Antibody that binds specifically to the N-terminal region of lysyl-tRNA synthetase exposed on the cell membrane.

Abstract

the present invention relates to an antibody binding specifically to an n-terminal region of lysyl-trna synthetase which is exposed on the cell membrane and a use thereof and, more particularly, to an antibody having a certain complementary determining region (cdr) stipulated in the specification and binding specifically to an epitope including the sequence of seq id no: 97 in a lysyl-trna synthetase (krs) n-terminal, or a fragment thereof, a use of the antibody or a fragment thereof in metastasis suppression and cancer diagnosis, and a pharmaceutical composition for prevention or treatment of an immune cell migration-related disease. far superior in specific binding affinity to a krs n-terminal region exposed on the cell membrane, the antibody or fragments thereof according to the present invention are effective for cancer diagnosis, have anti-cancer and metastasis inhibition effects, and can be very useful for preventing or treating an immune cell migration-related disease.

Description

1 / 67 Antibody that binds specifically to the N-terminal region of lysyl-tRNA synthetase exposed on the cell membrane. TECHNICAL FIELD OF THE INVENTION

[01] The present application claims priority over Korean patent application No. 10-2017-0038775 filed on March 27, 2017 and Korean patent applications Nos. 10-2017-0118890 and 10-2017-0118917 filed on September 15, 2017, respectively, the entire specifications of which are incorporated herein by reference in their entirety.

[02] The present invention relates to an antibody or fragment thereof that specifically binds to an N-terminal region of extracellularly exposed lysyl-tRNA synthetase and use thereof and, more specifically, to an antibody or fragment thereof having particular complementarity determination region (CDR) sequences defined in this descriptive report and that specifically binds to an epitope containing the SEQ ID NO: 97 sequence in the N-terminal lysyl-tRNA synthetase (KRS), to the use of the antibody or fragment thereof for the inhibition of cancer metastasis, to the use of the antibody or fragment thereof for cancer diagnosis and to a pharmaceutical composition for the prevention or treatment of a disease related to immune cell migration. PREVIOUS TECHNIQUE

[03] Recent studies have established that human lysyl-tRNA synthetase (KRS), usually present in the cytosol, is translocated to the plasma membrane (cell membrane) to interact with a 67 kDa laminin receptor (67LR) present in the plasma membrane, thereby promoting the migration of tumor (or cancer) cells to affect cancer metastasis (Dae Gyu Kim et al., Chemical inhibition of prometastatic lysyl-tRNA synthetase laminin receptor interaction, Nat Chem Biol. 2014 Jan; 10(1): 2934, Dae Gye Kim et. al. Interaction of two translational components, lysyl-tRNA synthetase and p40 / 37LRP, in plasma membrane promotes laminin-dependent cell migration, FASEB J. (2012)26, 41424159). Human KRS (GenBank accession number NP_005539.1, etc.) comprises an N-terminal extension (1-72), an anticodon-binding domain (73-209), and a catalytic domain (220-597). Human KRS is an essential enzyme for... Petition 870240042040, dated 05 / 17 / 2024, page 85 / 162 2 / 67 protein synthesis and normally resides within the multi-tRNA synthetase complex (MSC) in the cytosol. However, after the introduction of laminin signaling, p38 MAPK phosphorylates KRS at T52 residues and KRS is translocated to the cell membrane, where KRS protects 67LR against ubiquitin-mediated degradation. It has also been reported that KRS translocated to the cell membrane accelerates cancer metastasis by stabilizing and interacting with cancer metastasis-associated 67LR.

[04] Meanwhile, immune cells are involved in a primary defense mechanism in the body, but over-activation of immune cells has recently been reported as one of the main pathogeneses. Increased mobility of immune cells is normally observed upon activation of inflammatory immune cells and, specifically, such migration and invasion of immune cells are reported as being intimately involved in the disease pathology in the following diseases.

[05] For example, a cardiovascular disease whose lesions occur in the heart and major arteries includes atherosclerosis and coronary artery disease (Ross R et al., New Engl J Med, 1999;340(2):115-26, Poli G et al., Redox Biol 2013;1(1):125-30, Libby P et al., Circulation 2002;5;105(9):1135-43). Atherosclerosis is an inflammatory disease triggered by cholesterol and is caused by atheroma composed of cholesterol deposited on the inner membrane of an artery and immune cells that migrate from the blood into the artery. That is, atheroma is formed by the migration of immune cells, such as monocytes, to a site where oxidized cholesterol causes inflammation. The formation of atheroma roughens the inner surface of blood vessels and thickens the vessel walls, thus narrowing the internal diameter of the blood vessels and resulting in circulatory disorders.The rupture of the fibrous membranes surrounding the atheroma causes thrombi in the blood vessels and bleeding within the atheroma, thus rapidly narrowing the inner diameter of the blood vessels or causing blockages. This occurs primarily in blood vessels supplying blood to the heart, brain, kidneys, and peripheral blood vessels. Petition 870240042040, dated 05 / 17 / 2024, page 86 / 162 3 / 67 in this way, causing ischemic heart disease, cerebrovascular ischemic disease (stroke), kidney failure, and ischemic arterial disease of the limbs. It was known in the past that the chemokine ligand CC 2 (CCL2, MCP-1), which causes an inflammatory response by inducing monocyte migration, plays an important role in the occurrence and development of such cardiovascular diseases and, therefore, new measures to treat such cardiovascular diseases by inhibiting the action of CCL2 and the resulting monocyte migration have been suggested (Gu L et al., Mol Cell, 1998;2(2):275-81, Aiello RJ et al., Arterioscler Thromb Vasc Biol 1999;19(6):151825, Gosling J1 et al., Clin Invest 1999;103(6):773-8, Harrington JR et al., Stem Cells 2000;18(1):65-6, Ikeda U et al., Clin Cardiol 2002;25(4):143-7).

[06] Pulmonary arterial hypertension (PAH) is classified as Group 1 in the World Health Organization’s (WHO) clinical classification system (ESC Guidelines, European Heart Journal 2015) and is a rare disease clinically characterized by shortness of breath, an increase in mean pulmonary arterial pressure (mPAP, mPAP > 25 mm Hg) and right ventricular dysfunction. Several pre-existing factors, such as heredity, infection and related diseases, are involved in such pulmonary arterial hypertension, but the immune response resulting from endothelial cell injury has been known to act as a key pathological factor (Huertas et al., Circulation, 129:1332-1340, 2014).Regarding this phenomenon, a series of processes related to the invasion and dysfunction of immune cells have been known to be deeply associated with pathological phenomena, and especially the interaction between immune cells and vascular endothelial cells is known to be important in PAH. It has also been reported that monocyte and macrophage invasion accelerates the progression of Alport syndrome.

[07] In fibrosis-related diseases, continuous (chronic) inflammatory responses activate the wound healing program, leading to fibrosis. After tissue injury, inflammatory immune cells, such as monocytes / macrophages, neutrophils, eosinophils, and mast cells, rapidly invade the injured site while being activated and secreting various cytokines, which in turn activate surrounding fibroblasts, epithelial cells, or smooth muscle cells into cells Petition 870240042040, dated 05 / 17 / 2024, page 87 / 162 4 / 67 of the myoblast type, and these myoblast-type cells produce and secrete extracellular matrix proteins in large quantities, ultimately causing the accumulation of extracellular matrix proteins in large quantities and resulting in scar formation and fibrosis or hypertrophy of the tissue (Gurtner GC et al., Trends Cell Biol. 15: 599-607, 2005). This pathology is one of the fundamental causes of: scar formation in skin tissues, caused by skin wounds due to cuts, burns, pressure sores and the like; or sclerosing fibrosis of the liver, renal, vascular and pulmonary tissues. Fibrosis is also known to be a major pathological feature in chronic autoimmune diseases such as scleroderma, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis and systemic lupus erythematosus.It is also known that the activation of inflammatory immune cells contributes to pathology in atopic diseases, asthma, COPD, psoriasis, keloids, proliferative retinopathy, and similar conditions.

[08] Especially in the wound healing program, activated fibroblasts in myoblast-like cells are termed myofibroblasts. Since myofibroblasts are at the center of all fibrosis-associated disease pathologies, eliminating molecular or immunological mechanisms inducing myofibroblast activity is a key element in disease treatment. It is widely known that many types of innate or adaptive immunity are important in the activation and differentiation of fibroblasts, and therefore, eliminating an inflammatory response at the wound site is a key factor in halting tissue remodeling into fibrosis and maintaining normal tissue morphology. However, since the inflammatory response is not easily eliminated in practice, understanding the mechanisms of innate and adaptive immunity to find key mediators is important in delaying fibrosis.

[09] In some cases, monocytes, macrophages, and similar cells contribute to wound healing, but they secrete reactive oxygen, nitrogen, and similar substances, thus having detrimental effects on surrounding cells. Therefore, monocytes and macrophages, if not quickly removed, cause further tissue damage, resulting in fibrosis. Therefore, restricting monocytes and macrophages, which respond first in the early stages of the disease, is considered a Petition 870240042040, dated 05 / 17 / 2024, page 88 / 162 5 / 67 therapeutic strategy for various chronic inflammation- and fibrotic-related diseases.

[010] It is known that when the wound healing mechanism triggers a fibrosis response, platelet-derived growth factor (PDGF) associated with hemagglutination recruits other inflammatory immune cells into the wound site and TGF-β1 accelerates the synthesis of the extracellular matrix from local fibroblasts. However, it has been reported that factors involved in hemagglutination induce fibrosis even when the factors are deficient.

[011] Meanwhile, the fact that Myc-KRS41-597 (ΔN) with a deletion of 40 terminal residues in the N-terminal extension (N-ext) is not localized to the plasma membrane indicates that the N-ext region of KRS is an essential region in the translocation of KRS to the cell membrane. Regarding cancer metastasis specifically, it is known that the N-ext region of KRS is involved in the binding of KRS and 67LR in the interaction of the latter. To use this fact for therapeutic or diagnostic purposes, it is necessary to specifically target a particular site (especially the N-ext of KRS) in the KRS protein according to the characteristics of various domains that constitute the KRS protein.

[012] However, despite the importance of aminoacyl-tRNA synthetases (ARSs) including KRS as biomarkers, ARSs are similar in view of protein structure and, thus, antibodies obtained by immunizing animals with one ARS protein show cross-reactivity, for example, binding with other ARSs and, in many cases, highly sensitive antibodies are not even produced.

[013] In diseases caused by excessive activation of immune cells, as mentioned above, target factors for preventing the translocation (and invasion) of immune cells have been conventionally suggested, and attempts have been made to divert therapeutic methods to treat diseases that regulate target factors, but the respective limitations of this are being reported. Therefore, for the effective treatment of the disease, it is still a critical challenge to establish which is the key mediator and which strategy will control the key mediator in mitigating immune cells. DETAILED DESCRIPTION OF THE INVENTION Petition 870240042040, dated 05 / 17 / 2024, page 89 / 162 6 / 67 TECHNICAL PROBLEM

[014] While studying to construct an antibody that specifically binds to an extracellularly exposed N-terminal region of KRS, the present inventors found that antibodies having particular complementarity-determining region (CDR) sequences, defined in this descriptive report, showed very high binding specificity and affinity with the N-terminal region of KRS as well as inhibited cancer metastasis in vivo.Additionally, the present inventors have found that an increase in the level of KRS in the cell membrane of immune cells (monocytes / macrophages) is an important pathological phenomenon in diseases related to the migration and invasion of immune cells, and thus KRS has a particular correlation with laminin (especially laminin subtype α4β2γ1), and they have found that N-terminal KRS-binding antibodies provided in the present invention reduced the increased level of KRS in the cell membrane of immune cells and effectively inhibited the migration and infiltration of immune cells, and thus had a treatment effect on the related diseases; therefore, the present inventors have completed the present invention.

[015] Therefore, one aspect of the present invention is to provide an antibody or fragment thereof that binds specifically to an epitope containing the SEQ ID NO: 97 sequence at the N-terminal lysyl-tRNA synthetase (KRS).

[016] Another aspect of the present invention is to provide a polynucleotide encoding the antibody or fragment thereof of the present invention, a recombinant expression vector comprising the polynucleotide and a cell transformed with the recombinant vector.

[017] Yet another aspect of the present invention is to provide a method for producing an antibody or fragment thereof that binds specifically to an N-terminal region of extracellularly exposed lysyl-tRNA synthetase, the method comprising: (a) transforming host cells with the recombinant expression vector; (b) incubating the transformed host cells to produce an antibody or fragment thereof; and (c) collecting the antibody or fragment thereof produced in the host cells. Petition 870240042040, dated 05 / 17 / 2024, pp. 90 / 162 7 / 67

[018] Yet another aspect of the present invention is to provide a pharmaceutical composition comprising the antibody or fragment thereof of the present invention as an active ingredient for the inhibition of cancer metastasis.

[019] Yet another aspect of the present invention is to provide a pharmaceutical composition consisting of the antibody or fragment thereof of the present invention for the inhibition of cancer metastasis.

[020] Yet another aspect of the present invention is to provide a pharmaceutical composition consisting essentially of the antibody or fragment thereof of the present invention for the inhibition of cancer metastasis.

[021] Yet another aspect of the present invention is to provide a composition comprising the antibody or fragment thereof of the present invention as an active ingredient for cancer diagnosis.

[022] Yet another aspect of the present invention is to provide a composition consisting of the antibody or a fragment thereof of the present invention for cancer diagnosis.

[023] Yet another aspect of the present invention is to provide a composition consisting essentially of the antibody or fragment thereof of the present invention for cancer diagnosis.

[024] Yet another aspect of the present invention is to provide a pharmaceutical composition comprising the antibody or fragment thereof of the present invention as an active ingredient for the prevention or treatment of a disease related to immune cell migration.

[025] Yet another aspect of the present invention is to provide a pharmaceutical composition consisting of the antibody or fragment thereof of the present invention for the prevention or treatment of a disease related to the migration of immune cells.

[026] Yet another aspect of the present invention is to provide a pharmaceutical composition consisting essentially of the antibody or fragment thereof of the present invention for the prevention or treatment of a disease related to the migration of immune cells. Petition 870240042040, dated 05 / 17 / 2024, pp. 91 / 162 8 / 67

[027] Yet another aspect of the present invention is to provide use of the antibody or fragment thereof of the present invention for the preparation of an agent for the inhibition of cancer metastasis.

[028] Yet another aspect of the present invention is to provide a method for inhibiting cancer metastasis in an individual in need thereof, the method comprising administering the antibody or fragment thereof of the present invention to the individual in an amount effective for inhibiting cancer metastasis.

[029] Yet another aspect of the present invention is to provide use of the antibody or fragment thereof of the present invention for the preparation of an agent for cancer diagnosis.

[030] Yet another aspect of the present invention is to provide a method for diagnosing cancer in an individual in need thereof, the method comprising administering the antibody or fragment thereof of the present invention to the individual in an amount effective for cancer diagnosis.

[031] Yet another aspect of the present invention is to provide use of the antibody or fragment thereof of the present invention for the preparation of an agent for the treatment of a disease related to the migration of immune cells.

[032] Yet another aspect of the present invention is to provide a method for treating a disease related to the migration of immune cells in an individual in need thereof, the method comprising administering the antibody or fragment thereof of the present invention to the individual in an amount effective for treating a disease related to the migration of immune cells. TECHNICAL SOLUTION

[033] According to one aspect of the present invention, an antibody or fragment thereof is provided that binds specifically to an epitope containing the SEQ ID NO: 97 sequence at the N-terminal lysyl-tRNA synthetase (KRS).

[034] According to another aspect of the present invention, a polynucleotide encoding the antibody or fragment thereof of the present invention, a recombinant expression vector comprising the polynucleotide, and a cell transformed with the recombinant vector are provided. Petition 870240042040, dated 05 / 17 / 2024, pp. 92 / 162 9 / 67

[035] According to yet another aspect of the present invention, a method is provided for producing an antibody or fragment thereof that binds specifically to an N-terminal region of extracellularly exposed lysyl-tRNA synthetase, the method comprising: (a) transforming host cells with the recombinant expression vector; (b) incubating the transformed host cells to produce an antibody or fragment thereof; and (c) collecting the antibody or fragment thereof produced in the host cells.

[036] According to yet another aspect of the present invention, a pharmaceutical composition is provided comprising the antibody or fragment thereof of the present invention as an active ingredient for inhibiting cancer metastasis.

[037] According to yet another aspect of the present invention, a pharmaceutical composition is provided consisting of the antibody or a fragment thereof of the present invention for the inhibition of cancer metastasis.

[038] According to yet another aspect of the present invention, a pharmaceutical composition is provided which consists essentially of the antibody or fragment thereof of the present invention for the inhibition of cancer metastasis.

[039] According to yet another aspect of the present invention, a composition comprising the antibody or a fragment thereof of the present invention is provided as an active ingredient for cancer diagnosis.

[040] According to yet another aspect of the present invention, a composition is provided consisting of the antibody or a fragment thereof of the present invention for cancer diagnosis.

[041] According to yet another aspect of the present invention, a composition is provided which consists essentially of the antibody or fragment thereof of the present invention as an active ingredient for cancer diagnosis.

[042] According to yet another aspect of the present invention, a pharmaceutical composition is provided comprising the antibody or fragment thereof of the present invention as an active ingredient for the prevention or treatment of a disease related to the migration of immune cells.

[043] According to yet another aspect of the present invention, a pharmaceutical composition is provided consisting of the antibody or a fragment thereof from Petition 870240042040, dated 05 / 17 / 2024, pp. 93 / 162 10 / 67 present invention for the prevention or treatment of a disease related to the migration of immune cells.

[044] According to yet another aspect of the present invention, a pharmaceutical composition is provided which consists essentially of the antibody or fragment thereof of the present invention for the prevention or treatment of a disease related to the migration of immune cells.

[045] According to yet another aspect of the present invention, use of the antibody or fragment thereof of the present invention is provided for the preparation of an agent for the inhibition of cancer metastasis.

[046] According to yet another aspect of the present invention, a method is provided for inhibiting cancer metastasis in an individual in need thereof, the method comprising administering the antibody or fragment thereof of the present invention to the individual in an amount effective for inhibiting cancer metastasis.

[047] According to yet another aspect of the present invention, the antibody or fragment thereof of the present invention is used for the preparation of an agent for cancer diagnosis.

[048] According to yet another aspect of the present invention, a method is provided for the diagnosis of cancer in an individual in need thereof, the method comprising administering the antibody or fragment thereof of the present invention to the individual in an amount effective for the diagnosis of cancer.

[049] According to yet another aspect of the present invention, use is provided for the antibody or fragment thereof of the present invention for the preparation of an agent for the treatment of a disease related to the migration of immune cells.

[050] According to yet another aspect of the present invention, a method is provided for treating a disease related to the migration of immune cells in an individual in need thereof, the method comprising administering the antibody or fragment thereof of the present invention to the individual in an amount effective for the treatment of a disease related to the migration of immune cells. Petition 870240042040, dated 05 / 17 / 2024, pp. 94 / 162 11 / 67

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

[052] As used herein, the term “extracellularly exposed N-terminal lysyl-tRNA synthetase region” refers to a particular sequence exposed to the extracellular space or on the cell membrane surface when KRS produced in cells is translocated to the cell membrane (or plasma membrane) and may normally refer to a partial or full-length sequence of a 1 to 72 amino acid region in the N-terminal of KRS. In addition, there is sequence similarity between species in the N-terminal region of KRS and, especially, the N-terminal region of KRS may contain the amino acid sequence defined by SEQ ID NO: 97. Preferably, the N-terminal region of KRS contains the sequence defined by SEQ ID NO: 75 for humans, the sequence defined by SEQ ID NO: 113 for mice, and the sequence defined by SEQ ID NO: 114 for rats.

[053] As used herein, the term “KRS” refers to the full-length polypeptide known as lysyl-tRNA synthetase or any KRS fragment sequence comprising the N-terminal region. As described above, antibodies or fragments thereof according to the present invention specifically detect the extracellularly exposed N-terminal region of KRS and thus can also detect the aforementioned full-length KRS polypeptide or any KRS fragment sequence containing the N-terminal region. The specific KRS sequence is not particularly limited provided that the sequence contains the polypeptide defined by SEQ ID NO: 75 and is known as lysyl-tRNA synthetase in the art. For example, KRS of the present invention includes: a sequence derived from a human (Homo sapiens) and known as NCBI (Genbank) Accession No. NP_005539.1 or the like; a sequence derived from a mouse (Mus musculus) and known as NCBI (Genbank) Accession No. NP_444322.1 or similar; and a sequence derived from a rat (Rattus norvegicus) and known as NCBI (Genbank) Accession No. XP_006255692.1 or similar and, in addition, reference may be made to, but is not limited to, the following sequence information: XP_005004655.1 (guinea pig: Cavia porcellus), XP_021503253.1 (squirrel, Meriones unguiculatus), XP_002711778.1 (rabbit, Oryctolagus cuniculus), XP_536777.2. Petition 870240042040, dated 05 / 17 / 2024, pp. 95 / 162 12 / 67 (puppy, Canis lupus familiaris), XP_003126904.2 (pig, Sus scrofa), XP_011755768.1 (monkey, Macaca nemestrina), XP_008984479.1 (marmoset, Callithrix jacchus), XP_019834275.1 (cow, Bos indicus) and XP_511115.2 (chimpanzee, Pan troglodytes). With maximum preference, KRS can be a polypeptide consisting of the amino acid sequence defined by SEQ ID NO: 76 (Genbank Accession No. NP_005539.1).

[054] In the present invention, the antibody is also referred to as immunoglobulin (Ig) and is a generic term for proteins that are involved in biological immunity by acting selectively on antigens. A whole antibody found in nature usually consists of two pairs of light chain (LC) and heavy chain (HC), each of which is a polypeptide composed of several domains or has two HC / LC pairs as a basic unit. There are five types of heavy chains that constitute mammalian antibodies, which are denoted by the Greek letters: α, δ, ε, γ and μ, and different types of heavy chains constitute different types of antibodies: IgA, IgD, IgE, IgG and IgM, respectively. There are two types of light chains that constitute mammalian antibodies, which are denoted by λ and κ.

[055] The heavy and light chains of antibodies are structurally divided into a variable region and a constant region according to the variability of the amino acid sequence. The constant region of the heavy chain is composed of three or four constant regions of the heavy chain, such as CH1, CH2, and CH3 (IgA, IgD, and IgG antibodies) and CH4 (IgE and IgM antibodies), according to the type of antibody, and the light chain has a constant region CL. The variable regions of the heavy and light chains are each composed of a domain of a variable region of the heavy chain (VH) or a variable region of the light chain (VL). The light chain and the heavy chain are linked to each other by a covalent disulfide bond, while the variable and constant regions of the heavy chain are arranged in parallel, and two heavy chain molecules, which are linked to the light chains, are linked to each other by two covalent disulfide bonds, thus forming a complete antibody.The whole antibody binds specifically to an antigen through the variable regions of the heavy and light chains. The whole antibody is composed of two pairs of chains. Petition 870240042040, dated 05 / 17 / 2024, pp. 96 / 162 13 / 67 heavy and light (HC-LC) and thus a whole antibody molecule has divalent monospecificity in which a whole antibody molecule binds to two of the same antigens through two variable regions.

[056] The antibody variable regions, which comprise antigen-binding sites, are each divided into framework regions (FRs) with low sequence variability and complementarity-determining regions (CDRs), which are hypervariable regions with high sequence variability. In VH and VL, three CDRs and four FRs are arranged in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 in a direction from the N-terminal to the C-terminal. The CDRs, which have the highest sequence variability in the antibody variable regions, are sites that bind directly to an antigen and are very important in the antigenic specificity of the antibody.

[057] The present invention provides an antibody or fragment thereof that binds specifically to an epitope containing the SEQ ID NO: 97 sequence at the N-terminal lysyl-tRNA synthetase (KRS).

[058] As used herein, “epitope” refers to a protein determinant capable of specifically binding to an antibody. An epitope is usually composed of surface groups of molecules, such as amino acids or sugar side chains, and usually has specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished from each other by the fact that binding to conformational epitopes, but not to non-conformational epitopes, is lost in the presence of denaturing solvents.An epitope may comprise amino acid residues directly involved in binding (also called the immunogenic component of the epitope) and other amino acid residues not directly involved in binding, for example, amino acid residues effectively blocked by the antigen-specific binding peptide (in other words, the amino acid residue being within the occupied area of ​​the antigen-specific binding peptide).

[059] Preferably, the epitope is a site to which the N3 monoclonal antibody of the present invention derived from the N-terminal KRS sequence binds, and the specific sequence thereof is not particularly limited since the sequence is Petition 870240042040, dated 05 / 17 / 2024, pp. 97 / 162 14 / 67 a consecutive region comprising amino acids (klsknelkrrlka) defined by SEQ ID NO: 97 and may usually consist of a sequence of 13-52 amino acids, more preferably a sequence of 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, or 42 amino acids, comprising the amino acid sequence of SEQ ID NO: 97.

[060] Preferably, the epitope of the present invention may include the amino acid sequences defined by SEQ ID NO: 75, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101, which are derived from the human N-terminal of KRS; the amino acid sequences defined by SEQ ID NO: 113, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105 and SEQ ID NO: 106, which are derived from the mouse N-terminal of KRS; and the amino acid sequences defined by SEQ ID NO: 114, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110 and SEQ ID NO: 111, which are derived from the rat N-terminal of KRS. The epitope may most preferably be the amino acid sequence at positions 15 to 29 in the N-terminal region of human KRS defined by SEQ ID NO: 75 (SEQ ID NO: 75, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100 and SEQ ID NO: 101) and with maximum preference the amino acid sequence at positions 15 to 42 in the N-terminal region of human KRS defined by SEQ ID NO: 75 (SEQ ID NO: 101).

[061] The “antibody or fragment thereof that binds specifically to an extracellularly exposed N-terminal region of KRS” provided in the present invention comprises:

[062] a variable heavy chain region (VH) comprising: heavy chain complementarity determination region 1 (CDR1) containing the selected amino acid sequence from the group consisting of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 25 and SEQ ID NO: 37; heavy chain complementarity determination region 2 (CDR2) containing the selected amino acid sequence from SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 27 and SEQ ID NO: 39; and heavy chain complementarity determination region 3 (CDR3) containing the selected amino acid sequence from SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 29 and SEQ ID NO: 41; Petition 870240042040, dated 05 / 17 / 2024, pp. 98 / 162 15 / 67

[063] a variable light chain (VL) region comprising: light chain complementarity determination region 1 (CDR1) containing the selected amino acid sequence from the group consisting of SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 31 and SEQ ID NO: 43; light chain complementarity determination region 2 (CDR2) containing the selected amino acid sequence from SEQ ID NO: 9, SEQ ID NO: 21, SEQ ID NO: 33 and SEQ ID NO: 45; and light chain complementarity determination region 3 (CDR3) containing the selected amino acid sequence from SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 35 and SEQ ID NO: 47.

[064] Antibodies composed of CDR sequences have an excellent ability to bind specifically to the extracellularly exposed N-terminal region of KRS. This characteristic is well described in the examples of this descriptive report. In one example of the present invention, to construct scFv fragments that bind specifically to the extracellularly exposed N-terminal region of KRS, a total of five experimental steps were performed, starting from primary screening through screening of the scFv phage library to indirect ELISA (secondary screening), western blotting (tertiary screening), immunoprecipitation (quaternary screening) and immunofluorescence staining (quinary screening) to select scFv fragments showing high binding specificity and binding affinity towards the N-terminal KRS binding.A total of 1920 scFv clones were selected in the primary screening through screening of the scFv phage library, but four fragment types, N3 scFv, N5 scFv, N7 scFv, and N9 scFv, which have the highest specificity, were ultimately selected through five screening steps. Furthermore, the scFv fragments were converted into IgG antibodies, thus constructing IgG antibodies N3, N5, N7, and N9, and these antibodies were also found to show high binding specificity in view of the N-terminal KRS binding.

[065] The antibodies or fragments thereof that bind specifically to the extracellularly exposed N-terminal region of KRS according to the present invention are antibodies having the following CDR conformations of variable regions of Petition 870240042040, dated 05 / 17 / 2024, page 99 / 162 16 / 67 heavy and light chain, where (i), (ii), (iii) and (iv) below indicate combinations of CDRs of N3, N5, N7 and N9 antibodies in the respective examples: (1) a variable heavy chain region comprising heavy chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 3 and heavy chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 5 and a variable light chain region comprising light chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 7, light chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 9 and light chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 11; (2) a variable heavy chain region comprising heavy chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 13, heavy chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 15 and heavy chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 17 and a variable light chain region comprising light chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 19, light chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 21 and light chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 23; (3) a variable heavy chain region comprising heavy chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 25, heavy chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 27 and heavy chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: Petition 870240042040, dated 05 / 17 / 2024, pp. 100 / 162 17 / 67 and a variable light chain region comprising light chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 31, light chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 33 and light chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 35; (4) a variable heavy chain region comprising heavy chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 37, heavy chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 39 and heavy chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 41 and a variable light chain region comprising light chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 43, light chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 45 and light chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 47.

[066] Most preferably, the antibodies or fragments thereof according to the present invention are characterized by comprising the following variable heavy chain and light chain regions: in the antibodies or fragments thereof, the variable heavy chain region contains the amino acid sequence selected from the group consisting of SEQ ID NO: 49 (N3 VH), SEQ ID NO: 53 (N5 VH), SEQ ID NO: 57 (N7 VH) and SEQ ID NO: 61 (N9 VH) and the variable light chain region contains the amino acid sequence selected from the group consisting of SEQ ID NO: 51 (N3 VL), SEQ ID NO: 55 (N5 VL), SEQ ID NO: 59 (N7 VL) and SEQ ID NO: 63 (N9 VL).

[067] The antibody comprising the variable heavy chain (VH) region and the variable light chain (VL) region may be an antibody comprising a heavy chain containing the amino acid sequence selected from the group consisting of SEQ ID NO: 77, SEQ ID NO: 81, SEQ ID NO: 85 and SEQ ID NO: 89 and Petition 870240042040, dated 05 / 17 / 2024, pp. 101 / 162 18 / 67 a light chain containing the amino acid sequence selected from the group consisting of SEQ ID NO: 79, SEQ ID NO: 83, SEQ ID NO: 87 and SEQ ID NO: 91.

[068] Most preferably, antibodies may comprise: a heavy chain containing the amino acid sequence defined by SEQ ID NO: 77 and a light chain containing the amino acid sequence defined by SEQ ID NO: 79; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 81 and a light chain containing the amino acid sequence defined by SEQ ID NO: 83; a heavy chain containing the amino acid sequence defined by SEQ ID NO: 85 and a light chain containing the amino acid sequence defined by SEQ ID NO: 87; and a heavy chain containing the amino acid sequence defined by SEQ ID NO: 89 and a light chain containing the amino acid sequence defined by SEQ ID NO: 91.

[069] The “antibody that binds specifically to the N-terminal region of KRS exposed extracellularly” according to the present invention is not limited to this type since the antibody has the CDR combinations or VH and VL combinations mentioned above. As a specific example, the antibody can be selected from the group consisting of IgG, IgA, IgM, IgE and IgD antibodies and may preferably be an IgG antibody.

[070] The antibodies of the present invention may be monoclonal antibodies or polyclonal antibodies since the antibodies have the CDR combinations or VH and VL combinations above that bind specifically to the N-terminal region of KRS, but are preferably monoclonal antibodies, which are a group of antibodies each having substantially identical amino acid sequences in heavy and light chains.

[071] The antibody of the present invention may be derived from any animals, including mammals, including humans and birds, and may preferably be derived from humans. However, the antibody of the present invention may be a chimeric antibody including a portion of the antibody derived from humans and a portion of the antibody derived from a different animal species. That is, the present invention includes all antibodies. Petition 870240042040, dated 05 / 17 / 2024, pp. 102 / 162 19 / 67 chimeric, humanized antibodies and human antibodies and may preferably be human antibodies.

[072] Furthermore, the antibody fragment of the present invention refers to an antibody fragment that retains the antigen-specific binding capacity of a whole antibody. Preferably, the fragment retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% of the N-terminal KRS binding affinity of the parent antibody. Specifically, the fragment may be in the form of Fab, F(ab)2, Fab', F(ab')2, Fv, diacorpo, scFv or the like.

[073] Fab (antigen-binding fragment) is an antigen-binding fragment of an antibody and is composed of a heavy chain and a light chain, each consisting of a variable domain and a constant domain. F(ab')2 is a fragment produced by the pepsin hydrolysis of an antibody, and F(ab')2 has a form in which two Fab molecules are linked by disulfide bonds in the heavy chain linkage region. F(ab') is a monomeric antibody fragment in which a heavy chain linkage is added to a separate Fab fragment F(ab')2 by reducing its disulfide bonds. Fv (variable fragment) is an antibody fragment composed of only the respective variable regions of the heavy and light chains. scFv (single-chain variable fragment) is a recombinant antibody fragment in which a variable heavy chain (VH) region and a variable light chain (VL) region are linked to each other by means of a flexible peptide linker.The term "diabody" refers to a fragment in which the VH and VL of scFv are linked by a very short ligand and thus cannot be linked to each other, instead linking to the VL and VH of another scFv in the same way, respectively, to form a dimer.

[074] For the purposes of the present invention, the antibody fragment is not limited to the structure or conformation thereof, since the antibody fragment retains binding specificity to the N-terminal region of KRS, but may preferably be scFv. The scFv according to the present invention has a CDR conformation or VH and VL conformation specific to the N-terminal region of KRS, and its sequence is not particularly limited since the C-terminal of VH and the N-terminal of VL are linked through a linker. The linker is not particularly limited to its type since it is known as a ligand. Petition 870240042040, dated 05 / 17 / 2024, pp. 103 / 162 20 / 67 applied to scFv in the technique, but it can be a peptide containing the amino acid sequence defined by SEQ ID NO: 65. Specifically, the scFv of the present invention can contain the amino acid sequence selected from the group consisting of SEQ ID NO: 67 (N3 scFv), SEQ ID NO: 69 (N5 scFv), SEQ ID NO: 71 (N7 scFv) and SEQ ID NO: 73 (N9 scFv).

[075] The antibody or fragment thereof of the present invention may comprise a conservative amino acid substitution (also referred to as a conservative variant of the antibody) that does not substantially change its biological activity.

[076] Furthermore, the antibody or fragment thereof mentioned above in the present invention may be conjugated to an enzyme, a fluorescent material, a radioactive material, and a protein, but is not limited to these. Similarly, methods of conjugating the above materials with the antibody have been well known in the art.

[077] The present invention provides a polynucleotide encoding the antibody or fragment thereof mentioned above according to the present invention.

[078] In this descriptive report, a polynucleotide may be described as an oligonucleotide or a nucleic acid and includes: DNA or RNA analogs (e.g., peptide nucleic acids and unnaturally occurring nucleotide analogs) generated using DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA) or nucleotide analogs; and hybrids thereof. A polynucleotide may be single-stranded or double-stranded.

[079] The polynucleotide refers to a nucleotide sequence that encodes an antibody composed of heavy and light chains, each having a specific CDR conformation or VH and VL conformation of the N-terminal region of KRS. The polynucleotide of the present invention is not particularly limited to the sequence mentioned above, as the sequence encodes the antibody or fragment thereof of the present invention. The polynucleotides encoding the CDR sequences mentioned above in the antibodies described above according to the present invention are not particularly limited to the sequences mentioned above, but may preferably contain the nucleotide sequence defined by SEQ ID NO: 2 Petition 870240042040, dated 05 / 17 / 2024, pp. 104 / 162 21 / 67 (heavy chain CDR1), SEQ ID NO: 4 (heavy chain CDR2), SEQ ID NO: 6 (heavy chain CDR3), SEQ ID NO: 8 (light chain CDR1), SEQ ID NO: 10 (light chain CDR2), SEQ ID NO: 12 (light chain CDR3), SEQ ID NO: 14 (heavy chain CDR1), SEQ ID NO: 16 (heavy chain CDR2), SEQ ID NO: 18 (heavy chain CDR3), SEQ ID NO: 20 (light chain CDR1), SEQ ID NO: 22 (light chain CDR2), SEQ ID NO: 24 (light chain CDR3), SEQ ID NO: 26 (heavy chain CDR1), SEQ ID NO: 28 (heavy chain CDR2), SEQ ID NO: 30 (heavy chain CDR3), SEQ ID NO: 32 (CDR1 light chain), SEQ ID NO: 34 (CDR2 light chain), SEQ ID NO: 36 (CDR3 light chain), SEQ ID NO: 38 (CDR1 heavy chain), SEQ ID NO: 40 (CDR2 heavy chain), SEQ ID NO: 42 (CDR3 heavy chain), SEQ ID NO: 44 (CDR1 light chain), SEQ ID NO: 46 (CDR2 light chain), or SEQ ID NO: 48 (CDR3 light chain).

[080] Furthermore, the polynucleotides encoding the VH and VL mentioned above in the antibody according to the present invention are not particularly limited to the sequences thereto, but may preferably contain the nucleotide sequence defined by SEQ ID NO: 50 (VH), SEQ ID NO: 52 (VL), SEQ ID NO: 54 (VH), SEQ ID NO: 56 (VL), SEQ ID NO: 58 (VH), SEQ ID NO: 60 (VL), SEQ ID NO: 62 (VH) or SEQ ID NO: 64 (VL).

[081] In addition, the polynucleotide encoding the antibody fragment may preferably contain the nucleotide sequence of any one selected from the group consisting of SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72 and SEQ ID NO: 74, which encodes scFv fragments according to the present invention.

[082] The polynucleotides encoding the antibody or fragment thereof of the present invention can be obtained by a method known in the art. For example, based on DNA sequences encoding part or all of the antibody heavy and light chains or corresponding amino acid sequences, the polynucleotides can be synthesized by oligonucleotide synthesis methods known in the art, for example, a polymerase chain reaction (PCR) method. Petition 870240042040, dated 05 / 17 / 2024, pp. 105 / 162 22 / 67

[083] The present invention provides a recombinant expression vector comprising the polynucleotide encoding the antibody or a fragment thereof according to the present invention.

[084] As used herein, “recombinant” is used interchangeably with “genetic manipulation” and refers to the construction of a gene in a form that does not exist in nature, by the use of molecular cloning experiment techniques, such as genetic transformation, cleavage or linkage.

[085] As used herein, the term “expression” refers to the production of proteins or nucleic acids in cells.

[086] As used herein, the term “recombinant expression vector” is a vector that can express a target protein or nucleic acid (RNA) in a suitable host cell and refers to a gene construct comprising essential control elements that are operationally linked to be able to express a polynucleotide insert (gene). The term “operationally linked” refers to the functional linking of a nucleic acid expression control sequence and a nucleic acid sequence encoding a target protein or RNA so as to perform general functions, meaning the linking between them so as to allow a gene to be expressed by the expression control sequence. The expression control sequence refers to a DNA sequence that controls the expression of an operationally linked polynucleotide sequence in a particular host cell.Such an expression control sequence includes a promoter for transcription, any operator sequence for transcription control, a sequence to encode an appropriate mRNA ribosomal binding site, a sequence for transcription and translation termination control, an initiation codon, a termination codon, a polyadenylation A signal, an enhancer, and the like.

[087] The recombinant expression vector of the present invention is not particularly limited to this type, since the vector is commonly used in the field of cloning and examples of recombinant expression vectors include, but are not limited to, a plasmid vector, a cosmid vector, a bacteriophage vector, and a viral vector. Examples of plasmids may include plasmids Petition 870240042040, dated 05 / 17 / 2024, pp. 106 / 162 23 / 67 derived from Escherichia coli (pBR322, pBR325, pUC118, pUC119 and pET-22b(+)), plasmids derived from Bacillus subtilis (pUB110 and pTP5) and plasmids derived from yeast (YEp13, YEp24 and YCp50) and examples of viruses may include: animal viruses, such as retroviruses, adenoviruses or vaccinia viruses; and insect viruses, such as baculoviruses.

[088] The recombinant expression vector according to the present invention means a gene construct that is operationally linked so as to be able to express, in a suitable host cell, a polynucleotide encoding the antibody or fragment thereof of heavy and light chain compound having the CDR or VH and VL conformations mentioned above capable of specifically binding to the N-terminal region of KRS.

[089] The polynucleotides encoding antibody heavy and light chains according to the present invention may be contained in separate recombinant expression vectors, respectively, or may be contained in a recombinant expression vector. The present invention provides transformed cells with the recombinant expression vector described above.

[090] The cells of the present invention are not particularly limited to this type, as the cells can be used to express a polynucleotide encoding an antibody or a fragment thereof contained in the recombinant expression vector of the present invention. The cells (host cells) transformed with the recombinant expression vector according to the present invention can be prokaryotic cells (e.g., E. coli), eukaryotic cells (e.g., yeast or other fungi), plant cells (e.g., tobacco or tomato plant cells), animal cells (e.g., human cells, monkey cells, hamster cells, rat cells, mouse cells, or insect cells), or hybridomas derived therefrom. Preferably, the cells can be derived from mammals, including humans.

[091] Suitable exemplary prokaryotes for the present purpose include Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, for example, E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Petition 870240042040, dated 05 / 17 / 2024, pp. 107 / 162 24 / 67 Salmonella, for example, Salmonella typhimurium, Serratia, for example, Serratia marcescens and Shigella, as well as Bacilli, for example, B. subtilis and B. licheniformis, Pseudomonas, for example, P. aeruginosa and Streptomyces. The cells of the present invention are not particularly limited, since the cells may express the vector of the present invention, but may preferably be E. coli.

[092] Saccharomyces cerevisiae is most frequently used as a eukaryote for the cells of the present invention. However, several other genera, species and strains may be used, but are not limited to, for example, hosts of Schizosaccharomyces pombe; Kluyveromyces, such as K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, for example, hosts of Neurospora, Penicillium, Tolypocladium and Aspergillus, such as A. nidulans and A. niger.

[093] The term “transformation” refers to a modification of the genotype of a host cell due to the introduction of exotic polynucleotides and refers to the introduction of an exotic polynucleotide into a host cell regardless of the method used for the transformation. The exotic polynucleotide introduced into the host cell is either incorporated into and maintained in the host cell genome or is maintained without incorporation into it, and the present invention includes both.

[094] The recombinant expression vector capable of expressing the antibody or a fragment thereof that specifically binds to the N-terminal region of KRS according to the present invention can be introduced into cells for the production of the antibody or a fragment thereof by a method known in the art, for example, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE-dextran-mediated transfection, transfection mediated by Petition 870240042040, dated 05 / 17 / 2024, pp. 108 / 162 25 / 67 polybrene, electroporation, gene gun and known methods for introducing nucleic acids into cells and subsequently transforming the cells.

[095] The present invention provides a method for preparing an antibody or fragment thereof that binds specifically to an N-terminal region of extracellularly exposed lysyltRNA synthetase, the method comprising: (a) transformation of host cells with the recombinant expression vector; (b) incubation of the transformed host cells to produce an antibody or fragment thereof; (c) collection of the antibody or fragment thereof produced in the host cells.

[096] In step (a), in order to produce the antibody or fragment thereof according to the present invention, the host cells are transformed with the recombinant expression vector, in which the polynucleotide encoding the antibody or fragment thereof is operationally ligated.

[097] A person skilled in the art can perform this step by selecting the appropriate transformation method according to the host cells and recombinant expression vector selected as described above. Recombinant expression vectors comprising heavy and light chain nucleotide sequences can be co-transformed in the same host cell to allow the heavy and light chains to be expressed in one cell, or recombinant expression vectors comprising heavy and light chain nucleotide sequences can be transformed in separate host cells to allow the heavy and light chains to be expressed separately.

[098] In step (b), the transformed host cells are incubated to produce antibody heavy and light chain polypeptides or antibody fragment according to the present invention from the recombinant expression vector introduced into the host cells.

[099] The average composition, incubation conditions, and incubation time for the incubation of host cells can be appropriately selected according to a method commonly used in the art. Antibody molecules produced in the host cell can be accumulated in the cell cytoplasm, Petition 870240042040, dated 05 / 17 / 2024, pp. 109 / 162 26 / 67 can be secreted outside the cell or into the culture medium by a suitable signal sequence or can be targeted using a periplasm or similar. It is also preferable that the antibody according to the present invention has a functional conformation through protein refolding using a method known in the art so as to maintain binding specificity at the N-terminal of KRS. As for the production of IgG-type antibody, heavy and light chains can be expressed in separate cells and then contacted with each other in a separate step to constitute the whole antibody or heavy and light chains can be expressed in the same cell to form the whole antibody within the cell.

[0100] In step (c), the antibody or fragment thereof produced in the host cells is obtained.

[0101] A person skilled in the art may appropriately select and control the collection method by considering the polypeptide characteristics of the antibody or fragment thereof produced in the host cells, the characteristics of the host cells, the mode of expression, or whether or not the polypeptide is targeted. For example, the antibody or fragment thereof secreted into the culture medium may be collected by obtaining the culture medium in which the host cells are grown, removing impurities by centrifugation and the like. In order to, as needed, excrete the antibody present in specific organelles or cytoplasm in the cells out of the cells and collect the antibody, the cells may be lysed to a degree that does not affect the functional structure of the antibody or fragment thereof.The antibody obtained can be further subjected to a process of additional impurity removal and concentration, through chromatography, filtration using a filter, dialysis or similar methods.

[0102] The polypeptide in the manufacturing (production) method of the present invention may be the antibody or fragment thereof of the present invention itself and a polypeptide to which is attached yet another amino acid sequence other than the antibody or fragment thereof of the present invention. In this case, the amino acid sequence may be removed from the antibody or fragment thereof of the present invention by use of a method well known to a person skilled in the art. Petition 870240042040, dated 05 / 17 / 2024, pp. 110 / 162 27 / 67

[0103] The antibody or fragment thereof of the present invention binds specifically to the N-terminal region of KRS and is thus useful in diagnostic analysis for the detection and quantification of KRS proteins in, for example, particular cells, tissues, or serum. In particular, the extracellularly exposed N-terminal region of KRS can be specifically detected without cell lysis. Therefore, the present invention provides a method for the specific detection of an extracellularly exposed N-terminal region of lysyl-tRNA synthetase, the method comprising: contacting the antibody or fragment thereof with a sample; and detecting the antibody or fragment thereof.

[0104] The detection method of the present invention may comprise a sample preparation step, which must be measured for the presence or absence of KRS (or extracellularly exposed N-terminal KRS peptide) and the concentration thereof by using the antibody or fragment thereof according to the present invention (step (1)), before contact of the antibody or fragment thereof according to the present invention with the sample.

[0105] A person skilled in the art may appropriately select a known method of protein detection using an antibody and prepare a suitable sample for the selected method. Furthermore, the sample may be cells or tissues obtained by biopsy, blood, whole blood, serum, plasma, saliva, cerebrospinal fluid, or the like, collected from an individual to be examined for the presence or absence of cancer (especially breast cancer or lung cancer) or cancer metastasis. Examples of methods of protein detection using antibodies include, but are not limited to, western blotting, immune blotting, dot blotting, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, competitive binding assay, immunoprecipitation, and the like.For example, for western blotting, a preparation can be made by adding an additional buffer for electrophoresis to a sample or cell lysate, followed by boiling and immunohistochemistry; a treatment can be performed by immobilizing and blocking slices of cells or tissue, followed by blocking. Petition 870240042040, dated 05 / 17 / 2024, pages 111 / 162 28 / 67

[0106] Next, a contact step of the antibody or fragment thereof according to the present invention with the sample prepared in the step described above is performed (step (2)).

[0107] The antibody according to the present invention is an antibody or fragment thereof having the CDR or VH and VL conformations described above and specifically binds to the N-terminal region of KRS and specific types and sequence organization thereof are as described above.

[0108] The antibody or a fragment thereof may be labeled with a general detectable portion, for its “detection”. For example, the antibody or a fragment thereof may be labeled with a radioisotope or fluorescent label using the technique described in the literature [Current Protocols in Immunology, Volumes 1 and 2, 1991, Coligen et al., Ed. Wiley-Interscience, New York, NY, Pubs]. In addition, several enzyme-substrate labels are usable, and examples of enzymatic labels include: luciferase, such as drosophila luciferase and bacterial luciferase (U.S. Patent No. 4,737,456), luciferin, 2,3-dihydrophthalazine diionase, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, saccharide oxidase (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidase (e.g., uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.Techniques for conjugating enzymes to antibodies are described in, for example, the literature [O'Sullivan et al., 1981, Methods for the Preparation of Enzyme-Antibody Conjugates for use in Enzyme Immunoassay, in Methods in Enzym. (J. Langone & H. Van Vunakis, eds.), Academic press, NY, 73: 147-166]. Labels can be conjugated directly or indirectly to antibodies using various known techniques. For example, the antibody can be conjugated with biotin, and any labels belonging to the three generalized categories mentioned above can be conjugated with avidin or vice versa. Biotin can selectively bind to avidin, and therefore this label can be conjugated with an antibody in an indirect manner. Alternatively, in order to achieve indirect conjugation of a label with an antibody, the antibody can be conjugated with a small hapten (e.g., dioxin) and one of the different types of recited labels. Petition 870240042040, dated 05 / 17 / 2024, pages 112 / 162 29 / 67 above can be conjugated with an anti-hapten antibody (e.g., antidioxin antibody). Therefore, indirect conjugation of a label with an antibody can be achieved.

[0109] As used herein, “contact” is used in a general sense of this and refers to the mixing, binding, or touching of two or more substances. Contact may be carried out in vitro or in another container or may be carried out in situ, in vivo, in the individual, in the tissue, or in the cell.

[0110] Next, a detection step for the antibody or fragment thereof according to the present invention from the sample after performing step (2) is carried out (step (3)).

[0111] The “detection” is performed on a complex of the antibody or fragment thereof according to the present invention and an antigen, the complex being formed in the sample and refers to the detection of the presence or absence of the N-terminal KRS peptide (or a protein including the peptide, for example, KRS) or the measurement (including qualitative measurement, quantitative measurement or both) of the peptide level. Therefore, the detection method of the present invention may further comprise a step of removing extra antibodies or fragments thereof that did not form the complex with the N-terminal region of KRS, after performing step (2) before step (3) to be described later.

[0112] When the antibody or fragment thereof used in step (2) described above contains a detectable portion, such as fluorescence, radioactive isotope or enzyme, that directly labels the antibody or fragment thereof, detection may be performed by a detection method for the corresponding portion known in the art. For example, radioactivity may be measured by, for example, scintillation counting and fluorescence may be quantified using a fluorometer.

[0113] When the antibody or fragment thereof, per se, used in step (2) described above does not contain the detectable portion mentioned above, indirect detection using a labeled secondary antibody with fluorescence, radioactivity, enzyme or the like may be performed. The secondary antibody binds to the antibody or fragment thereof (primary antibody), according to the present invention. Petition 870240042040, dated 05 / 17 / 2024, pp. 113 / 162 30 / 67

[0114] Recent studies have established that human lysyl-tRNA synthetase (KRS) present in the cytosol is translocated to the plasma membrane (cell membrane) to interact with a 67 kDa laminin receptor (67LR) present in the plasma membrane, thereby promoting the migration of tumor (or cancer) cells to affect cancer metastasis (Dae Gyu Kim et al., Chemical inhibition of prometastatic lysyl-tRNA synthetaselaminin receptor interaction, Nat Chem Biol. 2014 Jan; 10(1): 2934.). Here, it is known that the N-terminal extension region of KRS (N-ext) is essential in the translocation of KRS to the cell membrane. Regarding cancer metastasis specifically, it is known that the Next region of KRS is involved in the binding of KRS and 67LR in the interaction of the latter.

[0115] The antibodies and fragments thereof according to the present invention are excellent in their ability to specifically bind to the N-ext region of KRS. In fact, the antibodies and fragments thereof according to the present invention bind to the N-ext region of KRS and thus inhibit binding (interaction) with a laminin receptor, thereby showing excellent ability to inhibit cancer metastasis. This is well described in the examples of the invention. One example in this descriptive report verified that, as a result of administering the antibody according to the present invention in in vivo cancer metastasis models with induced cancer, the antibody of the present invention showed excellent inhibitory ability of cancer metastasis in a dose-dependent manner.Specifically, the cancer metastasis-inhibiting capacity of the antibody of the present invention was excellent even compared with the compound YH16899, which is known to inhibit cancer metastasis by inhibiting the interaction between the laminin receptor (67LR) and KRS.

[0116] Therefore, the present invention provides a pharmaceutical composition for the inhibition of cancer metastasis and a composition for cancer diagnosis, each of the compositions comprising the antibody or fragment thereof mentioned above in the present invention as an active ingredient for the inhibition of cancer metastasis.

[0117] Additionally, the present invention provides a pharmaceutical composition for the inhibition of cancer metastasis and a composition for the diagnosis of Petition 870240042040, dated 05 / 17 / 2024, pp. 114 / 162 31 / 67 cancer, each of the compositions consisting of the antibody or fragment thereof mentioned above in the present invention.

[0118] Additionally, the present invention provides a pharmaceutical composition for the inhibition of cancer metastasis and a composition for cancer diagnosis, each of the compositions consisting essentially of the antibody or fragment thereof mentioned above in the present invention.

[0119] Cancer is not particularly limited to this type since cancer is known as a malignant tumor in the art and an example of this may be selected from the group consisting of breast cancer, large bowel cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, uterine cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer, kidney or ureter cancer,Renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma. Preferably, the cancer may be breast cancer or lung cancer.

[0120] The present invention provides a pharmaceutical composition comprising the antibody or fragment thereof of the present invention as an active ingredient for the prevention or treatment of a disease related to immune cell migration.

[0121] Additionally, the present invention provides a pharmaceutical composition consisting of the antibody or a fragment thereof of the present invention for the prevention or treatment of a disease related to the migration of immune cells.

[0122] Additionally, the present invention provides a pharmaceutical composition consisting essentially of the antibody or a fragment thereof of the present invention. Petition 870240042040, dated 05 / 17 / 2024, pp. 115 / 162 32 / 67 invention for the prevention or treatment of a disease related to the migration of immune cells.

[0123] As used herein, the term “immune cells” preferably refers to monocytes or macrophages.

[0124] As used herein, the term “immune cell migration-related disease” is not particularly limited to this specific type, since it is known in the art that excessive migration (and invasion) of immune cells is the main pathogenesis of the disease and that examples of this can be selected from the group consisting of a cardiovascular disease, a fibrotic disease, a chronic inflammatory disease and Alport syndrome.

[0125] Cardiovascular disease is not particularly limited to the following specific types of cardiovascular disease and it can be selected from the group consisting of pulmonary arterial hypertension, atherosclerosis, angina (angina pectoris), myocardial infarction, ischemic cerebrovascular disease, arteriosclerosis and mesenteric sclerosis.

[0126] Fibrotic disease is not particularly limited to the following specific types of fibrotic diseases and may be selected from the group consisting of scleroderma, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis, pulmonary fibrosis, hepatic fibrosis, hepatic cirrhosis, renal fibrosis, myofibrosis, cardiac fibrosis, systemic lupus erythematosus, hereditary fibrosis, infectious fibrosis (especially fibrosis caused by continuous infection), irritant fibrosis (fibrosis caused by repetitive exposure to irritants such as tobacco and toxic materials), fibrosis caused by chronic autoimmune fibrosis caused by antigenic incompatibility during organ transplantation, fibrosis due to hyperlipidemia, fibrosis due to obesity, diabetic fibrosis, fibrosis due to hypertension, and occlusion caused by fibrosis at stent insertion.

[0127] Chronic inflammatory disease may be selected from the group consisting of asthma, atopic dermatitis, eczema, psoriasis, osteoarthritis, gout, psoriatic arthritis, cirrhosis, non-alcoholic steatohepatitis, chronic obstructive pulmonary disease, rhinitis, diabetic retinopathy, diabetic renal failure, diabetic neuropathy and multiple sclerosis. Petition 870240042040, dated 05 / 17 / 2024, pp. 116 / 162 33 / 67

[0128] The pharmaceutical composition according to the present invention may comprise the antibody or a fragment thereof of the present invention alone or may further comprise at least one pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable” refers to a non-toxic composition that is physiologically acceptable, does not inhibit the action of an active ingredient when administered to humans, and does not normally cause an allergic response or similar responses, such as gastrointestinal problems and dizziness.

[0129] In the pharmaceutical composition according to the present invention, the antibody or fragment thereof can be administered in various oral and parenteral dosage forms during clinical administration. The antibody or fragment thereof, when formulated, can be prepared using a diluent or an excipient, such as a filler, extender, binder, wetting agent, disintegrant, or surfactant, which is commonly used. Solid formulations for oral administration include a tablet, pill, powder, granules, capsule, or similar form. These solid formulations can be prepared by mixing the antibody or fragment thereof of the present invention or a pharmaceutically acceptable salt thereof with at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, or gelatin. In addition, lubricants such as magnesium stearate and talc can be used alongside the simple excipients.Liquid formulations for oral administration include suspensions, solutions for internal use, emulsions, syrups, and similar products. In addition to simple diluents that are frequently used, such as water and liquid paraffin, various excipients, for example, a humectant, a sweetener, a flavoring, a preservative, and the like, may be contained in liquid formulations.

[0130] Exemplary formulations for parenteral administration include a sterile aqueous solution, a non-aqueous solvent, a suspension solvent, an emulsion, a lyophilization agent, and a suppository. The treatment composition of the present invention may be prepared in the form of a lyophilized cake or an aqueous solution for mixing and storing any vehicle. Petition 870240042040, dated 05 / 17 / 2024, pp. 117 / 162 34 / 67 physiologically acceptable, excipient or stabilizer (Remington: The Science and Practice of Pharmacy, 19th Edition, Alfonso, R., ed, Mack Publishing Co. (Easton, PA: 1995)) and an antibody with preferable purity.An acceptable vehicle, excipient, or stabilizer is non-toxic to a user at the dose and concentration used, and examples include: buffers, for example, phosphoric acid, citric acid, and other organic acids; antioxidants, including ascorbic acid; low molecular weight polypeptides (less than about 10 residues); proteins, for example, serum albumin, gelatin, or immunoglobulin; hydrophilic polymers, for example, polyvinylpyrrolidone; amino acids, for example, glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, for example, EDT; sugar alcohols, for example, mannitol or sorbitol; salt-forming counterions, for example, sodium; and / or nonionic surfactants, for example, Tween, Pluronics, or polyethylene glycol (PEG).

[0131] The antibody of the present invention can be administered in a pharmaceutically effective amount to an individual battling cancer or a disease related to immune cell migration. As used herein, the term “pharmaceutically effective amount” refers to an amount that shows a greater response compared to a negative control and preferably refers to an amount sufficient to treat cancer, an amount sufficient to inhibit cancer metastasis, and an amount sufficient to treat a disease related to immune cell migration. The total effective amount of the antibody or fragment thereof of the present invention can be administered to a patient as a single dose or can be administered by a fractionated treatment protocol, in which multiple doses are administered over a long period of time.The dose of the antibody or fragment thereof of the present invention to the human body can normally be 0.01-100 mg / kg / week, preferably 0.1-20 mg / kg / week and more preferably 510 mg / kg / week. However, regarding the dose of the antibody or fragment thereof of the present invention, an effective dose thereof in relation to a patient is determined by considering several factors, for example, the route of administration of the pharmaceutical composition, the number of times of treatment, the age of the patient. Petition 870240042040, dated 05 / 17 / 2024, pp. 118 / 162 35 / 67 patient, body weight, health condition and sex, disease severity, diet and excretion rate and, therefore, considering this fact, a person skilled in the art could determine an appropriate effective amount of the antibody or fragment thereof of the present invention according to the particular use as a cancer metastasis inhibitor. The pharmaceutical composition according to the present invention is not particularly limited to the dosage form, route of administration and method of administration thereof, since the composition shows effects of the present invention.

[0132] The route of administration of the composition of the present invention may be a known method of antibody administration, for example, injection or infusion via an intravenous, intraperitoneal, intracranial, subcutaneous, intramuscular, intraocular, intra-arterial, cerebrospinal or intralesional route, or injection or infusion via the sustained-release system described below. For example, the antibody of the present invention may be administered systemically or locally.

[0133] The pharmaceutical composition of the present invention can be used alone or in combination with surgery, hormone therapy, chemotherapy and methods using biological response control, for the prevention or treatment of cancer.

[0134] The pharmaceutical composition of the present invention can also be used alone or in combination with surgery, hormone therapy, chemotherapy and methods using biological response control, for the prevention or treatment of a disease related to immune cell migration.

[0135] The diagnosis and prognosis of cancer (or cancer metastasis) according to the present invention can be assessed by detecting KRS proteins (especially the extracellularly exposed N-terminal region of KRS) in the biological sample, and the diagnosis and prognosis of immune cell migration-related disease according to the present invention can be assessed by detecting KRS proteins (especially the extracellularly exposed N-terminal region of KRS) in the biological sample.

[0136] As used herein, the term “diagnosis” refers to the identification of the presence or characteristics of a pathological condition. In the present invention, the Petition 870240042040, dated 05 / 17 / 2024, pp. 119 / 162 36 / 67 Diagnosis is to identify the occurrence or probability (risk) of cancer and / or cancer metastasis or a disease related to the migration of immune cells.

[0137] The term “detection” is as described above, and the biological sample includes blood and other liquid samples having biological origins, biopsy specimens, solid tissue samples such as tissue cultures or cells derived therefrom. More specifically, examples of biological samples may include, but are not limited to, tissues, extracts, cell lysates, whole blood, plasma, serum, saliva, ocular fluid, cerebrospinal fluid, sweat, urine, milk, ascitic fluid, synovial fluid, peritoneal fluid, and the like. The sample may be obtained from animals, preferably mammals, and with the highest preference, humans. The sample may be pre-treated before use for detection. Examples of pre-treatment may include filtration, distillation, extraction, concentration, deactivation of interfering ingredients, reagent addition, and the like. In addition, nucleic acids and proteins isolated from the sample may be used for detection.

[0138] The antibody or fragment thereof according to the present invention may be provided as a diagnostic kit. The kit is not particularly limited to this type, as the kit is known in the art as an assay kit that provides a peptide having an antibody or a particular binding domain as a component, and examples thereof include a kit for western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, immunohistochemistry, immunoprecipitation assay, complement fixation assay, FACS, a protein microarray, or the like.

[0139] The antibody or fragment thereof of the present invention may be used in a kit, that is, a packaged combination of reagents in predetermined quantities with instructions for performing the diagnostic assay. Where the antibody is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme (e.g., a substrate precursor that provides the chromophore or fluorophore). In addition, other additives may be included such as stabilizers, buffers (e.g., a blocking buffer or lysis buffer) and the like. The relative quantities of various reagents may be Petition 870240042040, dated 05 / 17 / 2024, pages 120 / 162 37 / 67 vary widely to provide reagent solution concentrations that substantially optimize assay sensitivity. Reagents may be provided as dry powders, usually lyophilized, including excipients which, upon dissolution, will provide a reagent solution having an appropriate concentration.

[0140] The present invention provides use of the antibody or fragment thereof of the present invention for the preparation of an agent for the inhibition of cancer metastasis.

[0141] The present invention provides a method for inhibiting cancer metastasis in an individual in need thereof, the method comprising administering the antibody or fragment thereof of the present invention to the individual in an amount effective for inhibiting cancer metastasis.

[0142] The present invention provides use of the antibody or fragment thereof of the present invention for the preparation of an agent for cancer diagnosis.

[0143] The present invention provides a method for diagnosing cancer in an individual in need thereof, the method comprising administering the antibody or fragment thereof of the present invention to the individual in an amount effective for cancer diagnosis.

[0144] The present invention provides use of the antibody or fragment thereof of the present invention for the preparation of an agent for the treatment of a disease related to the migration of immune cells.

[0145] The present invention provides a method for treating a disease related to the migration of immune cells in an individual in need thereof, the method comprising administering the antibody or fragment thereof of the present invention to the individual in an amount effective for treating the disease related to the migration of immune cells.

[0146] As used herein, the term “effective quantity” refers to a quantity to show a cancer-relieving, treatment, prevention, detection or diagnostic effect or a cancer metastasis-inhibiting or metastasis-reducing effect and refers to a quantity to show a disease-relieving, treatment, prevention, detection or diagnostic effect related to immune cell migration. The term “individual” may be an animal, preferably a mammal, Petition 870240042040, dated 05 / 17 / 2024, pages 121 / 162 38 / 67 especially an animal including a human being and may be cells, tissue, an organ or similar derived from an animal. The individual may be a patient in need of the effect.

[0147] As used herein, the term “treatment” refers broadly to the relief of cancer, a cancer-related disease, or a disease related to the migration of immune cells, and may include the cure or substantially the prevention of such disease or the relief of a disease condition, and may include the relief, cure, or prevention of one or more of the symptoms resulting from cancer or a cancer-related disease, but is not limited to these.

[0148] As used herein, the term “comprising” is used synonymously with “containing” or “being characterized” and does not exclude additional ingredients or steps not mentioned in the composition or method. The term “consisting of” means the exclusion of additional elements, steps or ingredients not otherwise specified. The term “essentially consisting of” means including the elements or steps mentioned as well as any element or step that does not substantially affect the basic characteristics of the elements or steps mentioned in the scope of the compositions or methods. ADVANTAGEOUS EFFECTS

[0149] The antibodies or fragments thereof according to the present invention have particular complementarity-determining regions (CDRs) defined in this descriptive report and an excellent specific binding capacity to an N-terminal region of KRS exposed extracellularly. Additionally, the antibodies or fragments thereof according to the present invention are specifically targeted to the N-terminal region of KRS in vivo and thus inhibit the interaction between the laminin receptor and the N-terminal region of KRS, thereby exerting an excellent effect on the inhibition of cancer metastasis and can control the migration of immune cells, thus exerting a very considerable effect on the prevention, relief and treatment of a disease related to the migration of immune cells. BRIEF DESCRIPTION OF THE DRAWINGS Petition 870240042040, dated 05 / 17 / 2024, pages 122 / 162 39 / 67

[0150] Figure 1 shows the results of selection, using western blotting (WB), of scFv phage clones that bind to the full-length KRS sequence or N-terminal KRS fragments.

[0151] Figure 2 shows the results of selection, using immunoprecipitation (IP), of scFv phage clones that bind to the full-length KRS sequence or N-terminal KRS fragments.

[0152] Figure 3a shows the results, in which cells having an exposed N-terminal region of KRS on the cell membrane were constructed by expressing myc-KRS T52D (active mutant) and it was investigated whether the scFv N3, N5, N7 and N9 clones bound to the exposed region (KRS: meaning myc-KRS T52D).

[0153] Figure 3b shows that in the inactive mutant expression group (mycKRS T52A) or WT-KRS (untreated laminin), no detection signal was observed despite treatment with scFv N3, N5, N7 and N9 clones since the N-terminal region of KRS was not exposed on the cell membrane.

[0154] Figure 3c shows that when WT-KRS, T52D and T52A cells labeled with myc were treated with laminin and scFv staining was performed, staining was observed at similar sites in the membrane location of WT-KRS by laminin and mutant T52D and the transformed myc was also stained at the same site, but staining was not observed in T52A (scFv: green, myc: red).

[0155] Figure 4 shows the results of western blotting confirming whether scFv clones N3, N5, N7 and N9 specifically bound to the N-terminal of KRS using full-length KRS (denoted by F, SEQ ID NO: 76), a KRS fragment with amino acid deletions at positions 1-71 in the N-terminal (denoted by 1) and a KRS fragment composed of amino acid residues 1200 in the N-terminal (denoted by 2).

[0156] Figure 5a shows the western blotting results confirming the KRS binding capacity of IgG N3 and IgG N5 as representative antibodies of the present invention.

[0157] Figure 5b shows the immunoprecipitation confirmation results of the KRS binding capacity of IgG N3 and IgG N5 as representative antibodies of the present invention. Petition 870240042040, dated 05 / 17 / 2024, pp. 123 / 162 40 / 67

[0158] Figure 6a shows the SPR results of quantitative confirmation of the N-terminal KRS binding capacity of IgG N3.

[0159] Figure 6b shows the SPR results of quantitative confirmation of the N-terminal KRS binding capacity of IgG N5.

[0160] Figure 7a shows the results, in which cells transformed to express WT-KRS were treated with laminin to extracellularly expose the N-terminal region of KRS and then treated with the antibody of the present invention, IgG N3, to investigate the binding of IgG N3 to the exposed region by immunofluorescence staining.

[0161] Figure 7b shows the results, in which cells having an extracellularly exposed N-terminal region of KRS were constructed by expressing T52D-KRS (KRS labeled with an active mutant myc) and then treated with the antibody of the present invention, IgG N3, to investigate the binding of IgG N3 to the exposed region by immunofluorescence staining. It was also confirmed, through a cell membrane permeability test, that the antibody of the present invention translocates into cells and can bind to the KRS protein present inside the cells.

[0162] Figure 7c shows the results, in which cells transformed to express WT-KRS were treated with laminin to extracellularly expose the N-terminal region of KRS and then treated with the antibody of the present invention, IgG N5, to investigate the binding of IgG N5 to the exposed region by immunofluorescence staining (IgG N5: green).

[0163] Figure 8 shows the results of the MTT assay confirming that the antibody of the present invention IgG N3 had no cytotoxicity.

[0164] Figure 9a shows the confirmation results that cell migration was suppressed by treatment with the antibody of the present invention IgG N3.

[0165] Figure 9b shows confirmation results that the antibody of the present invention IgG N3 significantly suppressed cell migration in a dose-dependent manner.

[0166] Figure 10 schematically shows an experimental timeline for constructing mouse cancer metastasis models, administering a therapeutic substance (antibody or YH16899), and observing metastasis. Petition 870240042040, dated 05 / 17 / 2024, pages 124 / 162 41 / 67 lung in mouse models, in an experiment using in vivo cancer metastasis models.

[0167] Figure 11 shows mouse lung specimens capable of confirming that lung cancer metastasis was significantly suppressed by administration of the antibody of the present invention, IgG N3, in in-vivo cancer metastasis models. The degrees of progression and severity of cancer metastasis could be assessed from the count and condition of the nodules generated in the lung specimens.

[0168] Figure 12 shows the confirmation results that the generation of pulmonary nodules was significantly suppressed by the administration of the antibody of the present invention, IgG N3, compared with the control, in in-vivo cancer metastasis models (i.e., cancer metastasis in the lungs was significantly suppressed).

[0169] Figure 13 shows a comparative view of lung tissue in the control and IgG N3 treatment groups and confirmed that a significantly larger number of laminin receptors were expressed at metastasis nodule sites in the control group compared to the group treated with the antibody of the present invention.

[0170] Figure 14 shows lung specimens from the control group, YH16899 treatment group, and IgG N3 treatment group, and shows the results that lung nodule generation was significantly suppressed in the YH16899 treatment group and the IgG N3 treatment group compared to the control.

[0171] Figure 15 shows the efficiency of lung metastasis inhibition (pulmonary nodule formation inhibition efficiency) of the cancer metastasis inhibitor substances according to treatment concentration in the YH16899 treatment group and the IgG N3 treatment group.

[0172] Figure 16 shows the quantitative confirmation SPR results of the binding capacity of IgG N3 to N-terminal peptide fragments of human KRS (h) (F1, F2, F3, F4 and F5) (grey bars below the sequence indicate the binding capacity of N3 antibody to the corresponding regions (F1 to F5) and the darker the bar, the stronger the binding capacity). Petition 870240042040, dated 05 / 17 / 2024, pp. 125 / 162 42 / 67

[0173] Figure 17 shows the quantitative confirmation SPR results of the binding capacity of IgG N3 to N-terminal peptide fragments of KRS (F1, F3 and F5) from human (h), mouse (m) and rat (r) (grey bars below the sequences indicate the binding capacity of antibody N3 to the corresponding regions (F1 to F5) and the darker the bar, the stronger the binding capacity).

[0174] Figure 18a shows the results of the transcavity migration assay comparing the effects of collagen, fibronectin and laminin on immune cell (monocyte / macrophage) migration and provides microscopic images of the migrating cells.

[0175] Figure 18b is a graph showing cell counts measured (quantified) in the microscopic images of Figure 18a.

[0176] Figure 19a shows the results of the transcavity migration assay comparing the effects of various laminin subtypes (LN111, LN211, LN221, LN411, LN421, LN511 and LN521) on immune cell migration (monocyte / macrophage) and provides microscopic images of the migrating cells.

[0177] Figure 19b is a graph showing the cell count measured (quantified) in the microscopic images of Figure 19a.

[0178] Figure 19c shows the confirmatory western blotting results that KRS increased in the monocyte / macrophage membrane by treatment with LN421.

[0179] Figure 20a shows the results of the transcavity migration assay comparing the inhibitory effects of the antibody of the present invention, IgG N3, on the migration of LN421-specific monocytes / macrophages and provides microscopic images of the migrating cells.

[0180] Figure 20b is a graph showing the measured (quantified) cell counts in the microscopic images of Figure 20a.

[0181] Figure 20c shows the results of western blotting confirming that the KRS level increased by treatment with LN421 on the monocyte / macrophage membrane was reduced by treatment with the antibody of the present invention, IgG N3. Petition 870240042040, dated 05 / 17 / 2024, pp. 126 / 162 43 / 67

[0182] Figure 21a shows a change in right ventricular end-systolic pressure (RVESP) by administration of the antibody of the present invention, IgG N3, in pulmonary arterial hypertension (PAH) models (IgG in simulation: negative control, Ab 1 mpk: N3 antibody 1 mpk, Ab 10 mpk: N3 antibody 10 mpk, sildenafil: positive control).

[0183] Figure 21b shows a change in left ventricular end-systolic pressure (LVEPS) by administration of the antibody of the present invention, IgG N3, in models of pulmonary arterial hypertension (PAH) (IgG in simulation: negative control, Ab 1mpk: IgG N3 1 mpk, Ab 10 mpk: IgG N3 10 mpk, sildenafil: positive control).

[0184] Figure 22 shows the results of IHC staining confirming that administration of the antibody of the present invention, IgG N3, reduced the migration and invasion of immune cells in pulmonary arterial hypertension (PAH) models. METHOD FOR CARRYING OUT THE INVENTION

[0185] The present invention will now be described in detail.

[0186] However, the following examples are merely for illustration of the present invention and are not intended to limit the scope of the present invention. EXAMPLE 1 scFv Library Screening <1-1> phage screening scFv _ primary screening

[0187] In order to select scFv antibodies that bind specifically only to the N-terminal region of KRS (SEQ ID NO: 75) exposed extracellularly when KRS is translocated to the cell membrane by the laminin signal, in the full-length KRS sequence (SEQ ID NO: 76), repeated phage display panning was performed using the scFv phage library derived from HA-targeted human B cells. The scFv display phage library (library size: approx. 7.6 x 109, library produced by Prof. Hyunbo Shim) used in the present experiment is disclosed in Korean Patent No. 10-0961392. As shown in Table 1 below, full-length KRS sequences and KRS fragments with different particular N-terminal regions were used as antigenic proteins for repeated phage display panning. Petition 870240042040, dated 05 / 17 / 2024, pages 127 / 162 44 / 67

[0188] To an immune tube containing 1 ml of 1X PBS solution, 1-10 μg of antigenic proteins were added and incubated at 37 °C for 1 h at 200 rpm, thus coating the inner surface of the tube with antigens. The antigenic solution was drained and uncoated antigens were removed by washing once with running water. To prevent non-specific binding between antigenic proteins and phages, the immune tube and scFv library were incubated separately with 1X PBST (0.05% PBS containing Tween 20) containing 3% skim milk at room temperature for 1 h. After the skim milk was removed from the immune tube, the scFv library was added and incubated at 150 rpm for 1 h at 37 °C, thus binding scFv phages to the antigens. After the scFv phages were incubated in the tube, unbound scFv phages were removed by washing two or five times with 1X PBST.

[0189] ScFv phages that specifically bind to the respective KRS antigens were isolated within 10 min by adding 1 ml of triethylamine (100 mM) at room temperature and neutralized with Tris (1 M, pH 7.4). The filtered scFv phages were added to E. coli ER2537 cultured to OD<1, followed by infection with incubation at 120 rpm for 1 hour and 30 min at 37 °C. E. coli infected with the phages was centrifuged to partially remove the culture supernatant, followed by redispersion and then spread on a 15 cm diameter agarose plate containing ampicillin and glucose (2%). The following day, 5 ml of SB medium was applied to collect all cells developed on the plate, and glycerol (50%) was added at 0.5 times the total volume, followed by mixing. The mixture was then dispensed in 1 ml portions and stored at -80 °C (stock for repeated scFv cycle).Next, 20 μl of the prepared stock were seeded in 20 ml of SB solution, followed by incubation, and then constructed into a scFv phage library (1 ml) for the next step of repeated phage cycling using helper phages. The above procedure for isolating antigen-specific scFv-expressing phages was repeated two or three times. <1-2> Screening for specifically binding scFv antibodies via ELISA - secondary screening Petition 870240042040, dated 05 / 17 / 2024, pages 128 / 162 45 / 67

[0190] It was investigated by indirect ELISA whether the scFv-expressing phages selected in Example <1-1> bound to the N-terminal or full-length KRS fragments mentioned above.

[0191] The scFv product obtained from three repeated cycles was diluted and applied to a 10 cm diameter agarose plate. The following day, the respective colonies were selected and incubated in a 96-well plate containing 200 μl of SB medium. After the colonies were verified to be growing well overall, IPTG (1 mM) was added, followed by incubation at 30 °C for 16 h, thus inducing scFv production. The following day, the 96-well plate was centrifuged to isolate only cells, and then the cells were lysed with TES solution, followed by recentrifugation, thus separating only the supernatant. The supernatant obtained was subjected to indirect ELISA to select scFv that specifically binds to antigens.The plates were coated with full-length KRS antigens or N-terminal fragment antigens, respectively, incubated with culture supernatant containing scFv, and then incubated with anti-HA-HRP antibody (Roche Applied Science) as a secondary antibody. Color development was performed using tetramethylbenzimidine (TMB, Thermo Scientific) and then stopped using H2SO4 (1 M), and absorbance was read at 450 nm using an ELISA reader. ELISA was performed on control (blank) antigens and the antigens described above (Ag) simultaneously to select only colonies with positive values. Of the total 1920 colonies subjected to ELISA, 93 colonies were selected as positive (see Table 1). Table 1 Antigen (KRS fragment) Complete 1-20 13-36 31-46 1-29 5-34 10-38 15-42 24-49 Total Repeated cycle results 288 384 384 192 192 192 96 96 96 1920 Accesses 51 8 20 2 1 1 0 8 2 93 Petition 870240042040, dated 05 / 17 / 2024, pp. 129 / 162 46 / 67 <1-3> Sequencing

[0192] The sequences were analyzed to filter out the same Ab with overlapping CDR sequences from the 93 colonies selected through ELISA screening in Example <1-2>. Sequencing was specifically performed using the following method: after E. coli retention scFv clones were cultured, phagemids were obtained by miniprep. The phagemids were sequenced using Omp primer (Hye young Yang, et al., 2009, Mol. Cells 27, 225-235). The sequence thus obtained was used to verify the CDR region sequences of the phagemids using the Bioedit program. Outside of these, clones with overlapping CDR sequences were eliminated to verify scFv clones of the respective independent CDR sequences. Table 2 Antigen (KRS fragment) Complete 1- 20 13- 36 31- 46 1- 29 5- 34 10- 38 15- 42 24- 49 Total Accessions 51 8 20 2 1 1 0 8 2 93 Different Clones 10 8 11 1 1 1 0 4 2 38

[0193] As a result of screening antibodies with overlapping CDR sequences through sequencing, 38 scFv clones with different CDR sequences were obtained as shown in Table 2. <1-4> Screening for specifically binding scFv antibodies via western blotting - Tertiary screening

[0194] It was investigated by western blotting whether 38 scFv clones isolated in Example <1-3> specifically bound to KRS.

[0195] Single-colony positive scFv clones were incubated in 5 ml of SB medium containing kanamycin (Bactotritone 30 g, yeast extract 20 g, MOPS buffer 10 g / L) to initiate seed culture and after overnight incubation, the culture was transferred to 500 ml of SB medium containing kanamycin. When the OD value at 600 nm reached approximately 0.5, IPTG was added to achieve 1 mM, followed by overnight incubation at 30 °C, thus expressing scFv proteins in the periplasm of E. coli. On day Petition 870240042040, dated 05 / 17 / 2024, pages 130 / 162 47 / 67 next, E. coli obtained by centrifugation were suspended in 1X TES buffer (50 mM Tris, 1 mM EDTA, 20% sucrose, pH 8.0) and then 0.2X TES was added 1.5 times, followed by mixing and then the supernatant was removed by centrifugation, thus extracting the periplasm.

[0196] Finally, 5 mM MgSO4 was added to the scFv antibodies extracted from the periplasm, and the resulting material was mixed with Ni-NTA beads previously equilibrated with PBS, followed by agitation for 1 h in cold storage to bind the antibody to the Ni-NTA beads. After that, affinity chromatography was performed to sufficiently wash away unbound proteins with PBS. After sufficient additional washing with a buffer containing 5 mM imidazole, the bound scFv antibodies were eluted using 200 mM imidazole buffer. The eluted antibodies were dialyzed, and their purity was verified by electrophoresis. Protein quantification was performed by BCA assay, and the amount of purified antibodies was recorded; subsequently, a certain quantity was dispensed and then stored frozen.

[0197] As described above, scFv antibodies extracted from the periplasm were used to investigate using western blotting whether the scFv antibodies bound to the respective N-terminal fragments of KRS or to full-length KRS. Next, 30 μg of HCT116 cell lysate underwent electrophoresis via SDS PAGE, were transferred to a PVDF membrane, and then blocked with 3% skim milk. After that, the extracted scFv antibodies were added at 1.0 μg / μl, followed by incubation for 1 h. Unbound scFv antibodies were washed away, and for detection, scFv binding to antigens was incubated with horseradish peroxidase (HRP)-bound secondary anti-HA antibodies, and film sensitization was performed using ECL reagent as a substrate in a dark room. The sensitized bands were compared with standard molecule markers to identify bands that correspond to the full-length KRS sizes and their respective fragments.

[0198] Through western blotting, scFv clones with highly weak bands (weak bands) and non-specific bands (double bands) were excluded. Petition 870240042040, dated 05 / 17 / 2024, pp. 131 / 162 48 / 67 Therefore, 13 scFv clones were selected and these results are shown in Figure 1. < 1-5> Screening for scFv antibodies that bind specifically via immunoprecipitation - quaternary screening

[0199] Immunoprecipitation was performed to investigate whether the scFv clones selected in Example <1-4> actually bound to native KRS. The purified scFv clones and the HCT116 cell lysate were subjected to Ag-Ab binding and immunoprecipitation was performed using scFv marker HA.

[0200] Specifically, HCT116 cells were lysed in 20 mM TrisHCl buffer (pH 7.4, lysis buffer) containing 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS, and a protease inhibitor. Each scFv (5 μg) was added to 500 μg of HCT116 cell lysate and then incubated at 4 °C overnight. Next, 30 μl of anti-HA agarose beads were added, followed by incubation at 4 °C for 4 h. The supernatant was removed by centrifugation. The precipitate thus obtained was dissolved in SDS sample buffer and boiled for 7 min. The dissolution and boiling step was repeated twice.

[0201] Each of the immunoprecipitated samples prepared using the procedure described above was subjected to electrophoresis via SDS PAGE, transferred to a PVDF membrane, and then blocked with 3% skim milk. After that, polyclonal KRS antibodies (rabbit, Neomics, Co. Ltd. #NMS-01-0005) were added, followed by incubation for 1 h. After the unbound antibodies were washed away, secondary anti-rabbit antibodies (ThermoFisher Scientific, #31460) were added, followed by incubation. After incubation with the secondary antibodies, film sensitization was performed using ECL reagent with a substrate in a dark room. The sensitized bands were compared with standard molecule markers to identify bands corresponding to the full-length KRS sizes and respective fragments.

[0202] Therefore, 12 scFv clones were selected and these results are shown in Figure 2. < 1-6> Screening for specific binding scFv antibodies by immunofluorescence - quinary screening Petition 870240042040, dated 05 / 17 / 2024, pages 132 / 162 49 / 67

[0203] Immunofluorescence was performed to investigate whether the scFv clones selected in Example <1-5> actually bound to the N-terminal region of KRS exposed on the cell membrane. In order to create a phenomenon in which KRS is exposed on the membrane, the KRS-T52D mutant (active mutant) was used. Specifically, A549 cells were seeded on a glass coverslip (1 x 10⁵ cell, based on a 12-well plate) and after 24 h, myc-KRS WT / mycKRS T52D (active mutant) / myc-KRS T52A (inactive mutant) were overexpressed, respectively. After incubation for 24 h, the cells were incubated using serum-free media (RPMI 1640 media) at 37 °C for 1 h. After that, the cells were treated with 10 μg / ml of laminin (L2020; Sigma) and then incubated at 37 °C for 1 h.Each of the myc-KRS T52D (active mutant) and myc-KRS T52A (inactive mutant) vectors was constructed using the pcDNA3-myc-KRS WT vector as a primary structure via site-directed mutagenesis (QuikChange Π Site-Directed Mutagenesis kit, Agilent, #200523).

[0204] Prepared samples were washed with PBS (4 °C), fixed by treatment with 4% paraformaldehyde for 10 min, and then washed. Samples were blocked with CAS block for 10 min and treated with scFv and Myc antibodies for 2 h. After that, unbound antibodies were washed and incubated with secondary antibodies for 1 h in a dark room. DAPI staining was performed for 10 min before mounting.

[0205] As shown in Figure 3, experimental results showed that a total of four scFv clones (N3, N5, N7, and N9) not bound to the inactive mutant (T52A) and WT-KRS (untreated laminin), but bound to active mutant cells only, were selected. When laminin treatment was performed to induce WT-KRS membrane localization and scFv staining was performed, similar staining regions were observed in WT-KRS and T52D mutants.

[0206] It has been confirmed that these clones specifically bind to the cell surface (tip). < 1-7> Verification of specific binding capability to the KRS N-terminal

[0207] To investigate whether the scFv clones (N3, N5, N7 and N9) finally selected through Example <1-6> actually bound to the KRS N-terminal, Petition 870240042040, dated 05 / 17 / 2024, pp. 133 / 162 50 / 67 Full-length KRS (denoted by F, SEQ ID NO: 76), a KRS fragment with amino acid deletions at positions 1-71 in the N-terminus (defined by SEQ ID NO: 1), and a KRS fragment composed of 1200 amino acid residues in the N-terminus (defined by SEQ ID NO: 2) were used to conduct western blotting.

[0208] A549 cells were transformed using the method described in Example <1-6> using polynucleotides encoding full-length KRS and KRS fragments. After that, the cells were lysed and western blotting was performed using the same method as described in Example <1-4>.

[0209] As shown in Figure 4, the results confirmed that all selected scFv clones (N3, N5, N7, and N9) showed bands only in the fragment with amino acids at positions 1-200 in the N-terminal, while showing no bands in fragment 1 without amino acids at positions 1-72 in the N-terminal. It was therefore verified that all scFv clones N3, N5, N7, and N9 specifically bound to the N-terminal of KRS. < 1-8> Sequencing of scFv clones specific to the N-terminal bond of KRS

[0210] scFv clones (N3, N5, N7 and N9) finally selected using Example <1-6> were analyzed for CDR conformation and VH and VL sequences thereof. Sequencing was performed by the same method as described in Example <1-3>. [ 0211] As a result of sequencing, N3 scFv consists of the amino acid sequence defined by SEQ ID NO: 67, which contains the linker sequence of SEQ ID NO: 65 in the middle of this. Furthermore, N3 VH consists of the amino acid sequence defined by SEQ ID NO: 49 and N3 VL consists of the amino acid sequence defined by SEQ ID NO: 51. As a result of sequencing, the respective CDRs contained in VH and VL of N3 are: N3 VH comprises heavy chain CDR1 defined by SEQ ID NO: 1, heavy chain CDR2 defined by SEQ ID NO: 3, and heavy chain CDR3 defined by SEQ ID NO: 5; and N3 VL comprises light chain CDR1 defined by SEQ ID NO: 7, light chain CDR2 defined by SEQ ID NO: 9, and light chain CDR3 defined by SEQ ID NO: 11. Petition 870240042040, dated 05 / 17 / 2024, pages 134 / 162 51 / 67

[0212] The scFv N5 consists of the amino acid sequence defined by SEQ ID NO: 69, which contains the linker sequence of SEQ ID NO: 65 in the middle of it. In addition, N5 VH consists of the amino acid sequence defined by SEQ ID NO: 53 and N5 VL consists of the amino acid sequence defined by SEQ ID NO: 55. N5 VH comprises heavy chain CDR1 defined by SEQ ID NO: 13, heavy chain CDR2 defined by SEQ ID NO: 15 and heavy chain CDR3 defined by SEQ ID NO: 17 and N5 VL comprises light chain CDR1 defined by SEQ ID NO: 19, light chain CDR2 defined by SEQ ID NO: 21 and light chain CDR3 defined by SEQ ID NO: 23.

[0213] The scFv N7 consists of the amino acid sequence defined by SEQ ID NO: 71, which contains the linker sequence of SEQ ID NO: 65 in the middle of it. In addition, N7 VH consists of the amino acid sequence defined by SEQ ID NO: 57 and N7 VL consists of the amino acid sequence defined by SEQ ID NO: 59. N7 VH comprises heavy chain CDR1 defined by SEQ ID NO: 25, heavy chain CDR2 defined by SEQ ID NO: 27 and heavy chain CDR3 defined by SEQ ID NO: 29 and N7 VL comprises light chain CDR1 defined by SEQ ID NO: 31, light chain CDR2 defined by SEQ ID NO: 33 and light chain CDR3 defined by SEQ ID NO: 35.

[0214] scFv N9 consists of the amino acid sequence defined by SEQ ID NO: 73, which contains the linker sequence of SEQ ID NO: 65 in the middle of it. In addition, N9 VH consists of the amino acid sequence defined by SEQ ID NO: 61 and N9 VL consists of the amino acid sequence defined by SEQ ID NO: 63. N9 VH comprises heavy chain CDR1 defined by SEQ ID NO: 37, heavy chain CDR2 defined by SEQ ID NO: 39 and heavy chain CDR3 defined by SEQ ID NO: 41 and N9 VL comprises light chain CDR1 defined by SEQ ID NO: 43, light chain CDR2 defined by SEQ ID NO: 45 and light chain CDR3 defined by SEQ ID NO: 47. Example 2 Conversion of scFv antibodies to IgG antibodies and evaluation of their specific binding capacity < 2-1> Conversion of scFv antibodies to IgG antibodies Petition 870240042040, dated 05 / 17 / 2024, pp. 135 / 162 52 / 67

[0215] First, the polynucleotides encoding scFv were amplified by PCR from N3, N5, N7, and N9 phage genomes. The nucleotide sequences of the primers used to amplify a VH region gene from the scFv antibodies: Forward (AGA GAG TGT ACA CTC CCA GGC GGC CGA GGT GCA G, SEQ ID NO: 93), Reverse (CGC CGC TGG GCC CTT GGT GGA GGC TGA GCT CAC GGT GAC CAG, SEQ ID NO: 94). The nucleotide sequences of the primers used to amplify a VL gene region of the scFv antibodies: Forward (AAG CGG CCG CCA CCA TGG GAT GGA GCT GTA TCA TCC TCT TCT TGG TAG CAA CAG CTA CAG GTG TAC ACT CCC AGT CTG TGC TGA CTC AG, SEQ ID NO: 95), Reverse (CGC CGC CGT ACG TAG GAC CGT CAG CTT GGT, SEQ ID NO: 96)

[0216] PCR was performed with each phage DNA (50 ng) as a template using primers (10 pmol each) under the following conditions: 95 °C / 3 min; 95 °C / 30 s, 60 °C / 30 s, 72 °C / 30 s, 30 cycles; and 72 °C / 5 min, thus amplifying the VH or VL gene of scFv N3, N5, N7, or N9. The PCR product was inserted into the pcDNA3.4 vector used for IgG production using restriction enzymes. The heavy and light chain proteins of IgG were individually encoded on separate plasmids.

[0217] Vectors constructed comprising DNA encoding heavy and light chains of each of the IgGs (hereinafter referred to as IgG N3, IgG N5, IgG N7, and IgG N9, respectively) containing scFv variable regions were cotransformed into freestyle 293F cells to express the heavy and light chains together in the cells. The transformed 293F cells were incubated at 37 °C and 8% CO2 for 7 days, and the supernatant was obtained. The supernatant was filtered through a cellulose acetate membrane filter (pore size 0.22 μm, Corning) and purified using a CaptivA™ PriMAB protein A column (Repligen, USA). The concentrations of the obtained antibodies were measured using a BCA kit (Pierce, 23225), and the IgG antibody proteins produced under reduction and non-reduction conditions were analyzed. < 2-2> Verification of the KRS binding capacity of converted IgG - Western blotting and immunoprecipitation Petition 870240042040, dated 05 / 17 / 2024, pp. 136 / 162 53 / 67

[0218] The KRS binding capacity of IgGs constructed in Example <2-1> was investigated by western blotting (WB) and immunoprecipitation. Western blotting was performed in the same manner as described in Example <1-4> and immunoprecipitation was performed in the same manner as described in Example <15> .

[0219] The results verified that the IgGs constructed in the present invention bound to KRS and Figure 5 shows these results using IgG N3 and IgG N5 as representatives. < 2-3> Verification of the specific binding capacity of N-terminal KRS of converted IgGs - SPR

[0220] The quantitative binding capacity of purified antibody proteins (IgG N3 and IgG N5) to antigen (KRS 1-207 aa) was measured using a Biacore 2000 SPR (surface plasmon resonance) biosensor (GE healthcare, US). After KRS was immobilized on a sensor chip (CM5, GE healthcare, US), antibody proteins (6.25-100 nM), which were serially diluted with HES buffer solution (10 mM HEPES, pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20), were allowed to flow at a rate of 30 µl / min for 3 min, and 1 M NaCl / 20 mM NaOH was allowed to flow at a rate of 30 µl / min for 3 min, thus inducing the dissociation of proteins bound to the antigen. Simulated IgG was used as a control. The specific experimental conditions are as follows: Immobilized antigen: KRS Immobilized level: 185 RU Antibody: IgG N3 and IgG N5 Running buffer: HBS-N buffer Regeneration: 2 M NaCl, 20 mM NaOH (flow 30 µl / min 1 min) Table 3 ka (1 / Ms) kd (1 / s) KD (M) IgG N3 1.09E+05 0.009055 8.34E-08 IgG N5 3.13E+06 0.003282 1.05E-09

[0221] Table 3 shows rate kinetic constants and equilibrium dissociation constants measured for IgG N3 and IgG N5 using Biacore 2000 SPR. A Petition 870240042040, dated 05 / 17 / 2024, pages 137 / 162 Affinity of 54 / 67 was obtained from the kinetic rate constants (ka and kd) and equilibrium dissociation constants (KD) using BIA software evaluation ver. 3.2. Figure 6 shows the SPR plot results of IgG N3 and IgG N5, respectively. It was confirmed from Figure 6 and Table 3 that IgG N3 and IgG N5 of the present invention have a high specific binding capacity to the N-terminal region of KRS. No binding signal was observed in IgG in simulation as a control. <2-4> Verification of the specific binding capacity of the N-terminal KRS of converted IgG - immunofluorescence staining

[0222] To investigate whether the IgGs constructed in the present invention actually bound to the exposed KRS region on the cell membrane, immunofluorescence was performed. Immunofluorescence staining was performed using the same method as described in Example <1-6> using IgG N3 and IgG N5 as representatives.

[0223] As shown in Figure 7A, the results confirmed that IgG N3 bound favorably and specifically to the extracellularly exposed N-terminal region of KRS when the membrane localization of WT-KRS was induced by laminin treatment. As shown in Figure 7B, the experiment using T52D-KRS (active mutant myc-labeled KRS) also showed that the antibody of the present invention bound favorably to the extracellularly exposed N-terminal region of KRS, and when the experimental cells became permeabilized, the KRS proteins present in the cytosol were detected with high sensitivity. It was also confirmed, as shown in Figure 7C, that IgG N5 bound favorably and specifically to the extracellularly exposed N-terminal region of KRS when the membrane localization of WT-KRS was induced by laminin treatment.

[0224] Therefore, it has been verified that the antibodies provided in the present invention have high binding specificity in relation to the N-terminal region of KRS exposed extracellularly. Example 3 Verification of the inhibitory effect on cancer metastasis < 3-1> Cytotoxicity Assessment Petition 870240042040, dated 05 / 17 / 2024, pages 138 / 162 55 / 67

[0225] The MTT assay was performed on IgG N3 as a representative. A549 cells were seeded in 96-well plates (5,000 cells / well) and incubated. Cells were washed with serum-free media and then treated with simulated human IgG and IgG N3 at 0, 50, 100, 500 nM (in serum-free media). After 24 h of incubation, MTT solution was added at 50 μg / well, followed by incubation for 4 h. After the MTT solution was removed, the wells were treated with 100 μl of DMSO and then the absorbance was measured at 570 nm.

[0226] As shown in Figure 8, experimental results confirmed that the antibody of the present invention shows no cytotoxicity. < 3-2> Cell Migration Assay

[0227] Cell migration was measured using a 24-well Transwell chamber with a polycarbonate membrane (8.0 μm pore size, Costar) as disclosed in the previous technique (Park, SG et al., Human lysyl-tRNA synthetase is secreted to trigger pro-inflammatory response, Proc. Natl. Acad. Sci. USA 102, 6356-6361 (2005)). In the Transwell chamber, the lower well was coated with 10 μg of laminin (in gelatin) and UV-dried. After that, A549 cells were suspended in serum-free RPIM medium and then placed at a concentration of 1 x 10⁵ cells per well in the upper chamber. The chamber was treated with IgG N3 or human simulation IgG (control) at 100 nM or 500 nM, followed by incubation for 24 h. After that, the chamber was washed twice with PBS and treated with 70% MeOH (in PBS) for 30 min. The chamber was again washed twice with PBS and treated with hematoxylin solution for 30 min.The chamber was washed three times with DW, and the membrane in the chamber was cut and mounted on the sliding glass.

[0228] As shown in Figure 9A, experimental results showed that IgG N3 significantly inhibited A549 cell migration. Experimental results also showed that this inhibitory effect on cell migration was dose-dependent (see Figure 9B). < 3-3> Evaluation of the inhibitory effect on cancer metastasis in in-vivo cancer metastasis models Petition 870240042040, dated 05 / 17 / 2024, pages 139 / 162 56 / 67

[0229] Since KRS can accelerate cell migration through 67LR associated with cancer metastasis, animal tumor (cancer) models were constructed using 4T-1 mouse breast cancer cells (Korean Cell Line Bank), which are highly susceptible to lung metastasis. Orthotopic breast cancer animal models were constructed by injecting 4 x 104 4T1 cells into the fat pad of six 7-week-old BALB / cAnCr mice (Doo Yeol Biotech).

[0230] Cancer was injected into the mammary fat pad and after 10 days (Day 10), the cancerous tissues were resected from the fat pad. After one day (Day 11), IgG N3 (10 mg / kg) was administered twice a week for two weeks at 3-day intervals (a total of four times, Days 11, 14, 18, and 21) via intravenous injection via caudal vein, and the same dose of IgG in a control simulation (Thermo #31154) was also administered. One week after completion of the full antibody dosage schedule, i.e., 28 days after cancer injection, the mice were sacrificed to remove lung tissue. Upon autopsy of the lung tissue, the lung was inflated by injecting a physiological solution into the bronchus via syringe, collected, and then stored in Bouin's solution (Sigma #HT10132) for 24 h. After that, the metastatic nodules in each lobe of the lung were counted using a microscope.

[0231] As shown in Figures 11 and 12, experimental results confirmed that many nodules were generated in the lung due to cancer metastasis in the control group, and such nodules were significantly suppressed in the IgG N3 treatment group. Figure 13 shows a comparative view of lung tissues in the control and IgG N3 treatment groups and confirmed that a significantly larger quantity of laminin receptors was expressed at the metastatic nodule sites in the control group compared to the group treated with the antibody of the present invention. < 3-4> Comparison of the effect with an anticancer metastasis compound (YH16899) ​​in in-vivo cancer metastasis models

[0232] It is known from the literature mentioned above “Dae Gyu Kim et al., (2014)” that the compound YH16899 has an effect on inhibiting cancer metastasis by Petition 870240042040, dated 05 / 17 / 2024, pages 140 / 162 57 / 67 suppression of the interaction between 67LR and KRS. Next, the inhibitory effect on cancer metastasis was compared between YH16899 and IgG N3, the antibody of the present invention. The construction of in-vivo tumor models and the observation of lung metastasis were performed using the same method as in Example <3-3>. YH16899 was administered orally at 100 mpk daily. IgG N3 was injected intravenously at different concentrations (1 mpk, 10 mpk) through the mouse tails.

[0233] As shown in Figures 14 and 15, experimental results confirmed that the pulmonary nodule count was significantly reduced in a dose-dependent manner in the IgG N3 treatment group and treatment with just 1 mpk of IgG N3 significantly inhibited cancer metastasis compared with the YH16899 (100 mpk) treatment groups. Example 4 Verification of KRS antibody binding sites < 4-1> Human KRS binding sites of KRS monoclonal antibodies

[0234] To investigate the human IgG N3 KRS binding sites among the KRS antibodies constructed above, surface plasmon resonance (SPR) was performed as below.

[0235] First, the IgG N3 antibody was immobilized on a Biacore T200 (GE Healthcare) equipped with a Series S Sensor Chip CM5 (GE Healthcare) using an amine coupling kit (GE Healthcare). Next, the peptides shown in Table 4 dissolved below in PBS solution at corresponding concentrations were allowed to flow for 60 s. Then, the PBS was allowed to flow for 5 min. The binding capacity was then analyzed using Biacore T200 Evaluation software v2.0 (GE Healthcare). Table 4 Information about Peptides Name Sequence information Species MW SEQ ID NO F1 (1-29) MAAVQAAEVKVDGSEPLSKNELKRRLKA H 3168 97 F2 (5-34) QAAEVKVDGSEPLSKNELKRRLKAEKKVA H 3351 98 Petition 870240042040, of 17 / 05 / 2024, p. 141 / 162 58 / 67 F3 (10-38) KVDGSEPKLSKNELKRRLKAEKVAEKEA H 3310 99 F4 (15-42) EPKLSKNELKRRLKAEKVAEKEAKQKE H 3337 100 F5 (24-49) KRRLKAEKKWAEKQKELSEKQKEA H 3310 101-141 m 28) MATLQESEVKVDGEQKLSKNELKRRLKA M 3230 102 mF2 (3- 34) QESEVKVDGEQKLSKNELKRRLKAEKKLA M 3383 103 mF3 (10- 37) KVDGEQKLSKNELKRRLKA M 3383 103 mF3 (15- 41) QKLSKNELKRRLKAEKKLAEKEAKQKE M 3253 105 mF5 (24- 48) RRLKAEKKLAEKEAKQKELSEKQLN M 2997 106 rF1 (1-28) MATLREGEVKLDGEPKLSKNELKRRR3253 (1314) REGEVKLDGEPKLSKNELKRRLKAEKKLA R 3364 108 rF3 (10- 37) KLDGEPKLSKNELKRRLKAEKKLAEKEA R 3251 109 rF4 (15- 41) PKLSKNELKRRLKAEKKLAEKQKE R 125-248 (124) RRLKAEKKLAEAKQKELSEKQLN R 2997 111

[0236] As shown in Figure 16, the results showed that the antibody IgG N3 bound to epitopes F1, F2, F3, and F4, but not to epitope F5. Furthermore, the binding capacity to epitope F4 was stronger, and the binding capacity to F3, F2, and F1 was stronger in that order.

[0237] These results could confirm that the main IgG N3 antibody binding site corresponds to amino acid residues at positions 15 to 29 in the N-terminal region of KRS. <4-2> Interspecies cross-activity of KRS monoclonal antibody

[0238] The example above validated the human KRS binding site of the KRS IgG N3 antibody and to investigate whether IgG N3 showed cross-activity with other Petition 870240042040, dated 05 / 17 / 2024, pp. 142 / 162 59 / 67 species of mouse (m) and rat (r), surface plasmon resonance (SPR) was performed as below.

[0239] In the same manner as the experimental method described in Example 4-1 above, the IgG N3 antibody was immobilized on the chip using an amine coupling kit (GE Healthcare). Then, the peptides shown in Table 4 above dissolved in PBS solution at corresponding concentrations were allowed to flow for 60 min if PBS was allowed to flow for 5 min. The binding capacity was then analyzed using Biacore T200 Evaluation software v2.0 (GE Healthcare).

[0240] As shown in Figure 17, the results showed that the IgG N3 antibody bound to human (h), mouse (m), and rat (r) epitopes F1, F2, F3, and F4, but not the F5 epitope. The stronger binding capacity to the F3 epitope than to F1 was the same as among humans, mice, and rats (data for F2 and F4 not shown).

[0241] These results could confirm that the IgG N3 antibody is capable of interspecies cross-activity. Example 5 Verification of the role of the laminin signal in the migration and invasion of immune cells.

[0242] It was investigated which of the various extracellular matrices that constitute blood vessels accelerated the migration and invasion of monocytes / macrophages. The specific experimental methods for the transcavity migration assay using collagen, fibronectin, and laminin as extracellular matrices were as follows. The transcavity (Corning, #3421-5mm) was coated with gelatin (0.5 mg / ml) and then RAW 264.7 cells (1 x 105 cells / cavity) were seeded into the upper chamber. Each serum-free DMEM (500 μg / ml) containing laminin (laminin mixture, Biolamina), fibronectin, or collagen (10 μg / ml) was placed in the bottom chamber. After 24 h, non-migrating cells present on the upper part of the membrane were removed with cotton swabs. The cells in the bottom chamber were fixed by treatment with 70% methanol for 30 min and then stained with 50% hematoxylin for 30 min. After staining, the membrane was removed and mounted on the slide, and then the cells were placed on the slide. Petition 870240042040, dated 05 / 17 / 2024, pages 143 / 162 60 / 67 migrations present on the bottom surface of the membrane were observed and quantified using a high-resolution microscope.

[0243] As shown in Figures 18a and 18b, experimental results confirmed that apart from the various extracellular matrices, laminin accelerated monocyte / macrophage migration with more force. Example 6 Effects of Migration and Invasion of Immune Cells by Laminin Subtypes

[0244] The effects of laminin subtypes on immune cell migration and invasion were evaluated. The transcavity migration assay was performed using the same method as in Example 5 using LN111, LN211, LN221, LN411, LN421, LN511 and LN521 (10 μg / ml) as various laminin subtype proteins (obtained from Biolamina). The specific sequences of the laminin subtypes can be referenced as α4 chain of SEQ ID NO: 115, α2 chain of SEQ ID NO: 121, α5 chain of SEQ ID NO: 122, β2 chain of SEQ ID NO: 117, β1 chain of SEQ ID NO: 123 and γ1 chain of SEQ ID NO: 119, according to the chains that constitute the respective laminin subtypes.

[0245] RAW 264.7 cells (2 x 10⁶ cells) were incubated for 18 h, treated with 1 μg / ml of each laminin subtype in serum-free DMEM, and then harvested at 0 h, 12 h, and 24 h. RAW 264.7 cell protein was separated into cytosol and membrane fractions using the ProteoExtract Subcellular Proteome Extraction Kit (Calbiotech, cat# 539790). The obtained protein was subjected to electrophoresis, transferred to a PVDF membrane (Milipore), and blocked with 3% skim milk. After that, the KRS monoclonal antibody (rabbit, Neomics, Co. Ltd. #NMS-01-0005) was added, followed by incubation for 1 h. After the unbound antibodies were washed away, secondary anti-rabbit antibodies (ThermoFisher Scientific, #31460) were added, followed by incubation. After incubation with the secondary antibodies, film sensitization was performed using ECL reagent as a substrate in a dark room.The sensitized bands were compared with standard molecule markers to identify bands that corresponded to the KRS sizes. The antibodies Na+ / K+ ATPase (Abcam) and tubulin (Sigma) were used. Petition 870240042040, dated 05 / 17 / 2024, pages 144 / 162 61 / 67 for the identification of the plasma membrane and cytosol marker, respectively.

[0246] As shown in Figures 19a and 19b, experimental results confirmed that monocytes / macrophages migrate when responding specifically to the α4β2γ1 (LN421) subtype among all laminin subtypes tested. That is, it was confirmed that monocytes / macrophages migrated and invaded specifically in response to LN421. As shown in Figure 19c, it was confirmed that treatment of monocytes / macrophages with LN421 increased the amount of KRS detected in the cell membrane region, but partially decreased the amount of KRS detected in the cytosol region. These results indicate that KRS, which is generally present in the cytosol region after expression within monocytes / macrophages, is translocated to the cell membrane region by treatment with LN421 and that an increase in KRS in the immune cell membrane region corresponds to an important pathological phenomenon in diseases associated with the migration and invasion of immune cells. Example 7 Antibody construction to reduce KRS levels in the cell membrane and verify its effect on controlling the migration / invasion of immune cells.

[0247] The effect on the migration and invasion of immune cells was investigated using IgG N3 antibody as a representative among the antibodies constructed in Example 1 above. The specific experimental methods were as follows. The transcavity (Corning, #3421-5mm) was coated with gelatin (0.5 mg / ml) and then RAW 264.7 cells (1 x 105 cells / cavity) were seeded in the upper chamber. Serum-free DMEM (500 μg / ml) containing laminin 421 (1 μg / ml) was placed in the bottom chamber. The upper chamber was treated with each antibody at 100 nM. After 24 h, immobilization with 70% methanol was performed for 30 min, followed by staining with 50% hematoxylin for 30 min. Non-migrating cells present on the upper surface of the membrane were removed with cotton swabs, and then the membrane was removed and mounted on the slide. Migrating cells present on the bottom surface of the membrane were observed using a high-resolution microscope. Petition 870240042040, dated 05 / 17 / 2024, pages 145 / 162 62 / 67 (Figure 20a) and the cells were counted in the images obtained and plotted on the graph (Figure 20b).

[0248] RAW 264.7 cells were treated with laminin 421 (1 μg / ml) and antibody (100 nM), incubated for 24 hours, and harvested. Afterward, the harvest was separated into membrane and cytosol fractions using the ProteoExtract Subcellular Proteome Extraction Kit (Calbiochem), sampled, and then subjected to western blotting with respect to KRS. The specific method for this was as described in Example 6.

[0249] As shown in Figures 20a and 20b, experimental results confirmed that the antibody of the present invention effectively and specifically inhibited LN421-dependent monocyte / macrophage migration. As shown in Figure 20c, it was confirmed that treatment with LN421 increased the level of KRS in the monocyte / macrophage cell membrane and treatment with IgG N3 antibody effectively reduced the level of KRS in the cell membrane.

[0250] These results confirmed that the antibody of the present invention has potential as a novel therapeutic agent for diseases involving the migration of immune cells, such as monocytes / macrophages. Example 8 Verification of the effect on disease related to immune cell migration in in-vivo models.

[0251] As in the examples above, the following experiment was performed using IgG N3 antibody as a representative of the antibodies of the present invention. Methods 1. Construction of the pulmonary arterial hypertension (PAH) model and administration of the test substance.

[0252] To induce PAH in seven-week-old SD rats (Orient Bio), 60 mcg of monocrotaline (MCT) were injected subcutaneously. Afterward, the rats were divided into four groups (five animals per group) and administered 1 mcg of simulated human IgG (Thermo Fisher Scientific, negative control), 1 mcg of IgG N3, 10 mcg of IgG N3 10, and 25 mcg of sildenafil. Petition 870240042040, dated 05 / 17 / 2024, pages 146 / 162 63 / 67 (positive control) for three weeks. All antibodies were injected intravenously twice a week and sildenafil was administered orally every day. 2. Measurement of blood flow and blood pressure

[0253] After three weeks, the rats were anesthetized with isoflurane and blood flow and blood pressure were measured using a high-precision pneumatic measurement system (MPVS cardiovascular pressure and volume system, model name: MPVS Ultra, manufacturer: Millar Instruments). Right ventricular end-systolic pressure (RVESP), right ventricular end-diastolic pressure, left ventricular end-systolic pressure, and left ventricular end-diastolic pressure were measured using a dedicated catheter (Mikro-Tip rat pressure catheter, manufacturer: Millar Instruments). Cardiac output was measured using a perivascular blood flow probe (Transonic Flow probes, manufacturer: Millar Instruments), and the experimental method for this was the same as that described in the following literature: Pacher P, Nagayama T, Mukhopadhyay P, Batkai S, Kass DA.Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc 2008;3(9):1422-34. 3. Imuno-histoquímica (IHC)

[0254] The collected lungs were fixed in paraformaldehyde (PFA) according to the common procedure and then infiltrated and embedded with paraffin through washing, dehydration, and clearing. Paraffin blocks of rat lung tissue were microsectioned to a thickness of 6 μm and slides were fabricated. After that, staining was performed as below. The sample was first treated with xylene for 5 min three times, treated with 100% ethanol, 95% ethanol, 90% ethanol, and 70% ethanol and DW in that order for 2 min and washed with PBS for 5 min. After treatment with 0.3% H2O2, the sample was washed with PBS for 5 min twice. The sample was immersed in 0.01 M citrate buffer, heated, and washed with PBS-T (0.03% Tween 20). After that, the sample was blocked (2% BSA & 2% goat serum in PBS) at room temperature for 30 min. The sample was stained with anti-CD68 antibody (1:200, clone ED1, Abcam) at 4 °C overnight.The sample was washed with PBS-T for 5 min three times and then treated with polymer-HRP anti-mouse. Petition 870240042040, dated 05 / 17 / 2024, pages 147 / 162 The sample was infused using a 64 / 67 envision kit (DAKO) at 4 °C for 1 h. The sample was washed with PBS-T three times, and then the color was developed by treatment with DAB substrate buffer and DAB 20 chromogen. The stained tissue was treated with Mayer's hematoxylin (Sigma) for 1 min, followed by treatment with 70% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol in that order for 2 min each, twice. Finally, the tissue was treated with xylene three times for 5 min and then observed under a light microscope. Results <8-1> Verification of changes in blood pressure and cardiac output.

[0255] PAH models, a disease closely related to immune cell invasion and pathological phenomena, were treated with IgG N3 antibody (1 mpk or 10 mpk) for 3 weeks (IV, twice weekly) and then measured for right ventricular end-systolic pressure (RVESP), right ventricular end-diastolic pressure (RVEDP), left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP), and cardiac output (CO). The results are shown in Table 5. Table 5 MCT + IgG in simulation (n = 4) MCT + N3 Ab1mpk (n = 5) MCT + N3Ab10mpk (n = 5) MCT + Sildenafil (n = 5) VESP (mmHg) 62.5 ± 5.7 45.0 ± 8.1 41.2 ± 7.7 48.4 ± 9.6 VEDP (mmHg) 2.8 ± 1.5 1.4 ± 2.2 3.8 ± 1.3 2.6 ± 1.3 VESP (mmHg) 81.5 ± 11.4 95.8 ± 4.8 93.4 ± 11.3 83.2 ± 4.7 VEDP (mmHg) 1.0 ± 0.8 2.6 ± 1.9 4.6 ± 3.9 3.6 +- 2.3 CO (ml / min) 58 +- 4.7 (n = 4) 74.0 +- 10.9 (n = 5) 59.8 +- 12.9 (n = 5) 49.6 +- 17.7 (n = 4)

[0256] (No CO measurement for an animal in the MCT + IgG group in the simulation died of anesthesia and an animal in the sildenafil treatment group died during surgery)

[0257] Pulmonary arterial hypertension causes the end-pressure of the right ventricle to rise due to narrowing of the pulmonary artery, resulting in right ventricular failure. In addition, the reward mechanism of the right ventricle is destroyed. Petition 870240042040, dated 05 / 17 / 2024, pages 148 / 162 65 / 67 due to ongoing hypertension, resulting in right ventricular hypertrophy followed by right ventricular dilation. This causes compression of the left ventricle due to ventricular septal movement, resulting in reductions in left ventricular end-diastolic volume and cardiac output (WooSeok Lee, et al., Clinical Characteristics and Prognostic Factors of Patients with Severe Pulmonary Hypertension, Korean Circulation J 2007;37:265-270). As a result, pulmonary arterial hypertension is primarily associated with the right ventricle, but is also involved in left ventricular function.

[0258] Patients with PAH showed an increase in VESP, which was also observed in the animal models of PAH in the present experiment. In this regard, as shown in Figure 21a, the N3 antibody significantly reduced VESP at both concentrations and favorably reduced VESP more than sildenafil, the positive control drug.

[0259] Furthermore, a reduction in left ventricular end-systolic pressure (LVEPS) due to administration of the IgG N3 antibody was not observed and, instead, as shown in Figure 21b, LVEPS increased significantly in the group administered with the antibody of the present invention. Therefore, the antibody of the present invention is in contrast to sildenafil used as an existing therapeutic agent for pulmonary arterial hypertension in that sildenafil causes pulmonary arterial dilation and systemic arterial dilation, thereby risking a reduction in systemic blood pressure.

[0260] In other words, it was confirmed that the antibody of the present invention showed a tendency to have a low effect on systemic blood pressure compared to sildenafil, and this effect is considered a favorable characteristic of a therapeutic agent considering that the administration of sildenafil may have a risk of developing hypotension in clinical sites. Furthermore, severe pulmonary arterial hypertension causes systolic RV failure, which may be accompanied by low cardiac output and systemic hypotension.

[0261] On the other hand, a treatment to alleviate pulmonary arterial hypertension using the antibody of the present invention is expected to increase cardiac output. Petition 870240042040, dated 05 / 17 / 2024, pages 149 / 162 66 / 67 and systemic blood pressure, thus normalizing blood pressure.

[0262] In general, it has been confirmed that administration of the antibody of the present invention reduced the risk of side effects from existing therapeutic drugs and showed relief of PAH symptoms and treatment effects. Echocardiography <8-2>

[0263] The discovery of the left ventricle in a D shape indicating pressure overload in the right ventricle was observed in three animals in the MCT-alone administration group (i.e., PAH models without test substance administration) and three animals in the MCT + sildenafil administration group, but was not observed in the therapeutic antibody administration groups.

[0264] Furthermore, as shown in Table 6 below, the body weights of the respective groups increased to similar degrees, with no significant difference. That is, the findings did not indicate any abnormal signs, including abnormal weight loss, caused by the administration of the therapeutic antibody. Table 6 MCT + IgG in simulation (n = 4) MCT + Ab 1 mpk (n = 5) MCT + Ab 10 mpk (n = 5) MCT + Sildenafil (n = 5) Absolute change (g) 101.4 +14.2 113.5 +14.6 104.1 +- 12.3 104.1 +- 26.4 Relative change (%) 48.8 +- 7.8 43.6 +- 5.2 40.7 +5.0 49.8 +- 10.5 <8-3> Verification of monocyte / macrophage migration and degrees of infiltration

[0265] IHC staining was performed with respect to CD68, which is a monocyte / macrophage marker, using lung tissues from each experimental group. As shown in Figure 22, the experimental results confirmed that the groups treated with IgG N3 antibodies of the present invention explicitly reduced monocyte / macrophage infiltration in lung tissues, and this effect was significantly better than that of sildenafil. Industrial Applicability Petition 870240042040, dated 05 / 17 / 2024, pages 150 / 162 67 / 67

[0266] As described above, the antibodies or fragments thereof according to the present invention have particular CDR (complementarity-determining region) sequences defined in this descriptive report and excellent specific binding capacity to the extracellularly exposed N-terminal region of KRS and, thus, can be used in the diagnosis of a disease (e.g., cancer) known to be accompanied by specific KRS behaviors. Additionally, the antibodies or fragments thereof according to the present invention are also specifically targeted to the N-terminal region of KRS in vivo and thus inhibit the interaction between the laminin receptor and the N-terminal region of KRS to exert an excellent inhibitory effect on cancer metastasis and, therefore, can be used as a therapeutic agent.Additionally, antibodies or fragments thereof according to the present invention can control the migration of immune cells and thus can be very advantageously used in the prevention, relief and treatment of diseases related to the migration of immune cells and therefore have high industrial applicability. Petition 870240042040, dated 05 / 17 / 2024, pages 151 / 162

Claims

1 / 8 CLAIMS:

1. ANTIBODY OR FRAGMENT THEREOF, characterized by comprising: a variable heavy chain region (VH) comprising: heavy chain complementarity determination region 1 (CDR1) containing the selected amino acid sequence from the group consisting of SEQ ID NO: 1, SEQ ID NO: 13, SEQ ID NO: 25 and SEQ ID NO: 37; heavy chain complementarity determination region 2 (CDR2) containing the selected amino acid sequence from SEQ ID NO: 3, SEQ ID NO: 15, SEQ ID NO: 27 and SEQ ID NO: 39; and heavy chain complementarity determination region 3 (CDR3) containing the selected amino acid sequence from SEQ ID NO: 5, SEQ ID NO: 17, SEQ ID NO: 29 and SEQ ID NO: 41; a variable light chain (VL) region comprising: light chain complementarity determination region 1 (CDR1) containing the selected amino acid sequence from the group consisting of SEQ ID NO: 7, SEQ ID NO: 19, SEQ ID NO: 31 and SEQ ID NO: 43;Light chain complementarity determination region 2 (CDR2) containing the selected amino acid sequence from SEQ ID NO: 9, SEQ ID NO: 21, SEQ ID NO: 33 and SEQ ID NO: 45; and light chain complementarity determination region 3 (CDR3) containing the selected amino acid sequence from SEQ ID NO: 11, SEQ ID NO: 23, SEQ ID NO: 35 and SEQ ID NO:

47.

2. ANTIBODY OR FRAGMENT THEREOF, according to claim 1, wherein the antibody or fragment thereof is characterized by comprising a variable heavy chain region and a variable light chain region that are selected from the group consisting of: a variable heavy chain region comprising heavy chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 1, heavy chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 3 and heavy chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 5 and a variable light chain region comprising light chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 7,Light chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 9 and light chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 11; a variable heavy chain region comprising heavy chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 13, heavy chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 15 and heavy chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 17 and a variable light chain region comprising light chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 19, light chain complementarity determination region 2 containing the sequence of Petition 870260041396, dated 04 / 05 / 2026,pg. 22 / 31 3 / 8 amino acids defined by SEQ ID NO: 21 and light chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 23; a variable heavy chain region comprising heavy chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 25, heavy chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 27 and heavy chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 29 and a variable light chain region comprising light chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 31,Light chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 33 and light chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 35; and a variable heavy chain region comprising heavy chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 37, heavy chain complementarity determination region 2 containing the amino acid sequence defined by SEQ ID NO: 39 and heavy chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 41 and a variable light chain region comprising light chain complementarity determination region 1 containing the amino acid sequence defined by SEQ ID NO: 43, determination region of Petition 870260041396, dated 04 / 05 / 2026,pg. 23 / 31 4 / 8 light chain complementarity 2 containing the amino acid sequence defined by SEQ ID NO: 45 and light chain complementarity determination region 3 containing the amino acid sequence defined by SEQ ID NO: 47., 3. ANTIBODY OR FRAGMENT THEREOF, according to claim 1, characterized in that the variable heavy chain region contains the amino acid sequence selected from the group consisting of SEQ ID NO: 49, SEQ ID NO: 53, SEQ ID NO: 57 and SEQ ID NO: 61 and the variable light chain region contains the amino acid sequence selected from the group consisting of SEQ ID NO: 51, SEQ ID NO: 55, SEQ ID NO: 59 and SEQ ID NO:

63.

4. ANTIBODY OR FRAGMENT THEREOF, according to claim 1, characterized in that the antibody is selected from the group consisting of IgG, IgA, IgM, IgE and IgD and in that the fragment is selected from the group consisting of diacorpo, Fab, Fab', F(ab)2, F(ab')2, Fv and scFv.

5. ANTIBODY OR FRAGMENT THEREOF, according to claim 4, characterized in that the scFv contains the amino acid sequence selected from the group consisting of SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71 and SEQ ID NO:

73.

6. POLYNUCLEOTIDE characterized by comprising a nucleotide sequence selected from the group consisting of SEQ ID NO: 50, 52, 54, 56, 58, 60, 62 and 64, wherein said polynucleotide encodes the antibody or fragment thereof, as defined in any one of claims 1 to 5.

7. Recombinant expression vector characterized by comprising the polynucleotide as defined in claim 6. Petition 870260041396, dated 04 / 05 / 2026, page 24 / 31 5 / 8 8. TRANSGENIC MICROORGANISM characterized by being transformed with the recombinant vector, as defined in claim 7, wherein said microorganism is selected from the group consisting of bacteria of the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Bacillus, Pseudomonas and Streptomyces; and fungi and yeasts of the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Trichoderma, Neurospora, Schwanniomyces, Penicillium, Tolypocladium and Aspergillus.

9. METHOD FOR PRODUCING AN ANTIBODY OR FRAGMENT THEREOF THAT SPECIFICALLY BINDS TO AN EXTRACELLULARLY EXPOSED N-TERMINAL LYSYL-tRNA SYNTHETASE REGION, the method being characterized by comprising: transformation of selected transgenic microorganisms from the group consisting of bacteria of the genera Escherichia, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Bacillus, Pseudomonas and Streptomyces; and fungi and yeasts of the genera Saccharomyces, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Trichoderma, Neurospora, Schwanniomyces, Penicillium, Tolypocladium and Aspergillus with the recombinant expression vector, as defined in claim 7; Incubation of transgenic microorganisms transformed to produce an antibody or fragment thereof; and collection of the antibody or fragment thereof produced in the transgenic microorganisms. Petition 870260041396, dated 04 / 05 / 2026, page 25 / 31 6 / 8 10. METHOD FOR SPECIFIC DETECTION OF AN EXTRACELLULARLY EXPOSED LYSYL-tRNA SYNTHETASE N-TERMINAL REGION, the method being characterized by comprising: contact of the antibody or fragment thereof, as defined in claim 1, with a sample; and detection of the antibody or fragment thereof.

11. PHARMACEUTICAL COMPOSITION FOR INHIBITING CANCER METASTASIS characterized by comprising the antibody or a fragment thereof, as defined in claim 1, as an active ingredient.

12. USE OF THE ANTIBODY OR FRAGMENT THEREOF, as defined in claim 1, characterized by being intended for the preparation of an agent for the inhibition of cancer metastasis, wherein the cancer is selected from the group consisting of breast cancer, large intestine cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, uterine cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer,Kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

13. USE OF THE ANTIBODY OR FRAGMENT THEREOF, as defined in claim 1, characterized by being intended for the preparation of an agent for the diagnosis of cancer, wherein the cancer is selected from the group consisting of breast cancer, colon cancer, lung cancer, small cell lung cancer, gastric cancer, liver cancer, blood cancer, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, colon cancer, breast cancer, fallopian tube carcinoma, endometrial carcinoma, cervical cancer, vaginal cancer, vulvar carcinoma, Hodgkin's disease, esophageal cancer, small bowel cancer, endocrine cancer, thyroid cancer, parathyroid carcinoma, adrenal cancer, soft tissue sarcoma, uterine cancer, penile cancer, prostate cancer, chronic or acute leukemia, lymphocyte lymphoma, bladder cancer,Kidney or ureter cancer, renal cell carcinoma, renal pelvic carcinoma, CNS tumor, primary CNS lymphoma, spinal cord tumor, brainstem glioma, and pituitary adenoma.

14. USE OF AN ANTIBODY OR FRAGMENT THEREOF, as defined in claim 1, characterized by being intended for the preparation of an agent for the treatment of a disease related to the migration of immune cells, wherein the disease related to the migration of immune cells is selected from the group consisting of a cardiovascular disease, Petition 870260041396, dated 04 / 05 / 2026, page 27 / 31 8 / 8 a fibrotic disease, a chronic inflammatory disease and Alport syndrome; wherein the cardiovascular disease is selected from the group consisting of pulmonary arterial hypertension, atherosclerosis, angina pectoris, myocardial infarction, ischemic cerebrovascular disease, arteriosclerosis and mesenteric sclerosis;wherein fibrotic disease is selected from the group consisting of scleroderma, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis, pulmonary fibrosis, hepatic fibrosis, hepatic cirrhosis, renal fibrosis, myofibrosis, cardiac fibrosis, systemic lupus erythematosus, hereditary fibrosis, infectious fibrosis, irritant fibrosis, fibrosis caused by chronic autoimmune fibrosis caused by antigenic incompatibility during organ transplantation, fibrosis due to hyperlipidemia, fibrosis due to obesity, diabetic fibrosis, fibrosis due to hypertension, and occlusion caused by fibrosis at stent insertion; wherein chronic inflammatory disease is selected from the group consisting of asthma, atopic dermatitis, eczema, psoriasis, osteoarthritis, gout, psoriatic arthritis, cirrhosis, non-alcoholic steatohepatitis, chronic obstructive pulmonary disease, rhinitis, diabetic retinopathy, diabetic renal failure, diabetic neuropathy, and multiple sclerosis. Petition 870260041396, dated 04 / 05 / 2026, pages 28 / 31;

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