KR20180047928A - Biomarker composition for diagnosing sepsis comprising V-set and immunoglobulin-domain containing protein(VISG4) and method for diagnosing sepsis using the same - Google Patents

Abstract

The present invention relates to a biomarker composition for diagnosing sepsis comprising v-set and immunoglobulin-domain containing protein (VISG4) cell surface antigens as an active ingredient, and to a method for diagnosing sepsis by using the same. More specifically, the present invention relates to a biomarker composition for diagnosing sepsis by using VISG4 cell surface antigens expressed in blood cells in an animal model of sepsis induced by cecal ligation and puncture (CLP), and to a method for diagnosing sepsis by using the same. According to the present invention, after CLP induction, VISG4 expression on the surface of immune cells in blood, spleen, and thymus of mice increased as sepsis progressed, and more specifically, it was confirmed that VISG4 expression on the surface of CD4 T cells, CD8 T cells, B cells and CD11b cells increased and that apoptosis of immune cells in thymus was progressing. Accordingly, the composition of the present invention, which comprises the VISG4 cell surface antigens having the effects described above as an active ingredient, can be used in a biomarker composition for diagnosing sepsis, a pharmaceutical composition for preventing or treating sepsis, or a method for diagnosing sepsis.

Description

[0001] The present invention relates to a biomarker composition for diagnosing sepsis, which contains VISG4 (v-set and immunoglobulin-domain containing protein) cell surface antigen as an active ingredient, and a method for diagnosing sepsis using the VISG4 (VISG4 ) and method for diagnosing sepsis using the same}

The present invention relates to a biomarker composition for the diagnosis of sepsis, which contains VISG4 (v-set and immunoglobulin-domain containing protein) cell surface antigen as an active ingredient, and a method for diagnosing sepsis using the same.

Sepsis is a disease caused by bronchial infections, intraperitoneal infections, or various microbial infections caused by burns, leading to death due to systemic infection. Sepsis is an excessive host inflammatory reaction to infection and is severe due to organ damage. It is defined as a state of threatening disease. Sepsis is the leading cause of death from infections worldwide, accounting for 60% of infant mortality rates below 5 years of age, and one of the leading causes of death from pregnant and elderly infections. Sepsis is a complex clinical disease that accounts for the largest proportion of hospital deaths, leading to a high mortality rate of 30 to 50% and is the leading cause of death in ICUs. Sepsis is known to be the most common cause of mortality in most intensive care units (ICUs). Globally, there are 20 million sepsis patients each year, of which about 50% die from sepsis shock. In the United States, about 700,000 patients develop annually, of which more than 250,000 die. It is estimated that 37,000 people die annually from severe sepsis in the UK, and the annual NHS cost is over £ 1.5 billion.

Until a few years ago, sepsis was known to lead to death due to a "cytokine storm" caused by an immune response to an infection. Therefore, the treatment method focuses on this point and focuses on research to suppress excessive immune response. Despite extensive research on sepsis, however, there is much controversy over the realistic understanding of the immune response to sepsis. With respect to the increase in mortality and morbidity due to sepsis, some researchers claim to be the result of persistent immune activation accompanied by an inflammatory response, while others claim to be the result of immunosuppression to be. This discrepancy is due to the failure to improve the survival rate of patients with sepsis in the course of clinical application using various anti-inflammatory drugs. Recently, the major cause of death in sepsis has been reevaluated as a result of hyper-inflammatory response (Hotchkiss. Nat Rev Immunol. 2013).

In recent studies in sepsis patients with cecal ligation and puncture (CLP) and multiple organ damage, sepsis has been shown to induce extensive loss of lymphocytes through apoptosis. Since lymphocytes produce pro-inflammatory cytokines and activate macrophages, loss of lymphocytes may benefit survival rates by weakening or inhibiting excessive, ancillary inflammatory responses. From a different perspective, however, there are many studies that suggest that the loss of lymphocytes in sepsis may be detrimental because it impairs the ability of the immune system to fight pathogens.

Taken together, these results suggest that recent septicemia studies have led to major changes in the mechanism of sepsis. According to the researchers, sepsis is a systemic immune response due to infection, which is a complex immunosuppressive response to an early immune response and subsequent immune suppression. In addition, immunosuppression has been shown to decrease the function of adaptive immune cells or to decrease the number of T cells due to apoptosis, which is thought to play an important role in increasing the mortality rate as a cause of impaired immune function in the later stage of sepsis. However, to date, there has been no study on the causative agent of immune function or the immunosuppressive factor in the course of sepsis.

VSIG4 (V-set and Ig domain-containing 4) was first discovered by Dr. Campagne (Genentech) in 2006 and is known to have innate immunoregulatory ability as a complement receptor binding to C3b (Mol Immunol. 2008). Therefore, studies on VSIG4 molecules have been performed very limited and have recently been classified as cell surface proteins related to the B7 family. VSIG4 gene expression is highly expressed in hepatocytes, dendritic cells (DCs), neutrophils and macrophages, but is known to be very low or absent in other tissues, T cells and B cells (J Clin Invest. 2006). In contrast, VSIG4 protein is expressed only in resting macrophages, and it is known that VSIG4 also disappeared upon activation of these cells. In vivo studies have shown that macrophages in autoimmune tissues do not express VSIG4. Thus, VISG4 is mainly expressed in resting macrophages and disappears upon cell activation. In vitro experiments with VISG4-Ig strongly inhibited the proliferation of T-cells induced by anti-CD3 or anti-CD28 in VSIG4-Ig. It is also known as a negatively regulator of T cell activity because it greatly suppresses IL-2 expression, CD8 T cell IFN-y production, and Th cell-dependent IgG response in CD4 T cells. However, studies on the expression and function of VSIG4 in association with sepsis have not been made.

US registered patent No. 8088386 (registered on January 03, 2012)

It is an object of the present invention to provide a biomarker composition for diagnosing sepsis which contains VISG4 cell surface antigen as an active ingredient or a sepsis diagnosis kit comprising an antibody or an aptamer which specifically binds to VISG4 cell surface antigen .

Another object of the present invention is to provide a method for providing information necessary for diagnosis of sepsis by comparing the expression level of VISG4 cell surface antigen of a sample suspected of sepsis with a normal control.

It is still another object of the present invention to provide a pharmaceutical composition for preventing or treating sepsis, which contains a VISG4 inhibitor as an active ingredient.

It is still another object of the present invention to provide a method for screening candidate substances having reduced expression or activity level of VISG4 protein by contacting a candidate substance with cells containing VISG4 protein and comparing the expression or activity of VISG4 protein with a control sample A method for screening a therapeutic agent for sepsis.

In order to achieve the above object, the present invention provides a biomarker composition for diagnosing sepsis, comprising VISG4 cell surface antigen as an active ingredient.

In addition, the present invention provides a sepsis diagnosis kit comprising an antibody or an aptamer which specifically binds to VISG4 cell surface antigens.

The present invention also relates to a method for screening for VISG4 cell surface antigen, comprising the steps of: i) measuring the expression level of VISG4 cell surface antigen in a sample isolated from a patient suspected of sepsis, ii) comparing the expression level of VISG4 cell surface antigen with a sample isolated from normal, and iii) And a step of diagnosing sepsis when the expression level of VISG4 cell surface antigen of the sample suspected of sepsis is higher than that of the normal control sample.

The present invention also provides a pharmaceutical composition for preventing or treating septicemia which contains a VISG4 inhibitor as an active ingredient.

The present invention also relates to a method for producing a VISG4 protein, comprising the steps of: i) contacting a candidate substance with a cell containing VISG4 protein, ii) measuring the degree of expression or activity of VISG4 protein in the cell contacted with the candidate substance, and iii) And screening candidates for which the degree of expression or activity of the VISG4 protein is decreased.

According to the present invention, the expression of VISG4 on the surface of blood, spleen and thymic immune cells of mice after CLP induction was increased with the progress of sepsis, and more specifically, the expression of VISG4 on the surface of CD4 T cells, CD8 T cells, , The expression of VISG4 was increased, and the immune cells in the thymus were observed to be apoptotic.

Therefore, the composition of the present invention containing VISG4 cell surface antigen having the above-mentioned effect as an active ingredient can be applied to a biomarker composition for diagnosing sepsis, a pharmaceutical composition for preventing or treating sepsis, or a method for diagnosing sepsis.

1 is a microarray analysis of gene expression of immune cells isolated from the blood of an animal model of CLP induced sepsis.
FIG. 2 shows changes in the total number of immune cells isolated from the blood, spleen and thymus of an animal model of CLP induced sepsis.
FIG. 3 shows flow cytometric analysis of VISG4 expression of immune cells isolated from the blood of an animal model of CLP induced sepsis.
FIG. 4 shows the expression of VISG4 in immunocytes isolated from blood and spleen of an animal model of CLP induced sepsis by polymerase chain reaction.
FIG. 5 shows flow cytometric analysis of VISG4 expression in immunocytes isolated from the spleen of an animal model of CLP induced sepsis.
FIG. 6 shows the expression of VISG4 in CD4 T cells, CD8 T cells, B cells and CD11b cells in spleen immunocytes of an animal model of CLP induced sepsis by flow cytometry.
Figure 7 shows the expression of VISG4 in immune cells isolated from the thymus of an animal model of CLP induced sepsis by flow cytometry.
8 is a flow cytometric analysis of the apoptosis of immune cells isolated from the thymus of an animal model of CLP induced sepsis.

As used herein, the term " biomarker " refers to a substance capable of distinguishing a tissue or cell in which sepsis occurs from a normal cell or a tissue or cell treated with appropriate sepsis, An organic biomolecule such as a protein or nucleic acid, a lipid, a glycolipid, a glycoprotein, or the like that exhibits an increase or a decrease.

The term " antibody " as used herein refers to a specific immunoglobulin directed against an antigenic site, wherein an antibody in the present invention refers to an antibody that specifically binds to VISG4, Or those which are commercially available can be used. In addition, the antibody includes a polyclonal antibody, a monoclonal antibody, and a fragment capable of binding to an epitope. The antibody refers to a complete form having two full-length light chains and two long-length heavy chains, and also includes special antibodies such as humanized antibodies.

In addition, the kit of the present invention comprises an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a labeling substance that is colored by the reaction with the substrate, a coloring substrate solution that reacts with the labeling substance, Enzyme reaction termination solutions, and the like, and can be made from a number of separate packaging or compartments, including the reagent components used.

As used herein, the term " aptamer " is a single-stranded nucleic acid (DNA, RNA or modified nucleic acid) having a stable tertiary structure and capable of binding to a target molecule with high affinity and specificity . Aptamers are comparable to monoclonal antibodies due to their inherent high affinity (usually pM levels) and their ability to bind to target molecules with specificity, and there is a high likelihood of being an alternative antibody, especially as a "chemoantibody".

The term " patient " as used herein refers to a subject suspected of having sepsis, and includes any individual whose expression of VISG4 cell surface antigen changes due to sepsis, preferably human or rodent have.

As used herein, the term " patient sample " used herein refers to a tissue sample, cell, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid, or urine that differs from the normal control in the expression level of the VISG4 cell surface antigen as a biomarker for sepsis diagnosis But not limited to, the same sample. Preferably, the sample of the patient is a sample obtained from blood.

The present invention provides a biomarker composition for diagnosing sepsis, which contains VISG4 cell surface antigen as an active ingredient.

Preferably, the sepsis may be, but is not limited to, induced by cecal ligation and puncture (CLP).

In addition, the present invention provides a sepsis diagnosis kit comprising an antibody or an aptamer which specifically binds to VISG4 cell surface antigens.

Preferably, the antibody may be a monoclonal antibody or a polyclonal antibody.

An aptamer that specifically binds to the biomarker of the present invention may be used in place of the antibody of the present invention, and the aptamer may be an oligonucleic acid or a peptide molecule.

In addition, the kit of the present invention comprises an antibody that specifically binds to a marker component, a secondary antibody conjugate conjugated with a labeling substance that is colored by the reaction with the substrate, a coloring substrate solution that reacts with the labeling substance, Enzyme reaction termination solutions, and the like, and can be made from a number of separate packaging or compartments, including the reagent components used.

The marker of the secondary antibody conjugate is preferably a conventional coloring agent that undergoes a chromogenic reaction and is preferably selected from the group consisting of HRP (horseradish peroxidase), alkaline phosphatase, colloid gold, poly L-lysine-fluorescein fluorescein such as isothiocyanate and rhodamine-B-isothiocyanate, and dye may be used.

In addition, i) measuring the level of expression of the VISG4 cell surface antigen in a sample isolated from a patient suspected of sepsis, ii) comparing the expression level of the VISG4 cell surface antigen with a sample isolated from a normal person, and iii) The present invention provides a method for providing information necessary for diagnosis of sepsis including the step of diagnosing sepsis when the expression level of VISG4 cell surface antigen is higher than that of a normal control sample.

Preferably, the sample may be selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid and urine, but is not limited thereto.

Preferably, the level of expression of the VISG4 cell surface antigen can be measured through an antigen-antibody reaction.

Preferably, the antigen-antibody reaction is detected by enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay, western blot, immunoprecipitation, immunohistochemical staining, flow cytometry (FACS), enzyme substrate staining, and antigen-antibody aggregation.

The present invention also provides a pharmaceutical composition for preventing or treating septicemia which contains a VISG4 inhibitor as an active ingredient.

Preferably, the VISG4 inhibitor may be selected from the group consisting of antibodies and small molecules as an activity inhibitor, but is not limited thereto. In addition, the VISG4 inhibitor may be selected from the group consisting of NF-kB inhibitors and ERK inhibitors as an expression inhibitor, but it is not limited thereto.

When the composition of the present invention is a pharmaceutical composition, it may contain a pharmaceutically acceptable carrier in addition to the above-mentioned active ingredients. Such pharmaceutically acceptable carriers are those conventionally used in pharmaceutical preparations, and include lactose, dextrose, The present invention relates to a process for the preparation of a medicament for the treatment and / or prophylaxis of cancer, comprising administering a therapeutically effective amount of a compound selected from the group consisting of cross, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, Carboxymethylcellulose, carboxymethylcellulose, hydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. The pharmaceutical composition may further contain a filler, a weight agent, a binder, a lubricant, a wetting agent, a disintegrant, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a fragrance, a preservative and the like as an additive and an auxiliary agent.

The pharmaceutical composition may be formulated into tablets, pills, powders, granules, capsules, suspensions, solutions, emulsions, syrups, sterilized aqueous solutions, nonaqueous solutions, emulsions, lyophilized preparations, suppositories and sterile injectable solutions.

The pharmaceutical composition may be administered by intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, subcutaneous, intradermal, intranasal, intrapulmonary, intraocular, intramuscular, Rectally, topically, orally, and by inhalation.

An effective amount of the active ingredient of the above pharmaceutical composition means an amount required for prevention or treatment of the disease. Accordingly, the present invention is not limited to the particular type of the disease, the severity of the disease, the kind and amount of the active ingredient and other ingredients contained in the composition, the type of formulation and the patient's age, body weight, general health status, sex and diet, But are not limited to, various factors, including, for example, the rate of administration, the rate of administration, the duration of the treatment, the drugs used concurrently.

The present invention also provides a method for producing a VISG4 protein, comprising the steps of: i) contacting a candidate substance with a cell containing a VISG4 protein, ii) measuring the expression or activity of the VISG4 protein in the cell contacted with the candidate substance, and iii) And screening candidates for which the degree of expression or activity of the VISG4 protein is decreased.

Preferably, the expression or activity level of the VISG4 protein is determined by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunohistochemical staining, Western blotting western blot, flow cytometry (FACS), and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example  One : CLP  Induced sepsis animal model

Mice used in the CLP induced sepsis animal model were purchased from Daejeon, Korea for 8 weeks old female C57BL / 6, approved by the animal laboratory of Inje University College of Medicine.

An animal model of CLP-induced sepsis was referred to the standard protocol of the prior art (Current Protocols, Immunology, 2010). C57BL / 6 mice were anesthetized by intraperitoneal injection of a mixture of ketamine (80 mg / kg) and xylazine (12 mg / kg), followed by 1 cm incision of the abdominal wall and epidermis . After the cecum was removed, the intestine was pushed to the end of the cecum, and then the upper part of the cecum was tied to the upper part of the cecum. A 21-gauge needle was used to puncture the cecum, so that a portion of the stomach was excreted into the abdominal cavity, and then inserted into the abdominal cavity. The abdominal wall muscles and epidermis were sutured, and 1 ml of physiological saline was injected subcutaneously. After the operation, analgesic (diclofenac sodium) and antibiotics were subcutaneously injected into each mouse body weight and the changes of mice were observed. As a control group, a sham-operated mouse was used which did not damage the abdominal cavity-incised cecum.

Example  2 : CLP  Immune cells in the blood of an animal model of induced sepsis Microarray (microarray) analysis

Blood samples were collected in control and experimental groups (6 hours, 12 hours, 24 hours) after induction of CLP in mice to identify the expression patterns of the genes according to progression of sepsis and to find immune related genes. The blood was diluted 1: 1 with phosphate buffered saline (PBS), and 4 ml of diluted blood was added to 2 ml of Histopaque-1077 (Sigma- Aldrich) made. The centrifugal separator was centrifuged by setting the centrifuge at 400 g, 20 minutes, 20 ° C., and no brake. Then, peripheral blood mononuclear cells (PBMCs) were transferred to a new tube using a Pasteur pipette equipped with a syringe rubber (tube). After 10% RPMI medium was added, the cells were centrifuged again, and the supernatant was removed to obtain immune cells. After that, the degree of gene expression of isolated immune cells was analyzed by microarray analysis.

As a result, referring to FIG. 1, it was confirmed that many genes increased expression at the early stage, middle stage and late stage of CLP induction. In the process of classifying immune-related genes and classifying cell surface proteins among these genes, VSIG4 expression was significantly increased after 24 hours. The expression of VSIG4 was increased 3.3-fold compared to the control group at 24 hours, and the expression of VSIG4 was increased 4.1-fold and 6.3-fold, respectively, at 48 and 72 hours, respectively.

Example  3: CLP  Changes in blood, spleen and thymic immune cell counts in an animal model of induced sepsis

Immunocytes were isolated from the blood, spleen and thymus of control and experimental groups (24 hours, 48 hours, 72 hours) after induction of CLP in mice, and then the changes of immune cell numbers were checked with time.

As a result, referring to FIG. 2, the number of immune cells in the blood of the normal mice was 1.5 x 10 ^{6 on} average, whereas the immune cells in the blood of the CLP-induced mice were greatly reduced (about 1 / 3). At 72 hours, the number of immune cells was slightly increased to about 1/2. In addition, the number of spleen immune cells in normal mice showed an average of 1 x 10 ⁸ , while the number of spleen immune cells in CLP-induced mice decreased by about 1/2 from 24 hours, to about 1/5 from 48 hours , And slightly increased at 72 hours to maintain about 1/2 of the number of immune cells. The number of immune cells in the thymus rapidly decreased after the induction of CLP and decreased to about 1/3 from 24 hours. The number of cells in the thymus rapidly decreased to about 1/20 at 48 hours and to about 1/100 at 72 hours. Therefore, after the induction of CLP, blood and spleen immune cells showed a tendency to decrease at the early stage and to recover at 72 hours. However, most of the immune cells in the thymus were removed by apoptosis.

Example  4 : CLP  Immune Cells in the Blood of an Induced Sepsis Animal Model VSIG4  Expression analysis

After the induction of CLP in mice, immune cells were isolated from the blood of the control and experimental groups (24 hours, 48 hours, 72 hours) and the expression of VSIG4 was confirmed by flow cytometry (FACS).

As a result, referring to FIG. 3, the expression of VSIG4 in the immune cells of the normal mouse was very low, whereas the expression of VSIG4 was significantly increased in the immune cells of the CLP-induced mouse, Of the cells express VSIG4.

Example  5: CLP  Of splenic immune cells in an animal model of induced sepsis VSIG4  Expression analysis

Expression of VSIG4 in the spleen of the control and experimental groups (6 hours, 24 hours, 48 hours, 72 hours) after CLP induction in mice was analyzed by polymerase chain reaction (PCR). The primers (F: 5'-CTGGTAAGACACGGCTCTGACTCCG-3 ', R: 5'-CATCAGGAGTCTGCCAGGTGACCTC-3') used in the polymerase chain reaction were synthesized in bioneer using the base sequence of mouse VSIG4 gene.

As a result, referring to FIG. 4, it was confirmed that VSIG4 expression was increased in spleen immune cells of CLP-induced mice while VSIG4 expression in normal spleen immunocytes of normal mice was very low.

Next, VSIG4 expressed in spleen immunocytes was confirmed by flow cytometry. The VSIG4 antibody (sc82433) used for flow cytometry was purchased from Santa Cruz.

As a result, referring to FIG. 5, VSIG4 expression was significantly lower in spleen immunized cells of normal mice, whereas VSIG4 expression was significantly increased in splenic immune cells of CLP-induced mice, and about 40% RTI ID = 0.0 > VSIG4. ≪ / RTI >

6, the expression of VSIG4 was significantly increased in CD4 T cells, CD8 T cells and B cells after induction of CLP, and it was found that CD11 T cells, Respectively.

Example  6: CLP  Immune cells in the thymus of induced sepsis animal models VSIG4  Expression analysis

After the induction of CLP in mice, immune cells were isolated from the thymus of the control and experimental groups (24 hours, 48 hours, 72 hours) and the expression of VSIG4 was confirmed by flow cytometry.

As a result, referring to FIG. 7, VSIG4 expression in normal thymocytes of normal mice was very low, whereas expression of VSIG4 in the immunocytes of the thymus of CLP-induced mice was increased by about 13% at 24 hours, And VSIG4 was expressed in about 40% of the immune cells at 72 hours.

Example  7: CLP  Analysis of apoptotic cell death in the thymus of an animal model of induced sepsis

After the induction of CLP in mice, immune cells were isolated from the thymus of the control and experimental groups (24 hours, 48 hours, 72 hours), and cell death was confirmed by flow cytometry.

As a result, referring to FIG. 8, the degree of apoptosis of normal mouse was about 7%, whereas the degree of apoptosis of CLP-induced mouse was increased to about 45% Of the immune cells progressed to apoptosis process.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

The scope of the present invention is defined by the appended claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims (11)

VISG4 (v-set and immunoglobulin-domain containing protein) cell surface antigen as an active ingredient. The biomarker composition for diagnosing sepsis according to claim 1, wherein the sepsis is induced by cecal ligation and puncture (CLP). VISG4 (v-set and immunoglobulin-domain containing protein) A sepsis diagnosis kit comprising an antibody or an aptamer that specifically binds to a cell surface antigen. 4. The kit for diagnosing septicemia according to claim 3, wherein the antibody is a monoclonal antibody or a polyclonal antibody. i) measuring the level of VISG4 (v-set and immunoglobulin-domain containing protein) cell surface antigen expression in a sample isolated from suspected sepsis patients;
ii) comparing the level of expression of said VISG4 cell surface antigen with a sample isolated from normal persons; And
iii) diagnosing sepsis when the expression level of the VISG4 cell surface antigen of the sample suspected of sepsis is higher than that of the normal control sample;
The method comprising the steps < RTI ID = 0.0 > of: < / RTI > 6. The method according to claim 5, wherein the sample is selected from the group consisting of tissue, cells, whole blood, serum, plasma, saliva, sputum, cerebrospinal fluid and urine. 6. The method according to claim 5, wherein the expression level of the VISG4 cell surface antigen is measured through an antigen-antibody reaction. The method according to claim 7, wherein the antigen-antibody reaction is carried out by an enzyme immunoassay (ELISA), a radioimmunoassay (RIA), a sandwich assay, a western blot, an immunoprecipitation method, wherein the assay is performed by any one method selected from the group consisting of immunohistochemical staining, flow cytometry (FACS), enzyme substrate staining, and antigen-antibody aggregation. VISG4 (v-set and immunoglobulin-domain containing protein) inhibitor as an active ingredient. i) contacting the candidate material with a cell comprising VISG4 (v-set and immunoglobulin-domain containing protein) protein;
ii) measuring the degree of expression or activity of VISG4 protein in the cell in contact with the candidate substance; And
iii) selecting a candidate substance having decreased expression or activity level of the VISG4 protein as compared with a control sample. 11. The method according to claim 10, wherein the expression or activity level of the VISG4 protein is determined by reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (ELISA), immunohistochemical staining, Wherein the measurement is carried out by any one method selected from the group consisting of western blot and flow cytometry (FACS).

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