing phase, only one of the two identical objects is transformed into a different shape, and the rats are allowed to explore it for 5 minutes. The time spent with the novel object is recorded. The time spent exploring and tracking each object is recorded using smart 3.0 software (Panlab, Harvard Apparatus). As a result, it was observed that when β2m was injected into the brain, the exploration time for a novel object decreased and the number of times the novel object was entered into the area was reduced, whereas when Y03m antibody was co-injected, recovery was achieved (Fig. 4B to Fig. 4D).

[0267]

[0268] Example 5: Determination of Free β2m Levels in Alzheimer's Disease (AD) Patients and Mouse Models

[0269] We aimed to confirm the increase in the level of free β2m in the blood of Alzheimer's disease (AD) patients and mouse models by sandwich ELISA analysis using Y03m.

[0270] Y03m was diluted to 1 μg / mL in coating buffer and 50 μL was dispensed into each well of an ELISA half-plate. The plates were sealed and incubated overnight in a 4°C shaking incubator. The following day, the coating solution was removed, and each well was washed three times with PBS (wash buffer) containing 0.05% Tween 20. After the final wash, any residual solution was completely removed. Afterwards, 130 μL of PBS containing 3% BSA was added to each well to block nonspecific binding, and blocking was performed at 37°C for at least 1 hour.

[0271] Human or mouse blood samples were prepared by diluting them 1:100 in a commercial blood dilution buffer, and human or mouse recombinant β2m protein was used as a standard. After blocking, washing was repeated three times, and 50 μL of the prepared sample or standard was dispensed into each well. The plate was sealed and incubated in a shaking incubator at 37°C and 700 rpm for 2 hours, after which washing was repeated three times in the same manner.

[0272] In the next step, biotin-conjugated BBM.1 antibody was diluted to a concentration of 1 μg / mL in PBS containing 3% BSA, and 50 μL was treated to each well, and the reaction was performed at 37°C for 1 hour while shaking at 700 rpm. After washing three times, streptavidin-HRP was diluted 1:10,000 in PBS containing 3% BSA, and 50 μL was dispensed to each well, and the reaction was performed under the same conditions (37°C, 700 rpm) for 1 hour. After the reaction, the same washing was repeated three times.

[0273] For the colorimetric reaction for detection, 50 μL of R&D Systems' substrate solution (1:1 mixture) was added to each well and the reaction was allowed to proceed at room temperature until the solution color changed from clear to blue. After the colorimetric reaction had progressed appropriately, 25 μL of a stop solution diluted 1:2 in distilled water was added to each well, and the plate was gently tapped to ensure thorough mixing. Finally, the absorbance of each well was measured at 450 nm, and all measurements were performed within 30 minutes of the end of the reaction.

[0274] As a result, it was confirmed that free β2m increased in the serum of patients with probable Alzheimer's disease (pAD) compared to those with Subjective Memory Impairment (SMI), Normal Pressure Hydrocephalus (NPH), and Mild Cognitive Impairment (MCI) (Fig. 5A), and that free β2m increased in the plasma of an animal model of dementia (5xFAD mice) (Fig. 5B).

[0275]

[0276] Example 6: Amyloid pathology reduction effect of anti-free-β2m antibody

[0277] To confirm the dementia therapeutic efficacy of Y03m, the IgG control group and Y03m were intraperitoneally administered once a week at 25 mg / kg for 8 weeks to 5XFAD [B6.Cg Tg (APPSwFlLon, PSEN1*M146L*L286V) 6799Vas / Mmjax] mice (#034848-JAX), a dementia mouse model, and wild-type mice, and the decrease in amyloid pathology and inflammatory marker proteins was confirmed. That is, the mice used in the experiment were G1: WT-IgG (n=9), G2: 5XFAD-IgG (n=9), G3: 5XFAD-Y03 (n=9) (Fig. 6A). Amyloid pathology was confirmed in brain tissues (cerebral cortex and hippocampus) by western blot, and inflammatory marker proteins were confirmed in cerebral cortical brain tissues by qPCR.

[0278]

[0279] Example 6-1: Western blot

[0280] Mouse brain tissue (cortex, hippocampus) was lysed in lysis buffer (PBS, 1% Triton X-100) containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA), and protein concentration was measured using the Bradford assay. After mixing the protein with Laemmli Sample Buffer to a concentration of 2 μg / μl, western blotting was performed in the same manner as in Example 2-4. The antibodies used are shown in Table 6 below.

[0281]

[0282] As a result, it was confirmed that the β-Amyloid level was significantly reduced in the brain tissue (cerebral cortex and hippocampus) of 5xFAD mice administered Y03 antibody compared to IgG antibody (Fig. 6B).

[0283]

[0284] Example 6-2: Quantitative polymerase chain reaction

[0285] Mouse cortical tissue was homogenized using a QIAshredder (Qiagen #79654), RNA was extracted using the RNeasy Mini kit (Qiagen #74106), and DNA was removed using an RNase-Free DNase set (Qiagen #79254) to obtain pure RNA. Total RNA concentration was measured using a Nanodrop (Thermo Scientific #ND-one).

[0286] To synthesize complementary DNA from RNA samples, ReverTra Ace qPCR RT kit (TOYOBA #FSQ-101) was used. Master mix was prepared by mixing 5 ㎕ of Nuclease-free water (Thermo fisher #AM9937) in a total volume of 10 ㎕, 2 ㎕ of 5x RT Buffer provided in the kit, 0.5 ㎕ of RT Enzyme Mix, and 0.5 ㎕ of Primer Mix. Then, 1 ㎍ of each RNA sample was mixed with the Master mix. The reaction was performed at 37℃ for 15 minutes and then at 98℃ for 5 minutes to complete the reaction.

[0287] Quantitative polymerase chain reaction in complementary DNA was performed by mixing 10 ㎕ of IQ SYBR Green Supermix (Bio-rad #170-8882), 5 ㎛ / 1 ㎕ of forward and reverse primers, 100 ng / 1 ㎕ of DNA template, and 8 ㎕ of nuclease-free water in a total volume of 20 ㎕, and the reaction was performed according to the conditions in Table 7 below, and the Cq value was measured using CFX Connext software (Bio-rad).

[0288]

[0289] The primers used are as described in Table 8 below.

[0290]

[0291] As a result, it was confirmed that the levels of inflammation markers IL-1β and IL-18 were significantly reduced in the brain tissue of 5xFAD mice administered Y03 antibodies compared to 5xFAD mice administered IgG antibodies (Fig. 6C).

[0292]

[0293] Example 7: Confirmation of increased free β2m aggregates in fibrosis model mouse tissues (kidney, liver, lung)

[0294] Animal model mouse tissues (kidney, liver, lung) were lysed in lysis buffer (PBS, 1% Triton X-100) containing protease and phosphatase inhibitors (Sigma-Aldrich, St. Louis, MO, USA), and protein concentration was measured using the Bradford assay. After mixing the protein in Laemmli Sample Buffer to a concentration of 2 μg / μl, western blotting was performed using a free β2m-specific antibody clone in the same manner as in Example 2-4.

[0295] As a result, it was confirmed that free β2m aggregates increased in mouse kidney, liver, and lung tissues (Fig. 7).

[0296]

[0297] Example 8: Confirmation of increased free β2m in various tumors / cancers

[0298] To determine the expression of free β2m in various cancer tissues, we performed human tissue microarray (TMA) using a free β2m-specific antibody clone (#7). β2m expression was examined by immunohistochemical staining using a microarray (#BC001134b; a multi-organ tumor and normal tissue microarray comprising 26 human organs for pathological diagnosis).

[0299] Before immunohistochemical staining, paraffin was removed with xylene and ethanol. Tissue microarrays mounted on slides were subjected to antigen retrieval using IHC-Tek™ Epitope Retrieval Solution, Ready To Use (IHC World). Then, to block endogenous peroxidase activity and nonspecific antibody binding, sections were treated with a reagent containing 5% H₂O₂, 20% methanol, and 2% Triton X-100 in PBS. Biotin-conjugated antibodies (2 μg / mL) were reacted with pretreated tissue sections overnight at 4°C. Antibodies used for staining were IgG, BBM.1 (MA1-26040, Thermofisher) and clone #7 antibody. The following day, the reaction was performed using a standard avidin-biotin-peroxidase assay (VECTASTAIN® ABC Kits, Vector Lab.) and a DAB peroxidase (HRP) substrate kit (DAB Substrate Kits, Vector Lab.), and the immunostained sections were analyzed under a microscope (Motic EasyScan). As a result, free β2m expression was confirmed in various cancer tissues (Fig. 8).

[0300]

[0301] Example 9: Anti-free-β2m antibody reduces fibrosis in UUO kidney and CCL4 liver models.

[0302] To determine whether Y03m exhibits therapeutic efficacy in fibrotic diseases, we commissioned SMC Laboratories, Inc. (Japan), an animal efficacy test CRO company, to administer anti-free-β2m antibody to mouse animal models of renal fibrosis and liver fibrosis, and confirmed a decrease in fibrotic area in renal and liver tissues and changes in fibrotic biomarker proteins.

[0303]

[0304] Example 9-1: Efficacy evaluation test in mice with renal fibrosis

[0305] Unilateral ureteral obstruction (UUO) is a widely used disease model for studying renal fibrosis, and can induce tubular necrosis and inflammatory cell infiltration, which are major pathophysiological features of chronic kidney disease, in a relatively short period of time.

[0306] To verify the effect of Y03m on renal fibrosis in the UUO model, 5 mg / kg or 20 mg / kg of IgG and Y03m were administered intraperitoneally to UUO model mice on days 0, 5, and 10, and the mice were sacrificed on day 14 to analyze fibrosis indices.

[0307] To quantify the hydroxyproline content in renal tissue, the left kidney was treated with alkaline-acid hydrolysis. The tissue was alkaline-treated with NaOH, acid-hydrolyzed with HCl, and neutralized with NaOH containing activated charcoal. AC buffer (2.2 M acetic acid / 0.48 M citric acid mixture) was then added and centrifuged to collect the supernatant. Samples and hydroxyproline standards were reacted with chloramine T (Nacalai Tesque Inc., Japan) and Ehrlich's reagent to develop color, and the absorbance was measured at 560 nm. Protein concentration was quantified using the BCA assay, and the hydroxyproline content was expressed as μg per mg of protein. As a result, it was confirmed that Y03m reduced the level of hydroxyproline, a fibrosis marker protein, in mouse model kidney tissue (Fig. 9A).

[0308] For analysis of renal tissue fibrosis, the left kidney was fixed in Bouin's solution, embedded in paraffin, sectioned, and stained for collagen using picro-Sirius red solution (Waldeck, Germany). To quantify the area of ​​interstitial fibrosis, bright-field images of the cortico-medullary region were captured at 200x magnification using a digital camera (DFC295; Leica, Germany), and the positive areas in five fields of view per section were analyzed using ImageJ software (NIH, USA). As a result, it was confirmed that the area of ​​fibrosis in the mouse model kidney tissue was reduced by Y03m (Fig. 9B).

[0309]

[0310] Example 9-2: Efficacy evaluation test in liver fibrosis mice

[0311] Carbon tetrachloride (CCl₄)-induced liver fibrosis model is widely used to evaluate the efficacy and mechanism of therapeutic candidates. To evaluate the antifibrotic effect of Y03m in a CCl₄-induced liver fibrosis mouse model, IgG or Y03m was administered intraperitoneally at 5 mg / kg or 20 mg / kg, respectively, on days 0, 5, 10, 15, 20, and 25. Mice were sacrificed on day 28, and liver fibrosis indices were analyzed.

[0312] For visualization and quantitative analysis of collagen deposition, liver tissue from the left lateral lobe was prefixed in Bouin's solution, embedded in paraffin, and the sections were stained with picro-Sirius red solution (FUJIFILM Wako Pure Chemical Corporation, Japan). Sirius red staining is a method that specifically stains collagen fibers in liver tissue and can effectively visualize fibrotic areas. For quantitative analysis of the fibrotic area, bright-field images of Sirius red-stained tissue sections were captured in five random fields of view at 200x magnification using a digital camera (DFC280; Leica Microsystems, Germany). The acquired images were subjected to quantitative analysis of Sirius red-positive areas using ImageJ software (National Institute of Health, USA).

[0313] As a result, it was confirmed that the fibrotic area in the liver tissue of the liver fibrosis mouse model was reduced by Y03m (Fig. 9C).

[0314]

[0315] Example 10: Confirmation of species-specific reactivity of anti-free-β2m antibodies

[0316] To determine whether Y03m also shows reactivity to β2m proteins of animal species other than humans, cat and dog β2m recombinant proteins (MyBiosource, MBS1320466 / Cloud-Clone Corp., RPA260Ca01) were diluted in coating buffer (sodium bicarbonate buffer, pH 9.6) at concentrations (1,000, 333.3, 111.1, 37.04, 12.35, 4.16, 1.37, 0 ng / mL) and dispensed 50 μL per well into a 96-well plate. After coating for one day at 4°C, ELISA analysis was performed using the same procedure as in Example 2-6.

[0317] As a result, it was confirmed that Y03m also showed reactivity to β2m proteins of animal species other than humans (Fig. 10).

[0318]

[0319] The anti-β2m antibody according to the present invention specifically binds to free β2m protein dissociated from MHCI, and binds to both monomeric and amyloid forms of β2m. Since it was confirmed that free β2m monomeric and amyloid forms can be detected in the body fluids of an animal model of dementia and kidney, it can be usefully used as a diagnostic antibody for the above diseases. In addition, since the antibody protects against cognitive impairment induced by β2m in normal mice, and after intraperitoneal administration to a mouse model of dementia, it was confirmed that amyloid beta and inflammation in the brain were reduced, renal fibrosis and biomarkers were reduced in an animal model of renal fibrosis, and liver fibrosis was reduced in an animal model of liver fibrosis, it is useful for the prevention and / or treatment of the above diseases.

[0320]

[0321] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0322]

[0323] Electronic file attached.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to beta2-microglobulin (β2m).

2. An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof in claim 1 binds to an epitope comprising the amino acid sequence of SEQ ID NO:

59.

3. An antibody or antigen-binding fragment thereof, characterized in that the region containing the epitope in the second paragraph contains the amino acid sequence of SEQ ID NO: 49 or SEQ ID NO:

52.

4. An antibody or antigen-binding fragment thereof, characterized in that it comprises the following in the first paragraph: A heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 17; A heavy chain CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 18, and SEQ ID NO: 22; and A heavy chain variable region comprising a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 12, SEQ ID NO: 19, and SEQ ID NO: 23; and A light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6; A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7; and A light chain variable region comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

8.

5. An antibody or antigen-binding fragment thereof, characterized in that it comprises a heavy chain variable region and a light chain variable region selected from the group consisting of: (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (ii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 12; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; (iii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 17, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 18, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 19; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 8; and (iv) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 6, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 7, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

8.

6. An antibody or antigen-binding fragment thereof, characterized in that in paragraph 4, the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 13, SEQ ID NO: 20, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 36, SEQ ID NO: 38, and SEQ ID NO:

40.

7. An antibody or antigen-binding fragment thereof, characterized in that in paragraph 4, the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 15, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, and SEQ ID NO:

34.

8. An antibody or antigen-binding fragment thereof, characterized in that in paragraph 1, the antibody or antigen-binding fragment thereof does not bind to β2m bound to the MHCI alpha chain, but specifically binds only to free β2m.

9. An antibody or antigen-binding fragment thereof, characterized in that the antibody or antigen-binding fragment thereof in the 8th paragraph binds to both monomeric and amyloid forms of free β2m.

10. A nucleic acid encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9.

11. A recombinant expression vector comprising the nucleic acid of Article 10.

12. Cells transformed with the recombinant expression vector of Article 11.

13. A method for producing an anti-beta2-microglobulin (β2m) antibody or an antigen-binding fragment thereof, comprising a step of culturing the cell of claim 12.

14. An antibody-drug conjugate (ADC) in which a drug is conjugated to an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9.

15. A bispecific or multi-specific antibody comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 9.

16. A chimeric antigen receptor (CAR) comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 9.

17. An immune cell into which the chimeric antigen receptor of item 16 has been introduced.

18. A composition for diagnosing a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome, renal disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer, or inflammatory disease, comprising an antibody or antigen-binding fragment thereof of any one of claims 1 to 9.

19. A pharmaceutical composition for the prevention or treatment of a neurodegenerative disease, aging, Down syndrome, acquired immunodeficiency syndrome, renal disease, dialysis-related amyloidosis, liver fibrosis, pulmonary fibrosis, multiple myeloma, cancer, or inflammatory disease, comprising an antibody or antigen-binding fragment thereof of any one of claims 1 to 9.

20. An isolated peptide comprising an epitope of beta2-microglobulin (β2m), The above epitope is an isolated peptide comprising the amino acid sequence of SEQ ID NO: 59.

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