METHOD FOR PREDICTING THERAPEUTIC EFFECT OF BIOLOGICAL PREPARATION ON RHEUMATOID ARTHRITIS

Abstract

The objective of the present invention is to provide a method for simply, inexpensively and accurately assessing, before administering a biological preparation, the therapeutic effect thereof (in particular whether there will be a complete response) or the improvement of symptoms in patients having rheumatoid arthritis.

By using at least one serum concentration selected from the group consisting of sgp130, IP-10, sTNFRI, sTNFRII, GM-CSF, IL-1β, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, Eotaxin, VEGF, MCP-1, TNF-α, IFN-γ, FGF basic, PDGF-bb, sIL-6R and MIP-1α, the therapeutic effect (improvement of symptoms and possibility of response) of an inflammatory cytokine-targeting biological preparation on a patient having rheumatoid arthritis can be predicted in any type of facility in a simple, inexpensive, and highly accurate manner before administering the biological preparation.

Claims

1. A method of predicting and determining a therapeutic effect of a biological formulation targeting an inflammatory cytokine on a rheumatoid arthritis patient, characterized in comprising the step of measuring a concentration of at least one type of determination marker selected from the group consisting of sgp130, IP-10, sTNFRI, sTNFRII, GM-CSF, IL-1β, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, Eotaxin, VEGF, MCP-1, TNF-α, IFN-γ, FGFbasic, PDGF-bb, sIL-6R, and MIP-1α in a serum collected from the rheumatoid arthritis patient prior to the administration of the biological formulation.

2. The method of claim 1, wherein the method is a method of predicting and determining a possibility of remission with tocilizumab, and at least sgp130 is used as the determination marker.

3. The method of determining of claim 2, wherein a patient to be administered with tocilizumab is a rheumatoid arthritis patient who has not received anti-cytokine therapy in the past, and the determination marker is a combination of (i) sgp130, (ii) IP-10, (iii) sTNFRII, and (iv) IL-6, IL-7, MCP-1 or IL-1β.

4. The method of determining of claim 2, wherein a patient to be administered with tocilizumab is a rheumatoid arthritis patient who has received anti-cytokine therapy in the past, and the determination marker is a combination of (i) sgp130, (ii) IP-10, (iii) sTNFRII, and (iv) IL-6 or IL-1β.

5. The method of determining of claim 1, wherein the method is a method of predicting and determining a possibility of remission with etanercept in a rheumatism patient who has not received anti-cytokine therapy in the past, and the determination marker is a combination of IL-9 and TNF-α, a combination of VEGF and PDGF-bb, or a combination of MIP-1α and PDGF-bb.

6. The method of determining of claim 1, wherein the method is a method of predicting and determining a disease activity indicator after therapy with tocilizumab in a rheumatism patient who has not received anti-cytokine therapy in the past, and wherein the determination marker is a combination of sgp130, IL-8, Eotaxin, IP-10, sTNFR1, sTNFRII, and IL-6 or a combination of sgp130, IL-8, Eotaxin, IP-10, sTNFRI, sTNFRII, IL-6 and VEGF.

7. The method of determining of claim 1, wherein the method is a method of predicting and determining a value of a disease activity indicator after therapy with tocilizumab in a rheumatism patient who has received anti-cytokine therapy in the past, and the determination marker is a combination of sgp130, IP-10, and GM-CSF.

8. The method of determining of claim 1, wherein the method is a method of predicting and determining a value of a disease activity indicator after therapy with etanercept in a rheumatism patient who has not received anti-cytokine therapy in the past, and the determination marker is a combination of IL-9, TNF-α and VEGF or a combination of IL-6 and IL-13.

9. The method of determining of claim 1, wherein the method is a method of predicting and determining a level of improvement in a symptom after therapy with tocilizumab in a rheumatism patient who has not received anti-cytokine therapy in the past, and the determination marker is a combination of IL-1β, Il-7, TNF-α, and sIL-6R.

10. The method of determining of claim 1, wherein the method is a method of predicting and determining a level of improvement in a symptom after therapy with etanercept in a rheumatism patient who has not received anti-cytokine therapy in the past, and the determination marker is a combination of IL-2, IL15, sIL-6R, and sTNFRI or a combination of IL-6 and IL-13.

11. A method of selecting a more effective biological formulation for therapy in a rheumatism patient who has not received anti-cytokine therapy in the past from among biological formulations consisting of tocilizumab and etanercept, comprising: predicting and determining a possibility of remission with tocilizumab in accordance with the method of determining of claim 3; predicting and determining a possibility of remission with etanercept in accordance with the method of determining of claim 5; and comparing the possibility of remission with tocilizumab with the possibility of remission with etanercept that were predicted and determined in the aforementioned steps to select a biological formulation with a high possibility of remission.

12. A method of selecting a more effective biological formulation for therapy in a rheumatism patient who has not received anti-cytokine therapy in the past from among biological formulations consisting of tocilizumab and etanercept, comprising: predicting and determining a disease activity indicator after therapy with tocilizumab in accordance with the method of determining of claim 6; predicting and determining a disease activity indicator after therapy with etanercept in accordance with the method of determining of claim 8; and comparing the disease activity indicator after therapy with tocilizumab with the disease activity indicator after therapy with etanercept that were predicted and determined in the aforementioned steps to select a biological formulation with a low disease activity indicator after therapy.

13. A method of selecting a more effective biological formulation for therapy in a rheumatism patient who has not received anti-cytokine therapy in the past from among biological formulations consisting of tocilizumab and etanercept, comprising: predicting and determining a level of improvement in a symptom after therapy with tocilizumab in accordance with the method of determining of claim 9; predicting and determining a level of improvement in a symptom after therapy with etanercept in accordance with the method of determining of claim 10; and comparing the level of improvement in a symptom after therapy with tocilizumab with the level of improvement in a symptom after therapy with etanercept that were predicted in the aforementioned steps to select a biological formulation with a high level of improvement in a symptom after therapy.

14. A diagnostic agent for predicting and determining a therapeutic effect due to a biological formulation targeting an inflammatory cytokine on a rheumatoid arthritis patient, comprising a reagent capable of detecting at least one type of marker selected from the group consisting of sgp130, IP-10, sTNFRI, sTNFRII, GM-CSF, IL-1β, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, Eotaxin, VEGF, MCP-1, TNF-α, IFN-γ, FGFbasic, PDGF-bb, sIL-6R, and MIP-1α.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0049] FIG. 1 is a diagram showing trial profiles of tocilizumab therapy patients and etanercept therapy patients.

[0050] FIGS. 2-1 to 2-4 are diagrams showing clinical baseline individual group statistics for healthy individuals and rheumatoid arthritis patients with respect to serum concentrations of cytokines/chemokines/soluble receptors.

[0051] FIG. 3 is a diagram showing the relationship between DAS-28 values prior to therapy and DAS-28 values after 16 weeks of therapy in tocilizumab therapy patients and etanercept therapy patients.

[0052] FIG. 4 is a diagram showing the relationship between DAS-28 values prior to therapy (PreDAS-28 score) and values obtained from subtracting a DAS-28 value after 16 weeks from a DAS-28 value prior to therapy (PreDAS-28score−16W DAS-28 score) in naïve patients who received tocilizumab therapy.

[0053] FIG. 5 is a diagram showing results of comparing predicted DAS-28 values after 16 weeks of therapy calculated from regression equation (4) prior to therapy and actual DAS-28 values after 16 weeks of therapy subjecting naïve patients who received tocilizumab therapy.

[0054] FIG. 6 is a diagram showing results of comparing predicted DAS-28 values after 16 weeks of therapy calculated from regression equation (5) prior to therapy and actual DAS-28 values after 16 weeks of therapy subjecting switch patients who received tocilizumab therapy.

[0055] FIG. 7 is a diagram showing results of comparing predicted DAS-28 values after 16 weeks of therapy calculated from regression equation (7) prior to therapy and actual DAS-28 values after 16 weeks of therapy subjecting naïve patients who received etanercept therapy.

[0056] FIG. 8 is a diagram showing the relationship between actual values of DAS-28 after 16 weeks of etanercept therapy and predicted DAS-28 values after 16 weeks of therapy estimated by assuming a patient has received tocilizumab therapy in naïve patients who received etanercept therapy.

[0057] FIG. 9 is a diagram showing results of analyzing the relationship between serum sgp130 concentrations and DAS-28 values prior to therapy for patients in remission and non-remission.

[0058] FIGS. 10a-10d are a diagram showing results of tocilizumab naïve multiple logistic regression analysis.

[0059] FIG. 11a-11b are a diagram showing results of tocilizumab switch multiple logistic regression analysis.

[0060] FIG. 12 is a diagram showing results of etanercept naïve multiple logistic regression analysis.

DESCRIPTION OF EMBODIMENTS

1. Determining Method

[0061] The present invention is a method of determining a therapeutic efficacy of a biological formulation targeting an inflammatory cytokine on a rheumatoid arthritis patient, characterized in comprising the step of measuring a concentration of one or more types selected from the group consisting of sgp130, IP-10, sTNFRI, sTNFRII, GM-CSF, IL-1β, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, Eotaxin, VEGF, MCP-1, TNF-α, IFN-γ, FGFbasic, PDGF-bb, sIL-6R, and MIP-1α in a serum collected from the rheumatoid arthritis patient prior to the administration of the biological formulation. Hereinafter, the determining method of the present invention is discussed in detail.

[0062] Biological Formulation Subjected to Determination

[0063] The determining method of the present invention is a method of predicting and determining a therapeutic effect of a biological formulation targeting an inflammatory cytokine on a rheumatoid arthritis patient.

[0064] A biological formulation targeting an inflammatory cytokine is not particularly limited as long as it is a biological formulation used in rheumatoid arthritis therapy. A therapeutic effect can be predicted and determined in accordance with the type of biological formulation to be used in the determining method of the present invention. Examples of a biological formulation targeting an inflammatory cytokine include anti-IL-6 agents, anti-TNF-α agents and the like. Specific examples of anti-IL-6 agent include humanized anti-IL-6 receptor antibodies, anti-TNF-α antibodies, human soluble TNF/LTα receptors consisting of an Fc region of human IgG1 and a subunit dimer of an extracellular domain of a human tumor necrosis factor receptor II, and the like. More specific examples of the humanized anti-IL-6 receptor antibodies include tocilizumab. Further, examples of the human soluble TNF/LTα receptors more specifically include etanercept. Further, examples of the anti-TNF-α antibodies more specifically include adalimumab and infliximab.

[0065] Examples of optimal biological formulations thereamong which are applied in the determining method of the present invention include humanized anti-IL-6 receptor antibodies and humanized soluble TNF/LTα receptors, and still preferably tocilizumab and etanercept.

[0066] Patients Subjected to Determination

[0067] The determining method of the present invention determines whether administration of abiological formulation is effective in a rheumatoid arthritis patient prior to administration of the biological formulation.

[0068] Further, target rheumatoid arthritis patients in the determining method of the present invention are not particularly limited, as long as it is prior to administration of the biological formulation. In addition, whether DMARDs such as methotrexate are administered, past dosing history of anti-cytokine therapy (administration of infliximab etanercept, adalimumab, tocilizumab or the like) are not relevant. A therapeutic effect due to a biological formulation can be predicted and determined by selecting a desired determination marker in accordance with the past dosing history of the biological formulation in the determining method of the present invention.

[0069] Determination Markers

[0070] The determining method of the present invention uses one or two or more types of determination markers selected from the group consisting of sgp130 (soluble gp130), IP-10 (interferon-inducible protein 10), sTNFRI (soluble receptors for tumor necrosis factor type I), sTNFRII (soluble receptors for tumor necrosis factor type II), GM-CSF (granulocyte macrophage colony-stimulating factor), IL-1β (interleukin-1B), IL-2 (interleukin-2), IL-5 (interleukin-5), IL-6 (interleukin-6), IL-7 (interleukin-7), IL-8 (interleukin-8), IL-9 (interleukin-9), IL-10 (interleukin-10), IL-12 (interleukin-12), IL-13 (interleukin-13), IL-15 (interleukin-15), Eotaxin, VEGF (vascular endothelial growth factor), MCP-1 (monocyte chemotactic protein-1), TNF-α (tumor necrosis factor-α), IFN-γ (interferon-γ), FGFbasic (basic fibroblast growth factor), PDGF-bb (platelet-derived growth factor bb), sIL-6R (soluble receptors for interleukin-6), and MIP-1α(macrophage inflammatory protein-la) in the serum of the rheumatoid arthritis patient.

[0071] One type of the aforementioned specific cytokine, chemokine, and soluble receptor may be used alone as a determination marker in the determining method of the present invention. However, it is preferable to use two or more types from there among in combination as a determination marker, from the viewpoint of predicting and determining a therapeutic effect due to a biological formulation at a higher precision.

[0072] The determination marker is appropriately selected and used, depending on the therapeutic effect to be predicted and determined, type of biological formulation to be administered, past dosing history of biological formulation or the like. Specific optimal examples of determination marker are shown below for each therapeutic effect to be predicted and determined.

<Cases where level of improvement in symptom after therapy (level of improvement in value of disease activity indicator; value of disease activity indicator prior to therapy−value of disease activity indicator after therapy) is predicted and determined for biological formulation>

[0073] For a naïve patient administered with tocilizumab (hereinafter, also referred to as an “tocilizumab therapy naïve patient”), it is preferable to use at least one type selected from the group consisting of IL-7, IL-8, IL-12, IL-13, IP-10, VEGF, IL-1β, TNF-α, and sIL-6R as a determination marker. It is more preferable to use IL-1β, IL-7, TNF-α, and sIL-6R in combination as a determination marker.

[0074] For a switch patient administered with tocilizumab (hereinafter, also referred to as a “tocilizumab therapy switch patient”), it is preferable to use at least one type selected from the group consisting of IL-1β, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, FGFbasic, GM-CSF, IFN-γ, TNF-α, and VEGF as a determination marker.

[0075] For a naïve patient administered with etanercept (hereinafter, also referred to as an “etanercept therapy naïve patient”), it is preferable to use at least one type selected from the group consisting of IL-6, IP-10, IL-2, IL-13, IL-15, sIL-6R, and sTNFRI as a determination marker. It is more preferable to use a combination of IL-2, IL-15, sIL-6R, and sTNFRI as a determination marker.

<Cases where value of disease activity indicator after therapy itself is predicted and determined for biological formulation>

[0076] For a tocilizumab therapy naïve patient, it is preferable to use at least one type selected from the group consisting of sgp130, IL-8, Eotaxin, IP-10, sTNFRI, sTNFRII, IL-6, and VEGF as a determination marker. It is more preferable to use a combination of sgp130, IL-8, Eotaxin, IP-10, sTNFRI, sTNFRII, and IL-6, or a combination of sgp130, IL-8, Eotaxin, IP-10, sTNFRI, sTNFRII, IL-6, and VEGF as a determination marker.

[0077] For a tocilizumab therapy switch patient, it is preferable to use at least one type selected from the group consisting of sgp130, IL-1β, IL-2, IL-5, IL-15, GM-CSF, IFN-γ, TNF-α, and IP-10 as a determination marker. It is more preferable to use a combination of sgp130, IP-10, and GM-CSF as a determination marker.

[0078] For an etanercept therapy naïve patient, it is preferable to use at least one type selected from the group consisting of IL-9, IL-6, IL-13, TNF-α, and VEGF as a determination marker. It is more preferable to use a combination of IL-9, TNF-α, and VEGF, or a combination of IL-6 and IL-13 as a determination marker.

<Cases where possibility of remission (whether remission is reached) by therapy is predicted and determined for biological formulation>

[0079] For patients administered with tocilizumab (including both tocilizumab therapy naïve patients and tocilizumab therapy switch patients), it is preferable to use at least one type selected from the group consisting of sgp130, IP-10, sTNFRII, IL-6, IL-7, MCP-1 and IL-1β as a determination marker. It is more preferable to use at least sgp130, and even more preferable to use a combination of (i) sgp130, (ii) IP-10, (iii) sTNFRII, and (iv) IL-6, IL-7, MCP-1 or IL-1β. More specifically, for tocilizumab therapy naïve patients, a combination of (i) sgp130, (ii) IP-10, (iii) sTNFRII, and (iv) IL-6, IL-7, MCP-1 or IL-1β is especially preferable as a determination marker. Further, for tocilizumab therapy switch patients, a combination of (i) sgp130, (ii) IP-10, (iii) sTNFRII, and (iv) IL-6 or IL-1β is especially preferable as a determination marker.

[0080] For an etanercept therapy naïve patient, it is preferable to use at least one type selected from the group consisting of IL-9, TNF-α, VEGF, PDGF-bb, and MIP-1α as a determination marker. It is especially preferable to use a combination of IL-9 and TNF-α, a combination of VEGF and PDGF-bb, or a combination of MIP-1α and PDGF-bb as a determination marker.

[0081] It is known that serum concentration of each of the cytokines, chemokines, and soluble receptors used as a determination marker can be measured by a measurement system utilizing an antigen-antibody reaction such as ELISA. Such measuring kits are commercially available. Thus, the cytokines, chemokines, and soluble receptors can be measured with a known measuring kit by a known method in the determining method of the present invention.

[0082] Prediction and Determination of Therapeutic Effect Due to Biological Formulation

[0083] A therapeutic effect due to a biological formulation can be predicted and determined based on a measured value of the determination marker. For example, the prediction and determination include a method in which the determination marker is measured in advance for patients in full remission and patients who are not in remission from therapy with a biological formulation; a regression equation of a measured value of the determination marker (explanatory variable) and a therapeutic effect of biological formulation (objective variable) are found by regression analysis; and a measured value of a determination marker of a rheumatoid arthritis patient targeted for determination is applied to said regression equation. When finding a regression equation, it is preferable to use a log value of serum concentration (pg/ml) for the determination markers other than sgp130. For sgp130, a value of serum concentration (pg/ml) is preferably used. Further, it is preferable that a regression equation is derived by multiple regression analysis. The objective variable in the above-described regression equation may be appropriately determined based on the therapeutic effect to be predicted and determined.

[0084] For example, when predicting and determining the level of improvement in a symptom after therapy for a biological formulation, the objective variable may be set to “a value obtained by subtracting a value of a disease activity indicator after a predetermined period of therapy from a value of a disease activity indicator prior to therapy” for analysis by multiple linear regression analysis. For example, when predicting and determining a value of a disease activity indicator after therapy for a biological formulation, the objective variable may be set to “a value of disease activity indicator after a predetermined period of therapy” for analysis by multiple linear regression analysis. In this regard, specific examples of a value of a disease activity indicator include a DAS (Disease activity score)-28 value, CDAI (Clinical Disease Activity Index) value, SDAI (Simple Disease Activity Index) value and the like. A DAS-28 value, CDAI value and SDAI value are correlated with one another and reflect a symptom of rheumatoid arthritis. Thus, any of such disease activity indicator values may be used in the determining method of the present invention. Further, a disease activity indicator used in the determining method of the present invention is not limited to those exemplified above. Indicators that may be newly advocated in the future can be used.

[0085] Further, when predicting or determining the possibility of remission due to therapy with a biological formulation (result of whether there is remission or no remission), multiple logistic regression analysis may be used for analysis.

[0086] For regression analysis utilizing a measured value of the determination marker as an explanatory variable, a value of a disease activity indicator prior to therapy (DAS-28 value, CDAI value, SDAI value or the like) or a result of evaluation by a Boolean method may be utilized as an explanatory variable.

[0087] Hereinafter, therapeutic effects to be predicted and determined are separated into a level of improvement in a symptom after therapy, DAS-28 value after therapy, and possibility of remission to disclose specific methods for the determining method of the present invention. However, the determining method of the present invention should not be interpreted to be limited to the following specific methods.

<Prediction and Determination of Level of Improvement in Symptom after Therapy>

[0088] A level of improvement in a symptom after therapy due to a biological formulation can be predicted and determined by multiple linear regression analysis while setting an objective variable as “a value obtained by subtracting a value of a disease activity indicator after a predetermined period of therapy from a value of a disease activity indicator prior to therapy” and an explanatory variable as “a measured value of the determination marker”.

[0089] In Examples described below, the following equations (1) and (2) have been discovered as regression equations for predicting and determining a level of improvement in a symptom after 16 weeks of therapy due to a biological formulation (level of improvement in DAS-28 value; DAS-28 value prior to therapy−DAS-28 value after 16 weeks of therapy), separated by the past dosing history of a rheumatoid arthritis patient and type of biological formulation. A level of improvement in a symptom after 16 weeks of therapy can be predicted and determined by finding an objective variable from applying values to one of the following regression equations (1) and (2) depending on the past dosing history of a rheumatoid arthritis patient subjected to determination and type of biological formulation. A level of improvement in a symptom due to a biological formulation is predicted and determined to be large for the patient for larger values of the objective variable calculated by the following regression equation.

[0090] [Cases where level of improvement in symptom after 16 weeks of therapy (level of improvement in DAS-28 value; DAS-28 value prior to therapy−DAS28-value after 16 weeks of therapy) is predicted and determined for tocilizumab therapy naïve patient]

Determination markers: IL-1β, IL-7, TNF-α, and sIL-6R

Objective function (DAS-28 value prior to therapy−DAS28-value after 16 weeks of therapy)=5.505+(−3.618×A)+(3.255×B)+(1.475×C)+(−1.841×D)  Regression equation (1):

A: log value of serum IL-1β concentration (pg/ml)
B: log value of serum IL-7 concentration (pg/ml)
C: log value of serum TNF-α concentration (pg/ml)
D: log value of serum sIL-6R concentration (pg/ml)
[Cases where level of improvement in symptom after 16 weeks of therapy (level of improvement in DAS-28 value; DAS-28 value prior to therapy−DAS28-value after 16 weeks of therapy) is predicted and determined for etanercept therapy naïve patient]
Determination markers: IL-2, IL-15, sIL-6R, and sTNFRI

Objective function (DAS-28 value prior to therapy−DAS-28 value after 16 weeks of therapy)=7.325+(−1.567×E)+(1.632×F)+(−2.540×D)+(1.973×G)  Regression equation (2):

E: log value of serum IL-2 concentration (pg/ml)
F: log value of serum IL-15 concentration (pg/ml)
D: log value of serum sIL-6R concentration (pg/ml)
G: log value of serum sTNFRII concentration (pg/ml)

[0091] The regression equations (1) and (2) demonstrate an example of a regression equation used to predict and determine a level of improvement in DAS-28 value after 16 weeks of therapy due to a biological formulation. However, a level of improvement in a CDAI value or an SDAI value after 16 weeks of therapy due to a biological formulation (level of improvement in CDAI value or SDAI value; CDAI value or SDAI value prior to therapy−CDAI value or SDAI value after 16 weeks of therapy) can naturally be predicted and determined by multiple linear regression analysis by the same method using a CDAI value or SDAI value. Further, since a therapeutic effect stabilizes and appears after 16 weeks of therapy by a biological formulation, regression equations for predicting and determining a level of improvement in a symptom after 16 weeks of therapy are shown in the above-described regression equations (1) and (2). Naturally, a level of improvement in a symptom before or after 16 weeks of therapy due to the biological formulations can be predicted and determined by multiple linear regression analysis using the same method.

[0092] In Examples described below, the following equations (3)-(7) have been discovered as regression equations for predicting and determining a DAS-28 value of a symptom after 16 weeks of therapy due to a biological formulation, separated by the past dosing history of a rheumatism patient and type of biological formulation. ADAS-28 value after 16 weeks of therapy can be predicted and determined by finding an objective variable from applying values to one of the following regression equations (3)-(7), depending on the past dosing history of a rheumatoid arthritis patient subjected to determination and type of biological formulation. When the objective variable calculated by the following regression equation is 2.3 or less, the patient is predicted and determined to reach remission due to a biological formulation.

[Cases where DAS-28 value after 16 weeks of therapy is predicted and determined for tocilizumab therapy naïve patient]
Determination markers: sgp130, IL-8, Eotaxin, IP-10, sTNFRI, sTNFRII, IL-6, and VEGF

Objective function (DAS-28 value after 16 weeks of therapy)=6.909+(−5.341×H)+(3.940×I)(−1.039×J)+(−1.002×K)+(−2.580×L)+(1.407×G)+(0.744×M)+(−0.850×N)  Regression equation (3):

H: serum sgp130 concentration (pg/ml)
I: log value of serum IL-8 concentration (pg/ml)
J: log value of serum Eotaxin concentration (pg/ml)
K: log value of serum IP-10 concentration (pg/ml)
L: log value of serum sTNFRII concentration (pg/ml)
G: log value of serum sTNFRII concentration (pg/ml)
M: log value of serum IL-6 concentration (pg/ml)
N: log value of serum VEGF concentration (pg/ml)
[Cases where DAS-28 value after 16 weeks of therapy is predicted and determined for tocilizumab therapy naïve patient]
Determination markers: sgp130, IL-8, Eotaxin, IP-10, sTNFRI, sTNFRII, and IL-6

Objective function (DAS-28 value after 16 weeks of therapy)=4.731+(−5.433×H)+(2.551×I)(−0.937×J)+(−1.116×K)+(−2.010×L)+(1.630×G)+(0.577×M)  Regression equation (4):

H: serum sgp130 concentration (pg/ml)
I: log value of serum IL-8 concentration (pg/ml)
J: log value of serum Eotaxin concentration (pg/ml)
K: log value of serum IP-10 concentration (pg/ml)
L: log value of serum sTNFRII concentration (pg/ml)
G: log value of serum sTNFRII concentration (pg/ml)
M: log value of serum IL-6 concentration (pg/ml)
[Cases where DAS-28 value after 16 weeks of therapy is predicted and determined for tocilizumab therapy switch patient]
Determination markers: sgp130, IP-10, and GM-CSF

Objective function (DAS-28 value after 16 weeks of therapy)=2.837+(−6.037×H)+(0.714×K)+(−0.622×O)  Regression equation (5):

H: serum sgp130 concentration (pg/ml)
K: log value of serum IP-10 concentration (pg/ml)
O: log value of serum GM-CSF concentration (pg/ml)
[Cases where DAS-28 value after 16 weeks of therapy is predicted and determined for etanercept therapy naïve patient]
Determination markers: IL-6 and IL-13, DAS-28 value prior to etanercept administration is also used as an explanatory variable

Objective function (DAS-28 value after 16 weeks of therapy)=0.081+(0.522×a)+(−0.969×M)+(1.409×P)  Regression equation (6):

a: DAS-28 value prior to etanercept administration
M: log value of serum IL-6 concentration (pg/ml)
P: log value of serum IL-13 concentration (pg/ml)
[Cases where DAS-28 value after 16 weeks of therapy is predicted and determined for etanercept therapy naïve patient]
Determination markers: IL-9, TNF-α and VEGF

Objective function (DAS-28 value after 16 weeks of therapy)=0.703+(0.646×S)+(−0.551×C)+(0.858×N)  Regression equation (7):

S: log value of serum IL-9 concentration (pg/ml)
C: log value of serum TNF-α concentration (pg/ml)
N: log value of serum VEGF concentration (pg/ml)

[0093] Since regression equation (7) does not use a DAS-28 value prior to etanercept administration as an explanatory variable, a DAS-28 value after 16 weeks of therapy can be predicted while eliminating a subjective opinion of a physician. Thus, regression equation (7) is considered preferable over regression equation (6).

[0094] The regression equations (3)-(7) show examples of a regression equation used to predict and determine a DAS-28 value after 16 weeks of therapy due to a biological formulation. However, a CDAI value or SDAI value itself after 16 weeks of therapy due to a biological formulation can naturally be predicted and determined by multiple linear regression analysis by the same method using a CDAI value or SDAI value. Further, as discussed above, since a therapeutic effect stabilizes and appears after 16 weeks of therapy due to a biological formulation, regression equations for predicting and determining a value of a disease activity indicator after 16 weeks of therapy are shown in the above-described regression equations (3)-(7). However, a value of disease activity indicator prior to or after 16 weeks of therapy due to the biological formulations can naturally be predicted and determined by multiple linear regression analysis using the same method.

[0095] In Examples described below, the following equations (8)-(16) have been discovered as regression equations for predicting and determining the possibility of remission (either remission or not in remission) after 16 weeks of therapy due to a biological formulation, separated by the past dosing history of a rheumatoid arthritis patient and type of biological formulation. It is possible to predict and determine whether remission is reached after 16 weeks of therapy by finding the probability (p) of remission after 16 weeks of therapy from applying values to one of the following regression equations (8)-(16) depending on the past dosing history of a rheumatoid arthritis patient subjected to determination and type of biological formulation. The probability of remission estimated from the following regression equations (8)-(16) refers to the probability of a DAS-28 value being 2.3 or less. A p value computed from regression equations (8)-(16) closer to 1 indicates a higher possibility of remission after 16 weeks of therapy. For example, the p value of 0.5 or higher can predict and determine remission and less than 0.5 can predict and determine no remission for convenience's sake. In this regard, a DAS-28 value of 2.3 is used as the boundary between remission and non-remission to enhance the precision of prediction and determination of remission because a CRP value tends to decrease and DAS-28 value may decreases regardless of inflammation by inhibiting IL-6. The value is set at a lower value of DAS-28 value (2.6), which is generally considered the boundary between remission and non-remission.

[Cases where possibility of remission is predicted and determined for tocilizumab therapy naïve patient]
Determination markers: sgp130, IP-10, sTNFRII, and IL-6

p/(1−p)=exp{(−5.095)+(−36.648×H)+(−4.004×K)+(5.632×G)+(1.658×M)}  Regression equation (8):

p: probability of remission after 16 weeks of therapy
H: serum sgp130 concentration (pg/ml)
K: log value of serum IP-10 concentration (pg/ml)
G: log value of serum sTNFRII concentration (pg/ml)
M: log value of serum IL-6 concentration (pg/ml)
[Cases where possibility of remission is predicted and determined for tocilizumab therapy naïve patient]
Determination markers: sgp130, IP-10, sTNFRII, and IL-7

p/(1-p)=exp{(−3.467)+(−42.849×H)+(−4.430×K)+(5.736×G)+(2.705×B)}  Regression equation (9):

p: probability of remission after 16 weeks of therapy
H: serum sgp130 concentration (pg/ml)
K: log value of serum IP-10 concentration (pg/ml)
G: log value of serum sTNFRII concentration (pg/ml)
M: log value of serum IL-7 concentration (pg/ml)
[Cases where possibility of remission is predicted and determined for tocilizumab therapy naïve patient]
Determination markers: sgp130, IP-10, sTNFRII, and MCP-1

p/(1-p)=exp{(−2.834)+(−38.721×H)+(−4.664×K)+(5.369×G)+(2.502×B)}  Regression equation (10):

p: probability of remission after 16 weeks of therapy
H: serum sgp130 concentration (pg/ml)
K: log value of serum IP-10 concentration (pg/ml)
G: log value of serum sTNFRII concentration (pg/ml)
P: log value of serum MCP-1 concentration (pg/ml)
[Cases where possibility of remission is predicted and determined for tocilizumab therapy naïve patient]
Determination markers: sgp130, IP-10, sTNFRII, and IL-1β

p/(1-p)=exp{(−1.269)+(−39.538×H)+(−3.807×K)+(5.086×G)+(1.647×A)}  Regression equation (11):

p: probability of remission after 16 weeks of therapy
H: serum sgp130 concentration (pg/ml)
K: log value of serum IP-10 concentration (pg/ml)
G: log value of serum sTNFRII concentration (pg/ml)
A: log value of serum IL-1β concentration (pg/ml)
[Cases where possibility of remission is predicted and determined for tocilizumab therapy switch patient]
Determination markers: sgp130, IP-10, sTNFRII, and IL-6

p/(1-p)=exp{(−10.935)+(−29.051×H)+(4.466×K)+(2.067×G)+(−2.757×M)}  Regression equation (12):

p: probability of remission after 16 weeks of therapy
H: serum sgp130 concentration (pg/ml)
K: log value of serum IP-10 concentration (pg/ml)
G: log value of serum sTNFRII concentration (pg/ml)
M: log value of serum IL-6 concentration (pg/ml)
[Cases where possibility of remission is predicted and determined for tocilizumab therapy switch patient]
Determination markers: sgp130, IP-10, sTNFRII, and IL-1β

p/(1-p)=exp{(−9.671)+(−27.150×H)+(3.205×K)+(1.914×G)+(−2.540×A)}  Regression equation (13):

p: probability of remission after 16 weeks of therapy
H: serum sgp130 concentration (pg/ml)
K: log value of serum IP-10 concentration (pg/ml)
G: log value of serum sTNFRII concentration (pg/ml)
A: log value of serum IL-1β concentration (pg/ml)
[Cases where possibility of remission is predicted and determined for etanercept therapy naïve patient]
Determination markers: VEGF and PDGF-bb, DAS-28 value prior to etanercept administration is also used as an explanatory variable.

p/(1-p)=exp{(−19.058)+(1.390×a)+(−2.763×E)+(4.962×Q)  Regression equation (14):

p: probability of remission after 16 weeks of therapy
a: DAS-28 value prior to etanercept administration
E: log value of serum VEGF concentration (pg/ml)
Q: log value of serum PDGF-bb concentration (pg/ml)
[Cases where possibility of remission is predicted and determined for etanercept therapy naïve patient]
Determination markers: MIP-1α and PDGF-bb, DAS-28 value prior to etanercept administration is also used as an explanatory variable.

p/(1-p)=exp{(−18.491)+(1.107×a)+(−1.808×R)+(3.930×Q)}  Regression equation (15):

p: probability of remission after 16 weeks of therapy
a: DAS-28 value prior to etanercept administration
R: log value of serum MIP-1α concentration (pg/ml)
Q: log value of serum PDGF-bb concentration (pg/ml)
[Cases where possibility of remission is predicted and determined for etanercept therapy naïve patient]
Determination markers: IL-9 and TNF-α

p/(1-p)=exp{(−1.004)+(1.711×S)+(−1.031×C)}  Regression equation (16):

p: probability of remission after 16 weeks of therapy
S: log value of serum IL-9 concentration (pg/ml)
C: log value of serum TNF-α concentration (pg/ml)

[0096] Since the regression equation (16) does not use a DAS-28 value prior to etanercept administration as an explanatory variable, the possibility of remission can be predicted while eliminating a subjective opinion of a physician. Thus, regression equation (16) is considered preferable over regression equations (14) and (15).

[0097] The regression equations (6)-(16) show examples of a regression equation for predicting and determining the possibility of remission after 16 weeks of therapy, with a DAS-28 value after 16 weeks of therapy of 2.3 or lower considered remission and the value over 2.3 as non-remission. However, the possibility of remission after 16 weeks of therapy due to a biological formulation can naturally be predicted and determined by multiple logistic regression analysis with the same method using a CDAI value or SDAI value. Further, as discussed above, since a therapeutic effect stabilizes and appears after 16 weeks of therapy by a biological formulation, regression equations for predicting and determining the possibility of remission after 16 weeks of therapy are shown in the above-described regression equations (6)-(16). However, the possibility of remission prior to or after 16 weeks of therapy due to a biological formulation can be predicted and determined by multiple logistic regression analysis using the same method.

[0098] Selection of Biological Formulation to be Administered

[0099] The determining method of the present invention can predict the therapeutic effectiveness of a biological formulation prior to the administration thereof. Thus, the method can be utilized in selecting the optimal biological formulation that should be administered prior to starting therapy.

[0100] For example, for a level of improvement in a symptom after therapy of a naïve patient, cases in which tocilizumab is administered and cases in which etanercept is administered are each predicted by the aforementioned method and a biological formulation with a higher level of improvement is selected, so that an optimal biological formulation can be administered to the patient. Specifically, a level of improvement in a symptom after therapy with tocilizumab therapy, which is predicted by using regression equation (1) is compared to a level of improvement in a symptom after therapy with etanercept therapy, which is predicted by using regression equation (2), so that the biological formulation with a higher level of improvement can be selected as the optimal biological formulation.

[0101] For example, for a DAS-28 value after 16 weeks of therapy of a naïve patient, cases in which tocilizumab is administered and cases in which etanercept is administered are each predicted by the aforementioned method and a biological formulation with a lower DAS-28 value after 16 weeks of therapy is selected so that an optimal biological formulation can be administered to the patient. Specifically, a DAS-28 value after 16 weeks of therapy with tocilizumab therapy, which is predicted by using one of regression equations (3)-(5) is compared to a DAS-28 value after 16 weeks of therapy with etanercept therapy, which is predicted by using regression equation (6) or (7), so that the biological formulation with a smaller DAS-28 value can be selected as the optimal biological formulation.

[0102] For example, for the possibility of remission of a naïve patient, cases in which tocilizumab is administered and cases in which etanercept is administered are each predicted by the aforementioned method to select a biological formulation with a higher possibility of remission, so that the optimal biological formulation can be administered to the patient. Specifically, the possibility of remission with tocilizumab therapy, which is predicted by using one of regression equations (8)-(11), is compared to the possibility of remission, which is predicted by using one of regression equations (14)-(16), so that the biological formulation with a higher possibility of remission can be selected as the optimal biological formulation.

2. Diagnostic Agent

[0103] The present invention further provides a diagnostic agent for carrying out the above-described detection method. Specifically, the diagnostic agent of the present invention is a diagnostic agent for determining the effectiveness of therapy due to a biological formulation targeting an inflammatory cytokine for a rheumatoid arthritis patient, characterized by comprising a reagent capable of detecting at least one type of determination marker selected from the group consisting of sgp130, IP-10, sTNFRI, sTNFRII, GM-CSF, IL-1β, IL-2, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, Eotaxin, VEGF, MCP-1, TNF-α, IFN-γ, FGFbasic, PDGF-bb, sIL-6R, and MIP-1α.

[0104] The determination marker can be measured by a measurement system utilizing an antigen-antibody reaction such as ELISA. Specific examples of reagents capable of detecting the determination marker include antibodies that can specifically bind to the determination marker and fragments thereof. Further, antibodies that can specifically bind to the determination marker may be bound on a suitable support to be provided as an antibody array.

[0105] Furthermore, the diagnostic agent of the present invention may comprise a reagent (secondary antibody, color producing substance or the like) required for detecting the determination marker by an antigen-antibody reaction.

EXAMPLES

[0106] Hereinafter, the present invention is disclosed in detail while using Examples. However, the present invention is not limited thereby.

1. Patient and Experimental Method

(Patient)

[0107] Hereinafter, a rheumatism patient who has not received anti-cytokine therapy (administration of infliximab, etanercept, adalimumab, tocilizumab or the like) in the past is referred to as a naïve patient, and a rheumatism patient who has received anti-cytokine therapy in the past is referred to as a switch patient.

[0108] 155 rheumatoid arthritis patients, to whom methotrexate therapy was ineffective, were registered at the Higashihiroshima Memorial Hospital from March 2008 to June 2013. Among the 155 patients, 98 patients received therapy with tocilizumab and the remaining 57 patients received therapy with etanercept. Among the 98 patients who received therapy with tocilizumab, 58 patients were naïve patients who had not previously received anti-cytokine therapy and 40 patients were switch patients who had previously received anti-cytokine therapy 1-3 times. Among 57 patients who received etanercept therapy, 49 patients were naïve patients who had not previously received anti-cytokine therapy, and the remaining 8 patients were switch patients who had previously received anti-cytokine therapy. Informed consent was obtained prior to receiving a blood sample supply from all patients. Further, the tests were conducted with permission prior to the study from the ethics committee of the Higashihiroshima Memorial Hospital.

[0109] Table 1 shows the clinical baseline individual group statistics for group of individuals and clinical diagnosis. Further, FIG. 1 shows a trial profile of patients receiving therapy with tocilizumab and patients receiving therapy with etanercept. FIGS. 2-1 to 2-4 show serum concentration of cytokine/chemokine/soluble receptor prior to therapy. 9 naïve patients (8 patients who suffered from side effects or other diseases and 1 patient for whom data could not be obtained for the entire 16 weeks) among patients treated with tocilizumab were eliminated. Further, 1 switch patient suffering from a side effect (patient for whom data could not be obtained for the entire 16 weeks) among patients treated with tocilizumab was eliminated. There was hardly any difference in DAS-28 value, CRP, swollen joint count, tender joint count, Stage, and Class among the groups (Table 1). Further, the duration of disease was shorter for patients treated with etanercept in comparison to patients treated with tocilizumab (Table 1).

[0110] In order to create a baseline concentration of cytokines, serum was collected from healthy individuals (56 individual; 20 males and 36 females) without a history of suffering from hepatitis C or cancer. The healthy individuals underwent medical examination by the Louis Pasteur Center for Medical Research or the Higashihiroshima Memorial Hospital and informed consent was received in writing from the healthy individuals. The baseline concentration was used to find a distribution pattern of cytokines/chemokines/soluble receptors.

[0111] (Experimental Method)

[0112] Prior to therapy, concentrations of cytokines, chemokines, and soluble receptors in the serum of rheumatoid arthritis patients were measured.

[0113] FIG. 1 shows clinical results for naïve patients and switch patients administered with 8 mg/kg tocilizumab or 50 mg/kg etanercept once every 4 weeks. After 16 weeks of therapy (after 4 administrations), a therapeutic effect was determined based on DAS-28-CRP values and whether the patient is in remission or non-remission. Results for non-remission were further classified into low, medium and high based on DAS-28-CRP values of the patients. DAS-28-ESR values are extensively used to determine the symptom of rheumatoid arthritis patients. However, it is reported that DAS-28-CRP values are almost interchangeable with DAS-28-ESR values and the same results are derived therefrom (Ann Rheum Dis. 2007, March 407-409 Comparison of Disease Activity Score (DAS)28-erythrocyte Sedimentation rate and DAS-C-reactive protein threshold votes. Inoue E, Yamanaka H, et al.)

[0114] In the present tests, DAS-28-CRP values were used to determine the symptoms of rheumatoid arthritis patients. Remission was classified as DAS-28-CRP value<2.3 and non-remission was classified as DAS-28-CRP value≤2.3. Furthermore, DAS-28-CRP classification system developed by Inoue et al was used to classify non-remission patients as low (DAS-28-CRP value=2.3-2.6), medium (DAS-28-CRP value=2.7-4.1) and high (DAS-28-CRP value>4.1) depending of the severity of the symptoms. To obtain consistent determination of symptoms, the same physician at the Higashihiroshima Memorial Hospital determined the final symptoms of all patients. Further, FIG. 3 shows detailed clinical results of each patient shown in FIG. 1, i.e., results of determining DAS-28-CRP values prior to therapy and after 16 weeks of therapy for naïve patients who received tocilizumab therapy, switch patients who received tocilizumab therapy, and naïve patients who received etanercept therapy. Hereinafter, DAS-28-CRP values may be denoted simply as DAS-28 values.

[0115] (Analysis of cytokine/chemokine/soluble receptor) For all measurements of cytokines, a multiplex cytokine array system (Bio-Plex 200, Bio-Rad Laboratories) was used in accordance with the product protocol thereof. 1600 g of serum for all patients and healthy individuals were collected by 10 minutes of centrifugation. All serum samples were stored at −80° C. Bio-Plex Human Cytokine 27-Plex Panel is configured such that 27 types of cytokines (IL-1β, IL-1RA, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p70), IL-13, IL-15, IL-17, basic FGF, eotaxin, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, MIP-1B, PDGF-bb, RANTES, TNF-α, and VEGF) can be analyzed. In addition thereto, sIL-6R, sgp130, sTNF-RI and sTNF-RII were also analyzed (Milliplex®MAP, Human Soluble Cytokine Receptor Panel: Millipore Co. MA). In the present test, concentrations of cytokines, chemokines, and soluble receptors of 56 healthy individuals were simultaneously measured to find the distribution patterns thereof. Bio-Plex Manager software version 5.0 was used to conduct data collection and analysis.

(Statistical Analysis)

[0116] The distribution of cytokine/chemokine values in healthy individuals was analyzed. The log values of the values of the concentration (pg/ml) of cytokines, chemokines, and soluble receptors, other than sgp130, were used for the analysis. The values of concentration (μg/ml) were directly used for sgp130.

[0117] First, simple linear regression analysis and multiple linear regression analysis were performed to investigate the association between cytokine/chemokine/soluble receptor concentration or clinical test values and values obtained by subtracting DAS-28 values after 16 weeks from DAS-28 values of patients at 0 weeks. Next, DAS-28 values after 16 weeks were estimated from a value computed from regression introduced with the clinical test values. Furthermore, simple logistic regression analysis and multiple logistic regression analysis were preformed to analyze the relationship between serum cytokine concentration and remission or non-remission. The resulting parameter p value of <0.05 indicates the presence of a significant difference. All statistical analysis was conducted by using the JMP 9.0 software.

2. Results

(Clinical Evaluation)

[0118] Tables 1 and 2 show clinical baseline individual group statistics, clinical diagnosis and cytokine/chemokine/soluble receptor characteristics. FIGS. 2-1 to 2-4 shows clinical baseline individual group statistics for healthy individuals and rheumatoid arthritis patients with respect to serum concentrations of cytokines/chemokines/soluble receptors prior to therapy. In Table 1, Stage and Class are results of determination with respect to functional classification criteria for rheumatoid arthritis based on Steinbrocker (1949) classification (I-IV; Steinbrocker O et al: Therapeutic criteria in rheumatoid arthritis. JAMA 140:659, 1949) and Hochberg (1992) classification (I-IV; Hochberg M C et al, The American College of Rheumatology 1991 revised criteria for the Classification of global functional status in rheumatoid arthritis. Arthritis and Rheumatism, 35:498-502, 1992), respectively. The results indicate that there is no clinically significant difference among the three rheumatism patient groups and the serum concentrations of cytokines/chemokines/soluble receptors in most rheumatism patients are significantly higher in comparison to healthy individuals.

TABLE-US-00001 TABLE 1 Basic information data for patients Na ve patients who received  therapy Switch patients who received  therapy Na ve patients who received  therapy Clinical parameters  ±  ±  ±  ±  ±  ± WBC  ±  ±  ± Fe  ±  ±  ± Ferritin  ±  ±  ± RBC  ±  ±  ± Hb  ±  ±  ± Ht  ±  ±  ±  ±  ±  ± CRP  ±  ±  ± DAS28-CRP  ±  ±  ± RF  ±  ±  ± VAS  ±  ±  ± Swollen joint count  ±  ±  ± Tender joint count  ±  ±  ± Stage  ±  ±  ± Class  ±  ±  ± WBC White blood cell count (×10.sup.3/μl) Fe Serum iron (μg/dl) Ferritin Ferritin (ng/dl) CLIA method RBC Red blood cell count (×10.sup.6/μl) Hb Hemoglobin value (g/dl) Ht Hematocrit value (%) Plt Platelet count (×10.sup.3/μl) CRP C-reactive protein (mg/dl) DAS28-CRP Disease activity score obtained by .fwdarw. DAS is an evaluation method recommended by EULAR (The changing a variable for the erythrocyte European League Against Rheumatism). Absolute values of sedimentation rate to a variable for CRP disease activity are calculated. RF Rheumatoid factor concentration (IU/ml) DAS28 assessment is narrowed down to 28 joints. VAS Level of pain with 100 mm as the maximum pain DAS28 is calculated with the formula* by measuring the following 4 items experienced up to date (mm) (1) Tender joints Stage Functional classification criteria for rheumatoid arthritis (2) Swollen joints (mainly level of radiological progression) (I~IV) (3) Patient global health condition (in terms of VAS) Class Functional classification criteria for rheumatoid arthritis (4) CRP or ESR (mainly level of difficulty in terms of daily living) (I~IV) Formula* DAS28 = 0.56 ×  T28 +  s 28 + 0.7 × in(CRP) + 0.014 × G H <References> VanDer Heijde DMFM et al. Ann Rheum Dis 49, 916-920, 1990 Van Der Heijde DMFM et al. Ann Rheum Dis 51, 177-181, 1992 indicates data missing or illegible when filed

[0119] 49 naïve patients received 16 weeks of tocilizumab therapy. 56% of the patients thereof (27 patients) exhibited remission and the rest of the 21 patients exhibited non-remission (FIG. 1). Furthermore, 39 switch patients received 16 weeks of tocilizumab therapy, of which 9 patients exhibited remission and the rest of the 30 patients exhibited non-remission.

[0120] Further, 49 naïve patients received 16 weeks of etanercept therapy. 18 patients thereamong exhibited remission and the remaining 31 patients exhibited non-remission (FIG. 1). In the present test, the number of switch patients was extremely low. Thus, only naïve patients were used in the analysis for etanercept therapy.

[0121] FIG. 4 shows the relationship between a DAS-28 value prior to therapy and a value obtained from subtracting a DAS-28 value after 16 weeks from the DAS-28 value prior to therapy (PreDAS-28score−16W DAS-28 score) in a naïve patient who has received tocilizumab therapy (PreDAS-28 score). According to FIG. 4, improvement in DAS-28 values was observed after tocilizumab therapy in most naïve patients.

(Search for Biomarkers Based on DAS-28 Value after 16 Weeks of Therapy by Using Serum Concentration of Cytokines/Chemokines/Soluble Receptors in Rheumatism Patient Prior to Therapy)

[0122] According to the current results, about 55% of naïve patients and about 23% of switch patients are expected to exhibit remission after tocilizumab therapy. Although the final symptoms are not identical, some improvement in the symptom is observed in about 45% of naïve patients and about 77% of switch patients. Further, about 36.7% of naïve patients are expected to exhibit remission after etanercept therapy. In addition, some improvement in the symptom is observed after etanercept therapy in about 60% of the remaining patients.

[0123] It has been reported by Pers Y. M. et al (Rheumatology (2013) doi: 10.1093/rheumatology/ket301 First published online: Sep. 19, 2013) that when DAS-28 values after 12-24 weeks of tocilizumab therapy were assessed from general medical examination, 40% of patients exhibited remission, and the main prediction markers were young patients, high CRP value, and patients without a cardiovascular disorder. Further, Koike T (J. Rheumatology, November 2013) et al reported 47.6% remission with respect to DAS28-ESR after 28 weeks of tocilizumab therapy. Meanwhile for etanercept, it is reported by Markenson J A et al (J. Rheumatology, July 2011, p. 1273-81) in the RADIUS study and Curtis J R et al (Ann RheumDis. 2012. 71. 206-212) in the TEMPO study that patients achieving a low disease activity of DAS-28 value≤3.2 after 52 weeks and remission were 53%, and 63% when methotrexate is added. Further, Koike T et al (J. Rheumatology, October 2013, p. 1658-1668) have demonstrated that therapy of etanercept adding with methotrexate, when assessing DAS-28 values after 24 weeks, was more effective than therapy with etanercept alone or therapy adding an anti-rheumatism agent (DMARD) other than methotrexate. Furthermore, it is reported by Cannon G W et al (Clin Exp Rheumatol. November 2013) that 35% reached remission in a 3 year observation from TEMPO and RADIUS studies. Furthermore, Cannon G W et al demonstrated that patients with low disease activity are more likely exhibit remission. However, these reports do not reveal a marker for predicting and determining a therapeutic effect due to a biological formulation.

[0124] In this regard, serum concentrations of cytokines/chemokines were used to investigate whether it is possible to estimate the level of improvement in rheumatoid arthritis based on DAS-28 values after 16 weeks of therapy.

[0125] FIGS. 2-1 to 2-4 show comparisons of baseline individual group statistics of cytokines/chemokines/soluble receptors prior to therapy for healthy individuals and three groups (naïve patients who received tocilizumab therapy, switch patients who received tocilizumab therapy, naïve patients who received etanercept therapy). As can be seen from FIGS. 2-1 to 2-4, serum concentration other than those for sgp130, sIL-6R and sTNFRI in rheumatoid arthritis patients were significantly higher in comparison to healthy individuals. Further, serum concentrations of cytokines/chemokines were lower in naïve patients who received etanercept therapy in comparison to naïve patients who received tocilizumab therapy. Further, it was revealed from this result that naïve patients who received tocilizumab therapy have a higher CRP value prior to therapy in comparison to naïve patients who received etanercept therapy.

[0126] Simple linear regression analysis was performed to find the cytokine/chemokine involved in the level of improvement in DAS-28 values. The level of improvement in a DAS-28 value (DAS-28 value prior to therapy−DAS-28 value after 16 weeks of therapy) was used as an objective variable and serum concentration of cytokines/chemokines/soluble receptors were used directly, or by converting into a log value, as an independent variable. The results are shown in Table 2. As shown in Table 2, log IL-7, log IL-8, log IL-12, log IL-13, log IP-10 and log VEGF exhibiting p<0.05 significantly matched the level of improvement in DAS-28 values in naïve patients who received tocilizumab therapy. Further, for switch patients who received tocilizumab therapy, log IL-1β, log IL-5, log IL-6, log IL-7, log IL-10, log IL-12, log IL-13, log IL-15, log FGF, log GM-CSF, log IFN-γ, log TNF-α and log VEGF significantly matched the level of improvement in DAS-28 values. Meanwhile, log IL-6 and log IP-10 significantly matched the level of improvement in DAS-28 values for naïve patients who received etanercept therapy.

TABLE-US-00002 TABLE 2 Simple linear regression analysis Level of improvement in DAS-28 Objective variable: DAS-28 improvement (=0 week DAS-28 value-16 week DAS-28 value) Naïve patients who received Switch patients who received Naïve patients who received tocilizumab therapy tocilizumab therapy etanercept therapy Cytokine/Chemokine Estimates p value Estimates p value Estimates p value logHu IL-1b 0.211 0.513 0.944 0.006 0.290 0.321 logHu IL-1ra 0.298 0.189 0.386 0.142 0.240 0.341 logHu IL-2 0.294 0.213 0.522 0.278 0.240 0.246 logHu IL-4 0.836 0.158 0.789 0.238 0.453 0.344 logHu IL-5 0.534 0.133 1.337 0.003 0.198 0.598 logHu IL-6 0.067 0.701 0.018 0.040 logHu IL-7 0.890 0.035 1.204 0.207 0.578 logHu IL-8 1.603 0.043 0.439 0.743 0.230 logHu IL-9 0.136 0.169 0.182 0.460 logHu IL-10 0.011 0.054 0.865 logHu IL-12 0.010 0.008 0.004 0.990 logHu IL-13 0.036 0.930 −0.023 0.958 logHu IL-15 0.276 0.099 0.433 0.010 0.306 0.073 logHu IL-17 0.453 0.431 −0.174 0.873 logHu Eotoxin 0.574 0.084 0.763 0.570 0.122 logHu FGF basic 0.333 0.396 0.978 0.045 0.276 0.589 logHu G-CSF 0.290 0.576 1.331 0.084 0.347 0.479 logHu GM-CSF 0.143 0.573 0.002 0.675 logHu IFN- 0.297 0.397 1.069 0.005 0.289 0.381 logHu IP-10 1.119 0.008 0.582 0.049 logHu MCP-1 0.610 0.543 0.208 0.659 0.103 logHu MIP-1a 0.862 0.057 0.751 0.099 0.451 0.282 logHu PDGF-bb 0.104 0.650 −0.845 logHu MIP-1b 0.108 0.124 0.842 0.461 0.258 logHu RANTES 0.144 0.297 0.519 −0.826 0.348 logHu TNF-a 0.364 0.187 0.810 0.010 0.398 0.099 logHu VEGF 0.007 0.899 0.028 0.208 0.628  130 0.000 0.216 0.000 0.382 0.000 logHU −0.881 0.292 0.783 0.370 −0.788 0.332 logHU TNFRI −0.360 0.617 0.438 0.131 0.828 logHU TNFRB −0.895 0.312 −0.111 −0.083 CRP 0.081 0.025 0.064 0.265 0.014 0.841 DAS28-CRP 0.893 <0.0001 0.741 <0.0001 0.597 <0.0001 MMP 0.001 0.384 0.002 0.058 −0.001 0.473 RF 0.001 0.004 0.039 0.001 0.220 VAS 0.029 <0.0001 0.024 0.025 <0.0001 Swollen joint count 0.062 0.002 0.133 0.026 0.102 0.008 Tender joint count 0.106 <0.0001 0.009 0.121 0.000 indicates data missing or illegible when filed

[0127] Multiple linear regression analysis was performed to find the correlation between the level of improvement in DAS-28 value and cytokine/chemokine/soluble receptor concentration. As a result, it was found by phased multiple regression analysis that a combination of log IL-1β, log IL-7, log TNF-α and logs IL-6R is significantly correlated with the level of improvement in DAS-28 values in naïve patients who received tocilizumab therapy (Table 3).

[0128] Meanwhile, a combination of log IL-2, log IL-15, log IL-6R, and log TNFRI was found to have significant correlation with the level of improvement in DAS-28 values in naïve patients who received etanercept therapy (Table 4).

TABLE-US-00003 TABLE 3 Multiple linear regression analysis on naïve patients who received tocilizumab therapy Level of improvement in DAS-28 Objective variable: DAS-28 improvement (=0 week DAS-28 value-16 week DAS-28 value) Naïve patients who received tocilizumab therapy Multiple regression analysis (Objective value = 0 w-16 wDAS28) R{circumflex over ( )}2 0.376 ANOVA(Analysis of variance) p = 0.0004 Cytokine/Chemokine/ soluble receptor Estimate p value intercept 5.505 0.1216 logHu IL-1b −3.618 0.0002 logHu IL-7 3.255 0.0002 logHu TNF-a 1.475 0.0221 logHu-sIL-6R −1.814 0.0264

TABLE-US-00004 TABLE 4 Naïve patients who received etanercept therapy Multiple linear regression analysis Level of improvement in DAS-28 Objective variable: DAS-28 improvement (=0 week DAS-28 value-16 week DAS-28 value) Naïve patients who received etanercept therapy Multiple regression analysis (Objective value = 0 w-16 wDAS28) R{circumflex over ( )}2 0.343 ANOVA(Analysis of variance) p = 0.0037 Cytokine/Chemokine/ soluble receptor Estimate p value intercept 7.325 0.0231 logHu IL-2 −1.567 0.0058 logHu IL-15 1.632 0.0008 logHusIL-6R −2.540 0.0130 logHu-sTNFRI 1.973 0.0115

[0129] Simple linear regression analysis was performed to find the cytokine/chemokine/soluble receptor involved in the final assessment of a DAS-28 value after 16 weeks of therapy (16wDAS28). The DAS-28 value after 16 weeks of therapy was used as an objective variable and serum concentration of cytokines/chemokines/soluble receptors were used directly, or by converting into a log value, as an independent variable. As shown in Table 5, sgp130 exhibiting p<0.05 significantly matched DAS-28 values after 16 weeks of therapy in naïve patients who received tocilizumab therapy. Further, for switch patients who received tocilizumab therapy, log IL-1β, log IL-2, log IL-5, log IL-15, log GM-CSF, log IFN-γ, log TNF-α and sgp130 significantly matched DAS-28 values after 16 weeks of therapy. Meanwhile, log IL-9 significantly matched DAS-28 values after 16 weeks of therapy for naïve patients who received etanercept therapy.

TABLE-US-00005 TABLE 5 Simple linear regression analysis 16-week DAS-28 Objective variable: 16-week DAS-28 Simple linear regression analysis of cytokine/chemokine/soluble receptor based on DAS-28 16 w Simple linear regression analysis were performed to find the parameters related to 16 wDAS-28 (=16 wDAS28). Naïve Tocilizumab Switch Tocilizumab Naïve Etanercept Therapy Therapy Therapy Tocilizumab naïve Tocilizumab switch Etanercept naïve Cytokine/Chemokine Estimates p value Estimates p value Estimates p value logHu IL-1b pg/ml 0.094 0.681 0.035 −0.047 0.860 logHu IL-1ra pg/ml −0.178 0.269 0.041 0.850 0.053 0.817 logHu IL-2 pg/ml −0.078 0.644 −0.482 0.012 0.179 0.341 logHu IL-4 pg/ml 0.335 0.426 0.140 −0.190 0.661 logHu IL-5 pg/ml −0.131 0.606 −0.832 0.025 0.119 0.724 logHu IL-6 pg/ml 0.288 0.156 −0.301 0.216 0.095 0.712 logHu IL-7 pg/ml 0.026 0.933 −0.617 0.119 0.199 0.550 logHu IL-8 pg/ml 0.568 0.319 0.168 0.721 −0.175 logHu IL-9 pg/ml −0.174 0.291 −0.190 0.330 0.545 0.011 logHu IL-10 pg/ml −0.232 0.298 −0.395 0.163 0.351 0.217 logHu IL-12 pg/ml −0.202 0.438 −0.529 0.115 0.413 0.177 logHu IL-13 pg/ml −0.100 0.699 −0.533 0.467 0.236 logHu IL-15 pg/ml −0.053 0.660 −0.325 0.019 0.092 0.557 logHu IL-17 pg/ml −0.578 0.158 −0.619 0.262 −0.578 0.556 logHu Eotaxin pg/ml −0.363 0.124 −0.360 0.291 0.056 0.868 logHu FGF basic pg/ml −0.168 0.546 −0.688 0.085 0.493 0.281 logHu G-CSF pg/ml −0.321 0.380 −0.978 0.120 −0.032 0.943 logHu GM-CSF pg/ml −0.036 0.839 −0.589 0.001 0.191 0.368 logHu IFN-g pg/ml 0.038 0.879 −0.709 0.024 0.960 logHu IP-10 pg/ml −0.048 0.877 0.241 0.568 0.104 0.818 logHu MCP-1 pg/ml 0.144 0.623 −0.113 0.739 0.009 0.981 logHu MIP-1a pg/ml 0.196 0.591 −0.388 0.301 0.051 0.890 logHu PDGF-bb pg/ml 0.097 0.798 −0.165 0.720 0.794 0.301 logHu MIP-1b pg/ml 0.351 0.477 −0.284 0.573 −0.396 0.281 logHu RANTES pg/ml 0.249 0.444 −0.452 0.224 0.382 0.631 logHu TNF-a pg/ml −0.033 0.012 0.035 0.875 logHu VEGF pg/ml 0.400 0.139 −0.042 0.902 0.573 0.132 sgp130 μg/ml −3.785 0.046 −7.801 0.001 −3.005 0.207 logHu-sIL-6R pg/ml −0.866 0.187 −1.246 0.075 −0.754 0.336 logHu-sTNFRI pg/ml −1.028 0.039 0.033 0.955 0.902 logHu-sTNFRII pg/ml −0.179 0.766 0.078 0.728 0.690 0.115 DAS-28 0 w 0.306 0.000 0.259 0.097 0.403 0.003 MMP 0.000 0.645 0.000 0.464 0.002 0.023 RF 0.001 0.378 0.000 0.818 0.000 0.504 VAS 0.003 0.642 0.004 0.560 0.006 0.334 Swollen joint count 0.069 0.000 0.066 0.183 0.081 0.022 Tender joint count 0.067 0.000 0.074 0.042 0.193 Stage 0.092 0.418 0.393 0.162 −0.197 0.232 Class 0.188 0.483 0.130 0.680 0.453 0.096 indicates data missing or illegible when filed

[0130] Multiple linear regression analysis was performed to find the correlation between DAS-28 value after 16 weeks of therapy and cytokine/chemokine/soluble receptor concentration. As a result thereof, it was found by phased multiple regression analysis that a combination of sgp130, log IL-8, logEotaxin, log IP-10, log TNFRI, log TNFRII, log IL-6, and log IL-VEGF is significantly correlated with a DAS-28 value after 16 weeks of therapy in naïve patients who received tocilizumab therapy as shown in Table 6. Further, it was found that there is a very significant correlation even without using log IL-VEGF (Table 7).

[0131] Further, it was found that a combination of sgp130, log IP-10, and log GM-CSF is significantly correlated with a DAS-28 value after 16 weeks of therapy in switch patients who received tocilizumab therapy (Table 8).

[0132] Meanwhile, a combination of DAS-28 value prior to therapy, log IL-6 and log IL-13 was also found to be significantly correlated with the level of improvement in DAS-28 value for naïve patients who received etanercept therapy (Table 9). Further, a combination of log IL-9, log TNF-α, and log VEGF, even without using a DAS-28 value prior to therapy, is significantly correlated with a DAS-28 value after 16 weeks of therapy naïve patients who received etanercept therapy (Table 10).

TABLE-US-00006 TABLE 6 Tocilizumab naïve multiple linear regression analysis Objective variable 16-week DAS-28 Multiple linear regression anlysis of cytokine/chemokine/soluble receptor based on 16 w DAS-28 A. Multiple regression anlysis were performed to find the parameters related to 16 wDAS-28 (=16 wDAS28). Naïve Tocilizumab Therapy Tocilizumab naïve Multiple regression analysis (Objective value = 16 wDAS28) R{circumflex over ( )}2 0.646 ANOVA(Analysis of variance) p < 0.0001 Cytokine/Chemokine/ soluble receptor Estimate p value intercept 6.909 0.001 sgp130# −0.534 0.002 log IL-8 3.940 <.0001 log Eotaxin −1.039 <.0001 log IP-10 −1.002 0.002 log sTNFRI −2.580 <.0001 log sTNFRII 1.407 0.030 log IL-6 0.744 0.002 log VEGF −0.850 0.039 sgp130#: μg/ml others: pg/ml

TABLE-US-00007 TABLE 7 Tocilizumab naïve multiple linear regression analysis Objective variable 16-week DAS-28 Multiple linear regression anlysis of cytokine/chemokine/soluble receptor based on 16 w DAS-28 A. Multiple regression anlysis were performed to find the parameters related to 16 wDAS-28 (=16 wDAS28). Naïve Tocilizumab Therapy Tocilizumab naïve Multiple regression analysis (Objective value = 16 wDAS28) R{circumflex over ( )}2 0.605 ANOVA(Analysis of variance) p < 0.0001 Cytokine/Chemokine/ soluble receptor Estimate p value intercept 4.731 0.0127 sgp130# −0.543 0.003 log IL-8 2.551 <.0001 log Eotaxin −0.937 0.0004 log IP-10 −1.116 0.0007 log sTNFRI −2.010 0.0004 log sTNFRII 1.630 0.0152 log IL-6* 0.577 0.0096 sgp130#: μg/ml others: pg/ml

TABLE-US-00008 TABLE 8 Tocilizumab switch multiple linear regression analysis Objective variable 16-week DAS-28 Multiple linear regression anlysis of cytokine/chemokine/soluble receptor based on 16 w DAS-28 A. Multiple regression anlysis were performed to find the parameters related to 16 wDAS-28 (=16 wDAS28). Tocilizumab switch Multiple regression analysis (Objective value = 16 wDAS28) R{circumflex over ( )}2 0.486 ANOVA(Analysis of variance) p < 0.0001 Cytokine/Chemokine/ soluble receptor Estimate p value intercept 2.837 0.011 sgp130# −0.604 0.003 log IP-10 0.714 0.003 log GM-CSF −0.622 0.0003 sgp130#: μg/ml others: pg/ml

TABLE-US-00009 TABLE 9 Multiple linear regression analysis on naïve patients who received etanercept therapy Multiple linear regression anlysis of cytokine/chemokine/soluble receptor and DAS28-CRP before therapy on 16 week Das-28. Objective variable: 16-week DAS-28 Naïve Etanercept Therapy Multiple regression analysis (Objective value = 16 wDAS28) R{circumflex over ( )}2 0.321 ANOVA(Analysis of variance) p = 0.0016 Cytokine/Chemokine/ soluble receptor estimate p value intercept 0.081 0.907 DAS28-CRP (Prior to therapy) 0.522 0.000 logHu IL-6 −0.969 0.015 log HuIL-13 1.409 0.015

TABLE-US-00010 TABLE 10 Etanercept naïve multiple linear regression analysis Objective variable 16-week DAS-28 Multiple linear regression anlysis of cytokine/chemokine/soluble receptor based on 16 w DAS-28 A. Multiple regression anlysis were performed to find the parameters related to 16 wDAS-28 (=16 wDAS28). Tocilizumab switch Multiple regression analysis (Objective value = 16 wDAS28) R{circumflex over ( )}2 0.264 ANOVA(Analysis of variance) p = 0.0093 Cytokine/Chemokine/ soluble receptor Estimate p value intercept 0.703 0.348 log IL-9 0.646 0.007 log TNF-α −0.551 0.039 log VEGF 0.858 0.053 IL-9, TNF-α, VEGF: pg/ml

[0133] Further, regression equation (4) found based on the multiple linear regression analysis shown in Table 7 was used to find a predicted value of DAS-28 value after 16 weeks of therapy in naïve patients who received tocilizumab therapy. FIG. 5 shows the results of comparing predicted values of DAS-28 values after 16 weeks of therapy calculated by regression equation (4) and actual values of DAS-28 values after 16 weeks of therapy. It was confirmed from the results that DAS-28 values after 16 weeks of therapy estimated from the results of multiple linear regression analysis shown in Table 7 are very consistent with actual values of DAS-28 values after 16 weeks of therapy.

[0134] Further, regression equation (5) found based on the multiple linear regression analysis shown in Table 8 was used to find a predicted value of DAS-28 value after 16 weeks of therapy in switch patients who received tocilizumab therapy. FIG. 6 shows the results of comparing the predicted values of DAS-28 values after 16 weeks of therapy calculated by regression equation (5) and actual values of DAS-28 values after 16 weeks of therapy. It was confirmed from the results that DAS-28 values after 16 weeks of therapy estimated from the results of multiple linear regression analysis shown in Table 8 are very consistent with actual values of DAS-28 values after 16 weeks of therapy.

[0135] Regression equation (7) found based on the multiple linear regression analysis shown in Table 10 was used to find a predicted value of DAS-28 value after 16 weeks of therapy in naïve patients who received etanercept therapy. FIG. 7 shows the results of comparing predicted values of DAS-28 values after 16 weeks of therapy calculated by regression equation (7) and actual values of DAS-28 values after 16 weeks of therapy. It was confirmed from the results that DAS-28 values after 16 weeks of therapy can be estimated to a certain extent from the results of multiple linear regression analysis shown in Table 10.

[0136] Further, a predicted value of DAS-28 after 16 weeks of therapy was found by using the aforementioned regression equation (4) while assuming that naïve patients who received etanercept therapy had received tocilizumab therapy without receiving etanercept therapy. FIG. 8 shows the actual values of DAS-28 after 16 weeks of etanercept therapy and predicted values of DAS-28 values after 16 weeks of therapy while assuming that tocilizumab therapy was received. From this result, naïve patients who received etanercept therapy are classified into patients who are predicted to have a higher therapeutic effect when receiving tocilizumab therapy (FIG. 8 a), patients who are predicted to have barely any difference observed between etanercept therapy and tocilizumab therapy (FIG. 8 b), and patients who are predicted to have a higher therapeutic effect observed when receiving etanercept therapy (FIG. 8 c). For patients shown in FIG. 8 a, tocilizumab therapy is estimated to be more effective than etanercept therapy that was actually received. Thus, it was found that a more effective therapeutic agent can be selected by estimating DAS-28 values due to tocilizumab therapy and etanercept therapy prior to therapy by the present invention.

(Search for biomarkers for predicting and determining the possibility of remission by using serum concentration of cytokines/chemokines/soluble receptors in rheumatism patient prior to therapy)

[0137] In therapy of rheumatoid arthritis, it is desirable that even a partial improvement is observed in the symptom of a patient. However, it is most desirable to reach complete remission. In this regard, in addition to a search for various factors for estimating the final DAS-28 value, a search was conducted for cytokines/chemokines/soluble receptors for predicting whether a patient reaches complete remission.

[0138] Data for cytokine/chemokine/soluble receptor concentrations was analyzed for complete remission and non-remission patient groups by simple logistic regression analysis. Further, Table 10 shows the results of analyzing data for cytokine/chemokine/soluble receptor concentrations for naïve patients and switch patients who received tocilizumab therapy and naïve patients who received etanercept therapy. It was found by simple logistic regression analysis that swollen joint count and tender joint count and DAS-28 values were significantly different between complete remission and non-remission groups. Furthermore, sgp130 was significantly different between complete remission and non-remission groups in naïve and switch patients who received tocilizumab therapy (Table 11). Meanwhile, significant difference in sgp130 was not observed between remission and non-remission groups in naïve patients who received etanercept therapy (Table 11). Further, FIG. 9 shows the results of analyzing the relationship between serum sgp130 concentration and DAS-28 value prior to therapy for remission and non-remission patients. As is clear from FIG. 9, many patients who have reached remission had a high sgp130 concentration.

TABLE-US-00011 TABLE 11 Simple logistic regression analysis Naïve patients who received Switch patients who received Naïve patients who received tocilizumab therapy (n = 48) tocilizumab therapy (n = 40) etanercept therapy (n = 43) Whole Whole Whole Model Model Model Test Test Test Single Single Single logistic Parameter logistic Parameter logistic Parameter analysis, Estimates analysis, Estimates analysis, Estimates Cytokine/Chemokine p value Estimates p value Estimates p value Estimates logHU IL-1b pg/ml 0.378 0.486 0.147 0.577 −0.274 logHu IL-1ra pg/ml 0.087 1.573 0.323 0.478 logHu IL-2 pg/ml 0.858 0.074 −1.009 0.857 0.064 logHu IL-4 pg/ml 0.534 0.420 −1.241 0.965 −0.035 logHu IL-5 pg/ml 0.814 0.143 0.189 −1.276 0.896 −0.083 logHu IL-6 pg/ml 0.184 0.863 0.270 −0.710 0.950 −0.030 logHu IL-7 pg/ml 0.585 0.401 0.233 −1.231 0.660 0.280 logHu IL-8 pg/ml 0.432 1.088 0.864 −0.207 0.751 −0.333 logHu IL-9 pg/ml 0.545 −0.242 0.289 −0.540 0.020 1.075 logHu IL-10 pg/ml −0.281 0.196 0.572 0.310 logHu IL-12 pg/ml 0.773 −0.181 0.113 −1.457 0.833 0.123 logHu IL-13 pg/ml 0.963 0.029 0.432 −0.669 0.671 0.322 logHu IL-15 pg/ml 0.924 0.027 0.173 −0.519 0.942 0.021 logHu IL-17 pg/ml 0.197 −1.332 0.920 0.147 0.691 −0.730 logHu Eotaxin pg/ml 0.447 −0.441 0.512 −0.590 0.639 0.299 logHu FGF basic pg/ml 0.792 −0.177 0.725 −0.371 0.402 0.773 logHu G-CSF pg/ml 0.599 −0.471 0.786 −0.450 0.901 −0.104 logHu GM-CSF pg/ml 0.910 0.104 0.099 −0.930 0.798 −0.102 logHu IFN-g pg/ml 0.536 0.190 −1.081 −0.228 logHu IP-10 pg/ml 0.647 −0.344 0.604 0.557 0.733 logHu MCP-1 pg/ml 0.402 0.593 0.696 −0.366 0.960 0.035 logHu MIP-1a pg/ml 0.426 0.698 0.305 −0.963 0.885 logHu PDGF-bb pg/ml 0.751 0.290 0.456 0.894 0.358 1.357 logHu MIP-1b pg/ml 0.709 0.444 0.508 −0.882 0.161 −0.991 logHu RANTES pg/ml 0.748 0.252 0.166 0.823 −0.335 logHu TNF-a pg/ml 0.787 0.127 0.143 −1.020 0.694 −0.162 logHu VEGF pg/ml 0.400 0.558 0.389 −0.793 0.967 0.030 sgp130 μg/ml −18.182 0.003 −24.159 0.003 0.212 −5.882 logHu-sIL-6R pg/ml 0.118 −2.590 0.023 −5.922 0.679 0.590 logHu-sTNFRI pg/ml 0.302 −1.284 0.643 −0.306 0.566 0.591 logHu-sTNFRII pg/ml 0.719 −0.519 0.064 1.210 0.390 0.775 age 0.139 0.039 0.064 −0.073 0.444 0.019 Duration of disease 0.226 0.041 0.221 −0.059 0.414 0.033 WBC 0.173 0.000 0.434 0.000 0.057 0.000 DAS28-CRP 0.011 0.689 0.165 0.005 VAS 0.328 0.013 0.810 0.005 0.419 0.011 CRP 0.993 0.001 0.939 −0.009 0.019 0.342 RF 0.121 0.002 0.995 0.000 0.015 0.006 Swollen joint count 0.015 0.123 0.193 0.182 0.012 0.218 Tender joint count 0.014 0.123 0.363 0.137 0.046 0.147 Stage 0.237 0.328 0.352 0.651 0.615 −0.158 Class 0.459 0.481 0.806 −0.201 0.403 0.438 indicates data missing or illegible when filed

[0139] A multivariable model was examined as a prediction biomarker for remission and non-remission by phased multiple forward logistic regression analysis based on serum concentration of cytokines/chemokines/soluble receptors in patients prior to administration of tocilizumab. FIGS. 10 and 11 show optimal combinations of prediction biomarkers for remission and non-remission found based on phased multiple forward logistic regression analysis and ROC curves. It was found from the results of analysis that sgp130, log IP-10, logsTNFRII and log IL-6 can be prediction biomarkers for determining with high precision whether remission is reached for naïve patients who received tocilizumab therapy (p=0.0004) (Table 11a). Further, it was found that log IL-7 (p=0.0003), log IL-1β (p=0.0005) or log MCP-1 (p=0.0004), in combination with sgp130, log IP-10, and logsTNFRII, can be a prediction biomarker for determining with high precision whether remission is reached for naïve patients who received anti-IL-6 therapy (tocilizumab therapy) (FIGS. 10b-10d).

[0140] Further, it was found that a combination of sgp130, log IP-10, log sTNFRII and log IL-6 can be a prediction biomarker for determining whether remission is reached for switch patients who received tocilizumab therapy (p=0.002) (FIG. 11a). Furthermore, it was also found that a combination of sgp130, log IP-10, log sTNFRII and log IL-1β can also be a predication biomarker for determining with high precision whether remission is reached (p=0.003) (FIG. 11b).

[0141] Meanwhile, p value was 0.257 for biomarker groups for predicting and determining the possibility of remission found based on the ROC curve and multiple logistic regression analysis obtained in tocilizumab therapy for naïve patients who received etanercept therapy, thus demonstrating that this biomarker group cannot predict whether remission is reached (Table 14). Meanwhile, it was demonstrated that a combination of DAS-28 value prior to therapy (0wDAS-28), log VEGF, and log PDGF-bb can also predict and determine the possibility of remission to a certain extent, as shown in FIG. 12, by another multiple logistic regression analysis. Furthermore, it was found that a combination of log IL-9 and log TNF-α can also predict and determine the possibility of remission to a certain extent without using DAS-28 value (0wDAS-28) for naïve patients who received etanercept therapy as shown in Table 16. That is, it is suggested that the pathology of rheumatoid arthritis patients is diverse, and a biomarker for predicting and determining the possibility of remission is different for patients to whom IL-6 inhibition is effective and patients for whom TNF-α inhibition is effective.

TABLE-US-00012 TABLE 12 Results of multiple logistic regression analysis on naïve patients who received etanercept therapy by using biomarkers for predicting and determining the possibility of remission found based on results of multiple logistic regression analysis obtained from patients who received tocilizumab therapy Whole Model Test p = 0.257 Parameter Estimates Term Estimates p value(Prov > ChiSq) Intercept −6.489 0.179 sgp130 −9.591 0.150 logHu IL-6 −0.422 0.467 logHu IP-10 0.893 0.435 hgHu-sTNFR II 1.789 0.235

TABLE-US-00013 TABLE 13 Etanercept naïve Multiple logistic regression analysis multiple logistic analysis, Objective variable: remission vs non-remission Whole Model Test p = 0.0115 Parameter Estimates Term Estimates p value(Prob > ChiSq) Intercept −1.004 0.337 log IL-9 1.711 0.012 log TNF-α −1.031 0.079