TECHNICAL FIELD

The present invention relates to a composition for preventing or treating diseases caused by overexpression of chemokine CX₃CL1 (fractalkine) comprising a death receptor 5 (DR5) inhibitor as an active ingredient, and it can reduce expression of chemokine CX₃CL1 by inhibiting binding of FasL and DR5 on a cell surface using an agent controlling DR5 expression or activity, for example using an inhibitor, and thereby it can effectively prevent and treat diseases caused by overexpression of chemokine CX₃CL1.

BACKGROUND ART

Recently, as a target substance of cancer therapeutic agent development, the apoptosis route through TNF-related apoptosis inducing ligand (TRAIL or Apo2L) which selectively induces apoptosis of cancer cells only without affecting normal cells, and one of its receptors, DR5 (death receptors 5) system has been considered important (Ashkenazi et al., Nat Rev Cancer 2: 420, 2002). Currently, as a cancer cell therapeutic agent targeting DR5, recombinant TRAIL and apoptosis receptor-specific antibodies have been developed.

However, TRAIL has a problem of low specificity for DR5, since it binds to DR4 (Death receptor 4, TRAIL-Receptor 1), and DR5 (Death receptor 5, TRAIL-Receptor 2) which deliver apoptosis signals, as well as DcR1(Decoy Receptor 1, TRAIL-Receptor 3) and DcR2(Decoy Receptor 1, TRAIL-Receptor 4) which cannot deliver apoptosis signals. In addition, recombinant TRAIL is less stable and has side effects that cause apoptosis in normal cells such as astrocytes, hepatocytes, keratinocytes, and the like (Jo et al., Nature Medicine 6, 564-567, 2000).

Therefore, in recent years, the development of anti-DR5 to anti-DR4 antibodies which induce cancer cell selective apoptosis has been actively studied.

However, until now, research has focused on the development of cancer cell therapeutic agents using the apoptosis route though the DR5 system, and there have been no specific studies on the mechanism inducing inflammation of DR5 and the development of inflammation therapeutic agents using the same.

Fas ligands (FasL, CD95L, CD178, Apo-1) are one of Type II membrane protein, which belong to the tumor necrosis factor (TNF) system with TNF, CD40L, 4-1BBL and the like, and are mainly expressed in immune privileged sites such as activated T cells, NK cells, tumor cells and eyeballs and the like. Fas ligand (hereinafter, FasL) has a homotrimer structure, and is known to kill target cells through trimerization with Fas receptor (Fas; FasR; CD95; UniProt P25445), which is its receptor.

FasL can be divided into membrane FasL and soluble FasL (sFasL). Since apoptosis is induced by cell-cell contact, the membrane FasL plays a role in killing cells by forming a death inducing signaling complex (DISC) with Fas. sFasL is a cleavage of membrane FasL cut by serine matrix metalloproteinase-3 or -7 (MMP-3 or MMP-7), and has been known to inhibit apoptosis of target cells as opposed to function of membrane FasL, or function as a chemoattractant depending on the cellular microenvironment.

There are few reports on the specific role of FasL in inflammatory diseases, particularly, rheumatoid arthritis (RA). As fas as is known, as the result of comparing the amount of sFasL in Rap patients and Osteoarthritis patients, the amount of sFasL is increased in RA patients, and this is the only report that has been shown to play a role in inhibiting angiogenesis by reducing VEGF secreted from synovial fibroblasts. On the other hand, with respect to membrane FasL, there has been a report that apoptosis through Fas-FasL inhibits production of autoreactive cells in the early stage of rheumatoid arthritis to play a role in alleviating rheumatoid arthritis, in the CIA (collagen induced arthritis) model.

Considering that chemokine plays an important role in occurrence of various diseases, research on the development of an chemokine inhibitor which can effectively prevent or treat various diseases caused by chemokine and a therapeutic agent using the same is important, and in addition, specifically, it is necessary to reveal the mechanism inducing diseases such as inflammation of chemokine in cells and develop a therapeutic agent targeting a new target with excellent effects. However, specific research results are still insignificant.

DISCLOSURE
Technical Problem

Accordingly, the present inventors have newly determined the use of inhibiting expression of chemokine CX₃CL1 of a modulator which inhibits the activity or expression of DR5 inhibitor, thereby providing a composition for preventing or treating diseases caused by overexpression of chemokine CX₃CL1 and a composition for inhibiting expression of chemokine CX₃CL1, which contain a modulator that inhibits the activity or expression of DR5 inhibitor as an active ingredient.

Another embodiment provides a method of screening a modulator of chemokine expression and a therapeutic agent for diseases caused by overexpression of chemokine CX₃CL1, targeting DR5 protein.

Othere embodiment relates to a method for preventing and/or treating diseases caused by overexpression of chemokine CX₃CL1 comprising administering an inhibitor of expression or activity of DR5 to a patient in need of prevention and/or treatment of diseases caused by overexpression of chemokine CX₃CL1 in a therapeutically effective amount.

Other embodiment provides a use for prevention and/or treatment of diseases caused by overexpression of chemokine CX₃CL1 of a DR5 expression or activity inhibitor.

Technical Solution

The present invention provides an effective prevention and treatment method of diseases caused by overexpression of chemokine CX₃CL1, by reducing expression of CX₃CL1 that is one of inflammatory cytokines, by inhibiting binding FasL and DR5 on a cell surface, using an agent, for example, an inhibitor, which controls expression or activity of death receptor 5 (DR5).

Accordingly, the present invention provides a method for preventing and/or treating diseases caused by overexpression of chemokine CX₃CL1, comprising administering an inhibitor of expression or activity of DR5 in a therapeutically effective amount to a patient in need of prevention and/or treatment of diseases caused by overexpression of chemokine CX₃CL1. Another embodiment provides a use for preventing and/or treating diseases caused by overexpression of chemokine CX₃CL1 of an inhibitor of expression or activity of DR5.

DR5 is also called TRAIL receptor 2 (TRAILR2) or tumor necrosis factor receptor superfamily member 10B (TNFRSF10B), and is a cell surface receptor of TNF-receptor superfamily binding to TRAIL and delivers apoptosis signals to mediate apoptosis. DR5 is known to interact with Caspase 8, Caspase 10, FADD (Fas-Associated protein with Death Domain), TRAIL, and the like. The DR5 may be derived from mammals, and for example, it may be human DR5 (e.g., NCBI accession no. UniProtKB/Swiss-Prot: Q6FH58 etc.)

One specific embodiment of the present invention provides a composition for preventing or treating diseases caused by overexpression of chemokine CX₃CL1 containing an inhibitor of expression or activity of DR5 as an active ingredient.

The kind of the inhibitor of expression or activity of DR5 is not particularly limited, but it means all substances acting reducing, removing and/or blocking actions of DR5 including reducing or removing the activity of DR5, and inhibiting, removing and inactivating expression of DR5. For example, it may be one or more kinds selected from the group consisting of siRNA, shRNA, miRNA, ribozyme, DNAzyme, PNA (peptide nucleic acids), anti-sense oligonucleotide, peptide, an antibody, an aptamer, natural extract or a chemical substance or the like, and preferably, the antibody or siRNA may be used.

According to one embodiment of the present invention, the inhibitor of expression or activity of DR5 may bind to a CRD2 domain of DR5, or a CRD3 domain of DR5, or both a CRD2 domain and a CRD3 domain.

According to another embodiment of the present invention, the antibody may bind to the 53rd to 181th amino acid part in the amino acid sequence of SEQ ID NO: 1 as an antigen.

Preferably, the antibody may bind to a CRD2 domain of DR5 consisting of the 101th to 120th amino acid sequence of SEQ ID NO: 1, or a CRD3 domain of DR5 consisting of the 143th to 160th amino acid sequence of SEQ ID NO: 1, or both the CRD2 and CRD3 domains, and for example, it may be an antibody binding to a CRD2 domain of huDR5 or a CRD3 domain of huDR5, respectively, as an antigen epitope.

According to one embodiment of the present invention, the siRNA may be siRNA binding to a sequence encoding CRD2 domain of DR5 consisting of the 301st to 363th sequence of SEQ ID NO: 4, or a sequence encoding CRD3 domain of DR5 consisting of the 430th to 483th sequence of SEQ ID NO: 4, or both a sequence encoding CRD2 domain of DR5 and a sequence encoding CRD3 domain of DR5. Preferably, it may be siRNA binding to each of the sequence encoding CRD2 domain of DR5 and the sequence encoding CRD3 domain of DR5 to inhibit expression of CRD2 and CRD3 of DR5.

The chemokine CX₃CL1 is called fractalkine (FKN), and is known as chemokine (C—X₃—C motif) ligand 1, and is a chemokine involved in causing inflammation. CX₃CL1 is a unique type of chemokine which comprises an extracellular N-terminal domain, mucin-like stalk, transmembrane αhelix and short intracytoplasmic tail, and consists of 373 amino acids. In particular, an aqueous type of CX₃CL1 is known to show chemotactic activity in monocytes, NK cells and T cells. In addition, CX₃CL1 is expressed in macrophages, fibroblasts, endotheliocytes, and dendritic cells in rheumatoid arthritis (RA) synovial tissue, and acts as an adhesive molecule in white blood cells, and enhances extravasation of white blood cells through an endothelium. In addition, CX₃CL1 induced by TNF-α, IFN-γ, and IL-1β is known to be related to scleroma, rheumatoid arthritis (RA), HIV infection, cancer and other various diseases, complications, and the like, but until now, there is no clinical therapeutic method targeting CX₃CL1.

The kinds of the diseases caused by overexpression of chemokine CX₃CL1 are not particularly limited, but it may be one or more kinds selected from the group consisting of arthritis, cardiovascular disease, cancer, HIV infection, primary biliary cirrhosis, renal disorder, allograft rejection, hypertension, eye disease, chronic pancreatitis, neuropathic pain, Sjogren's syndrome, chronic obstructive pulmonary disease and emphysema (COPD and emphysema; Am J Pathol. 2008 October; 173(4):949-61), pulmonary fibrosis (Ann Rheum Dis. 2005 January; 64(1):21-8.), atopic dermatitis (J Allergy Clin Immunol. 2004 May; 113(5):940-8), and Lupus nephritis (Arthritis Rheum. 2005 May; 52(5):1522-33.), and in particular, it may be one of arthritis, cardiovascular disease, cancer, and HIV infection, and preferably, it may be arthritis.

Specifically, the arthritis may be osteoarthritis, degenerative arthritis, desquamative osteoarthritis, articular ligament damage, meniscal damage, joint malalignment, avascular necrosis, rheumatoid arthritis, juvenile idiopathic arthritis, trauma, inflammatory arthritis or arthritis caused by infection.

On the other hand, the cardiovascular diseases may be atherosclerosis, coronary artery disease, carotid artery disease, stroke or carotid atherosclerosis, and the cancer may be a colorectal cancer or lung cancer.

The composition for preventing or treating diseases may further comprise one or more of adjuvants selected from the group consisting of a carrier, an excipient, a disintegrating agent, a sweetener, a coating material, an inflating agent, a lubricant, a glydent, a flavoring agent, an antioxidant, a buffer solution, a bacteriostatic agent, a diluent, a dispersing agent, a surfactant, a binding agent and a lubricant.

Specifically, the carrier, excipient and diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxy benzoate, propyl hydroxy benzoate, talc, magnesium stearate, and mineral oil, and solid preparations for oral administration include tablets, pills, powder, granules, capsules, and the like, and such solid preparations may be prepared by mixing at least one of excipients, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and the like to the composition. In addition, other than simple excipients, lubricants such as magnesium stearate and talc may be used. Liquid preparations for oral administration include suspension, oral liquids, emulsion and syrup and the like, and various excipients in addition to the commonly used simple diluent, water and liquid paraffin, for example, a wetting agent, a sweetener, a flavoring agent, a preservative and the like may be comprised. Preparations for parenteral administration include a sterile aqueous solution, a non-aqueous solvent, suspension, emulsion, a lyophilized preparation, a suppository and the like. As the suspension, propylene glycol, polyethylene glycol, plant oil such as olive oil, injectable ester such as ethyl oleate and the like may be used. As a base material of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like may be used.

The formulation of the composition is not particularly limited, but the kind may be selected from the group consisting of granules, powder, coating tablets, tablets, pills, capsules, suppositories, gel, syrup, juice, suspension, emulsion, drops or liquids.

The range of the effective dose of the composition of the present invention may differ according to various factors such as gender, severity, age, administration method, target cell and expression level, and the like, and it may be easily determined by those skilled in the art.

Furthermore, the present inventors have confirmed that the amount of intracellular CX₃CL1 is increased when FasL and DR5 bind specifically as FasL-DR5 binding, differently from it when TRAIL commonly known as a ligand binding to DR5 is treated for TRAIL-DR5 binding reaction, through a specific experiment (See Example 4 as follows). In addition, it has been confirmed that the amount of secreted CX₃CL1 is decreased, when an anti-DR5 antibody, or siRNA for knocking-down DR5 gene is treated to FasL-DR5 to interrupt formation of FasL-DR5 complexes (See Example 5 as follows).

In other words, the present inventors have found that the expression of intracellular chemokine CX₃CL1 can be significantly inhibited when an inhibitor of expression or activity of DR5 is treated, and as aforementioned, they have found that the inhibitor of expression or activity of DR5 can be used as an active ingredient of the composition for preventing and treating diseases caused by overexpression of CX₃CL1 (See Example 6).

Based on the findings, in one other embodiment of the present invention, a composition for inhibiting expression of chemokine CX₃CL1 containing an inhibitor of expression or activity of DR5 as an active ingredient is provided.

DR5 protein binds to FasL and increases expression of intracellular CX₃CL1, and thus when the inhibitor of DR5 expression or activity of the present invention is treated, the expression of intracellular CX₃CL1 may be significantly inhibited.

The kinds of the DR5 inhibitor are not particularly limited, but it may be one or more kinds selected from the group consisting of siRNA, shRNA, miRNA, ribozyme, DNAzyme, PNA (peptide nucleic acids), anti-sense oligonucleotide, peptide, an antibody, an aptamer, natural extract or a chemical substance or the like, and preferably, an antibody or siRNA may be used.

According to one other embodiment of the present invention, the antibody may bind to the 53th to 181th amino acid part in the amino acid sequence of SEQ ID NO: 1 as an antigen.

Preferably, the antibody may bind to a CRD2 domain of DR5 consisting of the 101th to 120th amino acid sequence of SEQ ID NO: 1, or a CRD3 domain of DR5 consisting of the 143th to 160th amino acid sequence of SEQ ID NO: 1, or both the CRD2 and CRD3 domains, and for example, it may be an antibody binding to each of a CRD2 domain of DR5 or a CRD3 domain of huDR5 as an antigen epitope.

According to one embodiment of the present invention, the siRNA may be siRNA binding to a sequence encoding CRD2 domain of DR5 consisting of the 301th to 363th sequence of SEQ ID NO: 4, or a sequence encoding CRD3 domain of DR5 consisting of the 430th to 483th sequence of SEQ ID NO: 4, or both a sequence encoding CRD2 domain of DR5 and a sequence encoding CRD3 domain of DR5. Preferably, it may be siRNA binding to each of the sequence encoding CRD2 domain of DR5 and the sequence encoding CRD3 domain of DR5 to inhibit expression of CRD2 and CRD3 of DR5.

In addition, in other specific embodiment of the present invention, provided is

a screening method of an inhibitor of chemokine expression, comprising

reacting a candidate substance to a sample; and

measuring the DR5 activity or expression of the sample,

and comprising determining the candidate substance as an inhibitor of expression of chemokine CX₃CL1, when the activity or expression of DR5 in the sample treated with the candidate substance is decreased than DR5 in the sample not treated with the candidate substance.

The inhibitor of expression of chemokine CX₃CL1 selected by the screening method may be used as a prophylactic agent and/or a therapeutic agent for diseases caused by overexpression of chemokine CX₃CL1 such as arthritis, cardiovascular diseases, cancer, HIV infection, primary biliary cirrhosis, renal disorder, allograft rejection, hypertension, eye disease, chronic pancreatitis, neuropathic pain, Sjogren syndrome, chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, atopic dermatitis, and lupus nephritic, and the like. Therefore, the screening method may be a screening method of a prophylactic agent and/or a therapeutic agent for diseases caused by overexpression of chemokine CX₃CL1.

The sample may be an animal, preferably, a cell, tissue or organ obtained from a mammal, and preferably, may include RA (Rheumatoid arthritis) sites, immune privileged sites, and the like, and more preferably, T cells, NK cells, rheumatoid cells, tumor cells or eyeballs or the like.

The activity of DR5 may be measured by common methods, and this can be easily seen by those skilled in the art. For example, confirmation of the reaction between DR5 and the sample may use methods commonly used for confirming the reaction between protein-protein or between protein-compound. For example, a method for measuring the activity after reacting DR5 and a test subject substance, yeast two hybrid, a screening method using HTS (high throughput screening) using search of phage display peptide clones binding to DR5, natural products and chemical substances library, and the like, drug hit HTS or cell-based screening, or the like, but not limited to these methods.

Moreover, the expression of DR5 may be measured by confirming the reaction between a composition comprising a gene encoding DR5 and a sample, and the reaction confirmation may use commonly used methods for confirming the reaction between DNA-DNA, DNA-RNA and DNA-protein. For example, in vitro, a hybridization test for confirming binding between the gene and test subject substance, a method for measuring the expression rate of the gene through northern analysis after reacting a mammal cell and a test subject substance, or a method for connecting a reporter gene to the gene to introduce it inside of a cell and then reacting it with a test subject substance to measure the expression rate of the reporter protein, or the like may be used, but not limited to these methods.

In the screening method of the present invention, the sample may be estimated to have possibility to prevent or treat diseases caused by overexpression of chemokine CX₃CL1 (fractalkine) according to the common selection method, or may be randomly selected respective nucleic acid, protein, other extract or natural products, compounds, or the like.

Substances obtained by this screening method have advantages of having a prophylactic effect of occurrence and a therapeutic effect in the early stage of occurrence of diseases caused by overexpression of chemokine CX₃CL1, as well as excellent effects of alleviating and treating disease conditions in the middle and end stages with some progress, and may act as a leading compound in the process of development of a prophylactic or therapeutic agent for diseases caused by overexpression of chemokine CX₃CL1. By modifying and optimizing the structure of the leading compound, a new therapeutic agent may be developed, and this substance exhibits an inhibitory effect of expression of CX₃CL1 by inhibiting expression or activity of DR5, and therefore it may prevent or treat diseases caused by overexpression of chemokine CX₃CL1 such as arthritis, cardiovascular diseases, cancer, HIV infection, primary biliary cirrhosis, renal disorder, allograft rejection, hypertension, eye disease, chronic pancreatitis, neuropathic pain, Sjogren syndrome, chronic obstructive pulmonary disease, emphysema, pulmonary fibrosis, atopic dermatitis, and lupus nephritic and the like, in particular, arthritis including rheumatoid arthritis.

One embodiment of the present invention provides a method for preventing or treating diseases caused by overexpression of chemokine CX₃CL1, comprising administering an inhibitor of expression or activity of DR5 to a subject in need of prevention or treatment caused by overexpression of chemokine CX₃CL1 in a therapeutically effective amount.

The inhibitor of expression or activity of DR5 may be siRNA, shRNA, miRNA, ribozyme, DNAzyme, PNA (peptide nucleic acids), anti-sense oligonucleotide, peptide, an antibody, an aptamer, natural extract or a chemical substance, and preferably, it may be an antibody or siRNA.

According to one specific embodiment of the present invention, the inhibitor of expression or activity of DR5 may bind to a CRD2 domain of DR5, or a CRD3 domain of DR5, or both a CRD2 domain and a CRD3 domain.

In addition, the antibody may bind to the 53th to 181th amino acid part in the amino acid sequence of SEQ ID NO: 1, and preferably, may bind to a CRD2 domain of DR5 consisting of the 101th to 120th amino acid sequence of SEQ ID NO: 1, or a CRD3 domain of DR5 consisting of the 143th to 160th amino acid sequence of SEQ ID NO: 1, or both the CRD2 and CRD3 domains.

One other embodiment of the present invention provides a use for prevention or treatment of diseases caused by overexpression of chemokine CX₃CL1 of an inhibitor of expression or activity of DR5.

Thus, the technology of prevention and/or treatment of inflammation and diseases caused by overexpression of chemokine CX₃CL1, comprising the inhibitor of expression or activity of DR5 proposed herein as an active ingredient, and the technology of screening of a novel chemokine expression inhibitor using the same can not only effectively prevent occurrence of diseases caused by overexpression of chemokine CX₃CL1, but also obtain an excellent therapeutic effect even after some progress, and therefore they are very useful technologies for treating a lot of suffering patients, and they may be usefully used in investigation and development of a prophylactic or therapeutic agent of diseases caused by overexpression of chemokine CX₃CL1.

On the other hand, in other embodiment of the present invention, a method for decreasing expression of chemokine CX₃CL1 by administering a DR5 inhibitor to a patient in need of reduction of chemokine expression or a cell where chemokine is overexpressed may be provided, and also, a method of preventing or treating inflammation and diseases caused by overexpression of chemokine CX₃CL1, particularly, arthritis, by administering a DR5 inhibitor to a cell where chemokine CX₃CL1 is overexpressed.

The DR5 inhibitor is not particularly limited, unless it is a substance inhibiting expression of DR5 gene or inhibiting activity of DR5 protein, and the DR5 inhibitor may be siRNA, shRNA, miRNA, ribozyme, DNAzyme, PNA (peptide nucleic acids), anti-sense oligonucleotide, peptide, an antibody, an aptamer, natural extract or a chemical substance.

Advantegeous Effects

The present invention can effectively prevent and treat diseases caused by overexpression of chemokine CX₃CL1, by reducing expression of CX₃CL1 that is one of inflammatory cytokines, by inhibiting binding between FasL and DR5 on a cell surface using an agent controlling DR5 expression or activity, for example, an inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 (A) shows the result of confirming the expression of DR5 gene in each cell by real-time PCR, after isolating synovial cells (Adherent cell/Adh.) and normal immunocytes (Supernatant cell/Sup.) in the joint obtained from the normal mouse (Normal) and arthritis-induced mouse (RA), and FIG. 1 (B) shows the result of confirming the expression of DR5 surface protein in the synovial cells of the joint obtained from the arthritis-induced mouse (RA) by flow cytometry, and FIG. 1 (C) shows the result of confirming the expression of DR5 surface protein in hFLS (human fibroblast-like synoviocytes) by flow cytometry.

FIG. 2 shows the experimental result for confirming FasL-DR5 intercombination, and FIG. 2 (A) shows the result of verifying the interaction of FasL-DR5 by confirming that FasL-IgG binding is inhibited, when FasL binding to hFLS is inhibited by treating an anti-DR5 antibody, and FIG. 2 (B) is the result of confirming the IgG signal strength on the cell surface treated with IgG-combined FasL after DR5 knock down in hFLS by treating siRNA by flow cytometry.

FIG. 3 shows the result of confirming secretion of CX₃CL1 by ELISA after treating FasL and TRAIL to hFLS alone or in combination.

FIG. 4 (A) shows the result of measuring the amount of secreted CX₃CL1 by ELISA after inhibiting the interaction of FasL and DR5 by treating an anti-DR5 antibody to hFLS, and FIG. 4 (B) shows the result of measuring the amount of secreted CX₃CL1 by ELISA, after treating an anti-DR5 antibody to mouse synovial cells and then treating FasL.

FIG. 5 (A) is a graph showing the result of reduced expression of DR5 surface protein on the cell surface of hFLS due to DR5 siRNA treatment, and FIG. 5 (B) is a graph showing the result of reduced secretion of CX₃CL1 in hFLS due to DR5 siRNA treatment.

FIG. 6 shows the result of confirming that the disease symptoms induced by sFasL injection, when sFasL is injected and an anti-DR5 antibody is injected at the same time to a FasL-mutated gld mouse which is a FasL deficient mouse. It shows the changes of arthritis symptoms in gld mouse treated with K/B×N serum, gld mouse treated with K/B×N serum and sFasL, and gld mouse treated with K/B×N serum, sFas ligand and anti-DR5 antibody (anti-DR5), and (A) is the graph measuring the thickness of the joint and (B) is the graph measuring clinical indexes.

FIG. 7a shows huDR5-CRD2 and huDR5-CRD3 which are candidate sites where huDR5 protein amino acid sequence and sFasL are expected to bind, and the 111th, 114th, 119th, 120th, 148th, 150th, 153rd and 156th amino acids are Alanine-substituted mutation occurring sites.

FIG. 7b shows the 1st to 840th sequence of SEQ ID NO: 4 that is the huDR5 DNA sequence, and the polynucleotide consisting of the 334th to 363rd sequence and the polynucleotide consisting of the 445th to 471st sequence are mutation occurring sites.

FIG. 8a shows immunogen sites of the huDR5-CRD2 and huDR5-CRD3 and DR5 FACS antibody, which are candidate sites where huDR5 protein amino acid sequence, sFasL are expected to bind.

FIG. 8b shows the result of performing flow cytometry using human FasL where an immunoglobulin (IgG) that binds to huDR5 is combined, after expressing DR5 where huDR5-CRD2 or huDR5-CRD3 is mutated.

FIG. 9 shows a schematic diagram of a mechanism model which facilitates expression of sCX₃CL1 as sFasL binds to a DR5 receptor of human-derived FLS and induces inflammation as this sCX₃CL1 increases expression of chemokine such as CCL2, CCL3, CXCL10 and the like from immunocytes.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the prevent invention will be described in more detail by examples. However, these examples are intended to illustrate the present invention only, and the scope of the present invention is not limited by these examples.

Example 1. Identification Membrane Protein which Binds to sFas Ligand (DR5)

Until now, it has been known that FasL binds to its receptor, Fas, and induces apoptosis of target cells, and thereby it causes joint inflammation. In addition, in the previous research of the present inventors, it has been revealed that FasL controls expression of chemokine with a new mechanism different from conventional inflammation induction of apoptosis of Fas-FasL, and also, it has been determined that FasL itself acts as chemokine attracting inflammatory cells. However, in the previous research, it has not been specifically revealed how FasL controls occurrence of inflammation by binding to which membrane protein in the new inflammation causing mechanism by FasL.

Accordingly, the present inventors have performed the protein identification experiment in order to find a protein binding to sFasL (soluble Fas Ligand) in the new inflammation causing mechanism by FasL.

At first, after obtaining synovium of an arthritis patient in vitro and then digesting with collagenase and culturing for 3 days, adherent cells were isolated to obtain human fibroblast-like synoviocytes (hFLS). By reacting biotinylated sFasL obtained by biotinylation of the obtained hFLS and recombinant human Fas ligand (R&D Systems 126-FL-010) with Sulfo-NHS-SS-Biotin—Thermo Scientific #21331, sFasL and hFLS targeting receptor protein were combined. Then, after cross-linking using a cross-linking agent (bis(sulfosuccinimidyl)suberate; BS3), cell lysis was conducted.

Then, to isolate sFasL-combined hFLS target protein, avidin purification was performed, and thereby the biotinylated sFas ligand recombinant protein and hFLS targeting receptor protein combined thereto were isolated together. After that, the sample was separated by SDS-PAGE and the gel was fractionized, and then peptides were extracted by the in-gel digestion method.

After analyzing the extracted peptides repeatedly twice using high resolution Hybrid quadrupole-orbitrap mass spectrometer, proteins which were not shown in the control group (Biotinylated Fc) and were shown only in the experimental group at least once were selected using SEQUEST algorithm. Proteins known to be present in cell membrane or extracellular matrix among selected proteins were sorted using classification by Uniprot database, and among them, DR5 was identified.

Example 2. Confirmation of Expression of DR5 Surface Protein in hFLS and Joint Synovial Cells of Arthritis-Induced Mouse

In order to confirm the expression of DR5 surface protein in human-derived FLS (hFLS) and joint synovial cells of the arthritis-induced mouse, flow cytometry and real-time PCR were conducted. In real-time PCR, Applied Biosystems 7500 Real Time PCR System was used, and F: GGGCCACAGGGACACCTT/R: GCATCTCGCCCGGTTTT were used as primers, and they were reacted by 40 cycles of 50° C. for 2 minutes, 95° C. for 2 minutes, 95° C. for 15 seconds and then 60° C. for 1 minutes.

Then, after isolating synovial cells (Adherent cell/Adh.) and normal immunocytes (Supernatant cell/Sup.) in the joint obtained from the normal mouse (Normal) and arthritis-induced mouse (RA), respectively, expression of DR5 gene was compared in each cell. The experimental result was shown in FIG. 1.

As confirmed in FIG. 1 (A), the relative expression of DR5 surface protein in synovial cells of the joint obtained from the arthritis-induced mouse was increased than that in the normal mouse, and as confirmed by the flow cytometry result of FIG. 1 (B) and (C), it was confirmed that the DR5 surface protein was expressed in the arthritis-induced mouse synovial cells (Adherent cells) and hFLS.

Therefore, it could be seen that the expression of DR5 surface protein was increased in hFLS and cells obtained from a joint of arthritis-induced mouse, specifically in synovial cell, compared to the normal mouse.

Example 3. Confirmation of FasL-DR5 Intercombination

3-1. DR5 Antibody Treatment

In order to confirm whether FasL and membrane protein DR5 actually bind, an anti-DR5 antibody (R&D systems AF631) was treated to hFLS to interrupt binding to cell surface DR5 of FasL.

Then, IgG-combined FasL was treated, and the IgG signal strength on the surface was confirmed by flow cytometry, and the result was shown in FIG. 2 (A). As could be seen in FIG. 2 (A), it could be seen that the signal strength by IgG was decreased when the anti-DR5 antibody was treated, and FasL-DR5 binding was reduced when the DR5 activity was inhibited by the anti-DR5 antibody.

3-2. Knock-Down Using siRNA Against DR5

In order to confirm whether FasL and membrane protein DR5 actually bind, siRNA (Sigma Aldrich MISSION siRNA; SASI_Hs01_00040567) was treated to hFLS, and using an electroporator (Neon transfection system, Thermo Fisher Scientific), expression of DR5 was knocked-down.

Then, after treating IgG-combined FasL, the IgG signal strength on the surface was confirmed by flow cytometry, and the result was shown in FIG. 2 (B). As could be seen in FIG. 2 (B), it could be seen that the signal strength by IgG was decreased when siRNA was treated, that is the signal strength by IgG was decreased as binding of Fas ligands was reduced, when expression of DR5 was reduced on the hFLS surface.

From the above results, it was confirmed that FasL and DR5 interactively bound specifically each other.

Example 4. Confirmation of Increase of Chemokine CX₃CL1 Expression by FasL DR5 Binding

In order to confirm chemokine where its expression is increased by FasL-DR5 binding, 200 ng/ml of each of FasL and TRAIL was treated to hFLS alone or in combination for 24 hours, and then the secretion of CX₃CL1 was confirmed according to the protocol provided by the manufacturer of ELISA (R&D systems DY365), and the result was shown in FIG. 3. As could be seen in FIG. 3, it was confirmed that the amount of CX₃CL1 was not increased when TRAIL known as a common ligand of DR5 was treated, but the amount of CX₃CL1 was increased when FasL was treated.

From that, it could be seen that the expression of chemokine CX₃CL1 was increased in hFLS by binding of FasL and DR5. It was confirmed that CX₃CL1 was one of chemokines causing inflammation, and facilitated the secretion of CX₃CL1 of hFLS cells during FasL-DR5 binding to cause inflammation.

Example 5. Confirmation of Decrease of Chemokine Expression by FasL-DR5 Inhibition

In order to confirm whether the secretion of chemokine CX₃CL1 which is secreted when FasL is treated is decreased, when the interaction of FasL and DR5 is inhibited, the following experiment was performed.

5-1. DR5 Antibody Treatment

After treating an antibody against 0.5 ug/ml of DR5 (R&D Systems AF631) to hFLS or mouse synovial cells for 1 hour and treating FasL 200 ng/mL, the amount of CX₃CL1 secreted was confirmed by ELISA, and the result was shown in FIG. 4. FIG. 4 (A) shows the treatment of the DR5 antibody to hFLS and the expression of CX₃CL1 was reduced in the anti-DR5 than Isotype (Goat IgG, R&D Systems AB-108-C), and FIG. 4 (B) shows the treatment of the anti-DR5 antibody to mouse synovial cells and it could be seen that the expression of CX₃CL1 in the anti-DR5 was also reduced.

5-2. siDR5 Knock-Down

siRNA (Sigma Aldrich MISSION siRNA; SASI_Hs01_00040567) was treated to hFLS, and the expression of DR5 was knocked-down using an electroporator (Neon transfection system, Thermo Fisher Scientific), and as a result, as could be seen in FIG. 5 (A), it was confirmed that the expression of cell surface DR5 surface protein of hFLS was reduced by DR5 siRNA treatment.

Then, after treating FasL 200 ng/mL for 24 hours, and the amount of secreted CX₃CL1 was confirmed by ELISA, and as a result, as could be seen in FIG. 5 (B), it was confirmed that the amount of CX₃CL1 secreted by FasL treatment was decreased compared to the control group.

Taken the above results together, it could be seen that FasL bound to DR5 and the expression of CX₃CL1 was increased by this DR5 binding, and when interrupting FasL-DR5 interaction, the expression of CX₃CL1 was reduced. Thereby, it could be confirmed that when the DR5 antibody was treated, or DR5 was knocked-down to interrupt binding between DR5 and FasL, the expression of intracellular inflammation causing chemokine CX₃CL1 was reduced to inhibit inflammation occurrence, and it was effective in inhibiting arthritis symptoms not only in the early stage of occurrence of inflammation, particularly arthritis, but also after occurrence.

Example 6. Test of Alleviation of Arthritis Symptoms when Injecting the Anti-DR5 Antibody

In order to confirm the alleviation of arthritis symptoms when injecting the anti-DR5 antibody to the arthritis mouse model induced by FasL, at first, serum was collected and prepared from blood in FasL deficient gld mouse (Central experimental animal) and K/B×N mouse that arthritis was naturally caused (obtained by crossbreeding KRN TCR transgenic mouse and NOD mouse provided from Drs.D.Mathis and C.Benoist of Harvard medical school, Boston, Mass.).

To the gld mouse, 1) K/B×N serum obtained above was injected (−), and 2) K/B×N serum and sFas ligand ((−)+sFas ligand)) was injected, and 3) K/B×N serum, sFas ligand and an anti-DR5 antibody (anti-DR5) were injected for 10 days, and the thickness of each joint was measured with a caliper (Manostat, Switzerland), and a clinical index was measured.

The clinical index referred to the followings:

- 0: no joint swelling,
- 1: swelling of one finger joint,
- 2: mild swelling of wrist or ankle,
- 3: severe swelling of wrist or ankle.

The thickness of the joint and the clinical index measured as above were shown in FIG. 6. As confirmed in FIG. 6 (A), when the serum and sFasL were treated to the gld mouse, the joint began to be swell from the 4th day and the highest index could be observed at day 9˜10. However, when the serum and sFasL and the anti-DR5 were treated together to the gld mouse, little swelling of the joint could be observed. In addition, as confirmed in FIG. 6 (B), also in case of the clinical index, when treating sFasL and anti-DR5 together to the gld mouse, the clinical index remained at 3-4 points, and little swelling of the joint could be observed.

From the above results, it could be confirmed that the blockage of binding of DR5-FasL by anti-DR5 treatment inhibited the inflammation reaction, and it could be used as an agent for preventing and treating inflammation including arthritis.

Example 7. Confirmation of sFasL-DR5 Binding Sites Through DR5 Mutagenesis

In order to confirm the binding sites of sFasL and DR5, huDR5 (SEQ ID NO: 1 in Table 1)-cloned piRES3-Puro vector was used as a template, and using Overlap PCR method, in the candidate sites where sFasL was expected to bind in the huDR5 protein amino acid sequence (See FIG. 7a) (selecting sites where the ligand of DR5, TRAIL binds conventionally as the candidate sites), huDR5-CRD2 (334-363 in the total sequence) or huDR5-CRD3 (445-471 in the total sequence), the amino acids shown in blue in FIG. 7b were substituted with alanine and then were cloned, to prepare huDR5 mutation DNA (See FIG. 7b and Table 1).

After transfecting mutated huDR5 DNA to mouse cells and expressing each of DR5 where huDR5-CRD2 was mutated (Table 1, SEQ ID NO: 4), or DR5 where huDR5-CRD3 was mutated (Table 1, SEQ ID NO: 5), flow cytometry was performed using human FasL where an immunoglobulin (IgG) binding only to cells expressing huDR5 was combined (huFasL-IgG), and the result was shown in FIG. 8b.

As could be seen in FIG. 8b, huFasL-IgG did not bind to huDR5 where CRD2 or CRD3 was mutated, and therefrom, it could be confirmed that the binding sites between FasL-DR5 bound using CRD3 and CRD3 of DR5 similar to TRAIL.

TABLE 1

SEQ

Classification
Sequence
ID NO

huDR5 Protein
MEQRGQNAPAASGARKRHGP
1

GPREARGARPGPRVPKTLVL

VVAAVLLVSAESALITQQDL

APQQRAAPQQKRSSPSEGLC

PPGHHISEDGRDCISCKYGQ

DYSTHWNDLLFCLRCTRCDS

GEVELSPCTTTRNTVCQCEE

GTFREEDSPEMCRKCRTGCP

RGMVKVGDCTPWSDIECVHK

ESGIIIGVTVAAVVLIVAVF

VCKSLLWKKVLPYLKGICSG

GGGDPERVDRSSQRPGAEDN

Mutation huDR5-CRD2
MEQRGQNAPAASGARKRHGP
2

Protein
GPREARGARPGPRVPKTLVL

VVAAVLLVSAESALITQQDL

APQQRAAPQQKRSSPSEGLC

PPGHHISEDGRDCISCKYGQ

DYSTHWNDLLACLACTRCAA

GEVELSPCTTTRNTVCQCEE

GTFREEDSPEMCRKCRTGCP

RGMVKVGDCTPWSDIECVHK

ESGIIIGVTVAAVVLIVAVF

VCKSLLWKKVLPYLKGICSG

GGGDPERVDRSSQRPGAEDN

VLNEIVSILQPTQVPEQEME

VQEPAEPTGVNMLSPGESEH

LLEPAEAERSQRRRLLVPAN

EGDPTETLRQCFDDFADLVP

FDSWEPLMRKLGLMDNEIKV

AKAEAAGHRDTLYTMLIKWV

NKTGRDASVHTLLDALETLG

ERLAKQKIEDHLLSSGKFMY

LEGNADSAMS

Mutation huDR5-CRD3
MEQRGQNAPAASGARKRHGP
3

Protein
GPREARGARPGPRVPKTLVL

VVAAVLLVSAESALITQQDL

APQQRAAPQQKRSSPSEGLC

PPGHHISEDGRDCISCKYGQ

DYSTHWNDLLFCLRCTRCDS

GEVELSPCTTTRNTVCQCEE

GTFREEDAPAMCAKCATGCP

RGMVKVGDCTPWSDIECVHK

ESGIIIGVTVAAVVLIVAVF

VCKSLLWKKVLPYLKGICSG

GGGDPERVDRSSQRPGAEDN

VLNEIVSILQPTQVPEQEME

VQEPAEPTGVNMLSPGESEH

LLEPAEAERSQRRRLLVPAN

EGDPTETLRQCFDDFADLVP

FDSWEPLMRKLGLMDNEIKV

AKAEAAGHRDTLYTMLIKWV

NKTGRDASVHTLLDALETLG

ERLAKQKIEDHLLSSGKFMY

LEGNADSAMS

huDR5 DNA
atggaacaacggggacagaa
4

cgccccggccgcttcggggg

cccggaaaaggcacggccca

ggacccagggaggcgcgggg

agccaggcctgggccccggg

tccccaagacccttgtgctc

gttgtcgccgcggtcctgct

gttggtctcagctgagtctg

ctctgatcacccaacaagac

ctagctccccagcagagagc

ggccccacaacaaaagaggt

ccagcccctcagagggattg

tgtccacctggacaccatat

ctcagaagacggtagagatt

gcatctcctgcaaatatgga

caggactatagcactcactg

gaatgacctccttttctgct

tgcgctgcaccaggtgtgat

tcaggtgaagtggagctaag

tccctgcaccacgaccagaa

acacagtgtgtcagtgcgaa

gaaggcaccttccgggaaga

agattctcctgagatgtgcc

ggaagtgccgcacagggtgt

cccagagggatggtcaaggt

cggtgattgtacaccctgga

gtgacatcgaatgtgtccac

aaagaatcaggcatcatcat

aggagtcacagttgcagccg

tagtcttgattgtggctgtg

tttgtttgcaagtctttact

gtggaagaaagtccttcctt

acctgaaaggcatctgctca

ggtggtggtggggaccctga

gcgtgtggacagaagctcac

aacgacctggggctgaggac

aatgtcctcaatgagatcgt

gagtatcttgcagcccaccc

aggtccctgagcaggaaatg

gaagtccaggagccagcaga

gccaacaggtgtcaacatgt

tgtcccccggggagtcagag

catctgctggaaccggcaga

agctgaaaggtctcagagga

ggaggctgctggttccagca

aatgaaggtgatcccactga

gactctgagacagtgcttcg

atgactttgcagacttggtg

ccctttgactcctgggagcc

gctcatgaggaagttgggcc

tcatggacaatgagataaag

gtggctaaagctgaggcagc

gggccacagggacaccttgt

acacgatgctgataaagtgg

gtcaacaaaaccgggcgaga

tgcctctgtccacaccctgc

tggatgccttggagacgctg

ggagagagacttgccaagca

gaagattgaggaccacttgt

tgagctctggaaagttcatg

tatctagaaggtaatgcaga

ctctgccatgtcctaa

Mutation huDR5-
atggaacaacggggacagaa
5

CRD2 DNA
cgccccggccgcttcggggg

cccggaaaaggcacggccca

ggacccagggaggcgcgggg

agccaggcctgggccccggg

tccccaagacccttgtgctc

gttgtcgccgcggtcctgct

gttggtctcagctgagtctg

ctctgatcacccaacaagac

ctagctccccagcagagagc

ggccccacaacaaaagaggt

ccagcccctcagagggattg

tgtccacctggacaccatat

ctcagaagacggtagagatt

gcatctcctgcaaatatgga

caggactatagcactcactg

gaatgacctccttgcctgct

tggcctgcaccacgtgtgct

gcaggtgaagtggagctaag

tccctgcaccacgaccagaa

acacagtgtgtcagtgcgaa

gaaggcaccttccgggaaga

agattctcctgagatgtgcc

ggaagtgccgcacagggtgt

cccagagggatggtcaaggt

cggtgattgtacaccctgga

gtgacatcgaatgtgtccac

aaagaatcaggcatcatcat

aggagtcacagttgcagccg

tagtcttgattgtggctgtg

tttgtttgcaagtctttact

gtggaagaaagtccttcctt

acctgaaaggcatctgctca

ggtggtggtggggaccctga

gcgtgtggacagaagctcac

aacgacctggggctgaggac

aatgtcctcaatgagatcgt

gagtatcttgcagcccaccc

aggtccctgagcaggaaatg

gaagtccaggagccagcaga

gccaacaggtgtcaacatgt

tgtcccccggggagtcagag

catctgctggaaccggcaga

agctgaaaggtctcagagga

ggaggctgctggttccagca

aatgaaggtgatcccactga

gactctgagacagtgcttcg

atgactttgcagacttggtg

ccctttgactcctgggagcc

gctcatgaggaagttgggcc

tcatggacaatgagataaag

gtggctaaagctgaggcagc

gggccacagggacaccttgt

acacgatgctgataaagtgg

gtcaacaaaaccgggcgaga

tgcctctgtccacaccctgc

tggatgccttggagacgctg

ggagagagacttgccaagca

gaagattgaggaccacttgt

tgagctctggaaagttcatg

tatctagaaggtaatgcaga

ctctgccatgtcctaa

Mutation huDR5-
atggaacaacggggacagaa
6

CRD3 DNA
cgccccggccgcttcggggg

cccggaaaaggcacggccca

ggacccagggaggcgcgggg

agccaggcctgggccccggg

tccccaagacccttgtgctc

gttgtcgccgcggtcctgct

gttggtctcagctgagtctg

ctctgatcacccaacaagac

ctagctccccagcagagagc

ggccccacaacaaaagaggt

ccagcccctcagagggattg

tgtccacctggacaccatat

ctcagaagacggtagagatt

gcatctcctgcaaatatgga

caggactatagcactcactg

gaatgacctccttttctgct

tgcgctgcaccaggtgtgat

tcaggtgaagtggagctaag

tccctgcaccacgaccagaa

acacagtgtgtcagtgcgaa

gaaggcaccttccgggaaga

agatgctcctgcgatgtgcg

cgaagtgcgccacagggtgt

cccagagggatggtcaaggt

cggtgattgtacaccctgga

gtgacatcgaatgtgtccac

aaagaatcaggcatcatcat

aggagtcacagttgcagccg

tagtcttgattgtggctgtg

tttgtttgcaagtctttact

gtggaagaaagtccttcctt

acctgaaaggcatctgctca

ggtggtggtggggaccctga

gcgtgtggacagaagctcac

aacgacctggggctgaggac

aatgtcctcaatgagatcgt

gagtatcttgcagcccaccc

aggtccctgagcaggaaatg

gaagtccaggagccagcaga

gccaacaggtgtcaacatgt

tgtcccccggggagtcagag

catctgctggaaccggcaga

agctgaaaggtctcagagga

ggaggctgctggttccagca

aatgaaggtgatcccactga

gactctgagacagtgcttcg

atgactttgcagacttggtg

ccctttgactcctgggagcc

gctcatgaggaagttgggcc

tcatggacaatgagataaag

gtggctaaagctgaggcagc

gggccacagggacaccttgt

acacgatgctgataaagtgg

gtcaacaaaaccgggcgaga

tgcctctgtccacaccctgc

tggatgccttggagacgctg

ggagagagacttgccaagca

gaagattgaggaccacttgt

tgagctctggaaagttcatg

tatctagaaggtaatgcaga

ctctgccatgtcctaa