KR101410904B1 - Method for screening of anti-cancer agent and inflammatory disease agent - Google Patents

Abstract

The present invention relates to a method for screening an anticancer medicine and an inflammatory disease treatment medicine and, more specifically, to an inflammatory disease treatment medicine/anticancer medicine screening method which includes the steps of: forming a contact with a part or whole of an ARS-interacting multi-functional protein 1 (AIMP1), a CD23, and candidate agents; and selecting a candidate agent which hinders an interaction between the part or whole of the AIMP1 and the CD23. The method of the present invention is efficient to select an inflammatory disease treatment medicine which suppresses an AIMP1 medium inflammatory cytokine; and an anticancer medicine which increases the level of a free AIMP1 having an anticancer activity by suppressing the cell attachment of the AIMP1.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method of screening for an anti-cancer agent and an inflammatory disease agent,

The present invention relates to a therapeutic agent for an inflammatory disease or an anti-cancer agent screening method, and more particularly to a method for screening an anti-inflammatory agent or an anti-cancer agent, And a candidate agent which inhibits the interaction between the whole AIMP1 or a fragment thereof and CD23, and a method for screening an anti-cancer agent.

Protein synthase-binding multifunctional protein 1 (AIMP1) is one of the three cofactors that link the aminoacyl-tRNA synthetase (ARS). AIMP1 is secreted out of cells in monocytes, endothelial cells, fibroblasts, etc., and acts as a cytokine. It plays a role as a glucagon-like hormone in vivo, or it binds to gp96 protein intracellularly to regulate the immune response.

Functional relevance in the immune response of AIMP1 begins with the discovery of EMAP II (endothelial monocyte activating polypeptide II) with cytokine activity. AIMP1 (ARS-interacting multi-functional protein 1) has been conventionally renamed by the present inventors to be known as p43 protein (Sang Gyu Park, et al., Trends in Biochemical Sciences, 30: 569-574, 2005) .

This polypeptide was found to be the C-terminal part of AIMP1, and at first it was expected that AIMP1 would be an inactive precursor of EMAP II, but AIMP1 itself is a cytokine that is secreted by environmental changes in various cells to cause proinflammatory response It turned out to be a role. Secreted AIMP1 is known to act on a variety of target cells such as monocytes / macrophages, endothelial cells and fibroblasts.

In addition, AIMP1 has been shown to be highly potent in tumor size and weight in recent studies (Han et al., Cancer Letters, 287: 157-164, 2010) It turned out.

Among the inflammation-related molecules induced by AIMP1, tumor necrosis factor-alpha (TNF-alpha) is typical and is known to mediate signal transduction mechanisms such as mitogen-activated protein kinase (MAPK). Thus, the study of AIMP1-induced signaling mechanisms related to TNF-α production will play an important role in elucidating the physiological or pathological role of AIMP1 in inflammatory responses. In some reports, functional receptor candidates for AIMP1 or EMAP II have been proposed, but no receptor associated with AIMP1-induced production of TNF-α is yet known.

Accordingly, the present inventors have found that AIMP1 interacts with CD23 and inhibits the above-mentioned interaction, thereby decreasing cell adhesion of AIMP1 and significantly decreasing TNF-a, thus completing the present invention.

Accordingly, it is an object of the present invention to provide a method for the treatment of cancer, comprising: (a) contacting whole AIMP1 (ARS-interacting multi-functional protein 1) or a fragment thereof, CD23 and a candidate agent; And

(b) selecting a candidate agent that inhibits the interaction between the whole AIMP1 or a fragment thereof and CD23, and a method for screening a therapeutic agent for an inflammatory disease.

Another object of the present invention is to provide a pharmaceutical composition for the treatment of inflammatory diseases comprising a candidate agent selected by the above method.

Another object of the present invention is to provide a method for the treatment of cancer, comprising the steps of: (a) contacting whole AIMP1 (ARS-interacting multi-functional protein 1) or a fragment thereof, CD23 and a candidate agent; And

(b) screening a candidate agent that inhibits the interaction between the whole AIMP1 or a fragment thereof and CD23.

Another object of the present invention is to provide a pharmaceutical composition for treating cancer comprising a candidate agent selected by the above method.

(A) contacting all or a fragment thereof, CD23 and a candidate agent, AIMP1 (ARS-interacting multi-functional protein 1); And

(b) selecting a candidate agent that inhibits the interaction between the whole AIMP1 or a fragment thereof and CD23, and a method for screening a therapeutic agent for an inflammatory disease.

In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for the treatment of inflammatory diseases comprising a candidate agent selected by the above method.

According to another aspect of the present invention, there is provided a method of screening for a disease or condition selected from the group consisting of: (a) contacting whole ARIM-interacting multi-functional protein 1 (AIMP1) or a fragment thereof, CD23 and a candidate agent; And

(b) selecting a candidate agent that inhibits the interaction between the whole AIMP1 or a fragment thereof and CD23.

In order to accomplish another object of the present invention, the present invention provides a pharmaceutical composition for treating cancer comprising a candidate agent selected by the above method.

Hereinafter, the present invention will be described in detail.

The therapeutic agent for inflammatory diseases or the method for screening an anti-cancer agent of the present invention comprises the steps of: (a) contacting all or a fragment thereof, AIMP1 (ARS-interacting multi-functional protein 1), CD23 and a candidate agent; And (b) selecting a candidate agent which inhibits the interaction between the whole AIMP1 or a fragment thereof and CD23.

The screening method of the present invention will be described step by step as follows.

In step (a), the entire ARS-interacting multi-functional protein 1 (AIMP1) or a fragment thereof, CD23 and the candidate agent are contacted.

AIMP1 is also an ARS-binding multifunctional protein (EMAP2, EMAPII, HLD3, SCYE1, p43). AIMP1 is a proinflammatory cytokine that is secreted outside the cell and regulates the immune response. There is no known receptor and sub-signaling system of AIMP1, which is disclosed for the first time in the present invention.

In the screening method of the present invention, AIMP1 may be the entire AIMP1, preferably an amino acid sequence represented by SEQ ID NO: 23. Alternatively, in the screening method of the present invention, AIMP1 may be a fragment containing some amino acid sequence but not whole AIMP1. Preferably, a fragment of AIMP1 of the invention may be a fragment of AIMP1 comprising a site that interacts with CD23. The AIMP1 site interacting with CD23 is a site consisting of amino acid sequence 101 to 192 of the amino acid sequence shown in SEQ ID NO: 23, and it is disclosed for the first time in the present invention that the site specifically interacts with AIMP1. Accordingly, the AIMP1 fragment of the present invention preferably comprises the amino acid sequence of SEQ ID NO: 23, and more preferably the fragment of AIMP1 of the present invention comprises SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: SEQ ID NO: 26 and the like.

CD23 is also described as Fc epsilon RII (FcεRII), a "low-affinity" receptor for IgE, and is known to play an important role in IgE level regulation.

The candidate agent of the present invention is not particularly limited as long as it is a pharmaceutically usable substance. For example, the candidate agent may be a biologically derived substance such as protein, nucleic acid, and a modified or chemically synthesized substance thereof, preferably an antibody, Oligonucleotides, siRNA, mimotopes, peptidomimetic, foldamers, small molecules, natural or engineered lipocalin-based cognitive proteins, DARPin, fibronectin, May be selected from the group consisting of affibody, Kunitz-type inhibitor, peptidic aptamer, ribozyme, toxin and organic / inorganic compound.

Mimotopes are peptides that mimic proteins, hydrocarbons or lipid epitopes, and can be produced by phage display techniques.

Peptide mimetics are small protein-like chains designed to mimic peptides. This is typically done by modifying existing peptides to alter the properties of the molecule. For example, peptide mimetics can be prepared by modification to give stability to the molecule or change in biological activity. Such modifications involve changes to peptides that do not occur naturally (e.g., backbone modifications and the incorporation of non-natural amino acids).

Foldamers are individual chains or oligomers that have a secondary structure stabilized by noncovalent interactions (Gellman, Acc. Chem. Res (1998) 31 (4): 173-180; Hill et al., Chem Rev. (2001) 101 (12): 3893-4012).

It is an artificial molecule that mimics well-defined structures of proteins, nucleic acids and polysaccharides, such as helical structure and ability to fold into? -Sheet. Poddamer has been demonstrated to represent a number of interesting supramolecular features such as self-assembly of molecules, molecular recognition and host-guest chemistry. It has been studied as a model of biomolecules and has been shown to exhibit antimicrobial activity. In addition, it has tremendous potential utility in the development of new functional materials.

Small molecules are by definition low molecular weight organic compounds rather than polymers. The upper limit of the molecular weight of a small molecule is about 800 daltons, which is likely to disperse rapidly through the cell membrane and reach the intracellular action site. Small molecules exert a high affinity for biopolymers such as proteins, nucleic acids or polysaccharides, as well as to alter the activity or function of the biopolymer.

DARPin (Designated Ankyrin Repeat Proteins) is an artificial protein that can recognize antigen or antigenic structures. It is structurally derived from Ankyrin protein, is about 14 kDa (166 amino acids), and consists of three repeating motifs. This shows a similar level of affinity to the antigen for the antigen (Stumpp et al., Drug Discov. Today (2008) 13, Nr. 15-16, S. 695-701).

Affibody molecules are small, robust, highly affinity protein molecules that can be manipulated to specifically bind to a large number of target proteins.

The term "camelid" refers to an antibody produced in camelid and comprising heavy chain dimers and derivatives of these molecules (Muyldermans et al., Veterinary Immunology and Immunopathology, 128; 1-3; pp. 178-183 2009).

The contact may be carried out in vitro or in vivo, and the method comprising administering the CD13-containing cell either to the candidate preparation or the AIMP1 first and then the other, or to administer the AIMP1 and the candidate preparation mixture to the CD23- To the cells to which they are added.

In step (b), a candidate agent that inhibits the interaction between the whole AIMP1 or its fragment and CD23 is selected.

The interaction is not particularly limited thereto, but may be a direct or indirect combination of CD23 with whole AIMP1 or a fragment thereof.

Inhibiting the above-mentioned interactions includes the effects of eliminating, preventing or suppressing the interaction of AIMP1 and CD23.

Inhibition of such interactions may result in the candidate agent being able to alter the level of AIMP1 or CD23 by eliminating, producing or inhibiting the production, production or inhibition of AIMP1 or CD23, or the candidate agent may combine competitively or noncompetitively with AIMP1 or CD23, The method comprising: Competitively binding refers to the elimination, prevention or inhibition of the interaction of AIMP1 and CD23 by binding to the interacting site of AIMP1 or CD23, and uncompetitive binding refers to the binding of AIMP1 or CD23 to a site other than the interacting site of AIMP1 or CD23 Preventing, or inhibiting the interaction of AIMP1 and CD23.

Thus, the candidate agent of the present invention may be an inhibitor that inhibits the expression of AIMP1 or CD23, a competitive or noncompetitive binding agent to whole AIMP1 or a fragment thereof or CD23. However, since the anticancer drug screening method screened a candidate agent that increases the level of free form extracellular AIMP1 that is not adhered to cells through inhibition of interaction with CD23, the candidate agent that inhibits the expression of AIMP1 may have a limited anticancer activity.

Therefore, the candidate agent of the method for screening therapeutic agents for inflammatory diseases of the present invention is preferably a candidate agent, which may be an inhibitor that inhibits the expression of AIMP1 or CD23, competitively or noncompetitively binds to whole AIMP1 or its fragment or CD23, The candidate agent of the anticancer drug screening method of the present invention can preferably be an inhibitor that inhibits the expression of CD23, preferably a competitive inhibitor that binds to whole AIMP1 or its fragment or CD23 competitively or noncompetitively.

Selection of the present invention can be accomplished by a variety of biochemical and molecular biology techniques known in the art, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press , NY, Second (1998) and Third (2000) Editions; and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York (1987-1999).

The screening of the present invention can be carried out by, for example, labeling in vitro labeled protein-protein binding assays, electrophoretic mobility shift assays (EMSAs), immunoassays for detecting protein binding, functional assays (phosphorylation assays of sub- Biotechnology, 13: 115-122,1995; Ecker et al., Bio / Technology, 4: 376,199; 4,376,110; 4,517,288 and 4,837,168; and Bevan et al., Trends in Biotechnology, 13: 351-360, 1995; and Hodgson, Bio / Technology, 10: 973-980, 1992).

AIMP1 interacts with CD23 to induce TNF-a production. TNF-α is a major cytokine that causes inflammation and is reported to be involved in the pathophysiology of acute or chronic inflammation such as septic shock, AIDS, and arthritis.

As the inventors of the present invention newly found, inhibition of the interaction between AIMP1 and CD23 inhibits the production of AIMP1-mediated TNF-α. Therefore, the candidate agent for inhibiting the interaction of AIMP1 and CD23 as described above is effective for the treatment of inflammatory diseases and can be used as a therapeutic agent for inflammatory diseases.

On the other hand, AIMP1 is also involved in intracellular translation, but it is also secreted by cytokines with various physiological activities. In previous studies, vascular injected AIMP1 has been shown to exhibit anticancer activity (Han et al., Cancer Letters, 287: 157-164, 2010).

Therefore, when a candidate agent inhibiting the interaction of AIMP1 and CD23 is treated, the level of extracellular AIMP1 is enhanced and the antitumor activity of AIMP1 is increased by inhibiting binding of AIMP1 to CD23 of cells. Therefore, the candidate agent that inhibits the interaction of AIMP1 and CD23 as described above has therapeutic effect on cancer and can be used as a therapeutic agent for cancer.

Such inflammatory diseases include, but are not limited to, sepsis, arteriosclerosis, bacteremia, systemic inflammatory response syndrome, multiorgan dysfunction, osteoporosis, periodontitis, systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, chronic arthritis, (S), systemic sclerosis, idiopathic inflammatory muscle disorders, Sjoegren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immunomodulatory renal disease, central nervous system or peripheral nervous system Guillain-Barre syndrome, chronic inflammatory dehydration multinucleate neuritis, hepatic biliary disease, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis , Sclerosing cholangitis, inflammatory bowel disease (IBD), ulcerative colitis colitis, Crohn's disease, irritable bowel syndrome, gluten-sensitive bowel disease, Whipple's disease, autoimmune or immunomodulatory skin disease, Or at least one of the following: a disease selected from the group consisting of psoriasis, atopic dermatitis, psoriasis, allergic disease, asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, urticaria, pulmonary immune disease, eosinophilic pneumonia, idiopathic pulmonary fibrosis, hypersensitivity pneumonia, Lt; RTI ID = 0.0 > inflammatory < / RTI >

In addition, the anticancer agent of the present invention is not limited to, but is not limited to, anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, angioma, Osteoporosis, prostate cancer, lung cancer, colon cancer, pancreatic cancer, renal cancer, gastric cancer, skin cancer, basal cell carcinoma, melanoma, squamous cell carcinoma, ovarian cancer, A cancer selected from the group consisting of oral squamous cell carcinoma, colorectal cancer, glioblastoma, endometrial cancer and malignant glioma.

In addition, the candidate substance screened by the screening method for the therapeutic agent for inflammatory diseases of the present invention is effective for the treatment of inflammatory diseases and thus can be used for the pharmaceutical use for the treatment of inflammatory diseases. Further, the candidate substance screened by the anticancer agent screening method of the present invention has an anticancer effect and can be used for pharmaceutical use for cancer treatment.

The pharmaceutical composition according to the present invention may include a pharmaceutically effective amount of the candidate agent selected by the screening method alone, or may further include one or more pharmaceutically acceptable carriers. The term "pharmaceutically effective amount" as used herein refers to an amount exhibiting a further reaction than the negative control, preferably an amount sufficient to treat an inflammatory disease or cancer.

The pharmaceutically effective dose of the selected candidate agent according to the screening method of the present invention may be appropriately varied depending on various factors such as the disease and its severity, the patient's age, body weight, health condition, sex, .

The term " pharmaceutically acceptable " as used herein means a non-toxic composition which is physiologically acceptable and which, when administered to humans, does not inhibit the action of the active ingredient and does not normally cause an allergic reaction such as gastrointestinal disorder, dizziness, . The composition of the present invention can be variously formulated according to the route of administration by a method known in the art together with the pharmaceutically acceptable carrier. Routes of administration include, but are not limited to, oral or parenteral administration. Parenteral routes of administration include, for example, various routes such as transdermal, nasal, peritoneal, muscular, subcutaneous or intravenous.

When the pharmaceutical composition of the present invention is orally administered, the pharmaceutical composition of the present invention may be formulated into a powder, a granule, a tablet, a pill, a sugar, a tablet, a capsule, a liquid, a gel , Syrups, suspensions, wafers, and the like. Examples of suitable carriers include saccharides including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, Cellulose such as methylcellulose, sodium carboxymethylcellulose and hydroxypropylmethyl-cellulose and the like, fillers such as gelatin, polyvinylpyrrolidone and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may optionally be added as a disintegrant. Further, the pharmaceutical composition may further comprise an anti-coagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and an antiseptic agent.

In addition, when administered parenterally, the pharmaceutical composition of the present invention can be formulated together with a suitable parenteral carrier according to methods known in the art in the form of injection, transdermal drug delivery, and nasal inhalation. In the case of such injections, they must be sterilized and protected against contamination of microorganisms such as bacteria and fungi. Examples of suitable carriers for injectables include, but are not limited to, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), mixtures thereof and / or vegetable oils . More preferably, suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose Etc. may be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like may be further included. In addition, the injections may in most cases additionally include isotonic agents, such as sugars or sodium chloride. Examples of transdermal dosage forms include ointments, creams, lotions, gels, solutions for external use, pastes, liniments, and air lozenges. By " transdermal administration " as used herein, it is meant that the pharmaceutical composition is locally administered to the skin so that an effective amount of the active ingredient contained in the pharmaceutical composition is delivered into the skin. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975, Mack Publishing Company, Easton, Pennsylvania, which is a commonly known formulary in pharmaceutical chemistry. In the case of an inhalation dosage form, the compounds used according to the invention can be formulated into a pressurized pack or a pressurized pack using a suitable propellant, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gases. It can be conveniently delivered in the form of an aerosol spray from a nebulizer. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges used in inhalers or insufflators may be formulated to contain the compound and a powder mixture of a suitable powder base such as lactose or starch.

Other pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, Pa., 1995).

Furthermore, the pharmaceutical compositions for the treatment of inflammatory diseases or for the treatment of cancer of the present invention may be administered in combination with known compounds having an anti-inflammatory effect or anti-cancer effect, respectively.

In one embodiment of the present invention, an ELISA-based binding assay was performed using a soluble Fc-fusion receptor protein to select a partner protein specifically binding to AIMP1. As a result, six proteins, such as CD23, were screened as receptor candidates showing high affinity for AIMP1. The secretion amount of TNF-α, an inflammatory cytokine, was measured by administering AIMP1 to the candidate siRNA- And the expression of TNF- [alpha] was significantly decreased in the case of siRNA treatment.

Thus, it was confirmed that a candidate agent inhibiting the interaction of AIMP1 and CD23 could be used as a therapeutic agent for inflammatory diseases.

In another embodiment of the present invention, AIMP1 was administered to cells treated with siRNA for CD23 and other receptor candidates, and the amount of AIMP1 attached to the cell surface was measured and compared with the control group. As a result, it was confirmed that the amount of AIMP1 adhered to the surface was significantly reduced only in the case of cells treated with siRNA against CD23.

As a result, it was confirmed that a candidate agent inhibiting the interaction of AIMP1 and CD23 could be used as an anticancer agent.

In another embodiment of the present invention, the AIMP1-deficient body was prepared and its binding capacity to CD23 was measured to determine which part of AIMP1 has high affinity with CD23. As a result, it was confirmed that AIMP1- (1312), AIMP1- (1192), AIMP1- (47192), AIMP1- (101192) and the like containing 101th to 192th amino acids interact well with CD23.

In addition, it was confirmed that when the AIMP1 fragment was administered to siRNA-treated cells, the amount of secreted TNF-α decreased.

As a result, it was confirmed that the screening of the present invention can be carried out using the fragment of AIMP1 and CD23.

In another embodiment of the present invention, it was examined whether CD23 is involved in the mechanism of other cytokines other than AIMP1. As a result, it was confirmed that CD23 specifically binds to AIMP1 and does not interact with other cytokines (EMAP II).

In another embodiment of the present invention, what is the lower signal transduction mechanism of CD23 involved in AIMP1 was examined. As a result, it was confirmed that the signal transduction pathway of CD23 is phosphorylation of ERK1 / 2 as a downstream signal transduction mechanism.

As described above, the present invention provides a method for producing AIMP1 comprising contacting whole AIMP1 or a fragment thereof, CD23 and a candidate agent; And a candidate agent that inhibits the interaction between the whole AIMP1 or a fragment thereof and CD23, and a method for screening an anti-cancer agent. The method of the present invention is effective for the treatment of inflammatory diseases that inhibit AIMP1-mediated inflammatory cytokines and the selection of anticancer agents that increase the level of free AIMP1 with anti-cancer activity by inhibiting cell adhesion of AIMP1.

Figure 1 shows the results of primary screening of AIMP1 binding receptors. The signal is the OD490 value in the form of a heat map from 0 (green) to 3 (red).
Figure 2 shows the binding affinity of AIMP1 and bovine serum albumin (BSA and AIMP1: BSA and AIMP1-coated wells) for the receptor showing high affinity for the selected AIMP1 OD ₄₉₀ readings; Ratio: AIMP1 value divided by BSA value, Mock: internal control without added water-soluble receptor).
FIG. 3 is a graph showing that the receptor mRNA production is effectively inhibited after siRNA treatment for each receptor protein (VEGFR1: VEGFR2: Vascular endothelial growth factor receptor 2 (sFRP1: Soluble frizzled-related proteins ; TNFSF13: Tumor necrosis factor ligand superfamily member 13; PLAC9: Placenta-specific 9 protein; CLEC10A: C-type lectin domain family 10 member A; CD23: Fc epsilon RII; IL20Rb: interleukin 20 receptor beta; CXCR3: chemokine ) receptor 3; siCntr: control group, GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) siRNA treatment group, white bar: each receptor mRNA production in control siRNA treatment black bars: each receptor mRNA production in each receptor siRNA treatment Ratio: MRNA production rate of the receptor siRNA treatment group).
FIG. 4 is a graph showing the change in TNF-α secretion level of THP-1 cells when the selected partner protein is administered (vertical axis: ratio of secretion of TNF-α to control (Cntr): VEGFR1: vascular endothelial growth factor receptor 1; : PLAC9: Placenta-specific 9 protein: CLEC10A: C-type lectin domain family 10 member A; CD23: Fc epsilon; CXCR3: chemokine (CXC motif) receptor 3, Cntr: control group, Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) group. The asterisk indicates a significant change from the control (* P <0.05, ** P <0.01).
Figure 5 is a graph showing the result of measurement of the degree of binding of AIMP1 binding receptor concentration (KD: equilibrium constant, longitudinal axis: specific binding force (OD490 absorbance), transverse axis: administered binding receptor concentration; CD23: Fc epsilon RII; CLEC10A: type lectin domain family member A, sFRP1: Soluble frizzled-related proteins).
Figure 6 shows the results of measuring the concentration of AIMP1 attached to the surface of THP-1 cells when treated with siRNA for each AIMP1 binding receptor (Tubulin: internal control; siCntr: negative control (THP-1 transfected with Stealth universal RNAi) siCLEC10A: siRNA treatment group for C-type lectin domain family 10 member A siCD23: siRNA treatment group for Fc epsilon RII siFRP1 siRNA treatment group for soluble frizzled-related protein siPLAC9: Placenta-specific 9 protein Ratio: AIMP1 binding ratio of control (siCntr) to each receptor siRNA treated group).
FIG. 7 is a graph showing the results of experiments on the ability of AIMP1 to regulate TNF-α regulation according to CD23 inhibition (vertical axis: TNF-α secretion amount, transverse axis: administered AIMP1 concentration; siCntr: negative control (THP-1 transfected with Stealth universal RNAi) ; siCD23: si23 treated group against CD23 (Fc epsilon RII)). The asterisk indicates a significant change from the control (* P <0.05, ** P <0.01).
FIG. 8 is a graph showing changes in TNF-α regulation ability of AIMP1 according to the stimulation of CD23 (vertical axis: TNF-α secretion amount, transverse axis: administered AIMP1 concentration; IL-4: IL-4 treatment group as a CD23 inducer; 4 untreated group; IL-4 / antiCD23: IL-4 and anti-CD23 antibody simultaneously). The asterisk indicates a significant change from the control (* P <0.05, ** P <0.01).
Figure 9 is a graph showing the results of experiments on the ability of AIMP1 to modulate TNF-a regulation by CD23 inhibition (vertical axis: secretion amount of TNF-a; transverse axis: concentration of administered AIMP1; Cntr / Fc: control group; CD23 / Fc: Soluble form of Cd23, including amino acids). The asterisk indicates a significant change from the control (* P <0.05, ** P <0.01).
FIG. 10 is a photograph showing the degree of phosphorylation of a lower signal transduction substance according to AIMP1 treatment (ERK1 / 2: extracellular-signal-regulated kinases 1/2; JNK: c-Jun N-terminal kinases; AKT: protein kinase B ; p-: type of phosphorylation of each signaling substance; min: time (minutes) after AIMP1 treatment).
11 is a graph showing the degree of phosphorylation of the lower signal transduction substance according to the AIMP1 treatment (vertical axis: relative ratio value when the ratio of the phosphorylated / non-phosphorylated form of the initial angiogenesis signal is 1; 2), extracellular-signal-regulated kinases 1/2, JNK: c-Jun N-terminal kinases, AKT: protein kinase B, p-: phosphorylation form of each signal transduction substance).
Figure 12 shows the results of an experimental change in ERK1 / 2 activation according to CD23 inhibition (siCntr: negative control (THP-1 transfected with Stealth universal RNAi); siCD23: siRNA treated group against F23 epsilon RII; ERK1 / 2 : extracellular-signal-regulated kinases 1/2; p-ERK1 / 2: phosphorylation form of ERK1 / 2).
Figure 13 shows the results of measurement of changes in TNF-a secretion of THP-1 cells following ERK1 / 2 inhibition (upper graph: TNF-alpha secretion relative to control group (AIMP1 +, U0126-, SP600125-) ; U0126: ERK1 / 2 inhibitor; SP600125: JNK inhibitor; Tubulin: internal control). The asterisk indicates a significant change from the control (* P <0.05, ** P <0.01).

14 is a schematic (top) of the AIMP1 deletion used in the experiment (bottom) (sCD23 / Fc: an aqueous form of Cd23 containing 172 amino acids at the end of CD23 COOH; EV : empty vector; Input: loading control with sCD23 / Fc only).
FIG. 15 is a graph showing the TNF-α induction ability of AIMP1 and its deletion products (vertical axis: concentration of secreted TNF-α, transverse axis: AIMP1 (1-312) .
FIG. 16 is a graph showing the TNF-α induction ability of AIMP1 and its defective cells in CD23-inhibited THP-1 cells (vertical axis: concentration of secreted TNF-α; siCntr: negative control Infected THP-1; siCD23: siRNA treated group against CD23; NT: control group not treated with AIMP1; 1-312: AIMP1; 101-192: deficient AIMP1). Asterisk indicates a significant change from negative control (* P <0.05, ** P <0.01).
17 shows the result of a pull-down assay for confirming the binding of CD23 to EMAP II (Cntr / Fc: control group; CD23 / Fc: CD23 soluble CD23 containing 172 amino acids at the COOH end; EMAP II: endothelial monocyte -activating polypeptide-II; IB: AIMP1: blotted with AIMP1 antibody; IB: IgG: blotted with IgG).
Figure 18 shows the result of measuring TNF-α secretion amount to confirm the effect of CD23 inhibition on TNF-α secretion by AIMP1 or EMAP II (vertical axis: secretion amount of TNF-α; siCntr: negative control (Stealth universal RNAi) SiRNA 23: siRNA treatment group against CD23; EMAP II: EMAP II treatment concentration; AIMP1: AIMP1 treatment concentration). Asterisk indicates a significant change from negative control (* P <0.05, ** P <0.01).
Figure 19 shows the effect of EMAP II on ERK1 / 2 phosphorylation in THP-1 cells compared with AIMP1 (upper min: time (min) after AIMP1 or EMAP II treatment; ERK1 / 2: extracellular-signal -regulated kinases 1/2; p-ERK1 / 2: phosphorylation form of ERK1 / 2).
20 is a graph showing the results of measurement of TNF-α secretion amount to confirm the effect of CD23 inhibition on TNF-α secretion by AIMP1 or EMAP II (vertical axis: TNF-α secretion amount; white graph: negative control group (U0126 untreated group); Black graph: U0126 (ERK inhibitor) treated group, NT: EMAP II and AIMP1 untreated group on the abscissa, EMAP II: EMAP II treated group, AIMP1: AIMP1 treated group). The asterisk indicates a significant change from the negative control (* P <0.237, ** P <0.01).

Hereinafter, the present invention will be described in detail with reference to examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

&Lt; Example 1 >

Selecting a partner protein that specifically binds to AIMP1

<1-1> Selection of a partner protein specifically binding to AIMP1

In order to select partner proteins that specifically bind to AIMP1, 499 soluble receptor proteins were bound to AIMP1 coated plates.

An ELISA-based binding assay using a soluble Fc-fusion receptor protein was performed to select AIMP1 binding partners.

Human cDNAs encoding extracellular regions of cell membrane receptors, except for seven transmembrane proteins in the pYK602 vector, which is readily designed for Fc-fusion protein purification in animal cells, were cloned and transfected into HEK293 cell line for 24 hours. After incubation for 3 days in serum-free DMEM, the culture broth was collected and reacted with protein A agarose beads, and the extracellular domains of the Fc-fused receptor protein bound to the beads were isolated.

A MaxiSorp plate (Nunc, USA) was coated with recombinant AIMP1 (1 mg / ml in PBS) for 12 h at 4 ° C and blocked with 4% non-fat milk (in PBS). The extracellular domain of the Fc-fused receptor protein was applied to the coated plate at a concentration of 1 mg / ml, washed, and reacted with antihuman IgG1 Fc-HRP (horseradish peroxidase) (Thermo). After washing the plate, TMB (3,3,5,5-Tetramethylbenzidine) was added to develop color, and the absorbance was measured at a wavelength of 490 nm.

As a result, six proteins including CD23, CLEC10A, FRP1, IL20Rb, TNFSF13 and PLAC9 were selected as receptor candidates showing high affinity for AIMP1 (see FIG. 1).

To determine the binding specificity for AIMP1, binding affinities for AIMP1 and BSA were measured for six receptor candidates.

The binding affinities of AIMP1 and BSA were compared by inserting the six water soluble receptor proteins on a plate coated with AIMP1 or BSA (Bovine serum albumin), and measuring the absorbance in the same manner as above.

As a result, it was confirmed that CD23 (2.2631) and CLEC10A (2.6641) exhibited high binding specificity to AIMP1 (see FIG. 2).

<1-2> Confirmation of TNF-α secretion regulation of the selected partner protein

In order to confirm whether the 6 proteins firstly selected as binding partners of AIMP1 regulate TNF-α secretion in Example <1-1>, 9 kinds of proteins such as VEGFR1, VEGFR2 and CXCR3 which are already known as EMAP II receptors siRNA were treated with human monocytic THP-1 cells to measure changes in mRNA expression and TNF-a secretion level.

THP-1 cells were incubated for 12 hours, and the 9 kinds of siRNAs were transfected by electroporation using Microporator-mini of Digital Bio Technology Co., After 48 hours, total RNA was extracted with RNeasy Mini Kit (QIAGEN) and quantitative RT-PCR was performed to confirm whether the transfected siRNA effectively inhibited the target and the expression level of TNF-α. All siRNAs used in the experiments were purchased from Invitrogen.

Glyceraldehydephosphate dehydrogenase (GAPDH) mRNA levels were used as controls.

List of primers used for RT-PCR name order SEQ ID NO: VEGFR1-Forward 5'-ATGGTCTTTGCCTGAAATGGTGAG-3 ' One VEGFR1-Reverse 5'-CTGTGAAGCCAGTGTGGTTTGC-3 ' 2 VEGFR2-Forward 5'-GGAAATGACACTGGAGCCTACAAG-3 ' 3 VEGFR2-Reverse 5'-GGACCCGAGACATGGAATCACC-3 ' 4 sFRP1-Forward 5'-GGCGGAGGTGAAGCAGCAG-3 ' 5 sFRP1-Reverse 5'-CGAAGAGCGAGCAGAGGAAGAC-3 ' 6 TNFSF13-Forward 5'-CCTGGAAGCCTGGGAGAATGG-3 ' 7 TNFSF13-Reverse 5'-ATGTCACATCGGAGTCATCCTTGG-3 ' 8 PLAC9-Forward 5'-GCCGCTGCCGAACCCTTC-3 ' 9 PLAC9-Reverse 5'-CCACGGTCTTCTCTACCATCTCC-3 ' 10 CLEC10A-Forward 5'-GCTGGTCATCATCTGTGTGGTTG-3 ' 11 CLEC10A-Reverse 5'-TGCCTGCCGTTCCTGCTTG-3 ' 12 CD23-Forward 5'-TGCTGACTCTGCTTCTCCTGTG-3 ' 13 CD23-Reverse 5'-TCTGCGTGGACTGGGATTTCTG-3 ' 14 IL20Rb-Forward 5'-TCTTGATGTGGAGCCCAGTGATC-3 ' 15 IL20Rb-Reverse 5'-TCAGGACCTTCAGTGAGTGAGC-3 ' 16 CXCR3-Forward 5'-CCGACACCTTCCTGCTCCAC-3 ' 17 CXCR3-Reverse 5'-GCTCCTGCGTAGAAGTTGATGTTG-3 ' 18 GAPDH-Forward 5'-CGCTCTCTGCTCCTCCTGTTC-3 ' 19 GAPDH-Reverse 5'-TTGACTCCGACCTTCACCTTCC-3 ' 20 TNF-a-Forward 5'-GGCGTGGAGCTGAGAGATAAC-3 ' 21 TNF-a-Reverse 5'-GGTGTGGGTGAGGAGCACAT-3 ' 22

The ratios were obtained by dividing each candidate group mRNA level of cells transfected with each candidate receptor siRNA by the level of mRNA of each candidate group of the transfected siRNA-transfected cells. The level of TNF-α was determined by the relative ratio of TNF-α to the control group.

RT-PCR showed that the expression of each mRNA was reduced by at least 50% when 9 kinds of siRNA were treated (see [Fig. 3]), and only when treated with siRNA against CD23, TNF- (Fig. 4). &Lt; tb &gt;&lt; TABLE &gt;

<1-3> CD23 of AIMP1 Confirmation of specific binding with

In order to confirm whether AIMP1 specifically binds to CD23, the binding affinity between AIMP1 and CD23 was measured.

CD23, CLEC10A and sFRP1 in various concentrations of Fc-fusion forms were put into a plate coated with AIMP1 and reacted, and then the amount bound to AIMP1 was measured in the same manner as in Example <1-1>.

As a result, the maximum binding force (Bmax) of CD23 was 0.35 and the equilibrium constant (KD) was 4.3 nM. In contrast, the equilibrium constants (KD) of CLEC10A showing similar binding specificities to CD23 and FRP1 showing relatively low binding specificities were 159.1 and 325.2 nM, respectively (see FIG. 5).

It was confirmed that CD23 was a receptor for AIMP1.

< Example  2>

CD23 of TNF -α secretion regulation mechanism

&Lt; 2-1 > AIMP1 of THP -1 Surface bonding force adjustment test

In order to confirm whether AIMP1 binds to CD23 on the surface of THP-1 cells, the amount of AIMP1 attached to the surface of THP-1 cells transfected with siRNAs of proteins having high binding affinity for AIMP1 including CD23 was measured. The siRNA transfected THP-1 was prepared in the same manner as in Example < 1-2 >.

His-AIMP1 (1 mg) was reacted with 0.25 mg of Sulfo-NHS-SS-biotin (Pierce) dissolved in PBS buffer for 4 hours and reacted with 100 nM of Tris-Hcl (pH 7.4) to prepare biotin-conjugated AIMP1. THP-1 was cultured for 12 hours, then cultured in serum-free RPMI medium for 30 minutes, and biotinylated AIMP1 was added. The THP-1 cells were washed with PBS and incubated in 50 mM Tris buffer containing 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.2% sodium deoxycholate, 10 mM NaF, 1 mM sodium orthovanadate, 10% glycerol, -HCl lysis buffer, and centrifuged at 18,000 g. Proteins were separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and detected by western blotting with streptavidin-coupled HRP (Pierce). The relative amount of AIMP1 bound to THP-1 cells was measured by siRNA treatment of each candidate receptor with THP-1 transfected with Stealth universal RNAi (Invitrogen) as a negative control (siCntr).

As a result, it was confirmed that the amount of AIMP1 adhering to the surface of THP-1 cells was reduced only when CD23 was knocked down (see FIG. 6).

Thus, it was confirmed that CD23 specifically mediates the surface adhesion of AIMP1 in THP-1 cells, thereby regulating TNF-a secretion induced by AIMP1.

<2-2> CD23  Due to inhibition AIMP1 of TNF -α regulation ability change experiment

In order to confirm that the TNF-α secretion induced by AIMP1 is CD23-dependent, the expression of TNF-α was secreted by treatment with AIMP1 at various concentrations after artificially regulating the expression of CD23.

AIMP1 was treated with a constant concentration (0, 50, 100, 200 nM) and incubated for 3 hours in THP-1 cells transfected with CD23 siRNA or control siRNA, and TNF- Were measured in the same manner.

As a result, it was confirmed that when AIMP1 was treated, the secretion of TNF-α was reduced to a certain level regardless of the concentration of AIMP1 (see FIG. 7).

<2-3> CD23  Accelerated AIMP1 of TNF -α regulation ability change experiment

The effect of CD23 on AIMP1-mediated TNF-α secretion was studied by treatment of CD23 inducer IL-4. THP-1 cells were treated with IL-4 (20 mg / ml) for 72 hours and then treated with anti-CD23 antibody (10 mg / ml). AIMP1 was treated for 3 hours at a predetermined concentration (0, 50, 100, 150, 200 nM) and centrifuged (3000 rpm, 10 minutes) to recover the culture medium and then the amount of TNF-α was measured. The amount of secreted TNF- [alpha] was measured according to the manufacturer's protocol using the TNF-alpha ELISA Kit (R &amp; D Systems).

As a result, in the case of treatment with IL-4, the secretion of TNF-α was increased dependently on the concentration of AIMP1, and the treatment of anti-CD23 neutralizing antibody induced the induction of A23P1 in cells expressing CD23 by IL- Gt; TNF-a &lt; / RTI &gt; secretion increase (see FIG. 8).

These results indicate that when the expression of CD23 is limited, even a small amount of AIMP1 treatment is already saturated and the secretion of TNF-α is not dose-dependent. However, when the expression of CD23 is increased, the binding with AIMP1 is dose-dependently regulated.

<2-4> CD23  Due to inhibition AIMP1 of TNF -α regulation ability change experiment

To determine whether AIMP1 competitively interferes with binding to CD23 bound to cell surface by binding to AIMP1, a mixture of AIMP1 and a water-soluble receptor has been previously treated with THP-1 cells and TNF -α secretion was measured.

Fc-fused sCD23 (the soluble form of CD23 encoding the COOH-terminal 172 amino acids) and the control Fc-fused protein were prepared in the same manner as in Example <1-1>, and then 2 mg of AIMP1 and Fc- After mixing with 2 mg of control Fc-fused protein, THP-1 cells were reacted for 3 hours and then tested in the same manner as in Example <2-3>.

As a result, it was confirmed that the secretion of TNF-α was inhibited only in the case of treatment with the water-soluble receptor of CD23 (FIG. 9).

These results suggest that CD23 is a functional receptor for AIMP1, a proinflammatory cytokine in the THP-1 cell line, and the binding site for CD23 of AIMP1 is expected to overlap antigenic determinants of neutralizing antibodies against CD23.

< Example  3>

AIMP1 For CD23 Child Signaling  Confirm

In THP-1 cells, AIMP1 activates proteins involved in signaling mechanisms such as MAPK and NF-kB. ERK (extracellular signal-regulating kinase) in MAPK is rapidly activated by cross-linking CD antibody within 5-10 min, and JNK is known to be activated slowly after 30 min. In contrast, activation of p38 MAPK and NF-kB is not associated with the production of TNF-α induced by CD23.

<3-1> CD23  Of the relevant lower signaling path AIMP1  Check associativity

In order to investigate how the signaling pathway mediated by CD23 in the TNF-a secretion process of AIMP1-induced immune cells occurs, THP-1 cells are treated with AIMP1 in a time-dependent manner and CD23 And phosphorylation of ERK1 / 2 and JNK and AKT (PKB).

THP-1 cells were reacted with AIMP1 at a concentration of 100 nM and the degree of phosphorylation of ERK1 / 2, JNK and AKT was measured at regular intervals.

To determine the degree of phosphorylation, THP-1 cells were washed with PBS and incubated with 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.2% sodium deoxycholate, 10 mM NaF, 1 mM sodium orthovanadate, 10% glycerol, The protein was collected by centrifugation at 18,000 g and separated by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Each antibody (primary antibody: phospho-p44 / 42 Cell Signaling Technology, Inc. Secondary antibody: Goat (Thr202 / Tyr204) antibody, phospho-SAPK / JNK (Thr183 / Tyr185) Antibody, phospho-p38 MAPK anti-Rabbit IgG (H + L) (HRP), Polyclonal (Thermo Fisher Scientific, Inc.)).

For the quantitative analysis, the value measured using a densitometer was set to 1 for initial phosphorylation, and a relative value was obtained.

As a result, ERK1 / 2 started to phosphorylate 5 minutes after the treatment with AIMP1, increased to 30 minutes and then slightly decreased, but JNK and AKT were not phosphorylated until 60 minutes (FIG. 10, Reference). These results suggest that ERK1 / 2 is a downstream signaling pathway of CD23-mediated signaling pathway in the TNF-α secretion process of AIMP1-induced immune cells.

<3-2> CD23  Due to inhibition ERK1 / 2 activation change experiment

In order to confirm whether the phosphorylation of ERK1 / 2 was CD23-dependent, THP-1 cells were treated with siRNA of CD23 to confirm the degree of phosphorylation of AIMP1 binding and ERK1 / 2 in THP-1 cells.

THP-1 cells transfected with DC23 siRNA or control siRNA were prepared in the same manner as in Example < 1-2 &gt;, cultured for 48 hours, and reacted with AIMP1 for 30 minutes. To measure the amount of protein.

As a result, it was confirmed that AIMP1 binding and activation of ERK1 / 2 were all reduced in THP-1 cells (see FIG. 12).

<3-3> ERK1 / 2 inhibition  by TNF -α secretion change experiment

TNF-α secretion changes by ERK1 / 2 inhibition were performed.

AMP1 was treated for 30 minutes with THP-1 for 30 minutes with an inhibitor against MEK (mitogen-activated ERK kinase) (U0126) or with an inhibitor against JNK (SP60125) The amount of ERK1 / 2 and ERK1 / 2 protein was measured, centrifuged (3000 rpm, 10 minutes), and the amount of TNF-? Was measured after recovering the culture. The amount of secreted TNF- [alpha] was measured according to the manufacturer's protocol using the TNF-alpha ELISA Kit (R &amp; D Systems).

As a result, it was confirmed that treatment of the inhibitor (U0126) against mitogen-activated ERK kinase (MEK) not only reduced the activity of ERK1 / 2 by AIMP1 but also decreased TNF-α secretion (see FIG.

This decrease in TNF-a could not be confirmed by treatment with the inhibitor for JNK (SP60125). Therefore, it was found that TNF-α secretion induced by CD23 in AIMP1 treatment is associated with activation of ERK1 / 2 in the CD23 downstream signaling pathway in the immune cells.

< Example 4>

CD23 of AIMP1  Confirm binding site

<4-1> AIMP1 Deficient CD23  Bond strength measurement

In order to confirm which part of AIMP1 binds to CD23, AIMP1 deletion constructs were prepared and the binding force with CD23 was confirmed by pull-down assay.

AIMP1- (1-192) (AIMP1- (1-312), SEQ ID NO: 23) and AIMP1 deletion products AIMP1- - (101-192) (SEQ ID NO: 26), AIMP1- (1-46) (SEQ ID NO: 27) and AIMP1- (193-312) Structural segregation of different extracellular activities in aminoacyl-tRNA synthetase-interacting multi-functional protein, p43 / AIMP1. Biochem. Biophys. Res. Commun. 342,113- 118.).

All AIMP1 and AIMP1 defects were expressed in E. coli BL21 (DE3) as a GST-tag fusion protein, and were identified as glutathione S beads by the conventional method (Ko, YG, Park, H., Kim, T., (2001). A cofactor of tRNA synthetase, p43, is secreted to up-regulate proinflammatory genes. JW Park, SG Seol, W. Kim, JE Lee, WH Kim, SH Park, JE et al. J. Biol. Chem. 276, 23028-23033. To remove the lipopolysaccharide, the protein solution was dialyzed with pyrogen-free buffer (10 mM PBS, pH 6.0, 100 mM NaCl), loaded onto polymyxin resin (Bio-Rad) equilibrated with the same buffer, allowed to react for 20 min and eluted .

AIMP1 and its defective fragments were mixed with Fc-fused sCD23 protein and mixed with protein A / G agarose for immunoprecipitation. Anti-AIMP1 polyclonal antibody (Abcam) and human IgG-coupled HRP were used for detection.

As a result, AIMP1- (146) and AIMP1- (193312) do not bind to CD23-Fc fusion protein while AIMP1- (1312), AIMP1- (1192), AIMP1- (47192), AIMP1- In summary, it was estimated that the part bound to CD23 was the middle part of AIMP1 (amino acid sequence 101192) (see FIG. 14).

<4-2> AIMP1 Deficient TNF -α secretion regulation ability measurement

TNF-α secretion was induced by treating each defect with THP-1 cell line to confirm whether the region of AIMP1 binding to CD23 is directly related to TNF-α secretion. The amount of secreted TNF- [alpha] was measured according to the manufacturer's protocol using the TNF-alpha ELISA Kit (R &amp; D Systems).

As a result, AIMP1- (1312), AIMP1- (1192), AIMP1- (47192) and AIMP1- (101192) showed cytokine activity while AIMP1- (146) and AIMP1- (See Fig. 15).

<4-3> AIMP1 The defects 101-192 and CD23 of interaction  Confirm

To determine whether secretion of TNF-α by AIMP1 deficient cells was also reduced by inhibition of CD23, the amount of TNF-α secreted after treatment of AIMP1 deficient cells was measured by preparing siRNA-transfected THP-1. The siRNA transfected THP-1 was prepared in the same manner as in Example < 1-2 &gt;. The amount of secreted TNF- [alpha] was measured according to the manufacturer's protocol using the TNF-alpha ELISA Kit (R &amp; D Systems).

As a result, AIMP1- (101192), which is an intermediate part of AIMP1, treated siCD23 like AIMP1 and decreased TNF-a secretion in a state where CD23 expression was reduced (see Fig. 16).

These results indicate that the middle part of AIMP1 (amino acid sequence 101-192) in the THP-1 cell line is closely related to AIMP1-mediated cytokine activity by binding CD23.

< Example  5>

Of other cytokines CD23 On by TNF -α Confirmation of secretion promoting mechanism

It has been confirmed that the intermediate part of AIMP1 binds to CD23 and induces secretion of TNF-α secretion. Therefore, in order to confirm that CD23 is an AIMP1-specific receptor involved in the production of TNF-α, the binding strength of AIMP1 to CD23 is determined by the combination of EMAP II, which is a C-terminal fragment of AIMP1, which plays a role of cytokine such as neovascularization, .

<5-1> CD23 and EMAP II Coupling experiment with

Pull-down assay was performed to determine whether CD23 binds to EMAP II.

His-AIMP1 or His-EMAP II protein was reacted with a CD23 water soluble receptor (Fc-fused sCD23 protein) for 1 hour, then immunoprecipitated by mixing with protein A / G agarose, followed by anti-AIMP1 polyclonal antibody (Abcam) And human IgG-coupled HRP.

Experimental results show that the soluble receptor for CD23 does not bind to EMAP II unlike AIMP1 (see FIG. 17).

<5-2> AIMP1 and EMAP II in CD23  By inhibition TNF -α secretion control experiment

We compared the effects of AIMP1 and EMAP II on TNF-α secretion in THP-1 knockdown CD23 using CD23 siRNA. The siRNA transfected THP-1 was prepared in the same manner as in Example < 1-2 &gt;. The amount of secreted TNF- [alpha] was measured according to the manufacturer's protocol using the TNF-alpha ELISA Kit (R &amp; D Systems).

As a result, AIMP1 increased the amount of TNF-α secretion in the control group and inhibited the effect of CD23 knockdown in THP-1. However, when the EMAP II was treated at different concentrations, it was confirmed that the TNF-α secretion amount in the control group was not increased and the TNF-α secretion amount was inhibited in the THP-1 in which CD23 was knocked down (see FIG. 18).

<5-3> AIMP1 and EMAP II  At processing ERK1 / 2 phosphorylation acceleration experiment

In order to investigate whether ERK1 / 2 is involved in the stimulation of TNF-α secretion by EMAP II, such as the involvement of CD23 and ERK1 / 2 in the TNF-α secretion promoting mechanism by AIMP1.

THP-1 cells were treated with AIMP1 or EMAP II and ERK1 / 2 and p-ERK1 / 2 were measured over time. The experiment was conducted in the same manner as in Example <3-1>.

As a result, it was confirmed that phosphorylation of ERK1 / 2 was accelerated 30 minutes after treatment with AIMP1, but time-dependent phosphorylation of ERK1 / 2 was not observed even after 3 hours with EMAP II treatment ] Reference).

<5-4> ERK1 / 2 inhibition TNF -α secretion suppression effect

It was confirmed in Example <3-3> that the mechanism of TNF-α secretion by AIMP1 is inhibited by treatment with ERK1 / 2 inhibitor. To investigate whether ERK1 / 2 is involved in the mechanism of TNF-α secretion by EMAP II, comparative experiments were conducted. The experiment was carried out in the same manner as in Example <3-3>.

As a result of the experiment, it was confirmed that treatment of U0126, an ERK1 / 2 inhibitor, did not decrease the secretion of TNF-α induced by EMAP II (see FIG. 20).

This suggests that EMAP II, unlike AIMP1, does not bind to CD23 in the immune cells and does not induce TNF-α secretion through the ERK signal pathway.

Accordingly, the present invention provides a method for the treatment of AIMP1 comprising contacting all AIMP1 or a fragment thereof, CD23 and a candidate agent; And a candidate agent that inhibits the interaction between the whole AIMP1 or a fragment thereof and CD23, and a method for screening an anti-cancer agent. The method of the present invention is effective for the treatment of an inflammatory disease that inhibits AIMP1-mediated inflammatory cytokine and an anti-cancer agent that increases the level of free AIMP1 that inhibits cell adhesion of AIMP1,

<110> Medicinal Bioconvergence Research Center <120> Method for screening of anti-cancer agent and inflammatory          disease agent <130> NP13-0029 <160> 28 <170> Kopatentin 2.0 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for VEGFR1 <400> 1 atggtctttg cctgaaatgg tgag 24 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for VEGFR1 <400> 2 ctgtgaagcc agtgtggttt gc 22 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for VEGFR2 <400> 3 ggaaatgaca ctggagccta caag 24 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for VEGFR2 <400> 4 ggacccgaga catggaatca cc 22 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for sFRP1 <400> 5 ggcggaggtg aagcagcag 19 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for sFRP1 <400> 6 cgaagagcga gcagaggaag ac 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for TNFSF13 <400> 7 cctggaagcc tgggagaatg g 21 <210> 8 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for TNFSF13 <400> 8 atgtcacatc ggagtcatcc ttgg 24 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PLAC9 <400> 9 gccgctgccg aacccttc 18 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PLAC9 <400> 10 ccacggtctt ctctaccatc tcc 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for CLEC10A <400> 11 gctggtcatc atctgtgtgg ttg 23 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CLEC10A <400> 12 tgcctgccgt tcctgcttg 19 <210> 13 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for CD23 <400> 13 tgctgactct gcttctcctg tg 22 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CD23 <400> 14 tctgcgtgga ctgggatttc tg 22 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for IL20Rb <400> 15 tcttgatgtg gagcccagtg atc 23 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for IL20Rb <400> 16 tcaggacctt cagtgagtga gc 22 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for CXCR3 <400> 17 ccgacacctt cctgctccac 20 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for CXCR3 <400> 18 gctcctgcgt agaagttgat gttg 24 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for GAPDH <400> 19 cgctctctgc tcctcctgtt c 21 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for GAPDH <400> 20 ttgactccga ccttcacctt cc 22 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for TNF-alpha <400> 21 ggcgtggagc tgagagataa c 21 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for TNF-alpha <400> 22 ggtgtgggtg aggagcacat 20 <210> 23 <211> 312 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > AIMP1- (1-312) <400> 23 Met Ala Asn Asn Asp Ala Val Leu Lys Arg Leu Glu Gln Lys Gly Ala   1 5 10 15 Glu Ala Asp Gln Ile Ile Glu Tyr Leu Lys Gln Gln Val Ser Leu Leu              20 25 30 Lys Glu Lys Ala Ile Leu Gln Ala Thr Leu Arg Glu Glu Lys Lys Leu          35 40 45 Arg Val Glu Asn Ala Lys Leu Lys Lys Glu Ile Glu Glu Leu Lys Gln      50 55 60 Glu Leu Ile Gln Ala Glu Ile Gln Asn Gly Val Lys Gln Ile Pro Phe  65 70 75 80 Pro Ser Gly Thr Pro Leu His Ala Asn Ser Met Val Ser Glu Asn Val                  85 90 95 Ile Gln Ser Thr Ala Val Thr Thr Val Ser Ser Gly Thr Lys Glu Gln             100 105 110 Ile Lys Gly Gly Thr Gly Asp Glu Lys Lys Ala Lys Glu Lys Ile Glu         115 120 125 Lys Lys Gly Glu Lys Lys Glu Lys Lys Gln Gln Ser Ile Ala Gly Ser     130 135 140 Ala Asp Ser Lys Pro Ile Asp Val Ser Arg Leu Asp Leu Arg Ile Gly 145 150 155 160 Cys Ile Ile Thr Ala Arg Lys His Pro Asp Ala Asp Ser Leu Tyr Val                 165 170 175 Glu Glu Val Asp Val Gly Glu Ile Ala Pro Arg Thr Val Val Ser Gly             180 185 190 Leu Val Asn His Val Leu Glu Gln Met Gln Asn Arg Met Val Ile         195 200 205 Leu Leu Cys Asn Leu Lys Pro Ala Lys Met Arg Gly Val Leu Ser Gln     210 215 220 Ala Met Val Met Cys Ala Ser Ser Pro Glu Lys Ile Glu Ile Leu Ala 225 230 235 240 Pro Pro Asn Gly Ser Val Pro Gly Asp Arg Ile Thr Phe Asp Ala Phe                 245 250 255 Pro Gly Glu Pro Asp Lys Glu Leu Asn Pro Lys Lys Lys Ile Trp Glu             260 265 270 Gln Ile Gln Pro Asp Leu His Thr Asn Asp Glu Cys Val Ala Thr Tyr         275 280 285 Lys Gly Val Pro Phe Glu Val Lys Gly Lys Gly Val Cys Arg Ala Gln     290 295 300 Thr Met Ser Asn Ser Gly Ile Lys 305 310 <210> 24 <211> 192 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > AIMP1- (1-192) <400> 24 Met Ala Asn Asn Asp Ala Val Leu Lys Arg Leu Glu Gln Lys Gly Ala   1 5 10 15 Glu Ala Asp Gln Ile Ile Glu Tyr Leu Lys Gln Gln Val Ser Leu Leu              20 25 30 Lys Glu Lys Ala Ile Leu Gln Ala Thr Leu Arg Glu Glu Lys Lys Leu          35 40 45 Arg Val Glu Asn Ala Lys Leu Lys Lys Glu Ile Glu Glu Leu Lys Gln      50 55 60 Glu Leu Ile Gln Ala Glu Ile Gln Asn Gly Val Lys Gln Ile Pro Phe  65 70 75 80 Pro Ser Gly Thr Pro Leu His Ala Asn Ser Met Val Ser Glu Asn Val                  85 90 95 Ile Gln Ser Thr Ala Val Thr Thr Val Ser Ser Gly Thr Lys Glu Gln             100 105 110 Ile Lys Gly Gly Thr Gly Asp Glu Lys Lys Ala Lys Glu Lys Ile Glu         115 120 125 Lys Lys Gly Glu Lys Lys Glu Lys Lys Gln Gln Ser Ile Ala Gly Ser     130 135 140 Ala Asp Ser Lys Pro Ile Asp Val Ser Arg Leu Asp Leu Arg Ile Gly 145 150 155 160 Cys Ile Ile Thr Ala Arg Lys His Pro Asp Ala Asp Ser Leu Tyr Val                 165 170 175 Glu Glu Val Asp Val Gly Glu Ile Ala Pro Arg Thr Val Val Ser Gly             180 185 190 <210> 25 <211> 147 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > AIMP1- (47-192) <400> 25 Lys Leu Arg Val Glu Asn Ala Lys Leu Lys Lys Glu Ile Glu Glu Leu   1 5 10 15 Lys Gln Glu Leu Ile Gln Ala Glu Ile Gln Asn Gly Val Lys Gln Ile              20 25 30 Pro Phe Pro Ser Gly Thr Pro Leu His Ala Asn Ser Met Val Ser Glu          35 40 45 Asn Val Ile Gln Ser Thr Ala Val Thr Thr Val Ser Ser Gly Thr Lys      50 55 60 Glu Gln Ile Lys Gly Gly Thr Gly Asp Glu Lys Lys Ala Lys Glu Lys  65 70 75 80 Ile Glu Lys Lys Gly Glu Lys Lys Glu Lys Lys Gln Gln Ser Ile Ala                  85 90 95 Gly Ser Ala Asp Ser Lys Pro Ile Asp Val Ser Arg Leu Asp Leu Arg             100 105 110 Ile Gly Cys Ile Ile Thr Ala Arg Lys His Pro Asp Ala Asp Ser Leu         115 120 125 Tyr Val Glu Glu Val Asp Val Gly Glu Ile Ala Pro Arg Thr Val Val     130 135 140 Ser Gly Leu 145 <210> 26 <211> 92 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > AIMP1- (101-192) <400> 26 Ala Val Thr Thr Val Ser Ser Gly Thr Lys Glu Gln Ile Lys Gly Gly   1 5 10 15 Thr Gly Asp Glu Lys Lys Ala Lys Glu Lys Ile Glu Lys Lys Gly Glu              20 25 30 Lys Lys Glu Lys Lys Gln Gln Ser Ile Ala Gly Ser Ala Asp Ser Lys          35 40 45 Pro Ile Asp Val Ser Arg Leu Asp Leu Arg Ile Gly Cys Ile Ile Thr      50 55 60 Ala Arg Lys His Pro Asp Ala Asp Ser Leu Tyr Val Glu Glu Val Asp  65 70 75 80 Val Gly Glu Ile Ala Pro Arg Thr Val Val Ser Gly                  85 90 <210> 27 <211> 46 <212> PRT <213> Artificial Sequence <220> &Lt; 223 > AIMP1- (1-46) <400> 27 Met Ala Asn Asn Asp Ala Val Leu Lys Arg Leu Glu Gln Lys Gly Ala   1 5 10 15 Glu Ala Asp Gln Ile Ile Glu Tyr Leu Lys Gln Gln Val Ser Leu Leu              20 25 30 Lys Glu Lys Ala Ile Leu Gln Ala Thr Leu Arg Glu Glu Lys          35 40 45 <210> 28 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> AIMP1- (193-312) <400> 28 Leu Val Asn His Val Leu Glu Gln Met Gln Asn Arg Met Val Ile   1 5 10 15 Leu Leu Cys Asn Leu Lys Pro Ala Lys Met Arg Gly Val Leu Ser Gln              20 25 30 Ala Met Val Met Cys Ala Ser Ser Pro Glu Lys Ile Glu Ile Leu Ala          35 40 45 Pro Pro Asn Gly Ser Val Pro Gly Asp Arg Ile Thr Phe Asp Ala Phe      50 55 60 Pro Gly Glu Pro Asp Lys Glu Leu Asn Pro Lys Lys Lys Ile Trp Glu  65 70 75 80 Gln Ile Gln Pro Asp Leu His Thr Asn Asp Glu Cys Val Ala Thr Tyr                  85 90 95 Lys Gly Val Pro Phe Glu Val Lys Gly Lys Gly Val Cys Arg Ala Gln             100 105 110 Thr Met Ser Asn Ser Gly Ile Lys         115 120

Claims (12)

(a) contacting a fragment, CD23 and a candidate agent, wherein the fragment is composed entirely of AIMP1 (ARS-interacting multi-functional protein 1) or less than 312 amino acids comprising amino acids 101 to 192 of SEQ ID NO: 23; And
(b) a fragment consisting of less than 312 amino acids comprising the entire AIMP1 or amino acids 101 to 192 of SEQ ID NO: 23, and a candidate agent that inhibits the interaction between CD23 and inflammatory disease Screening method.
(a) contacting a fragment, CD23 and a candidate agent, wherein the fragment is composed entirely of AIMP1 (ARS-interacting multi-functional protein 1) or less than 312 amino acids comprising amino acids 101 to 192 of SEQ ID NO: 23; And
(b) the whole of AIMP1, or less than 312 amino acids including amino acids 101 to 192 of SEQ ID NO: 23, and a candidate agent that inhibits the interaction between CD23 and CD23. .
The composition according to claim 1, which is composed of the entire AIMP1 or less than 312 amino acids including amino acids 101 to 192 of SEQ ID NO: 23, and the candidate agent inhibiting interactions between CD23 and AIMP1 are all AIMP1 or SEQ ID NO: 23 or less than 312 amino acids, including 101 to 192 amino acids, or an inhibitor that competitively or noncompetitively binds to CD23 or an inhibitor that inhibits the expression of AIMP1 or CD23.
3. The fragment of claim 2, wherein the fragment is composed of less than 312 amino acids comprising the entire AIMP1 or amino acids 101 to 192 of SEQ ID NO: 23. A candidate agent inhibiting the interaction between CD23 and the fragment is AIMP1 whole or SEQ ID NO: Or less than 312 amino acids, including amino acids 101 to 192 of SEQ ID NO: 23, or an inhibitor that inhibits the expression of CD23, either competitively or non-competitively binding to CD23 or inhibiting the expression of CD23.
3. The method of claim 1 or 2, wherein the candidate agent is selected from the group consisting of an antibody, an antibody-derived molecule, a peptide, an oligonucleotide, an siRNA, a mimotope, a peptidomimetic, a foldamer, Or modified lipocalin-based cognitive proteins, DARPin, fibronectin, affibody, Kunitz-type inhibitor, peptidic aptamer, ribozyme ribozyme, toxin and organic / inorganic compound.
3. The method according to claim 1 or 2, wherein the AIMP1 comprises the amino acid sequence shown in SEQ ID NO: 23.
delete 3. The method according to claim 1 or 2, wherein the fragment of AIMP1 consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26.
The method of claim 1, wherein the inflammatory disease is selected from the group consisting of sepsis, arteriosclerosis, bacteremia, systemic inflammatory response syndrome, multiorgan dysfunction, osteoporosis, periodontitis, systemic lupus erythematosus, rheumatoid arthritis, osteoarthritis, (S), systemic sclerosis, idiopathic inflammatory muscle disorders, Sjoegren's syndrome, systemic vasculitis, sarcoidosis, autoimmune hemolytic anemia, autoimmune thrombocytopenia, thyroiditis, diabetes mellitus, immunomodulatory renal disease, central nervous system or peripheral nervous system Guillain-Barre syndrome, chronic inflammatory dehydration multinucleate neuritis, hepatic biliary disease, infectious or autoimmune chronic active hepatitis, primary biliary cirrhosis, granulomatous hepatitis , Inflammatory cholangitis, inflammatory bowel disease (IBD), ulcerative colitis, Inflammatory bowel disease, Crohn's disease, irritable bowel syndrome, gluten-sensitive intestinal disease, Whipple's disease, autoimmune or immunomodulatory skin disease, watery skin disease, polymorphic erythema, Inflammatory diseases selected from the group consisting of psoriasis, allergic diseases, asthma, allergic rhinitis, atopic dermatitis, food hypersensitivity, urticaria, lung disease, eosinophilic pneumonia, idiopathic pulmonary fibrosis, hypersensitivity pneumonia, &Lt; / RTI &gt;
3. The method of claim 2, wherein the anticancer agent is selected from the group consisting of anal cancer, astrocytoma, leukemia, lymphoma, head and neck cancer, liver cancer, testicular cancer, cervical cancer, sarcoma, angioma, Cancer, cancer, skin cancer, basal cell carcinoma, melanoma, squamous cell carcinoma, oral squamous cell carcinoma, ovarian cancer, prostate cancer, lung cancer, colon cancer, pancreatic cancer, kidney cancer, stomach cancer, Wherein the cancer is an anticancer agent against cancer selected from the group consisting of carcinoma, colorectal cancer, glioblastoma, endometrial cancer, and malignant glioma.
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