WO2018021624A1 - Biomarkers for diagnosis of rheumatoid arthritis - Google Patents

Abstract

The present invention relates to: biomarkers for diagnosis of rheumatoid arthritis, activity evaluation of rheumatoid arthritis, and/or prediction of the severity and prognosis of rheumatoid arthritis; and a use thereof.

Description

Biomarkers for the Diagnosis of Rheumatoid Arthritis

The present invention relates to a biomarker for diagnosing rheumatoid arthritis.

Rheumatoid Arthritis (RA) is a disease caused by inflammation of the tissue called the synovial tissue surrounding the joints, and approximately 1% of the population in Korea is a representative chronic disease.

Although the diagnosis of rheumatoid arthritis is primarily dependent on clinical symptoms, there is a limit to the diagnosis after the severe destruction of the joint. Rheumatoid factor (RF) is an international diagnostic standard as a serological marker of rheumatoid arthritis. Although included in the diagnostic criteria of the American College of Rheumatology (ACR), RF has negative sensitivity throughout the course of the disease in 20% of patients with rheumatoid arthritis. Chronic inflammation, malignant tumors, even in some healthy elderly have the disadvantage of low specificity.

In addition, as a treatment method for rheumatoid arthritis, a drug treatment method in which analgesic anti-inflammatory drugs are generally combined with various anti-rheumatic agents is generally used to minimize joint damage, prevent loss of function, and reduce pain. It has been developed and used in combination therapy with antirheumatic drugs. Surgical therapy is performed in cases of severe disease severity. However, despite the excellent effects, such treatments may continue to cause joint deformity, and may be difficult to treat properly due to side effects of the drug. The development of methods to properly diagnose and predict prognosis and severity is urgently needed.

In particular, although a number of diagnostic markers for rheumatoid arthritis detected from blood have been reported, the present invention is characterized in that it is a diagnostic marker for rheumatoid arthritis that can be detected in a urine sample.

There are about 50,000 proteins that can be detected in the blood, and most of these proteins are filtered in the kidneys and are rarely detected in the urine, so not all proteins are detected in the urine. In addition, in the case of using a blood sample, because the sample must be obtained by an invasive test, there is a problem that causes pain and takes a lot of time.

Particularly, in the case of rheumatoid arthritis activity test, the present invention provides a method for evaluating activity using urine samples only because samples must be regularly collected for several years and decades after the onset of rheumatoid arthritis. Compared to the method, the patient's pain and discomfort has been eliminated.

[Preceding technical literature]

[Patent Documents]

Domestic Patent Registration No. 0937144 (2010. 1. 8)

An object of the present invention is to measure the expression level of at least one protein selected from the group consisting of interleukin-6 (IL-6), Chemokine (CC motif) ligand 2 (CCL-2) and interleukin-8 (IL-8). It provides a diagnostic composition for rheumatoid arthritis, comprising the agent.

It is also an object of the present invention to provide a kit and protein chip for diagnosing rheumatoid arthritis comprising the composition.

It is also an object of the present invention to provide a method for detecting one or more proteins selected from the group consisting of IL-6, CCL-2 and IL-8 from the urine sample of the patient in order to provide information necessary for the diagnosis of rheumatoid arthritis will be.

It is also an object of the present invention to provide a composition for evaluating the activity of rheumatoid arthritis, comprising an agent for measuring the concentration of at least one protein selected from the group consisting of IL-6, CCL-2 and IL-8.

It is also an object of the present invention to provide a kit or protein chip for evaluating the activity of rheumatoid arthritis, comprising the composition.

It is also an object of the present invention to provide information for evaluating rheumatoid arthritis activity, comprising measuring the concentration of at least one protein selected from the group consisting of IL-6, CCL-2 and IL-8 from urine samples To provide a way.

It is also an object of the present invention to provide a composition for predicting the severity prognosis of rheumatoid arthritis, comprising an agent for measuring the expression level of IL-6.

It is also an object of the present invention to provide a kit for predicting the severity prognosis of rheumatoid arthritis, comprising the composition.

As one aspect for achieving the above object, the present invention comprises an agent for measuring the expression level of one or more proteins selected from the group consisting of IL-6, CCL-2 and IL-8, diagnostic composition for rheumatoid arthritis It is about.

In another aspect, the present invention relates to a kit for diagnosing rheumatoid arthritis, comprising the composition.

In another aspect, the present invention provides a method for detecting at least one protein selected from the group consisting of IL-6, CCL-2 and IL-8 from the urine sample of the patient to provide information necessary for the diagnosis of rheumatoid arthritis It is about.

As another aspect, the present invention relates to a composition for evaluating the activity of rheumatoid arthritis, comprising an agent for measuring the concentration of at least one protein selected from the group consisting of IL-6, CCL-2 and IL-8.

As another aspect, the present invention relates to a kit for assessing the activity of rheumatoid arthritis, comprising the composition.

As another aspect, the present invention relates to a protein chip for evaluating the activity of rheumatoid arthritis, comprising the composition.

As another aspect, the invention provides information for assessing rheumatoid arthritis activity, comprising measuring the concentration of one or more proteins selected from the group consisting of IL-6, CCL-2 and IL-8 from urine samples It is about how to provide.

As another aspect, the present invention relates to a composition for predicting the severity prognosis of rheumatoid arthritis, comprising an agent for measuring the expression level of IL-6.

As another aspect, the present invention relates to a kit for predicting the severity prognosis of rheumatoid arthritis, comprising the composition.

The present invention enables the early treatment by diagnosing rheumatoid arthritis using an agent that measures the expression level of one or more proteins selected from the group consisting of IL-6, CCL-2 and IL-8. In addition, the present invention can be used to measure the activity of rheumatoid arthritis by using an agent for measuring the concentration of one or more proteins selected from the group consisting of IL-6, CCL-2, and IL-8, and widely utilized in predicting the severity prognosis. Therefore, it is possible to perform the appropriate treatment according to the condition of the patient.

1 is a comparison of cytokine levels in the urine of normal and rheumatoid arthritis patients.

Figure 2 shows the relationship between clinical variables measured in rheumatoid arthritis patients and cytokine levels in urine.

Figure 3 shows low disease activity with a DAS28 score of less than 3.2 (medium), moderate disease activity (more than 3.2) and less than 5.1, and high disease activity (more than 5.1). IL-6, CCL-2 and IL-8 levels in urine were compared for rheumatoid arthritis disease activity defined as).

Figure 4 shows the ROC curve for assaying high disease activity [a: predicted disease activity between urine IL-6 and serum CRP, b: predicted disease activity between urine CCL2 and serum CRP, c: urine IL-8 Comparison of disease activity with serum CRP].

5 is Predictive probability plot of radiological progression.

Figure 6 shows the ROC curve to test the severity [a: comparison of the prediction of bone fracture after 3 years with IL-6, ESR, and CRP, b: comparison of the prediction of bone destruction after 3 years with CCL2, ESR, and CRP, c: Comparison of prediction of bone destruction after 3 years with IL-8, ESR and CRP].

Hereinafter, the present invention will be described in more detail.

As used herein, the term "diagnostic" means identifying the presence or characteristic of a pathological condition. For the purposes of the present invention, the diagnosis may mean confirming the development of rheumatoid arthritis or further confirming whether the disease is progressing or deepening.

As used herein, the term "diagnostic marker, diagnostic marker, or diagnostic marker" is used to distinguish rheumatoid arthritis from a condition that is not rheumatoid arthritis (for example, to distinguish from normal or to other arthritis such as osteoarthritis). Polypeptides or nucleic acids (e.g. mRNA), lipids, glycolipids, glycoproteins or sugars that increase or decrease in a sample of an individual with rheumatoid arthritis compared to a sample of an individual that is not rheumatoid arthritis. Organic biomolecules such as monosaccharides, disaccharides, oligosaccharides, and the like. For the purposes of the present invention, the diagnostic markers of the present invention refer to IL-6, CCL-2 and IL-8 which exhibit specifically increased expression in samples of patients with rheumatoid arthritis.

More specifically, the present invention provides IL-6, CCL-2 and / or IL-8 as diagnostic markers of rheumatoid arthritis.

In the present invention, "activity of rheumatoid arthritis" or "disease activity of rheumatoid arthritis" refers to the general degree of inflammation of rheumatoid arthritis patients, or the degree of progression of rheumatoid arthritis, and is useful for evaluating the response or remission to treatment. Can be used.

In addition, in the present invention, the "evaluation of rheumatoid arthritis activity" refers to evaluating the overall degree of inflammation or the progression of rheumatoid arthritis in patients with rheumatoid arthritis, and further based on whether the current treatment is effective for the patient. The concept includes making a judgment, selecting an optimal treatment regimen (such as drug therapy or surgery), and predicting responsiveness and remission to treatment.

DAS (Disease Activity Score) or DAS28 (Disease Activity Score 28), a representative method of measuring the activity of rheumatoid arthritis in the conventional clinical practice, is an exponential evaluation method of scoring. Along with some objective factors, subjective factors assessed by patients and physicians are evaluated together.

DAS28 is a composite index consisting of the number of tenderness joints, swelling joints, erythrocyte sedimentation rate, and systemic evaluation of the patients. 28 joints include both shoulder joints, elbow and wrist joints, and metacarpal joints. , Muscle joints, knee joints, and the like.

Rheumatoid arthritis disease activity surveyed with DAS28 can be calculated from 0 to a maximum of 9.4, generally with a DAS28 score of less than 2.6, with little disease activity (Remission), and with a disease score between 2.6 and 3.2 below Low Disease Activity is defined as Moderate Disease Activity (severity) of 3.2 or more and less than 5.1, and High Disease Activity (Severe) of 5.1 or more. However, as mentioned in the background art, the conventional method for assessing disease activity by DAS28 is invasive, causing pain in patients and requiring a lot of time.

The present invention can determine the current disease activity of the patient non-invasively without having to evaluate the rheumatoid arthritis activity, such as the conventional DAS28, based on this to determine the optimal treatment regimen and determine whether the appropriate treatment response is shown in the treatment To provide objective disease activity markers.

More specifically, the present invention provides IL-6, CCL-2 and / or IL-8 as markers as disease activity markers of rheumatoid arthritis. In the present invention, the proteins reflect the disease state of rheumatoid arthritis, have a higher concentration in the active group than the rheumatoid arthritis inactive group, and the conventional disease activity evaluation means such as DAS28, which is currently used for evaluation of disease activity in clinical trials. Not only does it show a strong positive correlation, it also shows a significant correlation with CRP, which is used as an important inflammatory marker in the assessment of DAS28.

Thus, IL-6, CCL-2 and / or IL-8 as markers are non-invasive biomarkers and can by themselves be useful for evaluating the inflammatory response of a patient and for predicting the therapeutic response. In addition, inflammatory activity markers such as CRP and ESR can be used as ancillary markers for judging the treatment response when the clinical outcomes of patients with rheumatoid arthritis differ. In particular, when used in combination with serum CRP evaluation is expected to be utilized as a biomarker of better activity.

In the present invention, the term "predicting the severity prognosis of rheumatoid arthritis" refers to the possibility of bone fracture or joint deformation due to high disease activity when assessing the general degree of inflammation, bone fracture, or progression of rheumatoid arthritis in patients with rheumatoid arthritis. Related to the likelihood of consequences. DAS (Disease Activity Score), which is a representative method of measuring disease severity of rheumatoid arthritis, or its modified form, DAS28 (Disease Activity Score 28), is an exponential evaluation method of scoring method. Along with some of the same objective elements, subjective factors assessed by patients and physicians are evaluated together. DAS28 is a composite index consisting of the number of tenderness joints, swelling joints, erythrocyte sedimentation rate, and systemic evaluation of the patients. 28 joints include both shoulder joints, elbow and wrist joints, and metacarpal joints. , Muscle joints, knee joints, and the like.

Rheumatoid arthritis disease activity surveyed with DAS28 can be calculated from 0 to a maximum of 9.4. Generally, if the DAS28 score is less than 2.6, there is little disease activity (Remission). Low Disease Activity is defined as Moderate Disease Activity (severity) of 3.2 or more and less than 5.1, and High Disease Activity (Severe) of 5.1 or more.

The present invention can easily predict the future disease severity of the patient in a non-invasive way, it can be used as information for the delay, alleviation and / or cure of the disease after the onset of rheumatoid arthritis by determining whether to use the additional treatment method.

The present invention provides IL-6 as a predictor of severity prognosis in rheumatoid arthritis patients. In the present invention, IL-6 is an index for predicting the disease state of rheumatoid arthritis, and it can be predicted that the risk of osteoporosis of rheumatoid arthritis increases when IL-6 concentration is increased in the urine sample of the patient. IL-6 showed a similar level of power to C-reactive protein (CRP), a known risk factor for bone destruction.

In the present invention, "IL-6" is an abbreviation for interleukin-6. Interleukin-6 is a glycoprotein having a molecular weight of 210,000 separated by B cell stimulating factor 2 (BSF-2), which induces the final differentiation of B cells into antibody producing cells. In addition, it is a cytokine produced by various cells such as T lymphocytes, B lymphocytes, macrophages, fibroblasts, and the same molecule as interferon β ₂ (IFN-β ₂ ). It is known to be involved in immune response, proliferation and differentiation of hematopoietic and nervous system cells, and acute reactions. The amino acid sequence of IL-6 can be confirmed by its sequence information in a known gene database. For example, the amino acid sequence of human IL-6 protein can be found in NCBI Genbank Accession No. AAD13886.1, which is set forth in SEQ ID NO: 1.

[SEQ ID NO 1]

1 mnsfstsafg pvafslglll vlpaafpapv ppgedskdva aphrqpltss eridkqiryi

61 ldgisalrke tcnksnmces skealaennl nlpkmaekdg cfqsgfneet clvkiitgll

121 efevyleylq nrfesseeqa ravqmstkvl iqflqkkakn ldaittpdpt tnaslltklq

181 aqnqwlqdmt thlilrsfke flqsslralr qm

In the present invention, "CCL-2 is an abbreviation of chemokine (CC motif) ligand 2. It is also called monocyte chemoattractant protein-1 (MCP-1). It belongs to CC chemokine group and is mainly secreted by monocytes, macrophages, and dendritic cells. It is a protein that is responsible for introducing monocytes, memory-related T cells, and dendritic cells into infected or inflamed areas, and has a molecular weight of 13 kDa, which is activated when cleaved by the metalloproteinase MMP12. Amino acid sequences can be found in known genetic databases, eg, amino acid sequences of human CCL-2 proteins can be found in NCBI Genbank Accession No. AAB20651.1. Number 2 is described.

[SEQ ID NO 2]

1 mkvsaallcl lliaatfipq glaqpdaina pvtccynftn rkisvqrlas yrritsskcp

       61 keavifktiv akeicadpkq kwvqdsmdhl dkqtqtpkt

In the present invention, "IL-8" is an abbreviation of interleukin-8. Interleukin-8, also called CXC ligand 8, is a type of chemokine. It is mainly secreted from macrophages, epithelial cells, airway smooth muscle cells and endothelial cells. IL-8's primary role is chemotaxis, which induces neutrophils into infected areas, and is also involved in innate immunity through angiogenesis, toll-like receptors (TLRs). You can check. For example, the amino acid sequence of human IL-8 protein can be found in NCBI Genbank Accession No. AAH13615.1, which is set forth in SEQ ID NO: 3.

[SEQ ID NO 3]

1 mtsklavall aaflisaalc egavlprsak elrcqcikty skpfhpkfik elrviesgph

       61 canteiivkl sdgrelcldp kenwvqrvve kflkraens

In a specific embodiment of the present invention, a protein expressing a specific expression difference was selected from urine samples of patients with rheumatoid arthritis (RA) and normal persons, and proteins of IL-6, CCL-2 and / or IL-8. The amount was found to be significantly higher in RA patients than normal (FIG. 1). These proteins have proven to be urine protein biomarkers for the diagnosis of rheumatoid arthritis because of constant changes in proteome profile.

In a specific embodiment of the present invention, the results of the ROC analysis to compare with the well-known serum index in RA patients (DAS28≥5.1) with high rheumatoid arthritis activity, the result of the area under curve (AUC) of serum CRP Was 0.669 (95% confidence interval; CI [0.614-0.765]), AUC of IL-6 in urine was 0.657 [0.572-0.742], AUC of CCL-2 was 0.642 [0.551-0.734], and AUC of IL-8 Showed 0.546 [0.457-0.636] not inferior to CRP (FIG. 4).

In a specific embodiment of the present invention, the AUC of serum ESR was 0.738 [0.648-0.828]) as a result of performing ROC analysis to compare the power for predicting the degree of bone destruction with ESR and CRP after 3 years. AUC was 0.7220 [0.636-0.808]. The AUC of IL-6 in urine was 0.724 [0.628-0.821], which was comparable to that of ESR and CRP (FIG. 6).

The above results indicate that the measurement of markers in the urine alone enables the diagnosis, activity and disease severity of rheumatoid arthritis without invasive blood collection.

Furthermore, (i) the potential as a biomarker by the mutual combination of each protein, such as the combination of IL-6 in urine and CRT in serum, was demonstrated.

Thus, IL-6, CCL-2 and / or IL-8 can be used as a non-invasive biomarker for diagnosing rheumatoid arthritis, assessing activity or predicting severity prognosis, and the combination of these proteins is a better diagnostic biomarker It was confirmed that it can be used as.

In a preferred embodiment, the agent for measuring the expression level of the protein may be an antibody specific for IL-6, CCL-2 and / or IL-8.

In the present invention, "antibody" means a protein molecule specific for an antigenic site. For the purposes of the present invention, an antibody means an antibody that specifically binds to the marker proteins IL-6, CCL-2 and / or IL-8, and includes all monoclonal antibodies, polyclonal antibodies and recombinant antibodies.

Monoclonal antibodies are well known in the art by hybridoma methods (Kohler and Milstein (1976) European journal og Immunology 6: 511-519), or phage antibody libraries (Clarkson et al, Nature, 352: 624-628, 1991; Marks et al, J. Mol. Biol., 222: 58, 1-597, 1991).

Polyclonal antibodies can be produced by methods well known in the art for injecting the above described protein antigens into animals and collecting blood from the animals to obtain serum comprising the antibodies. Such polyclonal antibodies can be prepared from any animal species host, such as goats, rabbits, sheep, monkeys, horses, pigs, cattle, dogs, and the like.

The antibodies of the present invention also include special antibodies such as chimeric antibodies, humanized antibodies, human antibodies and the like.

Furthermore, the antibodies used in the present invention include functional fragments of antibody molecules as well as complete forms having two full length light chains and two full length heavy chains. The functional fragment of an antibody molecule means the fragment which has at least antigen binding function, and can illustrate Fab, F (ab '), F (ab') 2, Fv etc.

As a method for measuring the expression level of IL-6, CCL-2 and / or IL-8 in a sample using such an antibody, a method of treating the sample with the antibody and confirming the generation of antigen-antibody complexes Can be used without limitation.

The "antigen-antibody complex" means a combination of IL-6, CCL-2 and / or IL-8 proteins and antibodies specific thereto, and the amount of antigen-antibody complex formed is a signal of a detection label. It can be measured quantitatively through the size of.

For example, Western blot, ELISA (enzyme linked immunosorbent assay), Immunoprecipitation Assay, Complement Fixation Assay, Fluorecence Activated Cell Sorter (FACS), or Protein Chip (protein chip) and the like, but is not limited thereto.

Kits of the present invention may include agents capable of measuring the expression levels of IL-6, CCL-2 and / or IL-8 in patient samples, as well as one or more compositions, solutions or devices suitable for expression level analysis. have. For example, the kit may include a substrate, an appropriate buffer solution, a secondary antibody labeled with a detection label, a chromogenic substrate, and the like for immunological detection of the antibody.

As a specific example, the kit for diagnosing, assessing activity or predicting the severity prognosis of rheumatoid arthritis is a kit comprising an essential element necessary to perform ELISA in order to implement various ELISA methods such as an ELISA kit and a sandwich ELISA. Can be.

ELISA kits contain antibodies specific for these proteins. Antibodies are antibodies that have high specificity and affinity for IL-6, CCL-2 and / or IL-8 proteins and have little cross-reactivity to other proteins, and may be monoclonal, polyclonal or recombinant antibodies. In addition, the ELISA kit can include antibodies specific for the control protein. Other ELISA kits may include reagents that can detect bound antibodies, such as labeled secondary antibodies, chromopores, enzymes, and substrates thereof, or other materials that can bind to antibodies.

In addition, the kit may be a kit for implementing western blot, immunoprecipitation assay, complement fixation assay, flow cytometry, or protein chip, and may further include additional components suitable for each assay method. Through these analytical methods, rheumatoid arthritis can be diagnosed by comparing the amount of antigen-antibody complex formation, and the patient can be treated appropriately.

As another aspect, the kit of the present invention may be provided in the form of a protein chip.

Protein chips are those in which one or more antibodies against IL-6, CCL-2 and / or IL-8 proteins are arranged in a fixed position and immobilized at high density. In the analysis method using a protein chip, the protein is separated from a sample sample and hybridized with the protein chip to form an antigen-antibody complex, which is read to confirm the expression level of the protein to diagnose rheumatoid arthritis or In addition, activity can be assessed or the severity can be predicted.

The amount of the antigen-antibody complex formed can be measured quantitatively through the magnitude of the signal of the detection label.

The detection label may be selected from the group consisting of enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules, and radioisotopes, but is not necessarily limited thereto.

As used herein, the term "sample sample" refers to tissues, cells, whole blood, plasma, serum, blood, saliva, sputum, lymph fluid, which differ in expression levels of the marker proteins IL-6, CCL-2 and / or IL-8. Samples such as cerebrospinal fluid, intracellular fluid or urine, and the like. In a more preferred embodiment, the sample sample may be a urine sample.

The process of separating the protein from the sample sample can be carried out using a known process, the protein level can be measured by the aforementioned various antigen-antibody complex measurement method.

In a more preferred embodiment, the present invention can be carried out by measuring the expression level of the protein using an antibody specific for IL-6, CCL-2 and / or IL-8.

In a more preferred embodiment, the antibody may be a monoclonal or polyclonal antibody specific for IL-6, CCL-2 and / or IL-8, but may also be a functional fragment of an antibody that retains antigen binding activity.

In the present invention, by measuring the expression level of IL-6, CCL-2 and / or IL-8 with an antibody, it is possible to quickly and accurately diagnose rheumatoid arthritis, evaluate the activity or predict the severity.

Hereinafter, the present invention will be described in more detail through examples according to the present invention, but the scope of the present invention is not limited to the examples given below.

Example 1 Comparison of Cytokine Concentrations in Urine in Patients with Rheumatoid Arthritis

In urine collected from 205 patients with rheumatoid arthritis and 62 health controls, the levels of IL-6, CCL-2 and IL-8, which are representative inflammatory cytokines, were measured. Although these cytokines are known to have elevated serum levels in inflammatory diseases, it is not known whether they are elevated in the urine of patients with rheumatoid arthritis. These three cytokines were corrected with serum creatinine and compared for differences in concentrations between the two groups.

As shown in FIG. 1, IL-6 (RA vs control: 0.06 [0.03-0.133] vs 0.03 [0.01-0.07], P <0.001), CCL-2 (RA vs control: 1.47 [0.58-2.51] vs 1.01 [ 0.54-1.77], P = 0.033), IL-8 (RA vs control: 0.09 [0.03-0.19] vs 0.06 [0.01-0.14], P = 0.026), all increased significantly in patients with rheumatoid arthritis.

Example  2: Rheumatoid arthritis  Investigation of the relationship between cytokine concentration in urine, inflammatory markers and disease activity

Next, we examined whether the measured urine levels of these three cytokines, IL-6, IL-8, and CCL-2, were related to inflammatory markers in 205 patients with rheumatoid arthritis.

First, the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), albumin (Alb), hemoglobin (Hb), white blood cell (WBC), and DAS28 (disease), which are the indicators of serum inflammation, activity 28) and the correlation with the Sharp van der Heijde score (SvdH), an indicator of severity of rheumatoid arthritis. At the same time, each urine cytokine correlated with serum IL-6, IL-8 and CCL-2. As shown in Figure 2, IL-6 in urine showed a significant correlation with serum CRP, DAS28, SvdH score, Alb, WBC, IL-6, IL-8 in urine serum ESR, CRP, DAS28, Alb, Hb and urine CCL-2 were significantly associated with serum ESR, CRP, DAS28, Alb and Hb. These results suggest that IL-6, IL-8, and CCL-2 cytokines in urine may significantly reflect the degree of inflammation in serum.

To further examine the association with disease activity, patients with rheumatoid arthritis were divided into low (DAS28 <3.2), moderate (3.2≤DAS28 <5.1), and high (DAS28≥5.1) according to disease activity to serum creatinine in each group. The corrected cytokine concentrations in urine were compared. IL-6 concentrations were 0.10 ± 0.04, 0.11 ± 0.02, 0.37 ± 0.12 in the low, moderate, and high disease activity groups, respectively, P = 0.029 in the low versus high disease activity group, and P = 0.013 in the moderate versus high disease activity group. There was a significant difference (FIG. 3A). CCL-2 concentrations were 1.78 ± 0.39, 1.71 ± 0.25, and 5.69 ± 2.19 (mean ± SEM) in the low, moderate, and high disease activity groups, respectively (P = 0.040 only in the moderate versus high disease activity group). 3B). IL-8 concentrations were 0.24 ± 0.16, 0.37 ± 0.12 and 1.06 ± 0.65 (mean ± SEM) in the low, moderate and high disease activity groups, respectively. 3C).

ROC analysis was performed to compare the well-known serum indices with the power to diagnose severe disease activity (DAS28≥5.1). Currently, the most widely used serum inflammatory markers are ESR and CRP. In this experiment, only the CRP was compared, which excludes ESR because DAS28, which was used to evaluate disease activity in this patient group, was calculated using ESR. The area under curve (AUC) of serum CRP was 0.669 (95% confidence interval; CI [0.614-0.765]), the AUC of IL-6 in urine was 0.657 [0.572-0.742], and the AUC of CCL-2 was 0.642 [0.551]. -0.734], and the AUC of IL-8 was 0.546 [0.457-0.636], which was not inferior to CRP (FIG. 4).

Example  3: concentration of cytokine in urine Rheumatoid arthritis  Severity ( Bone fracture  Predictive analysis

To evaluate the severity of rheumatoid arthritis, we analyzed the score of bone fracture after 3 years according to the urine concentration of the patients. The degree of bone fracture was calculated in both hands and feet X-ray using Sharp van der Heijde (SvdH) method. The predicted probability plot of bone breakdown according to the concentration of cytokines in urine was calculated, including the known severity indicators. As urine IL-6 concentration, serum ESR and CRP concentration increased, the predicted probability of bone fracture occurrence increased when showing anti-cyclic citrullinated peptide antibody (ACPA) positivity (FIG. 5).

To investigate which risk factors can independently predict the degree of bone destruction after 3 years, the cytokines in urine, and multivariate analysis revealed significant variables and known bone destruction risk factors such as age, disease disease Multivariate analyzes were performed to correct glomerular filtration rates that could affect duration, CRP-, and urine cytokine concentrations. Multivariate analysis showed an increased risk of bone destruction by 3.97 times with a 1-quarter increase in IL-6 in urine, 1.03 times with a 1 mm / hr increase in ESR, and a 5.87-fold increase in risk of bone destruction when ACPA positive. It could be confirmed (Table 1).

In addition, ROC analysis was performed to compare the power of ESR and CRP to predict the degree of bone destruction after 3 years. The AUC of serum ESR was 0.738 [0.648-0.828]) and the AUC of CRP was 0.7220 [0.636-0.808]. The AUC of IL-6 in urine was 0.724 [0.628-0.821], which was comparable to that of ESR and CRP (FIG. 6). The above results indicate that the measurement of markers in the urine can evaluate the activity and disease severity of rheumatoid arthritis without invasive blood collection.

Multivariate Logistic Regression to Predict Radiation Progression

Variables	Odds ratio	Estimate	P-value
Urianry IL-6, per 1 quarter increase	3.97	1.379	0.016
ESR, per 1 unit increase	1.03	0.031	0.001
ACPA, positivity	5.87	1.77	0.042

Logistic regression is used to obtain a baseline model odds ratio adjusted for the presence of radiation progression, which model is adjusted for age, disease duration, C-reactive protein and glomerular filtration rate.

Table 2 is divided into three groups according to IL-6 and CRP level, both low group means the first group, both IL-6 and CRP level is low at the same time, both high means all three groups at the same time, The high CRP group refers to group 3 with high CRP, and the high IL-6 group refers to group 3 with only IL-6. The remaining groups belong to both mid groups. Both high and high groups showed a 4.6-fold increase in the risk of bone destruction compared to both low and high-risk groups, which were 5.0 times higher in the anti-CCP antibody positive group and 6.7 times higher in the ESR group.

As shown in Table 3 below, after dividing each marker into three groups, when the risk of bone destruction was measured using only one marker, the risk increased by 2.9 times for IL-6 and 2.8 times for CRP. IL-6 and CRP combinations can be useful markers.

In addition, combination modeling was performed to investigate whether the combination of proteins in the urine can predict the degree of bone destruction better after 3 years. To this end, patients were divided into two groups according to the median value of each protein concentration in urine, and three cases (IL6 + CCL2, IL6 + IL8, IL8 + CCL2) combined with two proteins were investigated.

For example, in the case of IL-6 and IL-8, both proteins are low, all high, only IL-6 high, only UL-8 high, and none of the above four. Divided into five groups, both groups were examined for the risk of bone destruction in both groups when the low protein was referenced.

As a result, IL-6 and IL-8 increased the risk of bone destruction by 3.1 times higher in both groups than in the low concentration of both proteins. 6.2 times higher in all cases compared to the low group, and 5.0 times higher risk of bone destruction in both the high group and IL-8 and CCL2 than the low group. 4).

<110> THE CATHOLIC UNIVERSITY OF KOREA INDUSTRY-ACADEMIC COOPERATION FOUNDATION

<120> Biomarkers for diagnosing rheumatoid arthritis

<130> X16U11C0249

<150> KR 10-2016-0097185

<151> 2016-07-29

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<170> KopatentIn 2.0

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Ser Ser Glu Arg Ile Asp Lys Gln Ile Arg Tyr Ile Leu Asp Gly Ile

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Val Lys Ile Ile Thr Gly Leu Leu Glu Phe Glu Val Tyr Leu Glu Tyr

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            180 185 190

Leu Ile Leu Arg Ser Phe Lys Glu Phe Leu Gln Ser Ser Leu Arg Ala

        195 200 205

Leu Arg Gln Met

    210

<210> 2

<211> 99

<212> PRT

<213> human CCL-2

<400> 2

Met Lys Val Ser Ala Ala Leu Leu Cys Leu Leu Leu Ile Ala Ala Thr

  1 5 10 15

Phe Ile Pro Gln Gly Leu Ala Gln Pro Asp Ala Ile Asn Ala Pro Val

             20 25 30

Thr Cys Cys Tyr Asn Phe Thr Asn Arg Lys Ile Ser Val Gln Arg Leu

         35 40 45

Ala Ser Tyr Arg Arg Ile Thr Ser Ser Lys Cys Pro Lys Glu Ala Val

     50 55 60

Ile Phe Lys Thr Ile Val Ala Lys Glu Ile Cys Ala Asp Pro Lys Gln

 65 70 75 80

Lys Trp Val Gln Asp Ser Met Asp His Leu Asp Lys Gln Thr Gln Thr

                 85 90 95

Pro Lys Thr

<210> 3

<211> 99

<212> PRT

<213> human IL-8

<400> 3

Met Thr Ser Lys Leu Ala Val Ala Leu Leu Ala Ala Phe Leu Ile Ser

  1 5 10 15

Ala Ala Leu Cys Glu Gly Ala Val Leu Pro Arg Ser Ala Lys Glu Leu

             20 25 30

Arg Cys Gln Cys Ile Lys Thr Tyr Ser Lys Pro Phe His Pro Lys Phe

         35 40 45

Ile Lys Glu Leu Arg Val Ile Glu Ser Gly Pro His Cys Ala Asn Thr

     50 55 60

Glu Ile Ile Val Lys Leu Ser Asp Gly Arg Glu Leu Cys Leu Asp Pro

 65 70 75 80

Lys Glu Asn Trp Val Gln Arg Val Val Glu Lys Phe Leu Lys Arg Ala

                 85 90 95

Glu asn ser

Claims (27)

1. To measure the expression level of one or more proteins selected from the group consisting of interleukin-6 (IL-6), chemokine (CC motif) ligand 2 (CCL-2) and interleukin-8 (IL-8) present in urine samples Comprising a formulation, a diagnostic composition for rheumatoid arthritis.
2. The method of claim 1,

   The agent for measuring the expression level of the protein is specific for at least one selected from the group consisting of interleukin-6 (IL-6), Chemokine (CC motif) ligand 2 (CCL-2), and interleukin-8 (IL-8). Composition that is an antibody.
3. The method of claim 2,

   The antibody is a monoclonal antibody or polyclonal antibody.
4. The method of claim 1,

   A composition for measuring the expression level of IL-6 present in urine and CRP present in blood serum.
5. A diagnostic kit for rheumatoid arthritis, comprising the composition of any one of claims 1 to 4.
6.  A diagnostic protein chip for rheumatoid arthritis, comprising the composition of any one of claims 1 to 4.
7. To provide you with the information you need to diagnose rheumatoid arthritis,

   A method for detecting at least one protein selected from the group consisting of interleukin-6 (IL-6), Chemokok (C-C motif) ligand 2 (CCL-2) and interleukin-8 (IL-8) from a patient's urine sample.
8. The method of claim 7, wherein

   Detection of the protein using an antibody specific for one or more selected from the group consisting of interleukin-6 (IL-6), Chemokine (CC motif) ligand 2 (CCL-2) and interleukin-8 (IL-8) That is performed.
9. The method of claim 7, wherein

   The antibody is a monoclonal antibody or a polyclonal antibody.
10. The method of claim 7, wherein

    The detection of the protein is Western blot, ELISA (enzyme linked immunosorbent assay), immunoprecipitation assay, complement fixation assay, Fluorecence Activated Cell Sorter (FACS), or protein The method is carried out using a chip (protein chip).
11. The method of claim 7, wherein

    Detecting IL-6 in urine and CRP in blood serum.
12. Agents for measuring the concentration of one or more proteins selected from the group consisting of interleukin-6 (IL-6), chemokine (CC motif) ligand 2 (CCL-2) and interleukin-8 (IL-8) present in urine samples Containing, Composition for assessing the activity of rheumatoid arthritis.
13. The method of claim 12,

    The agent for measuring the concentration of the protein is specific for at least one selected from the group consisting of interleukin-6 (IL-6), Chemokine (CC motif) ligand 2 (CCL-2), and interleukin-8 (IL-8). The composition is an antibody.
14. The method of claim 13,

    The antibody is a monoclonal antibody or polyclonal antibody.
15. The method of claim 12,

    A composition for measuring the concentration of IL-6 present in the urine and CRP present in the blood serum.
16. A kit for assessing the activity of rheumatoid arthritis, comprising the composition of any one of claims 12-15.
17. A protein chip for evaluating the activity of rheumatoid arthritis, comprising the composition of any one of claims 12-15.
18. A method of providing information for assessing rheumatoid arthritis activity, comprising measuring the concentration of one or more proteins selected from the group consisting of IL-6, CCL-2 and IL-8 from urine samples.
19. The method of claim 18,

    Measuring the concentration of the protein is performed using an antibody specific for at least one selected from the group consisting of IL-6, CCL-2 and IL-8.
20. The method of claim 19,

    The antibody is a monoclonal antibody or a polyclonal antibody.
21. The method of claim 18,

    The protein concentration can be measured by Western blot, ELISA (enzyme linked immunosorbentassay), Immunoprecipitation Assay, Complement Fixation Assay, Fluorecence Activated Cell Sorter (FACS), or protein. The method is carried out using a chip (protein chip).
22. The method of claim 18,

    Measuring the concentration of IL-6 present in urine and CRP present in blood serum.
23. A composition for predicting the severity prognosis of rheumatoid arthritis, comprising an agent for measuring the expression level of interleukin-6 (IL-6) present in a urine sample.
24. The method of claim 22,

    The agent for measuring the expression level of the protein is an antibody specific for interleukin-6 (IL-6).
25. The method of claim 23,

    The antibody is a monoclonal antibody or polyclonal antibody.
26. The method of claim 22,

    A composition for measuring the expression level of IL-6 present in urine and CRP present in blood serum.
27. 27. A kit for predicting the severity prognosis of rheumatoid arthritis, comprising the composition of any one of claims 22 to 26.

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