HK40096939A - Pulmonary arterial hypertension patient selection method and biomarker - Google Patents

Description

Selection methods and biomarkers for patients with pulmonary hypertension

Technical Field

This invention relates to a biomarker and patient selection method for predicting the responsiveness of patients with pulmonary hypertension to targeted therapy on the IL-6 signaling pathway.

Background Technology

Pulmonary hypertension is a rare and difficult-to-treat condition designated by the Japanese Ministry of Health, Labour and Welfare, affecting thousands of patients in Japan. It is characterized by elevated pulmonary artery pressure, primarily due to narrowing or obstruction of the pulmonary arteries caused by factors such as constriction, thickening, or thrombosis. As symptoms progress, it can lead to heart failure and even death. Survival time after diagnosis or onset is generally short, indicating a poor prognosis.

Clinically, pulmonary hypertension is classified into four types: 1) idiopathic pulmonary hypertension, 2) hereditary pulmonary hypertension, 3) drug- or toxin-induced pulmonary hypertension, and 4) pulmonary hypertension associated with various diseases (connective tissue diseases, HIV infection, portal hypertension, congenital heart disease, schistosomiasis) (Guidelines for the Treatment of Pulmonary Hypertension (2017 Revised Edition)). However, the detailed mechanisms of pulmonary artery stenosis and obstruction are not fully understood. Current treatment involves a combination of symptomatic therapies, including medications to improve pulmonary artery stenosis and obstruction and to dilate blood vessels, medications to regulate body fluids, oxygen inhalation, and lung transplantation, based on the progression of pulmonary hypertension.

In recent years, the industry has reported the following: Elevated serum concentrations of inflammatory cytokines, including interleukin-6 (IL-6), are associated with worsening of pulmonary hypertension and poor prognosis in patients with pulmonary hypertension (Non-Patent Literature 1); IL-6 receptors are highly expressed in pulmonary artery vascular smooth muscle cells, and significant improvements in pulmonary hypertension have been observed by using anti-IL-6 receptor antibodies to inhibit the action of IL-6 (Non-Patent Literature 2), etc. In other words, reports have documented elevated serum IL-6 levels in pulmonary hypertension, high expression of IL-6 receptors in diseased organs, and the effectiveness of treatments based on IL-6 signal transduction pathway inhibitors for pulmonary hypertension.

Here, the treatment of IL-6 in the patient's blood and/or IL-6 receptors in organs is a direct target based on IL-6 signaling pathway inhibitors such as anti-IL-6 antibodies and anti-IL-6 receptor antibodies. That is, as mentioned above, pulmonary hypertension can be treated by inhibiting the action of IL-6, and the IL-6 level in patients with pulmonary hypertension is considered an indicator of responsiveness to treatment based on IL-6 signaling pathway inhibitors.

IL-6 is a cytokine also known as B cell stimulating factor 2 (BSF2) or interferon β2. It binds to the IL-6 receptor on the cell membrane to form the IL-6/IL-6 receptor complex, which then binds to gp130, thereby transmitting the biological activity signal of IL-6 into the cell (Non-Patent Literature 3).

In recent years, personalized medicine, a strategy aimed at providing the most appropriate treatment for each patient, has been active in various disease areas in the medical field. Among patients with pulmonary hypertension, there is also a desire to appropriately select and apply treatments targeting the IL-6 signaling pathway.

Existing technical documents

[Non-patent literature]

Non-Patent Literature 1: Ectopic upregulation of membrane-bound IL6R drives vascular remodeling in pulmonary arterial hypertension, Yuichi Tamura et al., J. Clin. Invest., Vol. 128, No. 5, May 2018, 1956-1970

Non-patent literature 2: Interleukin-6/interleukin-21signaling axis is critical in the pathogenesis of pulmonary arterial hypertension, Takahiro Hashimoto-Kataoka et al., PNAS, Published online May 4, 2015, E2677-E2686

Non-patent document 3: Interleukin-6triggers the association of its receptorwith a possible signal transducer, gp130, Tetsuya Taga et al., Cell, Vol.58, Issue3, August 11, 1989, 573-581

Summary of the Invention

The inventors have discovered: biomarkers for predicting IL-6 levels; and biomarkers for predicting responsiveness to treatment based on IL-6 signaling pathway inhibitors, and/or for selecting subjects with a high probability of responding to treatment based on IL-6 signaling pathway inhibitors.

The inventors further discovered: a new method for selecting biomarkers; and a method for selecting biomarkers that are used to predict the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor, and/or for selecting biomarkers that are highly likely to be responsive to treatment based on an IL-6 signaling pathway inhibitor.

Based on this insight, the following invention is provided.

[1-1] A novel biomarker selection method, comprising:

(a) Selecting biomarker candidates based on the similarity of vectors obtained using the content of reference biomarkers and the content of biomarker candidate substances in samples taken from the object group; and

(b) Cluster the object group based on the content of the biomarker candidate substances selected in (a).

[1-2] The selection method described in [1-1] further includes: (c) selecting the biomarker candidate selected in (a) as a biomarker candidate if the clustering results in (b) can identify the object with high reliability, in comparison with the clustering results in (b).

[1-3] The selection method as described in [1-2], wherein (c) includes:

(d) The results obtained by identifying objects using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) are compared with the clustering results in (b) to obtain the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) Reliability is achieved based on the sensitivity and specificity obtained in (d); and

(f) Determine whether biomarker candidates can identify objects with high reliability.

[1-4] The selection method described in any of [1-1] to [1-3], wherein the similarity of vectors is cosine similarity.

[1-5] The selection method described in any of [1-1] to [1-4] is based on the selection of biomarker candidates when the similarity of the vectors is about 0.6 or higher.

[1-6] The selection method described in any of [1-1] to [1-5] clusters the object group using the K-medoids method.

[1-7] The selection method described in any of [1-1] to [1-6] clusters the object group into 3 clusters.

[1-8] The selection method described in any of [1-2] to [1-7], wherein reliability is represented by the AUC of the ROC curve obtained based on sensitivity and specificity.

[1-9] The selection method described in [1-8] wherein, when the AUC is approximately 0.8 or higher, the candidate biomarker can be identified with high reliability.

[1-10] The selection method described in any of [1-1] to [1-9] is a computer installation method.

[2-1] A novel biomarker selection device, comprising:

(a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the content of a reference biomarker and the content of biomarker candidate substances in samples taken from the object group; and

(b) Clustering unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a).

[2-2] The selection device described in [2-1] further includes (c) a selection unit that, in comparison with the clustering results in (b), selects the biomarker candidate selected in (a) as a biomarker candidate if the target can be identified with high reliability.

[2-3] The selection device as described in [2-2], wherein the selection unit of (c) includes:

(d) The acquisition unit compares the results obtained by identifying the object using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) with the clustering results in (b), thereby obtaining the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) The acquisition unit, which acquires reliability based on the sensitivity and specificity obtained in (d); and

(f) Judgment unit, which determines whether biomarker candidate substances can identify objects with high reliability.

[2-4] The selection device described in any of [2-1] to [2-3], wherein the similarity of the vectors is cosine similarity.

[2-5] The selection device described in any of [2-1] to [2-4] selects biomarker candidates based on the similarity of vectors being approximately 0.6 or higher.

[2-6] The selection device described in any of [2-1] to [2-5] clusters the object group by the K-medoids method.

[2-7] The selection device described in any of [2-1] to [2-6] clusters the object group into 3 clusters.

[2-8] The selection device described in any of [2-2] to [2-7], wherein reliability is represented by the AUC of the ROC curve obtained based on sensitivity and specificity.

[2-9] The selection device described in [2-8] wherein, when the AUC is about 0.8 or higher, the candidate biomarker can be identified with high reliability.

[3-1] A method for selecting a biomarker for predicting the level of IL-6 in an object, comprising:

(a) Biomarker candidates are selected based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from the subject group; and

(b) Cluster the object group based on the content of the biomarker candidate substances selected in (a).

[3-2] A method for selecting biomarkers to predict the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor, comprising:

(a) Biomarker candidates are selected based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from the subject group; and

(b) Cluster the object group based on the content of the biomarker candidate substances selected in (a).

[3-3] A method for selecting biomarkers for subjects highly likely to respond to treatment based on substances that block the IL-6 signaling pathway, comprising:

(a) Biomarker candidates are selected based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from the subject group; and

(b) Cluster the object group based on the content of the biomarker candidate substances selected in (a).

[3-4] The selection method described in any of [3-1] to [3-3] further includes:

(c) In contrast to the clustering results in (b), the biomarker candidates selected in (a) are selected as biomarker candidates if they can identify objects with high reliability.

[3-5] The selection method as described in [3-4], wherein (c) includes:

(d) The results obtained by identifying objects using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) are compared with the clustering results in (b) to obtain the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) Reliability is achieved based on the sensitivity and specificity obtained in (d); and

(f) Determine whether biomarker candidates can identify objects with high reliability.

[3-6] The selection method described in any of [3-1] to [3-5], wherein the subject suffers from PAH.

[3-7] The selection method described in any of [3-1] to [3-6], wherein the sample is whole blood, plasma or serum.

[3-8] The selection method described in any of [3-1] to [3-7], wherein the similarity of vectors is cosine similarity.

[3-9] The selection method described in any of [3-1] to [3-8] is based on the case where the similarity of the vectors is about 0.6 or higher to select biomarker candidates.

[3-10] The selection method described in any of [3-1] to [3-9] clusters the object group using the K-medoids method.

[3-11] The selection method described in any of [3-1] to [3-10] clusters the object group into 3 clusters.

[3-12] The selection method described in any of [3-4] to [3-11], wherein reliability is represented by the AUC of the ROC curves obtained based on sensitivity and specificity.

[3-13] The selection method described in [3-12] is such that when the AUC is about 0.8 or higher, the candidate biomarker can be identified with high reliability.

[3-14] The selection method described in any of [3-1] to [3-13] is a computer installation method.

[3-15] The selection method described in any of [3-2] to [3-14], wherein the IL-6 signal transduction pathway inhibitor is an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway.

[3-16] The selection method described in [3-15], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[4-1] A biomarker selection device for predicting the content of IL-6 in a subject, comprising:

(a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the content of IL-6 in samples taken from the target group and the content of biomarker candidate substances; and

(b) Clustering unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a).

[4-2] A biomarker selection device for predicting the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor, comprising:

(a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the content of IL-6 in samples taken from the target group and the content of biomarker candidate substances; and

(b) Clustering unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a).

[4-3] A selection device for selecting biomarkers in subjects highly likely to respond to treatment based on substances that block the IL-6 signaling pathway, comprising:

(a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the content of IL-6 in samples taken from the target group and the content of biomarker candidate substances; and

(b) Clustering unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a).

[4-4] The selection device described in any of [4-1] to [4-3] further comprises:

(c) Selection section: In comparison with the clustering results in (b), the biomarker candidate selected in (a) is selected as the biomarker candidate if it can identify the object with high reliability.

[4-5] The selection device as described in [4-4], wherein the selection unit of (c) includes:

(d) The acquisition unit compares the results obtained by identifying an object using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) with the clustering results in (b), thereby obtaining the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) The acquisition unit, based on the sensitivity and specificity acquired in (d), acquires reliability; and

(f) Judgment unit, which determines whether the biomarker candidate can identify the target with high reliability.

[4-6] The selection device described in any of [4-1] to [4-5], wherein the subject suffers from PAH.

[4-7] The selection device as described in any of [4-1] to [4-6], wherein the sample is whole blood, plasma, or serum.

[4-8] The selection device described in any of [4-1] to [4-7], wherein the similarity of the vectors is cosine similarity.

[4-9] The selection device described in any of [4-1] to [4-8] selects biomarker candidates based on the similarity of vectors being approximately 0.6 or higher.

[4-10] The selection device described in any of [4-J] to [4-9] clusters the object group by the K-medoids method.

[4-11] The selection device described in any of [4-1] to [4-10], wherein the object group is clustered into 3 clusters.

[4-12] The selection device described in any of [4-4] to [4-11], wherein reliability is represented by AUC of ROC curves obtained based on sensitivity and specificity.

[4-13] The selection device described in [4-12] wherein, when the AUC is about 0.8 or higher, the biomarker candidate can be identified with high reliability.

[4-14] The selection device described in any of [4-2] to [4-13], wherein the IL-6 signal transduction pathway inhibitor is an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway.

[4-15] The selection device as described in [4-14], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[5-1] Use of a biomarker for predicting the level of IL-6 in a subject, the biomarker comprising one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[5-2] Use of biomarkers as described in [5-1], wherein the aforementioned biomarkers comprise IL-1β/IL-1F2 and/or IL-4.

[5-3] Use of biomarkers as described in [5-1] or [5-2], wherein the cytokines are derived from the blood, plasma, or serum of the subject.

[5-4] Use of a biomarker as described in any of [5-1] to [5-3], wherein the subject has PAH.

[5-5] Use of a biomarker for predicting a subject’s response to treatment based on an IL-6 signaling pathway inhibitor, the biomarker comprising one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, IL-6, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[5-6] Use of a biomarker for predicting a subject’s response to treatment based on an IL-6 signaling pathway inhibitor, the biomarker comprising IL-6 and a combination of one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, JL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[5-7] Use of biomarkers as described in [5-6], wherein the biomarkers comprise IL-6 and a combination of one or more cytokines selected from IL-1β/IL-1F2 and IL-4.

[5-8] Uses of biomarkers as described in [5-6], wherein the aforementioned biomarkers include IL-6, IL-1β/IL-1F2, and IL-4.

[5-9] Use of biomarkers as described in any of [5-5] to [5-8], wherein the cytokine is derived from the blood, plasma, or serum of the subject.

[5-10] Use of a biomarker as described in any of [5-5] to [5-8], wherein the subject has PAH.

[5-11] The use of the biomarker as described in any of [5-5] to [5-10], wherein the IL-6 signal transduction pathway inhibitor is an antibody or an antigen-binding fragment thereof having the function of inhibiting the IL-6 signal transduction pathway.

[5-12] The use of biomarkers as described in [5-11], wherein the antibody or antigen-binding fragment thereof is an anti-IL-6 receptor antibody or antigen-binding fragment thereof.

[5-13] Use of a biomarker for selecting subjects with a high probability of responding to treatment based on an IL-6 signaling pathway inhibitor, the biomarker comprising one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, IL-6, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[5-14] Use of a biomarker for selecting subjects with a high probability of responding to treatment based on an IL-6 signaling pathway inhibitor, the biomarker comprising IL-6 and a combination of one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[5-15] Use of biomarkers as described in [5-14], which include IL-6, in combination with one or more cytokines selected from IL-1β/IL-1F2 and IL-4.

[5-16] Uses of biomarkers as described in [5-14], including IL-6, JL-1β/IL-1F2, and IL-4.

[5-17] Use of biomarkers as described in any of [5-13] to [5-16], wherein the cytokine is derived from the blood, plasma, or serum of the subject.

[5-18] Use of a biomarker as described in any of [5-13] to [5-17], wherein the subject has PAH.

[5-19] Use of a biomarker as described in any of [5-13] to [5-18], wherein the IL-6 signal transduction pathway inhibitor is an antibody or an antigen-binding fragment thereof having the function of blocking the IL-6 signal transduction pathway.

[5-20] The use of biomarkers as described in [5-19], wherein the antibody or antigen-binding fragment thereof is an anti-IL-6 receptor antibody or antigen-binding fragment thereof.

[6-1] A biomarker for predicting the level of IL-6 in a subject, the biomarker comprising one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[6-2] Biomarkers as described in [6-1], which include IL-1p/IL-1F2 and/or IL-4.

[6-3] Biomarkers as described in [6-1] or [6-2], wherein the cytokines are derived from the blood, plasma, or serum of the subject.

[6-4] A biomarker described in any of [6-1] to [6-3], wherein the subject has PAH.

[7-1] A combination of biomarkers for predicting the level of IL-6 in a subject, comprising two or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[7-2] Combinations of biomarkers as described in [7-1], which include IL-1β/IL-1F2 and IL-4.

[7-3] Combinations of biomarkers as described in [7-1] or [7-2], wherein the cytokines are derived from the subject’s blood, plasma, or serum.

[7-4] A combination of biomarkers as described in any of [7-1] to [7-3], wherein the subject has PAH.

[8-1] A biomarker for predicting a subject's response to treatment based on an IL-6 signaling pathway inhibitor, comprising:

One or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, IL-6, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[8-2] A biomarker for predicting a subject's response to treatment based on an IL-6 signaling pathway inhibitor, comprising:

IL-6, and a combination of one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[8-3] Biomarkers as described in [8-2] include IL-6 and a combination of one or more cytokines selected from IL-1β/IL-1F2 and IL-4.

[8-4] Biomarkers such as those described in [8-2] include IL-6, IL-1β/IL-1F2, and IL-4.

[8-5] A biomarker as described in any of [8-1] to [8-4], wherein the cytokine is derived from the blood, plasma, or serum of the subject.

[8-6] A biomarker described in any of [8-1] to [8-5], wherein the subject has PAH.

[8-7] The biomarker described in any of [8-1] to [8-6], wherein the IL-6 signal transduction pathway inhibitor is an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway.

[8-8] As described in [8-7], the biomarker is an antibody or an antigen-binding fragment thereof that is an anti-IL-6 receptor antibody or an antigen-binding fragment thereof.

[9-1] A combination of biomarkers for predicting the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor, comprising two or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, IL-6, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[9-2] A combination of biomarkers for predicting the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor, comprising: IL-6, and one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/JL-1F2, GDF-15, JL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[9-3] A combination of biomarkers as described in [9-2], comprising IL-6 and one or more cytokines selected from IL-1β/IL-1F2 and IL-4.

[9-4] The combination of biomarkers described in [9-2] includes IL-6, IL-1β/IL-1F2, and IL-4.

[9-5] A combination of biomarkers as described in any of [9-1] to [9-4], wherein the cytokines are derived from the blood, plasma, or serum of the subject.

[9-6] A combination of biomarkers as described in any of [9-1] to [9-5], wherein the subject has PAH.

[9-7] A combination of biomarkers as described in any of [9-1] to [9-6], wherein the IL-6 signal transduction pathway inhibitor is an antibody or an antigen-binding fragment thereof that has the function of inhibiting the IL-6 signal transduction pathway.

[9-8] Combinations of biomarkers as described in [9-7], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[10-1] A biomarker for selecting subjects that are likely to be responsive to treatment based on an IL-6 signaling pathway inhibitor, comprising one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, IL-6, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[10-2] A biomarker for selecting subjects that are likely to respond to treatment based on an IL-6 signaling pathway inhibitor, comprising IL-6 and a combination of one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[10-3] Biomarkers as described in [10-2] contain IL-6 and a combination of one or more cytokines selected from IL-1β/IL-1F2 and IL-4.

[10-4] Biomarkers such as those described in [10-2] include IL-6, IL-1β/IL-1F2, and IL-4.

[10-5] A biomarker as described in any of [10-1] to [10-4], wherein the cytokine is derived from the blood, plasma, or serum of the subject.

[10-6] Biomarkers such as any of [10-1] to [10-5], wherein the subject has PAH.

[10-7] The biomarker described in any of [10-1] to [10-6], wherein the IL-6 signal transduction pathway inhibitor is an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway.

[10-8] Biomarkers as described in [10-7], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[11-1] A combination of biomarkers for selecting subjects that are highly likely to respond to treatment based on IL-6 signaling pathway inhibitors, comprising two or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, IL-6, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[11-2] A combination of biomarkers for selecting subjects with a high probability of responding to treatment based on IL-6 signaling pathway inhibitors, comprising IL-6 and one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

[11-3] Combinations of biomarkers as described in [11-2], comprising IL-6 and one or more cytokines selected from IL-1β/IL-1F2 and IL-4.

[11-4] Combinations of biomarkers as described in [11-2], including IL-6, IL-1β/IL-1F2, and IL-4.

[11-5] A combination of biomarkers described in any of [11-1] to [11-4], wherein the cytokines are derived from the blood, plasma, or serum of the subject.

[11-6] A combination of biomarkers described in any of [11-1] to [11-5], wherein the subject has PAH.

[11-7] A combination of biomarkers described in any of [11-1] to [11-6], wherein the IL-6 signal transduction pathway inhibitor is an antibody or an antigen-binding fragment thereof that has the function of inhibiting the IL-6 signal transduction pathway.

[1J-8] Combinations of biomarkers as described in [11-7], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[12-1] A method for predicting the content of IL-6 in a target, comprising:

In samples taken from the subject, the content of biomarkers as described in [6-1] or [6-2] is determined.

[12-2] The method described in [12-1], wherein the sample is blood, plasma, or serum.

[12-3] The method described in [12-1] or [12-2], wherein the subject suffers from PAH.

[12-4] A predictive method for predicting the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor, comprising:

In samples taken from the subject, the content of the biomarkers described in any of [8-1] to [8-4] is determined;

Based on the levels of biomarkers, we can determine whether a subject is highly likely to respond to treatments based on substances that block the IL-6 signaling pathway.

[12-5] The method described in [12-4], wherein the sample is blood, plasma, or serum.

[12-6] The method described in [12-4] or [12-5], wherein the subject suffers from PAH.

[12-7] The method described in any of [12-4] to [12-6], wherein the IL-6 signal transduction pathway inhibitor is an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway.

[12-8] The method described in [12-7], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[12-9] A method for selecting subjects that are highly likely to respond to treatment based on an IL-6 signaling pathway inhibitor, comprising:

In samples taken from the subject, the content of the biomarkers described in any of [10-1] to [10-4] is determined;

Based on the levels of biomarkers, we can determine whether a subject is highly likely to respond to treatments based on substances that block the IL-6 signaling pathway.

[12-10] The method described in [12-9] further includes: selecting subjects who are judged to be highly likely to respond to treatment based on IL-6 signal transduction pathway inhibitors.

[12-11] The method described in [12-9] or [12-10], wherein the sample is blood, plasma, or serum.

[12-12] The method described in any of [12-9] to [12-11], wherein the subject suffers from PAH.

[12-13] The method described in any one of [12-9] to [12-12], wherein the IL-6 signal transduction pathway inhibitor is an antibody or an antigen-binding fragment thereof having the function of blocking the IL-6 signal transduction pathway.

[12-14] The method described in [12-13] wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[13-1] A kit for predicting the content of IL-6 in a subject, comprising components for determining biomarkers as described in [6-1] or [6-2].

[13-2] A kit for predicting the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor, comprising means for determining a biomarker as described in any of [8-1] to [8-4].

[13-3] A kit for selecting subjects who are more likely to respond to treatment based on IL-6 signaling pathway inhibitors, comprising means for determining biomarkers as described in any of [10-1] to [10-4].

[13-4] A kit as described in any of [13-1] to [13-3] includes components for determining biomarkers in blood, plasma, or serum taken from a subject.

[13-5] As described in any of [13-1] to [13-4], wherein the subject suffers from PAH.

[13-6] As described in any of [13-1] to [13-5], the kit contains an antibody that specifically binds to a biomarker.

[13-7] As described in any of [13-1] to [13-6], wherein the IL-6 signal transduction pathway inhibitor is an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway.

[13-8] As described in [13-7], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[14-1] A medicine for treating PAH comprising an IL-6 signal transduction pathway inhibitor, the medicine being administered to a subject who is selected by any of the selection methods described in [12-9] to [12-14] as a subject who is highly likely to respond to treatment based on the IL-6 signal transduction pathway inhibitor.

[14-2] As described in [14-1], the IL-6 signal transduction pathway inhibitor is an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway.

[14-3] As described in [14-2], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[14-4] A drug for treating PAH contains an IL-6 signaling pathway inhibitor.

[14-5] A drug for treating PAH contains an IL-6 signaling pathway inhibitor, which is administered to PAH patients with a serum IL-6 concentration of 2.73 pg/mL or higher.

[14-6] The medicine described in [14-4] or [14-5], wherein the IL-6 signal transduction pathway inhibitor is an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway.

[14-7] As described in [14-6], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[14-8] The medicine described in [14-3] or [14-7], wherein the anti-IL-6 receptor antibody or its antigen-binding fragment comprises:

The amino acid sequence containing at least one of the three supervariable regions (CDRs) in the heavy chain variable region amino acid sequence containing sequence number 1, and/or containing at least one of the three supervariable regions (CDRs) in the light chain variable region amino acid sequence containing sequence number 2.

[14-9] As described in [14-8], the anti-IL-6 receptor antibody or its antigen-binding fragment includes:

The heavy chain contains the amino acid sequence number 3, and the light chain contains the amino acid sequence number 4.

[14-10] The drug described in [14-9], wherein the anti-IL-6 receptor antibody or its antigen-binding fragment is Satralizumab or its antigen-binding fragment.

[15-1] A treatment for PAH includes administering a therapeutically effective amount of an IL-6 signaling pathway inhibitor to a subject, the subject being selected by a selection method described in any one of [12-9] to [12-14] as a subject with a high probability of being responsive to treatment targeting the JL-6-related cell signaling system.

[15-2] The treatment method described in [15-1] is an IL-6 signal transduction pathway inhibitor that is an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway.

[15-3] The treatment method described in [15-2], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[15-4] A treatment for PAH includes administering a therapeutically effective amount of an IL-6 signaling pathway inhibitor to a PAH subject requiring treatment.

[15-5] A treatment for PAH includes administering an IL-6 signaling pathway inhibitor to a PAH subject requiring treatment, wherein the serum IL-6 concentration is 2.73 (pg/mL) or higher.

[15-6] The treatment methods described in [15-4] or [15-5], wherein the IL-6 signal transduction pathway inhibitor is an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway.

[15-7] The treatment methods described in [15-6], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[15-8] The treatment methods described in [15-3] or [15-7], wherein the anti-IL-6 receptor antibody or its antigen-binding fragment comprises:

The amino acid sequence containing at least one of the three supervariable regions (CDRs) in the heavy chain variable region amino acid sequence containing sequence number 1, and/or containing at least one of the three supervariable regions (CDRs) in the light chain variable region amino acid sequence containing sequence number 2.

[15-9] The treatment methods described in [15-8], wherein the anti-IL-6 receptor antibody or its antigen-binding fragment comprises:

The heavy chain contains the amino acid sequence number 3, and the light chain contains the amino acid sequence number 4.

[15-10] The treatment method described in [15-9], wherein the anti-IL-6 receptor antibody or its antigen-binding fragment is Satralizumab or its antigen-binding fragment.

[16-1] An IL-6 signaling pathway inhibitor for use in the treatment of PAH, which is administered to a subject selected by any of the selection methods described in [12-9] to [12-14] as a subject with a high probability of being responsive to treatment based on the IL-6 signaling pathway inhibitor.

[16-2] IL-6 signal transduction pathway inhibitors as described in [16-1], including antibodies or antigen-binding fragments of which have IL-6 signal transduction pathway inhibitory functions.

[16-3] IL-6 signal transduction pathway inhibitors as described in [16-2], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[16-4] An IL-6 signaling pathway inhibitor used for the treatment of PAH.

[16-5] An IL-6 signaling pathway inhibitor for the treatment of PAH in subjects with PAH whose serum IL-6 levels are 2.73 (pg/mL) or higher.

[16-6] IL-6 signal transduction pathway inhibitors as described in [16-4] or [16-5], antibodies or antigen-binding fragments thereof having IL-6 signal transduction pathway inhibitory functions.

[16-7] IL-6 signal transduction pathway inhibitors as described in [16-6], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[16-8] IL-6 signaling pathway inhibitors as described in [16-3] or [16-7], wherein anti-IL-6 receptor antibodies or antigen-binding fragments thereof comprise:

The amino acid sequence containing at least one of the three supervariable regions (CDRs) in the heavy chain variable region amino acid sequence containing sequence number 1, and/or containing at least one of the three supervariable regions (CDRs) in the light chain variable region amino acid sequence containing sequence number 2.

[16-9] IL-6 signal transduction pathway inhibitors as described in [16-8], wherein anti-IL-6 receptor antibodies or their antigen-binding fragments include:

The heavy chain contains the amino acid sequence number 3, and the light chain contains the amino acid sequence number 4.

[16-10] IL-6 signal transduction pathway inhibitors as described in [16-9], wherein the anti-IL-6 receptor antibody or its antigen-binding fragment is Satralizumab or its antigen-binding fragment.

[17-1] Use of an IL-6 signal transduction pathway inhibitor in the manufacture of a medicament for treating PAH, wherein the medicament is administered to a subject who is selected by any of the selection methods described in [12-9] to [12-14] as being highly likely to respond to treatment based on the IL-6 signal transduction pathway inhibitor.

[17-2] As described in [17-1], wherein the signal transduction pathway blocker is an antibody or its antigen-binding fragment that has the function of blocking the JL-6 signal transduction pathway.

[17-3] As described in [17-2], wherein the antibody or antigen-binding fragment thereof is an anti-IL-receptor antibody or antigen-binding fragment thereof.

[17-4] Use of an IL-6 signaling pathway inhibitor in the manufacture of a pharmaceutical for the treatment of PAH.

[17-5] Use of an IL-6 signal transduction pathway inhibitor in the manufacture of a medicament for treating PAH, wherein the medicament is administered to a PAH patient whose serum IL-6 concentration is 2.73 (pg/mL) or higher.

[17-6] As described in [17-4] or [17-5], wherein the signal transduction pathway blocker is an antibody or an antigen-binding fragment thereof that has the function of blocking the IL-6 signal transduction pathway.

[17-7] As described in [17-6], wherein the antibody or its antigen-binding fragment is an anti-IL-6 receptor antibody or its antigen-binding fragment.

[17-8] As described in [17-3] or [17-7], wherein the anti-IL-6 receptor antibody or its antigen-binding fragment comprises:

The amino acid sequence containing at least one of the three supervariable regions (CDRs) in the heavy chain variable region amino acid sequence containing sequence number 1, and/or containing at least one of the three supervariable regions (CDRs) in the light chain variable region amino acid sequence containing sequence number 2.

[17-9] As described in [17-8], wherein the anti-IL-6 receptor antibody or its antigen-binding fragment comprises:

The heavy chain contains the amino acid sequence number 3, and the light chain contains the amino acid sequence number 4.

[17-10] As described in [17-9], wherein the anti-IL-6 receptor antibody or its antigen-binding fragment is Satralizumab or its antigen-binding fragment.

[18-1] A method for determining the content of a biomarker, the method comprising:

In a sample taken from the subject, the content of the biomarker described in any of [6-1] to [6-4], [8-1] to [8-8] and [10-1] to [10-8] is determined.

[18-2] As described in [18-1], the assay method uses an antibody that specifically binds to the biomarker to determine the biomarker content.

The effects of the invention

According to the present invention, a method for selecting novel biomarkers can be provided.

According to the present invention, a novel biomarker selection device can be provided.

According to the present invention, a method for selecting biomarkers for predicting the content of IL-6 in a subject can be provided.

According to the present invention, a method for selecting biomarkers for predicting the responsiveness of a subject to treatment based on substances that block the IL-6 signaling transduction pathway can be provided.

According to the present invention, a method for selecting biomarkers for subjects that are highly likely to respond to treatment based on substances that block the IL-6 signaling transduction pathway can be provided.

According to the present invention, a biomarker selection device for predicting the content of IL-6 in a subject can be provided.

According to the present invention, a device for selecting biomarkers for predicting the responsiveness of a subject to treatment based on a substance that blocks the IL-6 signaling pathway can be provided.

According to the present invention, a selection device for selecting biomarkers in subjects that are highly likely to be responsive to treatment based on substances that block the IL-6 signaling pathway can be provided.

According to the present invention, a biomarker for predicting the content of IL-6 in a subject can be provided.

According to the present invention, a biomarker for predicting the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor can be provided.

According to the present invention, it is possible to provide a use for selecting a biomarker for subjects that are highly likely to respond to treatment based on substances that block the IL-6 signaling transduction pathway.

According to the present invention, a biomarker for predicting the content of IL-6 in a subject can be provided.

According to the present invention, a biomarker can be provided for predicting the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor.

According to the present invention, a biomarker can be provided for selecting subjects that are more likely to respond to treatment based on substances that block the IL-6 signaling pathway.

According to the present invention, a combination of biomarkers for predicting the level of IL-6 in a subject can be provided.

According to the present invention, a combination of biomarkers can be provided for predicting the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor.

According to the present invention, a combination of biomarkers can be provided for selecting subjects that are highly likely to respond to treatment based on substances that block the IL-6 signaling transduction pathway.

According to the present invention, a method for predicting the IL-6 content in a subject can be provided.

According to the present invention, a method for predicting the response of a subject to treatment based on an IL-6 signaling pathway inhibitor can be provided.

According to the present invention, a method for selecting subjects that are highly likely to respond to treatment based on substances that block the IL-6 signaling transduction pathway can be provided.

According to the present invention, a kit can be provided that can be used to predict the IL-6 content in an object.

According to the present invention, a kit can be provided for predicting the response of a subject to treatment based on an IL-6 signaling pathway inhibitor.

According to the present invention, a kit can be provided for selecting subjects who are more likely to respond to treatment based on substances that block the IL-6 signaling pathway.

According to the present invention, a medicament for treating PAH can be provided.

According to the present invention, a treatment method for PAH can be provided.

According to the present invention, an IL-6 signaling pathway inhibitor for the treatment of PAH can be provided.

According to the present invention, there is a use for an IL-6 signal transduction pathway inhibitor, which is used in the manufacture of a medicament for treating PAH.

According to the present invention, a method for determining the content of a biomarker can be provided.

Attached Figure Description

Figure 1 shows 49 cytokines detected in serum samples taken from 145 specimens in Example 1.

Figure 2 shows the cosine similarity calculated for JL-6 and each of the 45 cytokines other than IL-6 in Example 2.

Figure 3 shows a histogram created using the cosine similarity obtained for each of IL-6 and cytokines other than IL-6 in Example 2.

Figure 4 shows a network diagram based on the cosine similarity obtained in Example 2 for each of the cytokines other than IL-6 included in the IL-6 analog group.

Figure 5 shows a summary of the results obtained in Example 2 by clustering the content data of 145 specimens containing cytokines in the IL-6 analogue into 3 clusters using the K-medoids method.

Figures 6A-B show the results of clustering the content data of 145 specimens containing cytokines in the IL-6 analogue into 3 clusters using the K-medoids method in Example 2, and the results of plotting the individual cytokines located relatively close to IL-6 in the network diagram of Figure 4.

Figures 7A-B show the results of clustering the content data of 145 specimens containing cytokines in the IL-6 analogue group into 3 clusters using the K-medoids method in Example 2, and plotting the individual cytokines located relatively close to IL-1β in the network diagram of Figure 4.

Figure 8 shows the sensitivity and specificity calculated by comparing the threshold candidate values of IL-6 content with the results of clustering the results of identifying objects using the K-medoids method in Example 3.

Figure 9 shows the sensitivity and specificity calculated by comparing the threshold candidate values of IL-1β content with the results of clustering the results of identifying objects using the K-medoids method in Example 3.

Figure 10 shows the reliability (AUC) of each cytokine contained in the IL-6 analogue obtained in Example 3.

Detailed Implementation

The embodiments of the present invention will now be described in detail. The present invention is not limited to the following embodiments, and may be implemented with appropriate modifications without impairing the effects of the present invention.

==Explanation of Terms==

[Biomarkers]

In this specification, unless otherwise stated, "biomarker" means a substance derived from the object that reflects the state of the object or changes thereof, and serves as an indicator of the state of the object, and/or data derived from the object.

[Responsiveness]

In this specification, unless otherwise stated, "responsiveness" has the same meaning as treatment sensitivity. It is not limited to any state in which some preferred results, whether sustained or temporary, can be obtained through the treatment of the subject's disease. Examples of "preferred results" include, for example, the inhibition or improvement of the progression of the subject's systemic symptoms, the inhibition or improvement of the progression of pathological symptoms, the inhibition or improvement of the deterioration of examination data reflecting the disease state, and/or the improvement of the subject's prognosis.

[IL-6 related diseases]

In this instruction manual, "interleukin-6 (IL-6) related diseases," unless otherwise specified, include diseases in which at least a portion of the direct or indirect causes of the disease or its onset are related to IL-6. Examples include: rheumatoid arthritis, juvenile idiopathic arthritis, systemic juvenile idiopathic arthritis, giant lymphadenopathy, systemic lupus erythematosus (SLE), lupus nephritis, Crohn's disease, lymphoma, ulcerative colitis, anemia, vasculitis, Kawasaki disease, Still's disease, amyloidosis, multiple sclerosis, transplantation, age-related macular degeneration, ankylosing spondylitis, psoriasis, psoriatic arthritis, chronic obstructive pulmonary disease (COPD), IgA nephropathy, osteoarthropathy, asthma, diabetic nephropathy, and graft-versus-host disease (GVH). D) Endometrial disease, hepatitis (NASH), myocardial infarction, arteriosclerosis, sepsis, osteoporosis, diabetes, multiple myeloma, prostate cancer, kidney cancer, B-cell non-Hodgkin's lymphoma, pancreatic cancer, lung cancer, esophageal cancer, colorectal cancer, cancer cachexia, cancerous nerve infiltration, myopic choroidal revascularization, idiopathic choroidal revascularization, uveitis, chronic thyroiditis, delayed onset hypersensitivity, contact dermatitis, atopic dermatitis, mesothelioma, polymyositis, dermatomyositis, panuveitis, anterior uveitis, intermediate uveitis, scleritis, keratitis, orbital inflammation, optic neuritis, diabetic retinopathy, proliferative vitreoretinopathy, dry eye syndrome, postoperative inflammation, neuromyelitis optica, myasthenia gravis, and pulmonary hypertension.

[IL-6 signaling pathway inhibitors]

The biological activity signal of IL-6 is transmitted via the following pathway: IL-6 binds to the IL-6 receptor on the cell membrane to form the IL-6/IL-6 receptor complex, and then binds to gp130. Therefore, in this specification, "IL-6 signal transduction pathway inhibitor" unless otherwise stated, refers to any molecule that can neutralize, block, inhibit, suppress, reduce, or interfere with at least a portion of the function or activity of IL-6, the IL-6 receptor, and/or gp130, such as antibodies, antibody fragments, binding peptides, peptides, or non-peptide molecules.

In one embodiment, the IL-6 signaling pathway inhibitor may be an anti-IL-6 antibody, an anti-IL-6 receptor antibody, and/or an anti-gp 130 antibody, or an IL-6 antagonist compound.

In one embodiment, the IL-6 signal transduction pathway inhibitor is preferably an anti-IL-6 antibody or its antigen-binding fragment, or an anti-IL-6 receptor antibody or its antigen-binding fragment, which are antibodies or their antigen-binding fragments that have the function of blocking the IL-6 signal transduction pathway, and more preferably an anti-IL-6 receptor antibody or its antigen-binding fragment.

In one embodiment, an anti-IL-6 receptor antibody may be exemplified as an antibody comprising: at least one of three supervariable regions (CDRs) in the amino acid sequence of the heavy chain variable region containing sequence number 1, and/or at least one of three supervariable regions (CDRs) in the amino acid sequence of the light chain variable region containing sequence number 2.

In one embodiment, an anti-IL-6 receptor antibody may be exemplified as an antibody comprising: a heavy chain containing an amino acid sequence of sequence number 3, and a light chain containing an amino acid sequence of sequence number 4.

In one embodiment, the anti-IL-6 receptor antibody may be exemplified by, for example, satralizumab, tocilizumab, and/or sarilumab, preferably satralizumab and/or tocilizumab, more preferably satralizumab.

Antibodies can be chimeric antibodies, humanized antibodies, human antibodies, antibodies derived from antibody libraries, or antibody fragments thereof. Antigen-binding fragments comprise antibody fragments that are missing a portion of the full-length antibody while maintaining antigen-binding ability; examples include Fab, Fab′, F(ab′) ₂ , Fv, etc. Furthermore, specific examples of low-molecular-weight antibodies include Fab, Fab′, F(ab′) ₂ , Fv, scFv, bispecific antibodies, sc(Fv) ₂ , etc.

Antibodies can be obtained using known methods, either in the form of polyclonal or monoclonal antibodies. The source of the antibody is not particularly limited, but antibodies derived from mammals are preferred, and antibodies derived from humans are more preferred. Monoclonal antibodies derived from mammals include those produced in hybridomas and those produced in converted hosts using expression vectors containing antibody genes through genetic engineering methods.

Antibodies or their antigen-binding fragments can also be conjugated antibodies formed by binding to any molecule such as polyethylene glycol (PEG), radioactive substances, toxins, or therapeutic compounds.

Antibodies include not only bivalent antibodies, represented by IgG, but also monovalent antibodies, or multivalent antibodies, represented by IgM. The multivalent antibodies of this invention include: multivalent antibodies having all identical antigen-binding sites, or multivalent antibodies having partially or completely different antigen-binding sites.

Antibodies can also be bispecific antibodies. Bispecific antibodies are antibodies that have a variable region within the same antibody molecule that recognizes different epitopes, but these epitopes can exist in different molecules or in the same molecule.

Methods for manufacturing bispecific antibodies are well known. For example, bispecific antibodies can be produced by combining two antibodies that recognize different antigens. The combined antibodies can be half molecules of antibodies with both H and L chains, or a quarter molecule of an antibody consisting only of an H chain. Alternatively, bispecific antibodies can be produced by fusing hybridomas that produce different monoclonal antibodies to create fusion cells. Furthermore, bispecific antibodies can be produced using genetic engineering methods.

==Methods for Selecting New Biomarkers==

The biomarker selection method of this embodiment is as follows: a novel biomarker selection method, which includes:

(a) Selecting biomarker candidates based on the similarity of vectors obtained using the content of reference biomarkers and the content of biomarker candidate substances in samples taken from the object group; and

(b) Cluster the object group based on the content of the biomarker candidate substances selected in (a).

In one implementation, the method for selecting new biomarkers further includes:

(c) In contrast to the clustering results in (b), the biomarker candidates selected in (a) are selected as biomarker candidates if they can identify objects with high reliability.

In one implementation, the method for selecting the new biomarker includes, in (c):

(d) The results obtained by identifying objects using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) are compared with the clustering results in (b) to obtain the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) Reliability is achieved based on the sensitivity and specificity obtained in (d); and

(f) Determine whether biomarker candidates can identify objects with high reliability.

[Object Group]

The term "subject" in the method for selecting new biomarkers is not particularly limited. It can refer to any organism in which the biomarker is present in the organism either homeostatically or temporarily. For example, it can be a mammal or any other animal. Mammals can be humans or non-human animals. Non-human animal species can include, for example, monkeys, dogs, cats, horses, cattle, pigs, sheep, goats, rabbits, guinea pigs, hamsters, mice, and/or rats, but are not limited to the purpose of domestic animals, pets, or laboratory animals. Mammals are preferred, and humans are more preferred.

An object group is a group of objects that can be clustered in (b) with at least two objects. For example, an object group containing at least three objects could be clustered into three clusters.

The object group can be a group of objects selected based on the state of the objects, a group of randomly selected objects, or a group of all objects that can be used when implementing the new biomarker selection method. Based on the purpose of selecting which biomarker, those skilled in the art can appropriately determine the new biomarker selection method.

Here, the object state can be any state of the object, such as a state related to a specific disease or a state unrelated to a specific disease.

In the case of a state related to a specific disease, the state of the object is not limited, as long as it is at least partly related to the specific disease. For example, the following states can be exemplified: the state in which the object is suspected of having a specific disease; the state in which the object does not have a specific disease; the state in which the object has any subtype of a specific disease; the state in which the object with a specific disease is responsive to a specific treatment; the state in which the specific disease of the object is progressing; and/or the state in which the symptoms of the specific disease of the object are improving, etc.

In cases where the state is not related to a specific disease, the state as an object can be exemplified by, for example, physiological characteristics, genetic status such as sex or race, age, and/or geographical status such as place of residence or range of movement that are not related to any disease.

[Reference Biomarkers]

In one implementation, in the method for selecting new biomarkers, the reference biomarker may be a substance whose relevance to the state of the object is unknown, or it may be a substance known to reflect the state of the object, i.e., the reference biomarker is a substance known to be useful as an indicator of the state of the object.

In one implementation, when the reference biomarker is a substance whose correlation with the state of the object is unknown, the method for selecting a new biomarker can be used for purposes such as selecting a new biomarker for predicting the content of the reference biomarker in the object.

In another embodiment, where the reference biomarker is a substance known to be useful as an indicator of the state of an object, the method for selecting a new biomarker may be used for purposes such as: obtaining a biomarker for predicting the content of the reference biomarker in the object; and/or obtaining a new biomarker that can be used in conjunction with or in place of the reference biomarker to indicate the state of an object for which the known reference biomarker is useful as an indicator.

In this context, the method for selecting new biomarkers can be used for purposes such as: obtaining new biomarkers that are superior to reference biomarkers as indicators; obtaining new biomarkers that, when used in conjunction with reference biomarkers, can provide more comprehensive and/or more reliable information about the state of an object; and/or obtaining new biomarkers that are also more economical, convenient, and/or versatile than reference biomarkers.

[Process(a)]

Methods for selecting new biomarkers include selecting biomarker candidates based on the similarity of vectors obtained using the content of a reference biomarker and the content of biomarker candidate substances in samples taken from the subject group.

Here, the sample is a biological sample taken from the subject group, and there is no limitation on the range in which the content of the reference biomarker and biomarker candidate substances can be measured. For example, it can be a solid tissue sample obtained during biopsy or treatment, or it can be blood (whole blood), plasma, or serum.

Reference biomarkers and biomarker candidates can originate from the same tissue or from different tissues of the same individual.

The content of reference biomarkers and biomarker candidate substances in the sample can be determined based on what the biomarker is and using methods commonly used in the field.

For example, when the reference biomarker and the biomarker candidate are cytokines, the method described in the section on "[Determination of biomarkers in samples]" under "Methods for the determination of biomarkers" can be used as an example.

Here, the content of the reference biomarker and biomarker candidate can be any of the following: absolute value, relative concentration, weight per unit volume, raw data measured to obtain the absolute concentration, etc.

In one embodiment, when plasma or serum is used as a sample for determination, the weight per unit volume of the sample, such as 1 mL or 1 μL, is preferred.

In one implementation, the similarity of vectors is not limited in the range commonly used in the field to represent the behavioral similarity between a reference biomarker and a biomarker candidate based on the abundance of the reference biomarker and the abundance of the biomarker candidate. For example, it can be cosine similarity as expressed by the following formula, or it can be Euclidean distance.

In the above formula, x represents the content of the reference biomarker, and y represents the content of the biomarker candidate substance (or vice versa). n is the number of objects constituting the object group.

Cosine similarity is equivalent to cosθ when the angle between the vectors of x and y is taken as θ. The closer the cosine similarity is to 1, the closer the vectors of x and y are to having the same orientation. Vector similarity represents the similarity of behavior between a reference biomarker and various biomarker candidate substances.

In one implementation, the values of the content of the reference biomarker used to obtain the similarity of the vectors and the values of the content of the biomarker candidate substance can also be processed in advance by methods commonly used in the field.

In one embodiment, processing for excluding extremely low and/or extremely high values may also be performed. The method for such processing can be determined by those skilled in the art through methods commonly used in the field, taking into account the number of objects constituting the object group (the number of measured values), the distribution of the content data, the purpose of implementing the selection method, etc.

For example, the processing for excluding extremely low and/or extremely high values can be performed by excluding values detected as outliers using any method commonly used in the field of data analysis, or by excluding values before and/or after an arbitrarily set proportion or value.

As a more specific implementation, in order to exclude extremely low values, all values of content below the displayed measurement sensitivity can be set to 0 (zero).

In another embodiment, in order to exclude extremely high values, for example, the values of the first 30, first 20, first 10, or first 5 can be processed by taking the 30th, 20th, 10th, or 5th value from the top, respectively.

Regarding vector similarity, for example, in the case of cosine similarity, as mentioned above, the closer it is to 1, the closer the vectors of x and y are to having the same orientation. That is, when the cosine similarity is 1, the reference biomarker and the biomarker candidate exhibit the same behavior; the closer it is to 1, the higher the similarity of behavior.

Regarding the similarity of the vector used as the selection criterion for biomarker candidates, it can be appropriately set by those skilled in the art using common methods in the field, taking into account the purpose of implementing the new biomarker selection method and the subsequent steps to be implemented. If the goal is to obtain an effective new biomarker that can predict the content of the reference biomarker, it is preferable to set it to, for example, about 0.5 or more, about 0.6 or more, about 0.7 or more, about 0.8 or more, or about 0.9 or more, and more preferably about 0.6 or more.

[Process (b)]

Methods for selecting new biomarkers include clustering the object group based on the content of the biomarker candidates selected in (a).

In one implementation, clustering is preferably a teacherless machine learning-based classification of data on the content of all biomarker candidates selected in (a).

As one implementation method in this case, it can be based on hierarchical clustering, or on non-hierarchical clustering methods such as k-means or K-medoids. There is no limitation on the range of clustering that can be performed, but non-hierarchical clustering is preferred, and K-medoids is more preferred.

The number of clusters into which a group of objects can be clustered can be determined appropriately by those skilled in the art using methods commonly found in the field; the objects can be clustered into two, three, or four clusters.

As an implementation method, for example, when using the k-means or K-medoids method for clustering, the number of clusters needs to be set during implementation.

[Process (c)]

In one embodiment, the method for selecting new biomarkers preferably further includes: selecting the biomarker candidate selected in (a) as a biomarker candidate if the clustering results in (b) can identify the object with high reliability, based on the results in (b).

In one implementation, the method for selecting the new biomarker may also include in (c):

(d) The results obtained by identifying objects using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) are compared with the clustering results in (b) to obtain the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) Reliability is achieved based on the sensitivity and specificity obtained in (d); and

(f) Determine whether biomarker candidates can identify objects with high reliability.

In one implementation, reliability is preferably represented by the area under the curve (AUC) of the ROC curve (Receiver Operating Curve) obtained based on sensitivity and specificity.

In one embodiment, in (f), it is preferable that the AUC is about 0.7 or more, about 0.8 or more, or about 0.9 or more, and more preferably that the AUC is about 0.8 or more, so that the biomarker candidate can be identified with high reliability.

In one implementation, the method for selecting new biomarkers can also be a computer-installed method implemented via a computer.

Here, we will explain the case that includes (d) to (f) as one implementation of (c), for example, in (b) we assume that the object group is clustered into 3 clusters based on the content of biomarker candidate substances.

The cluster with the highest content of biomarker candidate substances among the three clusters obtained through clustering (containing one or two clusters) can be set as the "object with high content of reference biomarker", and the other clusters can be set as the "object with low content of reference biomarker". This is the result of clustering in "(b)".

In (d), the “sensitivity” and “specificity” of the “biopter candidate material selected in (a)” can be obtained, for example, as follows.

First, for the content of a certain biomarker candidate substance selected in (a), an arbitrary series of threshold candidate values are set to identify each object as either an "object with a high content of reference biomarker" (the content of which is above the threshold candidate value) or an "object with a low content of reference biomarker" (the content of which is below the threshold candidate value). Then, the identification results of "objects with a high content of reference biomarker" and "objects with a low content of reference biomarker" obtained using each threshold candidate value are compared with the clustering results of "objects with a high content of reference biomarker" and "objects with a low content of reference biomarker" (the clustering results in (b)). Thus, for the case where each threshold candidate value is used, the sensitivity and specificity of the identification of "objects with a high content of reference biomarker" and "objects with a low content of reference biomarker" can be obtained.

More specifically, the identification results of "objects with high levels of reference biomarkers" based on a certain threshold candidate value are compared with the clustering results of "objects with high levels of reference biomarkers" (the clustering results in (b)). This yields the probability (sensitivity) that "objects with high levels of reference biomarkers" can be accurately identified based on that threshold candidate value. Furthermore, the identification results of "objects with low levels of reference biomarkers" based on a certain threshold candidate value are compared with the clustering results of "objects with low levels of reference biomarkers" (the clustering results in (b)). This yields the probability (specificity) that "objects with low levels of reference biomarkers" can be accurately identified based on that threshold candidate value.

By repeatedly performing this method, the sensitivity and specificity corresponding to each of any series of threshold candidate values can be obtained.

In (e), reliability is obtained based on the sensitivity and specificity obtained in (d). Reliability can be represented, for example, by the area under the ROC curve (AUC) plotted based on sensitivity and specificity.

That is, the AUC obtained in this way represents the reliability of the identification results obtained by using the biomarker candidate to identify the object compared with the clustering results in (b).

In (f), it is determined whether the biomarker candidate can identify the target with high reliability. This determination is preferably based on the reliability expressed by AUC, as described above. Then, if the AUC is about 0.7 or more, about 0.8 or more, or about 0.9 or more, and more preferably if the AUC is about 0.8 or more, it is determined that the biomarker candidate can identify the target with high reliability.

By using the above methods for selecting new biomarkers, effective new biomarkers can be selected for predicting the content of reference biomarkers in samples obtained from the subject.

==Selection Device for New Biomarkers==

The novel biomarker selection device of this embodiment is an apparatus for implementing the method described in the above-described "method for selecting novel biomarkers".

That is, the novel biomarker selection device of this embodiment includes:

(a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the content of a reference biomarker and the content of biomarker candidate substances in samples taken from the object group; and

(b) Clustering unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a).

In one embodiment, the selection device for the novel biomarker further comprises:

(c) Selection section: In comparison with the clustering results in (b), the biomarker candidate selected in (a) is selected as the biomarker candidate if it can identify the object with high reliability.

In one embodiment, in the selection device for the new biomarker

(c) The selection section has:

(d) The acquisition unit compares the results obtained by identifying the object using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) with the clustering results in (b), thereby obtaining the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) The acquisition unit, which acquires reliability based on the sensitivity and specificity obtained in (d); and

(f) Judgment unit, which determines whether biomarker candidate substances can identify objects with high reliability.

In the new biomarker selection apparatus, the embodiments and preferred embodiments of the "object group", "reference biomarker", etc., and each step in (a) to (f) are as described in the section on "method for selecting new biomarkers" above.

==Selection Methods for Biomarkers Used to Predict IL-6 Levels==

The method for selecting biomarkers for predicting IL-6 levels in a target according to this embodiment includes:

(a) Biomarker candidates are selected based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from the subject group; and

(b) Cluster the object group based on the content of the biomarker candidate substances selected in (a).

In one embodiment, the method for selecting biomarkers for predicting IL-6 levels further includes:

(c) In contrast to the clustering results in (b), the biomarker candidates selected in (a) are selected as biomarker candidates if they can identify objects with high reliability.

In one implementation, the method for selecting biomarkers for predicting IL-6 levels includes, in (c):

(d) The results obtained by identifying objects using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) are compared with the clustering results in (b) to obtain the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) Reliability is achieved based on the sensitivity and specificity obtained in (d); and

(f) Determine whether biomarker candidates can identify objects with high reliability.

[Object Group]

The term "subject" in the method for selecting biomarkers for predicting IL-6 levels is not particularly limited, and can be any organism in which IL-6 is present either homeostatically or transiently. For example, it can be a mammal, or any other animal. Mammals can be humans or non-human animals. Non-human animal species include, for example, monkeys, dogs, cats, horses, cattle, pigs, sheep, goats, rabbits, guinea pigs, hamsters, mice, and/or rats, but are not limited to the use as livestock, pets, or laboratory animals. Mammals are preferred, and humans are more preferred.

An object group is a group of objects that can be clustered according to (b), and that group contains at least two objects. For example, an object group containing at least three objects could be clustered into three clusters.

The object group may be a group of objects selected based on the state of the objects, a group of randomly selected objects, or a group of all objects that can be used when implementing a biomarker selection method for predicting IL-6 levels. Based on the purpose of selecting which biomarker, those skilled in the art can appropriately determine the biomarker selection method for predicting IL-6 levels.

Here, the object state can be any state of the object, such as a state related to a specific disease or a state unrelated to a specific disease.

In the case of states related to a specific disease, the state of the object is not limited, as long as it is at least partly related to the specific disease. For example, the following states can be exemplified: the state of the object being suspected of having a specific disease; the state of the object not having a specific disease; the state of the object not having any subtype of a specific disease; the state of the object having a specific disease being responsive to a certain treatment; the state of the progression of the specific disease the object has; and/or the state of the improvement of the symptoms of the specific disease the object has, etc.

In cases where the state is not related to a specific disease, the state as an object can be exemplified by, for example, physiological characteristics, genetic status such as sex or race, age, and/or geographical status such as place of residence or range of movement that are not related to any disease.

[IL-6 related diseases]

In one embodiment, the target group in the method for selecting biomarkers for predicting IL-6 levels is preferably a target group suspected of having IL-6-related disease, or a target group with IL-6-related disease. Here, "IL-6-related disease" is as described in the "Explanation of Terms" section above.

In one embodiment, an IL-6-related disease can be a disease in which the IL-6 content or presence of IL-6 in the sample changes depending on whether the subject is associated with the disease, or it can be a disease in which the IL-6 content or presence of IL-6 in the sample changes based on the status of each subject in a subject group with an IL-6-related disease, and the disease can be classified into subtypes based on such changes in the status of the subject group.

In one implementation, pulmonary hypertension is preferred as an example of IL-6-related disease, i.e., the subject group is preferably a subject group suffering from pulmonary hypertension.

Here, pulmonary hypertension is clinically classified as: pulmonary arterial hypertension (PAH, Group 1), pulmonary hypertension associated with left heart disease (Group 2), pulmonary hypertension associated with lung disease and/or hypoxemia (Group 3), CTEPH (Group 4), and pulmonary hypertension associated with multifactorial mechanisms of unknown details (Group 5) (Guidelines for the Treatment of Pulmonary Hypertension (2017 Revised Edition)).

In one embodiment, pulmonary hypertension is more preferably pulmonary arterial hypertension (PAH), that is, the subject group is more preferably a subject group suffering from PAH.

This report details the elevated serum IL-6 levels in PAH patients and the high expression of IL-6 receptors in diseased organs, as well as the effectiveness of treating PAH using IL-6 signaling pathway inhibitors. Specifically, PAH can be treated by inhibiting the action of IL-6, and serum IL-6 levels in PAH patients are considered an indicator of responsiveness to treatment based on IL-6 signaling pathway inhibitors.

Therefore, in one embodiment, when the subject group in the method for selecting biomarkers for predicting IL-6 levels is a group of subjects with PAH, the selection method can be used for the following purposes: to obtain biomarkers for predicting IL-6 levels; and/or to obtain biomarkers used in conjunction with or in place of IL-6 to represent the subject's responsiveness to treatment based on IL-6 signaling pathway inhibitors. Here, "IL-6 signaling pathway inhibitor" is as described in the "Explanation of Terms" section above.

[Process(a)]

Methods for selecting biomarkers to predict IL-6 levels include selecting biomarker candidates based on the similarity of vectors obtained using the IL-6 levels in samples taken from the subject group and the levels of biomarker candidates.

Here, the sample is a biological sample taken from the target group, and is not limited to the range in which the content of IL-6 and biomarker candidate substances can be measured. For example, it can be a solid tissue sample obtained during biopsy or treatment, or it can be blood (whole blood), plasma, or serum, but is preferably blood (whole blood), plasma, or serum.

IL-6 and biomarker candidates may be derived from the same tissue or from different tissues in the same individual, but are preferably derived from the same tissue in the same individual, i.e., preferably from blood (whole blood), plasma or serum in the same individual.

The content of IL-6 and biomarker candidate substances in the sample can be determined by methods commonly used in the field, such as those described in the section on "Determination of Biomarkers in Samples" under "Methods for the Determination of Biomarkers".

Here, the content of IL-6 and biomarker candidates can be any of the following: absolute value, relative concentration, weight per unit volume, raw data measured to obtain the absolute concentration, etc.

In one embodiment, when plasma or serum is used as a sample for determination, the weight per unit volume of the sample, such as 1 mL or 1 μL, is preferred.

In one implementation, the similarity of vectors is not limited to the range of behavioral similarity between IL-6 and biomarker candidates based on the content of IL-6 and the content of biomarker candidates. For example, it can be cosine similarity as expressed by the following formula, or it can be Euclidean distance, but cosine similarity is preferred.

In the above formula, x is the content of IL-6, y is the content of biomarker candidate substances (or vice versa), and n is the number of objects constituting the object group.

Cosine similarity is equivalent to cosθ when the angle between the vectors of x and y is taken as θ. The closer the cosine similarity is to 1, the closer the vectors of x and y are to having the same orientation. Vector similarity represents the similarity of behavior between IL-6 and various biomarker candidate substances.

In one implementation, the values of IL-6 content used to obtain the similarity of vectors and the values of biomarker candidate content can also be processed in advance using methods commonly used in the field.

In one embodiment, processing for excluding extremely low and/or extremely high values may also be performed. The method for such processing can be determined by those skilled in the art through methods commonly used in the field, taking into account the number of objects constituting the object group (the number of measured values), the distribution of the content data, the purpose of implementing the selection method, etc.

For example, the processing for excluding extremely low and/or extremely high values can be performed by excluding values detected as outliers using any method commonly used in the field of data analysis, or by excluding values before and/or after an arbitrarily set proportion or value.

As a more specific implementation, in order to exclude extremely low values, all values of content below the displayed measurement sensitivity can be set to 0 (zero).

In another embodiment, in order to exclude extremely high values, for example, the values of the first 30, first 20, first 10, or first 5 can be processed by taking the 30th, 20th, 10th, or 5th value from the top, respectively.

Regarding vector similarity, for example, in the case of cosine similarity, as mentioned above, the closer the similarity is to 1, the closer the vectors of x and y are to having the same orientation. That is, when the cosine similarity is 1, IL-6 exhibits the same behavior as the biomarker candidate, and the closer it is to 1, the higher the similarity of behavior.

Regarding the similarity of vectors used as the selection criterion for biomarker candidates, it can be appropriately set by those skilled in the art using common methods in the field, taking into account the purpose of implementing the biomarker selection method for predicting IL-6 content and the subsequent steps to be implemented. If the goal is to obtain an effective biomarker that can predict IL-6 content, it is preferably set to, for example, about 0.5 or more, about 0.6 or more, about 0.7 or more, about 0.8 or more, or about 0.9 or more, and more preferably about 0.6 or more.

[Process (b)]

The selection method for biomarkers used to predict IL-6 levels includes clustering the object group based on the levels of the biomarker candidates selected in (a).

In one implementation, clustering is preferably a teacherless machine learning-based classification of data on the content of all biomarker candidates selected in (a).

As one implementation method in this case, it can be based on hierarchical clustering, or on non-hierarchical clustering methods such as k-means or K-medoids. There is no limitation on the range of clustering that can be performed, but non-hierarchical clustering is preferred, and K-medoids is more preferred.

The number of clusters into which a group of objects can be clustered can be determined appropriately by those skilled in the art using methods commonly found in the field. The objects can be clustered into 2, 3, or 4 clusters, with 3 clusters being preferred.

As an implementation method, for example, when using the k-means or K-medoids method for clustering, the number of clusters needs to be set during implementation.

[Process (c)]

In one embodiment, the method for selecting biomarkers for predicting IL-6 levels further includes: selecting the biomarker candidate selected in (a) as a biomarker candidate if the clustering results in (b) can identify the target with high reliability, based on the clustering results in (b).

In one implementation, the method for selecting the biomarker for predicting IL-6 levels may also include in (c):

(d) The results obtained by identifying objects using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) are compared with the clustering results in (b) to obtain the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) Reliability is achieved based on the sensitivity and specificity obtained in (d); and

(f) Determine whether biomarker candidates can identify objects with high reliability.

In one implementation, reliability is preferably represented by the AUC of the ROC curve obtained based on sensitivity and specificity.

In one embodiment, in (f), it is preferable that the AUC is about 0.7 or more, about 0.8 or more, or about 0.9 or more, and more preferably that the AUC is about 0.8 or more, so that the biomarker candidate can be identified with high reliability.

In one implementation, the method for selecting biomarkers to predict IL-6 levels can also be a computer-implemented method.

Here, we will explain the case that includes (d) to (f) as one implementation of (c), for example, in (b) we assume that the object group is clustered into 3 clusters based on the content of biomarker candidate substances.

The cluster with higher levels of biomarker candidate substances (containing one or two clusters) among the three clusters obtained through clustering can be designated as the "objects with higher IL-6 levels", and the other clusters can be designated as the "objects with lower IL-6 levels". This is the result of clustering in "(b)".

In (d), the “sensitivity” and “specificity” of the “biopter candidate material selected in (a)” can be obtained, for example, as follows.

First, for the content of the biomarker candidate substances selected in (a), an arbitrary series of threshold candidate values are set to identify each object as either "object with high IL-6 content" (content above or higher than the threshold candidate value) or "object with low IL-6 content" (content below the threshold candidate value). Then, the identification results of "objects with high IL-6 content" and "objects with low IL-6 content" obtained using each threshold candidate value are compared with the clustering results of "objects with high IL-6 content" and "objects with low IL-6 content" (clustering results in (b)). Thus, for the case where each threshold candidate value is used, the sensitivity and specificity of the identification of "objects with high IL-6 content" and "objects with low IL-6 content" can be obtained.

More specifically, the identification results of "objects with high IL-6 levels" based on a certain threshold candidate value are compared with the clustering results of "objects with high IL-6 levels" (the clustering results in (b)). This yields the probability (sensitivity) that "objects with high IL-6 levels" can be accurately identified based on that threshold candidate value. Furthermore, the identification results of "objects with low IL-6 levels" based on a certain threshold candidate value are compared with the clustering results of "objects with low IL-6 levels" (the clustering results in (b)). This yields the probability (specificity) that "objects with low IL-6 levels" can be accurately identified based on that threshold candidate value.

By repeatedly performing this method, the sensitivity and specificity corresponding to each of any series of threshold candidate values can be obtained.

In (e), reliability is obtained based on the sensitivity and specificity obtained in (d). Reliability can be represented, for example, by the area under the ROC curve (AUC) plotted based on sensitivity and specificity.

That is, the AUC obtained in the above manner represents the reliability of the identification results obtained by comparing the identification results obtained after using the biomarker candidate substance to identify the object with the clustering results in (b).

In (f), it is determined whether the biomarker candidate can identify the target with high reliability. This determination is preferably based on the reliability expressed by AUC, as described above. Then, if the AUC is about 0.7 or more, about 0.8 or more, or about 0.9 or more, and more preferably if the AUC is about 0.8 or more, it is determined that the biomarker candidate can identify the target with high reliability.

By using the above methods for selecting biomarkers to predict IL-6 content, effective biomarkers can be selected for predicting IL-6 content in samples obtained from the subject.

==Selection device for biomarkers used to predict IL-6 levels==

The biomarker selection apparatus for predicting IL-6 content in this embodiment is an apparatus for implementing the method described in the above-described "method for selecting biomarkers for predicting IL-6 content".

That is, the biomarker selection device for predicting IL-6 levels according to this embodiment includes:

(a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the content of IL-6 in samples taken from the target group and the content of biomarker candidate substances; and

(b) Clustering unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a).

In one embodiment, the biomarker selection device for predicting IL-6 levels further comprises:

(c) Selection section: In comparison with the clustering results in (b), the biomarker candidate selected in (a) is selected as the biomarker candidate if it can identify the object with high reliability.

In one embodiment, the selection unit of the biomarker selection device (c) for predicting IL-6 levels includes:

(d) The acquisition unit compares the results obtained by identifying the object using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) with the clustering results in (b), thereby obtaining the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) The acquisition unit, which acquires reliability based on the sensitivity and specificity obtained in (d); and

(f) Judgment unit, which determines whether biomarker candidate substances can identify objects with high reliability.

In the biomarker selection apparatus for predicting IL-6 content, the embodiments and preferred embodiments of "subject group", "IL-6 related disease", etc., and each step of (a) to (f) are as described in the section "Method for selecting biomarkers for predicting IL-6 content" above.

==Selection Methods for Biomarkers to Predict Subject Response to Therapies Based on IL-6 Signaling Pathway Blockers==

In one embodiment, the above-described "method for selecting biomarkers for predicting IL-6 levels" can be used as a method for selecting biomarkers that can use IL-6 as a biomarker to predict the responsiveness of a subject to treatment based on substances that block the IL-6 signaling pathway.

That is, methods for selecting biomarkers to predict a subject's responsiveness to treatment based on substances that block the IL-6 signaling pathway include:

(a) Biomarker candidates are selected based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from the subject group; and

(b) Cluster the object group based on the content of the biomarker candidate substances selected in (a).

In one embodiment, the method for selecting biomarkers for predicting a subject's responsiveness to treatment based on an IL-6 signaling pathway inhibitor further includes:

(c) In contrast to the clustering results in (b), the biomarker candidates selected in (a) are selected as biomarker candidates if they can identify objects with high reliability.

In one implementation, the method for selecting biomarkers for predicting a subject's responsiveness to treatment based on an IL-6 signaling pathway inhibitor includes, in (c):

(d) The results obtained by identifying objects using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) are compared with the clustering results in (b) to obtain the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) Reliability is achieved based on the sensitivity and specificity obtained in (d); and

(f) Determine whether biomarker candidates can identify objects with high reliability.

Therefore, unless otherwise specified, the following methods for selecting biomarkers to predict the responsiveness of a subject to treatment based on substances that block the IL-6 signal transduction pathway, including the "subject group," "IL-6-related diseases," and the various embodiments and preferred embodiments of each step in (a) to (f), are as described in the section "Method for selecting biomarkers to predict IL-6 levels" above.

Additionally, “IL-6 signal transduction pathway inhibitors” are described in the “Explanation of Terms” section above.

Here, in one embodiment, the subject group in the method for selecting biomarkers for predicting the responsiveness of subjects to treatment based on IL-6 signaling pathway inhibitors is preferably a subject group suspected of having IL-6-related disease or a subject group with IL-6-related disease.

Therefore, in one embodiment, when the subject group is a group of subjects suspected of having IL-6-related diseases, the method for selecting biomarkers to predict the responsiveness of subjects to treatment based on IL-6 signal transduction pathway inhibitors can be used as a method for selecting biomarkers to predict whether each subject in the group of subjects suspected of having IL-6-related diseases has IL-6-related diseases.

In this case, IL-6-related diseases are preferably diseases in which the IL-6 content in the sample or the presence or absence of IL-6 varies depending on whether the subject has the disease.

In another embodiment, when the subject group is a subject group suffering from IL-6-related disease, the method for selecting biomarkers to predict the responsiveness of subjects to treatment based on IL-6 signaling pathway inhibitors can be used as a method for selecting biomarkers to predict whether individuals constituting the subject group suffering from IL-6-related disease are, in particular, responsive to treatment based on IL-6 signaling pathway inhibitors.

In this case, IL-6-related diseases are preferably diseases in which the IL-6 content in the sample or the presence or absence of IL-6 changes based on the responsiveness of each subject in the group of subjects with IL-6-related diseases to treatment based on substances that act on the IL-6 signaling pathway.

In one implementation, the group of subjects in the method for selecting biomarkers to predict the responsiveness of subjects to treatment based on IL-6 signaling pathway inhibitors is preferably a group of subjects with PAH.

This report details the elevated serum IL-6 levels and high IL-6 receptor expression in diseased organs during PAH, as well as the effectiveness of treating PAH using IL-6 signaling pathway inhibitors. Specifically, PAH can be treated by inhibiting the action of IL-6, and the IL-6 level in PAH patients is considered an indicator of responsiveness to treatment based on IL-6 signaling pathway inhibitors.

Therefore, the biomarker selection method of this embodiment can be used for the following purposes: to obtain biomarkers for predicting the responsiveness of PAH subjects to treatment based on IL-6 signaling pathway inhibitors; and/or to obtain biomarkers that can be used in conjunction with or in place of IL-6 to represent the responsiveness of subjects to treatment based on IL-6 signaling pathway inhibitors.

==Selection device for biomarkers used to predict the responsiveness of subjects to treatments based on substances that block the IL-6 signaling pathway==

The biomarker selection device for predicting the responsiveness to treatment based on IL-6 signal transduction pathway inhibitors in this embodiment is an apparatus for implementing the method described in the above-described "Method for Selecting Biomarkers for Predicting the Response to Treatment Based on IL-6 Signal Transduction Pathway Inhibitors".

Furthermore, the selection device described in the above-mentioned item "Selection device for biomarkers for predicting IL-6 content" can be used as a selection device for biomarkers for predicting the responsiveness to treatment based on substances that block the IL-6 signal transduction pathway.

That is, the biomarker selection device for predicting the response to treatment based on substances that block the IL-6 signaling transduction pathway according to this embodiment includes:

(a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the content of IL-6 in samples taken from the target group and the content of biomarker candidate substances; and

(b) Clustering unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a).

In one embodiment, the biomarker selection device for predicting the response to treatment based on an IL-6 signaling pathway inhibitor further comprises:

(c) Selection section: In comparison with the clustering results in (b), the biomarker candidate selected in (a) is selected as the biomarker candidate if it can identify the object with high reliability.

In one embodiment, the selection unit of the biomarker selection device (c) for predicting the response to treatment based on an IL-6 signaling pathway inhibitor includes:

(d) The acquisition unit compares the results obtained by identifying the object using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) with the clustering results in (b), thereby obtaining the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) The acquisition unit, which acquires reliability based on the sensitivity and specificity obtained in (d); and

(f) Judgment unit, which determines whether biomarker candidate substances can identify objects with high reliability.

In the apparatus for selecting biomarkers for predicting the responsiveness to treatment based on substances that block the IL-6 signal transduction pathway, the embodiments and preferred embodiments of "subject group", "IL-6 related disease", etc., and each step in (a) to (f) are as described in the section "Method for selecting biomarkers for predicting the responsiveness to treatment based on substances that block the IL-6 signal transduction pathway".

==A method for selecting biomarkers that are highly likely to be responsive to therapy based on substances that block the IL-6 signaling pathway==

By predicting the responsiveness of subjects to treatments based on IL-6 signaling pathway blockers, subjects with a higher probability of responding to such treatments can be selected.

Therefore, in one embodiment, the above-described "method for selecting biomarkers for predicting the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor" can be used as a method for selecting biomarkers for subjects that are highly likely to be responsive to treatment based on an L-6 signaling pathway inhibitor.

That is, selection methods for biomarkers used to select subjects with a high probability of responding to treatment based on substances that block the L-6 signaling pathway include:

(a) Biomarker candidates are selected based on the similarity of vectors obtained using the content of IL-6 and the content of biomarker candidates in samples taken from the object group.

(b) Cluster the object group based on the content of the biomarker candidate substances selected in (a).

In one embodiment, the method for selecting biomarkers for selecting subjects more likely to respond to treatment based on L-6 signaling pathway inhibitors further includes:

(c) In contrast to the clustering results in (b), the biomarker candidates selected in (a) are selected as biomarker candidates if they can identify objects with high reliability.

In one implementation, the method for selecting biomarkers for subjects more likely to respond to treatment based on L-6 signaling pathway inhibitors includes (c):

(d) The results obtained by identifying objects using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) are compared with the clustering results in (b) to obtain the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) Reliability is achieved based on the sensitivity and specificity obtained in (d); and

(f) Determine whether biomarker candidates can identify objects with high reliability.

Therefore, unless otherwise specified, in the method for selecting biomarkers for subjects that are highly likely to respond to treatment based on IL-6 signal transduction pathway inhibitors, the embodiments and preferred embodiments of "subject group", "IL-6 related disease", etc., and each step in (a) to (f) are as described in the section "Method for selecting biomarkers for predicting the responsiveness of subjects to treatment based on IL-6 signal transduction pathway inhibitors" above.

==Selection device for biomarkers used to select subjects with a high probability of responding to treatment based on substances that block the IL-6 signaling pathway==

The biomarker selection device of this embodiment for selecting subjects that are highly likely to respond to treatment based on IL-6 signal transduction pathway inhibitors is an apparatus for implementing the method described in the above-described "method for selecting biomarkers for selecting subjects that are highly likely to respond to treatment based on IL-6 signal transduction pathway inhibitors".

By predicting the responsiveness of subjects to treatments based on IL-6 signaling pathway blockers, subjects with a higher probability of responding to such treatments can be selected.

Therefore, in one embodiment, the above-described "selection device for biomarkers for predicting the responsiveness of a subject to treatment based on IL-6 signal transduction pathway inhibitors" can be used as a selection device for biomarkers of subjects that are highly likely to be responsive to treatment based on IL-6 signal transduction pathway inhibitors.

That is, the biomarker selection device of this embodiment for selecting subjects with a high probability of responding to treatment based on substances that block the IL-6 signaling transduction pathway includes:

(a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the content of IL-6 in samples taken from the target group and the content of biomarker candidate substances; and

(b) Clustering unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a).

In one embodiment, the selection device for selecting biomarkers for subjects more likely to respond to treatment based on IL-6 signaling pathway inhibitors further comprises:

(c) Selection section: In comparison with the clustering results in (b), the biomarker candidate selected in (a) is selected as the biomarker candidate if it can identify the object with high reliability.

In one embodiment, the selection unit of the selection device (c) for selecting biomarkers of subjects more likely to respond to treatment based on substances that block the IL-6 signaling pathway includes:

(d) The acquisition unit compares the results obtained by identifying the object using each of any series of threshold candidate values of the biomarker candidate substance selected in (a) with the clustering results in (b), thereby obtaining the sensitivity and specificity of the identification using each threshold candidate value of the biomarker candidate substance.

(e) The acquisition unit, which acquires reliability based on the sensitivity and specificity obtained in (d); and

(f) Judgment unit, which determines whether biomarker candidate substances can identify objects with high reliability.

In the biomarker selection device for selecting subjects who are highly likely to respond to treatment based on IL-6 signal transduction pathway inhibitors, the embodiments and preferred embodiments of "subject group", "IL-6 related disease", and each step in (a) to (f) are as described in the above-mentioned "Biomarker Selection Device for Predicting Subject Responsiveness to Treatment Based on IL-6 Signal Transduction Pathway Inhibitors" or "Biomarker Selection Method for Predicting Subject Responsiveness to Treatment Based on IL-6 Signal Transduction Pathway Inhibitors".

==Biomarkers for predicting IL-6 levels in subjects==

The biomarker used in this embodiment to predict the IL-6 content in a subject includes one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

Another embodiment of the use of the biomarker is for predicting the level of IL-6 in a subject, the biomarker comprising one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

In one embodiment of the above-mentioned “biomarker” and “use”, the biomarker may include one or more cytokines selected based on any conditions such as predicted reliability, use, economy and/or ease of measurement, or may include a combination of two or more cytokines.

For example, taking into account, in particular ease of measurement and/or economy, biomarkers for predicting IL-6 levels in subjects preferably contain IL-1β/IL-1F2 or IL-4, and may also contain a combination of IL-1β/IL-1F2 and IL-4.

[object]

Here, the term "object" in the biomarker used to predict the IL-6 content in the object is not limited. It can be any organism in which IL-6 and at least one of the aforementioned biomarkers other than IL-6 are present habitually or temporarily. For example, it can be a mammal or any other animal. Mammals can be humans or non-human animals. Non-human animal species can include, for example, monkeys, dogs, cats, horses, cattle, pigs, sheep, goats, rabbits, guinea pigs, hamsters, mice, and/or rats, but are not limited to the use of livestock, pets, laboratory animals, etc. Mammals are preferred, and humans are more preferred.

That is, in one embodiment, the cytokine used to predict the IL-6 content in the subject is preferably a human-derived cytokine.

In addition, the IL-6 content is preferably the IL-6 content in the blood (whole blood), plasma, or serum of the subject, that is, more preferably the IL-6 content in the blood (whole blood), plasma, or serum of a human subject.

Furthermore, the cytokines used to predict IL-6 levels are preferably cytokines contained in the blood (whole blood), plasma, or serum of the subject, that is, more preferably cytokines contained in the blood (whole blood), plasma, or serum of a human subject.

The object can be selected based on the object's state or can be randomly selected, and can be appropriately determined by those skilled in the art within the range where at least one of the above biomarkers can be used as an indicator for predicting IL-6 levels.

Here, "state" can refer to any state of an object; for example, it can be a state related to a specific disease or a state unrelated to a specific disease.

In the case of a state related to a specific disease, the state is not limited, as long as it is at least partially related to the specific disease. For example, the following states can be exemplified: the state in which the subject is suspected of having a specific disease; the state in which the subject does not have a specific disease; the state in which the subject does not have any subtype of a specific disease; the state in which the subject with a specific disease is responsive to a specific treatment; the state in which the subject's specific disease is progressing; and/or the state in which the symptoms of the subject's specific disease are improving, etc.

In cases where the state is not related to a specific disease, the state as an object can be exemplified by, for example, physiological characteristics, genetic status such as sex or race, age, and/or geographical status such as place of residence or range of movement that are not related to any disease.

[IL-6 related diseases]

In one implementation, the subject is preferably a subject suspected of having IL-6-related disease, or a subject who has IL-6-related disease. Here, "IL-6-related disease" is as described in the "Explanation of Terms" section above.

In one embodiment, an IL-6-related disease can be a disease in which the IL-6 content or presence of IL-6 in the sample changes depending on whether the subject is associated with the disease, or it can be a disease in which the IL-6 content or presence of IL-6 in the sample changes based on the state of the subject suffering from an IL-6-related disease, and the disease can be classified into subtypes based on such changes.

In one implementation, the IL-6-related disease is preferably pulmonary hypertension, that is, the subject is preferably a subject suffering from pulmonary hypertension.

Here, pulmonary hypertension is clinically classified as: pulmonary arterial hypertension (PAH, Group 1), pulmonary hypertension associated with left heart disease (Group 2), pulmonary hypertension associated with lung disease and/or hypoxemia (Group 3), CTEPH (Group 4), and pulmonary hypertension associated with multifactorial mechanisms of unknown details (Group 5) (Guidelines for the Treatment of Pulmonary Hypertension (2017 Revised Edition)).

In one embodiment, pulmonary hypertension is preferably pulmonary arterial hypertension (PAH), that is, the subject is more preferably a subject suffering from PAH.

The inventors of this application have discovered that in patients with PAH, CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1 exhibit behavior similar to IL-6 and serve as indicators of responsiveness to treatments based on substances that utilize the IL-6 signaling pathway.

Therefore, in patients with PAH, IL-6 levels can be predicted by one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

This report describes elevated serum IL-6 levels and high IL-6 receptor expression in diseased organs during PAH, as well as the effectiveness of treating PAH using IL-6 signaling pathway inhibitors. Specifically, PAH can be treated by inhibiting IL-6 activity, and the IL-6 level in PAH patients is considered an indicator of responsiveness to treatment based on IL-6 signaling pathway inhibitors.

Therefore, in one embodiment, when the subject is a PAH patient, the biomarker of this embodiment can be used for the purpose of predicting IL-6 levels; and/or for predicting the subject's responsiveness to treatment based on IL-6 signaling pathway inhibitors, either in combination with or in place of IL-6.

==Methods for predicting IL-6 levels in an object==

The biomarkers mentioned above used to predict the IL-6 content in a subject can be used to predict the IL-6 content in a subject.

Therefore, the method for predicting the IL-6 content in a subject in this embodiment includes: determining the content of the biomarker described in the above-mentioned "Biomarker for predicting the IL-6 content in a subject" in a sample taken from the subject.

In one embodiment, the method for predicting the IL-6 content in a subject preferably includes: predicting the IL-6 content based on the content of the aforementioned biomarkers.

The various embodiments and preferred embodiments of "determination" in this embodiment are described in the section on "methods for determining biomarkers".

Furthermore, the various embodiments and preferred embodiments of "object", "IL-6 related disease", etc. in this embodiment are as described in the section "Biomarker for predicting the content of IL-6 in the object" above.

More specifically here, the levels of IL-6 and the levels of the biomarker can be pre-determined to obtain corresponding data, targeting at least one biomarker described in the item “Biomarkers for predicting the levels of IL-6 in an object” which serves as an indicator of the behavior of IL-6 in the object.

Therefore, by measuring the content of biomarkers in the test subjects, the corresponding IL-6 content can be predicted.

The baseline value (threshold) of the biomarker content that serves as the benchmark for predicting IL-6 content can be appropriately set by those skilled in the art based on the desired level of sensitivity and/or specificity for predicting responsiveness.

For example, when the subject is a person suffering from PAH, i.e., the method of this embodiment is a method for predicting the IL-6 content in a person suffering from PAH, the threshold for the blood content of IL-1β as a biomarker can be referenced with respect to the threshold, sensitivity, and specificity shown in FIG9, and the appropriate selection can be made by those skilled in the art, considering how and to what extent they want to identify the subject group by IL-1β. For example, sensitivity and specificity can be selected in a way that maximizes both and/or maximizes the specificity, or in a way that maximizes the sum of sensitivity and specificity.

Specifically, in one embodiment, the threshold for the blood concentration of IL-1β is preferably one that corresponds to a high level of both sensitivity and specificity. For example, it is preferably a threshold corresponding to a sensitivity and specificity sum of about 1.3 to about 1.6, and more preferably a threshold corresponding to a sensitivity and specificity sum of about 1.4 to about 1.6. Furthermore, it is also possible to consider how and to what extent the IL-1β is used to identify the target group, selecting sensitivity and specificity in a manner that results in a relatively high level of either sensitivity or specificity.

More specifically, the threshold for serum IL-1β levels can be approximately 3.0 to approximately 9.0 pg/mL, approximately 3.5 to approximately 7.0 pg/mL, or approximately 4.0 to approximately 6.0 pg/mL. In one embodiment, referring to Figure 9, the threshold for serum IL-1β levels can also be selected as 4.49 pg/mL, 4.50 pg/mL, 5.04 pg/mL, 5.13 pg/mL, 5.59 pg/mL, or 5.76 pg/mL.

==Biomarkers for predicting a subject's responsiveness to treatments based on substances that block the IL-6 signaling pathway==

In one implementation, the aforementioned "biomarker for predicting the level of IL-6 in a subject" and IL-6 can be used as a biomarker for predicting the subject's responsiveness to treatment based on substances that block the IL-6 signaling pathway.

That is, the biomarker used in this embodiment for predicting the responsiveness of a subject to treatment based on an IL-6 signaling pathway inhibitor comprises one or more cytokines selected from IL-6, CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

Another embodiment of the use of the biomarker is for predicting the subject's response to treatment based on an IL-6 signaling pathway inhibitor, wherein the biomarker comprises one or more cytokines selected from IL-6, CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

In the above-mentioned “biomarkers” and “uses”, in one embodiment, the biomarker for predicting the responsiveness of a subject to treatment based on an IL-6 signal transduction pathway inhibitor may include one or more cytokines selected based on any conditions such as the reliability of prediction, use, economy, and/or ease of measurement, or may include a combination of two or more cytokines.

For example, taking into account, in particular ease of measurement and/or cost-effectiveness, biomarkers used to predict a subject's responsiveness to treatment based on substances that block the IL-6 signaling pathway preferably comprise IL-6, IL-1β/IL-1F2, or IL-4, and may also comprise combinations of two or three of these cytokines. That is, combinations of IL-6 and IL-1β/IL-1F2, IL-6 and IL-4, IL-1β/IL-1F2 and IL-4, or combinations of IL-6, IL-1β/IL-1F2, and IL-4 may also be included.

In this embodiment, unless otherwise specified, the various embodiments and preferred embodiments of "object" and "IL-6 related diseases" are as described in the section "Biomarkers for predicting the content of IL-6 in an object" above.

Additionally, “IL-6 signal transduction pathway inhibitors” are explained in the “Explanation of Terms” section.

The biomarker in this embodiment is used to predict the subject's responsiveness to treatment based on IL-6 signaling pathway inhibitors, thus placing the subject in a state where treatment based on IL-6 signaling pathway inhibitors is applicable.

In one implementation, such a state may be exemplified as: a state of suspected illness for which treatment based on IL-6 signaling pathway inhibitors is applicable; or a state of illness for which treatment based on IL-6 signaling pathway inhibitors is applicable.

In one embodiment, the disease is PAH. Therefore, the biomarker of this embodiment can be suitably used as a biomarker for predicting the responsiveness of PAH subjects to treatment based on substances that block the IL-6 signaling pathway.

This report details the elevated serum IL-6 levels and high IL-6 receptor expression in diseased organs during PAH, as well as the effectiveness of PAH treatment based on IL-6 signaling pathway inhibitors. Specifically, PAH can be treated by inhibiting the action of IL-6, and the IL-6 level in PAH patients is considered an indicator of responsiveness to treatment based on IL-6 signaling pathway inhibitors.

Furthermore, the inventors of this application have discovered that in patients with PAH, CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-IF2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1 exhibit behaviors similar to IL-6, serving as indicators of responsiveness to treatment based on substances from the IL-6 signaling pathway.

Therefore, in subjects with PAH, the responsiveness to treatment based on IL-6 signaling pathway inhibitors can be predicted by using biomarkers containing one or more cytokines selected from IL-6, CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1.

==Methods for predicting the responsiveness of subjects to treatments based on substances that block the IL-6 signaling pathway==

The aforementioned "biomarkers for predicting the responsiveness of subjects to treatment based on IL-6 signal transduction pathway inhibitors" can be used to predict the responsiveness of subjects to treatment based on IL-6 signal transduction pathway inhibitors.

Therefore, the method for predicting the responsiveness of a subject to treatment based on an IL-6 signal transduction pathway inhibitor according to this embodiment includes: determining the content of the biomarker described in the item “Biomarker for predicting the responsiveness of a subject to treatment based on an IL-6 signal transduction pathway inhibitor” in a sample taken from the subject.

In one embodiment, the method for predicting the responsiveness of a subject to treatment based on an IL-6 signal transduction pathway inhibitor preferably includes: determining, based on the content of the aforementioned biomarker, whether the likelihood of being responsive to treatment based on an IL-6 signal transduction pathway inhibitor is high.

The various embodiments and preferred embodiments of "determination" in this embodiment are described in the section on "methods for determining biomarkers".

In this embodiment, the various embodiments and preferred embodiments of "subject", "IL-6 related disease", "IL-6 signal transduction pathway inhibitor", etc. are as described in the section "Biomarker for predicting the subject's response to treatment based on IL-6 signal transduction pathway inhibitor" above.

Here, more specifically, for at least one biomarker described in the item “Biomarker for predicting the responsiveness of a subject to treatment based on IL-6 signal transduction pathway inhibitors”, which serves as an indicator of the subject’s responsiveness to treatment based on IL-6 signal transduction pathway inhibitors, corresponding data for the responsiveness to IL-6 signal transduction pathway inhibitors and the content of the biomarker are obtained in advance.

Therefore, by measuring the content of biomarkers for the test subject, the corresponding responsiveness can be predicted.

The baseline value (threshold) of the biomarker content that serves as the benchmark for responsiveness can be appropriately set by those skilled in the art based on the desired level of sensitivity and/or specificity for predicting responsiveness.

For example, when the subject is a person suffering from PAH, i.e., the method of this embodiment is for predicting the response of a person suffering from PAH to treatment based on an IL-6 signal transduction pathway inhibitor, the threshold for the blood concentration of IL-6 as a biomarker can be referenced to the threshold, sensitivity, and specificity shown in Figure 8, and should be appropriately selected by those skilled in the art, considering how and to what extent they want to identify the subject group using IL-6. For example, sensitivity and specificity can be selected in a way that maximizes both and/or maximizes the sum of sensitivity and specificity.

Specifically, in one embodiment, the threshold for the blood concentration of IL-6 is preferably one that corresponds to a high level of both sensitivity and specificity. For example, it is preferably a threshold corresponding to a sensitivity and specificity of about 1.3 to about 1.6, and more preferably a threshold corresponding to a sensitivity and specificity of about 1.4 to about 1.6. Furthermore, it is also possible to consider how and to what extent the IL-6 is used to identify a target group, selecting sensitivity and specificity in a manner that results in a relatively high level of either sensitivity or specificity.

More specifically, the threshold for the serum IL-6 concentration can be about 1.0 to about 5.0 pg/mL, about 1.5 to about 4.0 pg/mL, or about 2.0 to about 3.0 pg/mL. In one embodiment, referring to Figure 8, the threshold for the serum IL-6 concentration can also be selected as 2.12 pg/mL, 2.29 pg/mL, 2.43 pg/mL, 2.54 pg/mL, 2.73 pg/mL, or 2.79 pg/mL.

Furthermore, for example, when the subject is a person suffering from PAH, i.e., the method of this embodiment is for predicting the response of a person suffering from PAH to treatment based on an IL-6 signaling pathway inhibitor, the threshold for the blood concentration of IL-1β as a biomarker can be selected appropriately by those skilled in the art, taking into account the desired manner or extent to which the subject group is identified by IL-1β. For example, sensitivity and specificity can be selected in a manner where sensitivity and/or specificity are maximized, or in a manner where the sum of sensitivity and specificity is maximized.

Specifically, in one embodiment, the threshold for the blood concentration of IL-1β is preferably a threshold corresponding to a high level of both sensitivity and specificity, for example, a threshold corresponding to a sensitivity and specificity summed to about 1.3 to about 1.6, and more preferably a threshold corresponding to a sensitivity and specificity summed to about 1.4 to about 1.6. Furthermore, it is also possible to consider how or to what extent the IL-1β is used to identify the target group, selecting sensitivity and specificity in a manner that results in a relatively high level of either sensitivity or specificity.

More specifically, the threshold for serum IL-1β levels can be approximately 3.0 to approximately 9.0 pg/mL, approximately 3.5 to approximately 7.0 pg/mL, or approximately 4.0 to approximately 6.0 pg/mL. In one embodiment, referring to Figure 9, the threshold for IL-1β levels can also be selected as 4.49 pg/mL, 4.50 pg/mL, 5.04 pg/mL, 5.13 pg/mL, 5.59 pg/mL, or 5.76 pg/mL.

==Biomarkers for selecting subjects with a high probability of responding to treatments based on substances that block the IL-6 signaling pathway==

By predicting the responsiveness of subjects to treatments based on IL-6 signaling pathway blockers, subjects with a higher probability of responding to such treatments can be selected.

Therefore, in one embodiment, the aforementioned "biomarker for predicting the responsiveness of a subject to treatment based on IL-6 signaling pathway inhibitors" can be used as a biomarker for selecting subjects that are more likely to be responsive to treatment based on IL-6 signaling pathway inhibitors.

Among the biomarkers used to select subjects that are highly likely to respond to treatment based on IL-6 signal transduction pathway inhibitors, the various embodiments and preferred embodiments of "subject", "IL-6-related disease", "IL-6 signal transduction pathway inhibitor", etc. are as described in the section "Biomarkers for predicting the responsiveness of subjects to treatment based on IL-6 signal transduction pathway inhibitors" above.

As described above, in patients with PAH, the responsiveness to treatment based on IL-6 signaling pathway inhibitors can be predicted by any combination of one or more cytokines selected from IL-6, CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1, or a combination of two or more of these cytokines, as illustrated above. For example, taking into account ease of measurement and/or economy, the combination may preferably include IL-6, IL-1β/IL-1F2, or IL-4, or a combination of two or three of these cytokines, i.e., examples may include: a combination of IL-6 and IL-1β/IL-1F2, a combination of IL-6 and IL-4, a combination of IL-1β/IL-1F2 and IL-4, or a combination of IL-6, IL-1β/IL-1F2, and IL-4.

Then, based on this prediction, subjects that are more likely to respond to treatment based on substances that block the IL-6 signaling pathway can be selected.

==A selection method for choosing subjects with a high probability of responding to treatment based on substances that block the IL-6 signaling pathway==

The aforementioned "biomarker for selecting subjects with a high probability of responding to treatment based on IL-6 signal transduction pathway inhibitors" can be used to select subjects with a high probability of responding to treatment based on IL-6 signal transduction pathway inhibitors.

Therefore, the method for selecting subjects that are more likely to respond to treatment based on IL-6 signal transduction pathway inhibitors in this embodiment includes: measuring the content of the biomarkers described in the above-mentioned item "Biomarkers for selecting subjects that are more likely to respond to treatment based on IL-6 signal transduction pathway inhibitors" in a sample taken from the subject.

In one embodiment, the method for selecting subjects that are more likely to respond to treatment based on IL-6 signal transduction pathway inhibitors preferably further includes: determining whether the likelihood of responding to treatment based on IL-6 signal transduction pathway inhibitors is high based on the content of the aforementioned biomarker.

In one embodiment, the method for selecting subjects that are more likely to be responsive to treatment based on IL-6 signal transduction pathway inhibitors more preferably includes: selecting subjects that are judged to be more likely to be responsive to treatment based on IL-6 signal transduction pathway inhibitors.

Here, the various embodiments and preferred embodiments of "object", "IL-6 related disease", "IL-6 signal transduction pathway inhibitor" etc. in this embodiment are as described in the above-mentioned section "Biomarker for selecting objects that are highly likely to respond to treatment based on IL-6 signal transduction pathway inhibitor".

In addition, the various embodiments and preferred embodiments of "determination" in this embodiment are described in the section on "method for determination of biomarkers".

More specifically here, for at least one biomarker described in the item “Biomarker for selecting subjects with a high probability of responding to treatment based on IL-6 signal transduction pathway inhibitors”, which serves as an indicator of whether the likelihood of responding to treatment based on IL-6 signal transduction pathway inhibitors is high, corresponding data corresponding to the responsiveness to IL-6 signal transduction pathway inhibitors and the content of the biomarker can be obtained in advance.

Therefore, the levels of biomarkers can be measured for the subjects being tested, and their corresponding responsiveness can be predicted. Based on this predicted responsiveness, subjects with a higher probability of responding to treatments based on substances that block the IL-6 signaling pathway can be selected.

The baseline value (threshold) of the biomarker content that serves as the benchmark for responsiveness can be appropriately set by those skilled in the art based on the desired level of sensitivity and/or specificity to predict responsiveness.

For example, when the target is a patient with PAH, i.e., when the method of this embodiment is for selecting PAH patients who are highly likely to respond to treatment based on an IL-6 signal transduction pathway inhibitor, the threshold for the blood concentration of IL-6 as a biomarker can be referenced to the threshold, sensitivity, and specificity shown in Figure 8, and appropriately selected by those skilled in the art, considering how and to what extent they want to identify the target group using IL-6. For example, sensitivity and specificity can be selected in a way that maximizes both and/or maximizes the sum of sensitivity and specificity.

Specifically, in one embodiment, the threshold for the blood concentration of IL-6 is preferably one that corresponds to a high level of both sensitivity and specificity. For example, it is preferably a threshold corresponding to a sensitivity and specificity of about 1.3 to about 1.6, and more preferably a threshold corresponding to a sensitivity and specificity of about 1.4 to about 1.6. Furthermore, it is also possible to consider how and to what extent the IL-6 is used to identify a target group, selecting sensitivity and specificity in a manner that results in a relatively high level of either sensitivity or specificity.

More specifically, the threshold for the serum IL-6 concentration can be about 1.0 to about 5.0 pg/mL, about 1.5 to about 4.0 pg/mL, or about 2.0 to about 3.0 pg/mL. In one embodiment, referring to Figure 8, the threshold for the serum IL-6 concentration can also be selected as 2.12 pg/mL, 2.29 pg/mL, 2.43 pg/mL, 2.54 pg/mL, 2.73 pg/mL, or 2.79 pg/mL.

Furthermore, for example, when the target is a patient with PAH, i.e., when the method of this embodiment is for selecting PAH patients who are highly likely to respond to treatment based on IL-6 signaling pathway inhibitors, the threshold for the blood concentration of IL-1β as a biomarker can be referenced to the threshold, sensitivity, and specificity shown in FIG9, and appropriately selected by those skilled in the art considering how and to what extent they want to identify the target group by IL-1β. For example, sensitivity and specificity can be selected in a way that maximizes both and/or maximizes the sum of sensitivity and specificity.

Specifically, in one embodiment, the threshold for the blood concentration of IL-1β is preferably one that corresponds to a high level of both sensitivity and specificity. For example, it is preferably a threshold corresponding to a sensitivity and specificity sum of about 1.3 to about 1.6, and more preferably a threshold corresponding to a sensitivity and specificity sum of about 1.4 to about 1.6. Furthermore, it is also possible to consider how and to what extent the IL-1β is used to identify the target group, selecting sensitivity and specificity in a manner that results in a relatively high level of either sensitivity or specificity.

More specifically, the threshold for serum IL-1β levels can be approximately 3.0 to approximately 9.0 pg/mL, approximately 3.5 to approximately 7.0 pg/mL, or approximately 4.0 to approximately 6.0 pg/mL. In one embodiment, referring to Figure 9, the threshold for IL-1β levels can also be selected as 4.49 pg/mL, 4.50 pg/mL, 5.04 pg/mL, 5.13 pg/mL, 5.59 pg/mL, or 5.76 pg/mL.

==Methods for determining biomarkers==

The method for determining the biomarker in this embodiment includes: determining the content of a biomarker selected as one or more cytokines from IL-6, CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1 in a sample taken from the subject.

In one embodiment, the assay method can measure one or more cytokines selected based on any conditions such as predictive reliability, application, economy, and/or ease of measurement, or it can measure a combination of two or more cytokines.

For example, considering, in particular ease of measurement and/or economy, a method for measuring any one of IL-6, IL-1β/IL-1F2, or IL-4 is preferred, or a method for measuring two or three of these cytokines in combination. That is, a method for measuring a combination of IL-6 and IL-1β/IL-1F2, a combination of IL-6 and IL-4, a combination of IL-1β/IL-1F2 and IL-4, or a combination of IL-6, IL-1β/IL-1F2, and IL-4 is also possible.

The biomarker determination method of this embodiment can determine at least one biomarker described in the items "biomarker for predicting the IL-6 content in a subject", "biomarker for predicting the responsiveness of a subject to treatment based on an IL-6 signal transduction pathway inhibitor", and/or "biomarker for selecting subjects with a higher probability of responding to treatment based on an IL-6 signal transduction pathway inhibitor". Therefore, the biomarker determination method of this embodiment can collect data for the following purposes: predicting the IL-6 content in a subject; predicting the responsiveness of a subject to treatment based on an IL-6 signal transduction pathway inhibitor; and/or selecting subjects with a higher probability of responding to treatment based on an IL-6 signal transduction pathway inhibitor.

[object]

The “subject” in this assay method can be appropriately selected based on the purpose for which data is collected. Unless otherwise specified, the embodiments and preferred embodiments described below are as follows: “Biomarker for predicting the IL-6 content in the subject”, “Biomarker for predicting the responsiveness of the subject to treatment based on IL-6 signal transduction pathway inhibitors”, and/or “Biomarker for selecting subjects that are more likely to be responsive to treatment based on IL-6 signal transduction pathway inhibitors”.

[Determination of biomarkers in samples]

The sample is a biological sample taken from the subject group, and is not limited to the range in which at least one of the above-mentioned biomarkers is present in a measurable amount. For example, it may be a solid tissue sample obtained during a biopsy or treatment, or it may be blood (whole blood), plasma, or serum.

In one embodiment, where the subject is a human patient with PAH, the sample is preferably blood (whole blood), plasma, or serum, more preferably plasma or serum.

It is preferable to pretreat the sample before it is used for testing. For example, when using plasma or serum, it is preferable to treat the whole blood by standing or centrifugation to separate the plasma or serum from other blood components.

The content of biomarkers for cytokines in a sample taken from an object can be determined using methods commonly used in the field for measuring cytokines, without limitation on the range that can be measured.

In one embodiment, the cytokine levels are preferably determined, for example, using an antigen-antibody reaction-based method that utilizes antibodies that specifically bind to each cytokine. Examples of antigen-antibody reaction-based methods include, for instance, Western blotting, ELISA (enzyme-conjugated immunosorbent assay), FCM (flow cytometry), antibody array assays, etc., which may be appropriately selected by those skilled in the art depending on the purpose of the assay.

In one embodiment, the method for visualizing and quantifying antigen-antibody reactions can be any method commonly used in the field. For example, as a visualization method, radioisotope methods, fluorescence methods, enzymatic methods, and/or chemiluminescence methods can be used. Additionally, for quantification, direct or indirect methods can be used, or sensitization methods utilizing biotin-avidin complexes or labeled avidin can be employed.

Here, the content of the cytokine as a biomarker can be any of the following: the absolute value of the biomarker, the relative concentration, the weight per unit volume, or the raw data measured to obtain the absolute concentration.

In one embodiment, when plasma or serum is used as a sample for determination, the weight per unit volume of the sample, such as 1 mL or 1 μL, is preferred.

==Set==

The kit of this embodiment includes components for implementing the determination method described in the "Method for Determination of Biomarkers" above.

In one embodiment, the kit preferably includes at least one of the biomarkers described in the items “biomarker for predicting the level of IL-6 in a subject”, “biomarker for predicting the responsiveness of a subject to treatment based on an IL-6 signal transduction pathway inhibitor”, and/or “biomarker for selecting subjects that are more likely to be responsive to treatment based on an IL-6 signal transduction pathway inhibitor”.

Therefore, it can be used to predict the IL-6 level in a subject; to predict the subject's responsiveness to treatment based on IL-6 signaling pathway inhibitors; and/or to select subjects that are more likely to respond to treatment based on IL-6 signaling pathway inhibitors.

Here, regarding this kit, the various embodiments and preferred embodiments of "subject", "IL-6 related disease", "IL-6 signal transduction pathway inhibitor", etc., are as described above in the items "Biomarker for predicting the IL-6 content in the subject", "Biomarker for predicting the subject's responsiveness to treatment based on IL-6 signal transduction pathway inhibitor", and/or "Biomarker for selecting subjects with a high probability of responding to treatment based on IL-6 signal transduction pathway inhibitor".

Here, in one embodiment, the kit preferably includes an antibody that specifically binds to at least one of the biomarkers described in the items "Biomarker for predicting the level of IL-6 in a subject," "Biomarker for predicting the responsiveness of a subject to treatment based on an IL-6 signal transduction pathway inhibitor," and/or "Biomarker for selecting subjects with a higher probability of responding to treatment based on an IL-6 signal transduction pathway inhibitor," as a component for performing the assay method described in the item "Method for Assaying Biomarkers." That is, it preferably includes an antibody that specifically binds to at least one cytokine contained in the biomarkers described in the items "Biomarker for predicting the level of IL-6 in a subject," "Biomarker for predicting the responsiveness of a subject to treatment based on an IL-6 signal transduction pathway inhibitor," and/or "Biomarker for selecting subjects with a higher probability of responding to treatment based on an IL-6 signal transduction pathway inhibitor."

By using this antibody, the content of cytokines in a sample can be determined using antigen-antibody reaction-based methods such as Western blotting, ELISA (enzyme-conjugated immunosorbent assay), and antibody array methods.

In one embodiment, the kit is a reagent for visualization and quantification of antigen-antibody reactions, and may include, for example, radioactive isotopes, fluorescent dyes, enzymes, beads, and/or chemiluminescent matrices.

In another embodiment, the kit may also contain various buffer solutions or concentrates thereof required for the determination of biomarkers.

In one embodiment, the kit may also contain standard biomarker samples.

In one embodiment, the kit may also include containers for storing and processing samples taken from the object.

==Drugs, treatment methods, IL-6 signal transduction pathway inhibitors, uses of IL-6 signal transduction pathway inhibitors==

The subjects selected by the above-described "selection method for subjects with a high probability of responding to treatment based on IL-6 signal transduction pathway inhibitors" are highly likely to respond to treatment based on IL-6 signal transduction pathway inhibitors.

Therefore, this method allows for the exclusion of individuals in advance from treatments based on IL-6 signal transduction pathway inhibitors, whose therapeutic effects may be unattainable or very low. Instead, treatment based on IL-6 signal transduction pathway inhibitors can be applied only to individuals who are highly likely to respond to the treatment, which is effective for personalized medicine.

The medicine of this embodiment is a medicine containing an IL-6 signal transduction pathway inhibitor, which is administered to a subject that is selected as a subject that is highly likely to respond to treatment based on an IL-6 signal transduction pathway inhibitor by means of the selection method described above in "A method for selecting a subject that is highly likely to respond to treatment based on an IL-6 signal transduction pathway inhibitor".

Another embodiment of the treatment method includes: administering a therapeutically effective amount of an IL-6 signal transduction pathway inhibitor to a subject that is selected as a subject that is highly likely to respond to treatment based on an IL-6 signal transduction pathway inhibitor by means of the selection method described above in "A method for selecting a subject that is highly likely to respond to treatment based on an IL-6 signal transduction pathway inhibitor".

Here, "therapeutic effective amount" refers to the amount that can continuously or temporarily obtain some preferred results in the subject. Examples of "preferred results" include: inhibition or improvement of the progression of the subject's systemic symptoms, inhibition or improvement of the progression of pathological symptoms, inhibition or improvement of the deterioration of examination data reflecting the disease state, and/or improvement of the subject's prognosis.

In another embodiment, the IL-6 signal transduction pathway inhibitor is administered to a subject that is selected as a subject that is highly likely to respond to treatment based on the IL-6 signal transduction pathway inhibitor by means of the selection method described in the above-described "Selection Method for Selecting Subjects with High Probability of Responding to Treatment Based on IL-6 Signal Transduction Pathway Inhibitor".

Another application of this embodiment is the use of the IL-6 signal transduction pathway inhibitor in the manufacture of a pharmaceutical product, which is administered to an object that is selected as having a high probability of responding to treatment based on the IL-6 signal transduction pathway inhibitor by means of the selection method described above in "A method for selecting an object that is highly likely to respond to treatment based on the IL-6 signal transduction pathway inhibitor".

In each embodiment of the above-mentioned “medicine,” “treatment method,” “IL-6 signal transduction pathway inhibitor,” and/or “use of IL-6 signal transduction pathway inhibitor,” the embodiments and preferred embodiments of “subject,” “IL-6-related disease,” “IL-6 signal transduction pathway inhibitor,” etc., are described in the above-mentioned “method for selecting subjects that are more likely to respond to treatment based on IL-6 signal transduction pathway inhibitor.”

That is, as a preferred embodiment of the "medicine", an example of a medicine for treating PAH can be given, which contains an IL-6 signal transduction pathway inhibitor and is administered to a PAH patient, i.e., the patient is selected as having a high probability of responding to treatment based on the IL-6 signal transduction pathway inhibitor by the selection method described above in "Selection Method for Selecting a Subject with a High Probability of Responding to Treatment Based on the IL-6 Signal Transduction Pathway Inhibitor".

In addition, PAH can be treated by inhibiting the action of IL-6. Therefore, substances that block the IL-6 signaling pathway are effective in treating PAH.

As another embodiment of "medicine", examples include medicines for treating PAH that contain substances that block the IL-6 signal transduction pathway.

As another embodiment of the "medicine", one example is a drug for treating PAH containing an IL-6 signal transduction pathway inhibitor, which is administered to PAH patients in whom the IL-6 level in the serum is 2.73 (pg/mL) or higher.

The serum content of IL-6 can be determined, for example, by the method described in the section on [Determination of Biomarkers in Samples] of "Methods for the Determination of Biomarkers".

Here, the "IL-6 signal transduction pathway inhibitor" in the pharmaceutical product, as described in the "Explanation of Terms" section above, is preferably an antibody or its antigen-binding fragment that has the function of inhibiting the IL-6 signal transduction pathway, and more preferably an anti-IL-6 receptor antibody or its antigen-binding fragment.

In one embodiment, as an anti-IL-6 receptor antibody, an antibody comprising at least one of the three supervariable regions (CDRs) in the amino acid sequence of the heavy chain variable region containing sequence number 1, and/or comprising at least one of the three supervariable regions (CDRs) in the amino acid sequence of the light chain variable region containing sequence number 2 can be exemplified.

In one embodiment, an antibody against the IL-6 receptor may be exemplified as comprising a heavy chain containing an amino acid sequence of sequence number 3 and a light chain containing an amino acid sequence of sequence number 4.

In one embodiment, examples of anti-IL-6 receptor antibodies include: salidomide, tocilizumab, and/or salidomide, preferably salidomide and/or tocilizumab, more preferably salidomide.

In one embodiment, the medicine may be a medicine that is administered simultaneously, continuously, or separately with other therapeutic agents that can be used to treat PAH, or it may be formulated with such therapeutic agents into a single formulation.

In addition, pharmaceutical products can also be formulated into tablets, granules, powders, capsules, emulsions, suspensions, syrups, or sterile solutions, suspensions, and other injectable forms containing any pharmacologically permissible carriers, excipients, or stabilizers.

In addition, as a preferred embodiment of the "treatment method", a treatment method for PAH can be listed as follows, which includes: administering a therapeutically effective amount of an IL-6 signal transduction pathway inhibitor to a PAH patient, i.e., being selected as a patient who is highly likely to respond to treatment targeting the IL-6-related cell signaling system by means of the selection method described above in "Selection Method for Selecting Patients with High Probability of Responding to Treatment Based on IL-6 Signal Transduction Pathway Inhibitor".

As another implementation of the "treatment method", the following treatment method for PAH can be listed, which includes: delivering a therapeutically effective amount of IL-6 signal transduction pathway inhibitor to the PAH subject who needs treatment.

As another implementation of the "treatment method", the following treatment method for PAH can be listed, which includes: administering an IL-6 signal transduction pathway inhibitor to a PAH patient who requires treatment and whose serum IL-6 concentration is 2.73 (pg/mL) or higher.

The serum level of IL-6 can be determined by methods described, for example, in the section on "Determination of Biomarkers in Samples" under "Methods for the Determination of Biomarkers".

Here, the "IL-6 signal transduction pathway inhibitor" in the treatment method, as described in the "Explanation of Terms" section above under "[IL-6 signal transduction pathway inhibitor]", is preferably an antibody or its antigen-binding fragment that has the function of inhibiting the IL-6 signal transduction pathway, and more preferably an anti-IL-6 receptor antibody or its antigen-binding fragment.

In one embodiment, an anti-IL-6 receptor antibody may be exemplified as an antibody comprising at least one of three supervariable regions (CDRs) in the amino acid sequence of the heavy chain variable region containing sequence number 1, and/or at least one of three supervariable regions (CDRs) in the amino acid sequence of the light chain variable region containing sequence number 2.

In one embodiment, an anti-IL-6 receptor antibody may be exemplified, for example, by comprising a heavy chain containing an amino acid sequence of sequence number 3 and a light chain containing an amino acid sequence of sequence number 4.

In one embodiment, examples of anti-IL-6 receptor antibodies include: salidomide, tocilizumab, and/or salidomide, preferably salidomide and/or tocilizumab, more preferably salidomide.

In one embodiment, in the treatment method, a therapeutically effective amount of the IL-6 signaling inhibitor may be administered simultaneously, continuously, or separately with other therapeutic agents that can be used to treat PAH, or it may be administered in a single formulation with such therapeutic agents.

As a preferred embodiment of the "IL-6 signal transduction pathway inhibitor", an IL-6 signal transduction pathway inhibitor for treating PAH can be listed below. The inhibitor is administered to a PAH patient who is selected as a patient who is highly likely to respond to treatment based on the IL-6 signal transduction pathway inhibitor by the selection method described in the above-described "Selection Method for Selecting a Subject with a High Probability of Responding to Treatment Based on the IL-6 Signal Transduction Pathway Inhibitor".

As another implementation of "IL-6 signal transduction pathway inhibitors", examples of IL-6 signal transduction pathway inhibitors used in the treatment of PAH can be listed.

As another embodiment of the "IL-6 signal transduction pathway inhibitor", the following IL-6 signal transduction pathway inhibitors can be listed for the treatment of PAH in subjects with PAH whose serum IL-6 content is 2.73 (pg/mL) or higher.

The serum content of IL-6 can be determined, for example, by the method described in the section on [Determination of Biomarkers in Samples] of "Methods for the Determination of Biomarkers".

Here, "IL-6 signal transduction pathway inhibitor" as described in the "Explanation of Terms" section above, is preferably an antibody or its antigen-binding fragment that has the function of blocking the IL-6 signal transduction pathway, and more preferably an anti-IL-6 receptor antibody or its antigen-binding fragment.

In one embodiment, an anti-IL-6 receptor antibody may be exemplified as an antibody comprising at least one of three supervariable regions (CDRs) in the amino acid sequence of the heavy chain variable region containing sequence number 1, and/or at least one of three supervariable regions (CDRs) in the amino acid sequence of the light chain variable region containing sequence number 2.

In one embodiment, an anti-IL-6 receptor antibody may be exemplified, for example, by comprising a heavy chain containing an amino acid sequence of sequence number 3 and a light chain containing an amino acid sequence of sequence number 4.

In one embodiment, examples of anti-IL-6 receptor antibodies include: salidomide, tocilizumab, and/or salidomide, preferably salidomide and/or tocilizumab, more preferably salidomide.

Here, in one embodiment, the IL-6 signaling pathway inhibitor can be used in combination with other therapeutic agents that can be used to treat PAH, administered simultaneously, continuously, or separately, or it can be used as a single formulation with such therapeutic agents.

As a preferred embodiment of the "use", the use of the IL-6 signal transduction pathway inhibitor in the manufacture of a medicine for treating PAH can be listed. The medicine is administered to a PAH patient who is selected as a patient who is highly likely to respond to treatment based on the IL-6 signal transduction pathway inhibitor by the selection method described in the above-described "Selection Method for Selecting a Subject with a High Probability of Responding to Treatment Based on the IL-6 Signal Transduction Pathway Inhibitor".

As another embodiment of “uses”, the use of IL-6 signal transduction pathway inhibitors in the manufacture of pharmaceuticals for the treatment of PAHs can be cited.

As another embodiment of the “use”, the use of an IL-6 signal transduction pathway inhibitor in the manufacture of a drug for treating PAH, which is administered to PAH patients whose serum IL-6 levels are 2.73 (pg/mL) or higher.

The serum content of IL-6 can be determined, for example, by the method described in the section on [Determination of Biomarkers in Samples] of "Methods for the Determination of Biomarkers".

Here, the "IL-6 signal transduction pathway inhibitor" used in the context of the above-mentioned "Explanation of Terms" [IL-6 signal transduction pathway inhibitor] is preferably an antibody or its antigen-binding fragment that has the function of inhibiting the IL-6 signal transduction pathway, and more preferably an anti-IL-6 receptor antibody or its antigen-binding fragment.

In one embodiment, an anti-IL-6 receptor antibody may be exemplified as an antibody comprising at least one of three supervariable regions (CDRs) in the amino acid sequence of the heavy chain variable region containing sequence number 1, and/or at least one of three supervariable regions (CDRs) in the amino acid sequence of the light chain variable region containing sequence number 2.

In one embodiment, an anti-IL-6 receptor antibody may be exemplified, for example, by comprising a heavy chain containing an amino acid sequence of sequence number 3 and a light chain containing an amino acid sequence of sequence number 4.

In one embodiment, examples of anti-JL-6 receptor antibodies include: salidomide, tocilizumab, and/or salidomide, preferably salidomide and/or tocilizumab, and more preferably salidomide.

In one embodiment, the use may be in the manufacture of a pharmaceutical product that is administered simultaneously, continuously, or separately in combination with other therapeutic agents that can be used to treat PAH. In this case, the pharmaceutical product may also be a single formulation of such a therapeutic agent.

Example

The following examples illustrate in detail the embodiments of the present invention based on the above insights. It should be noted that the objectives, features, advantages, and ideas of the present invention are readily apparent to those skilled in the art based on the description in this specification, and can be easily reproduced by those skilled in the art according to the description in this specification. The embodiments and specific examples described below represent preferred embodiments of the invention and are shown for illustrative purposes only, not intended to limit the invention to these embodiments. It will be apparent to those skilled in the art that various changes and modifications can be made based on the description in this specification within the intent and scope of the invention disclosed herein.

[Example 1] Determination of biomarker candidate substances

Serum samples were obtained from 145 patients who were pulmonary hypertension patients at six participating facilities of the JAPHR (Journal of Pulmonary Hypertension Registry, www.japanph.com/japhr). Samples were collected in January 2021 and consent was obtained for publication of this study.

For each of the 145 specimens, the levels of 49 cytokines (Figure 1) were determined using the Human Magnetic Luminex Assay Kit (R&D Systems) via commissioned analysis by Filgen Inc.

Of the 49 cytokines, IL-12p70, LIF, and lymphotoxin-α/TNF-β were below the detection limit and could not be measured. Results were obtained for 46 cytokines, including IL-6.

[Example 2] Clustering of biomarker candidate substances

==Preprocessing of Measurement Data==

For the content of each cytokine measured in Example 1, cases with a measurement sensitivity below (OOR) were considered as 0 (zero), and the fifth value from the first five digits of the 145 data points was processed.

==Obtaining Cosine Similarity==

The data on the levels of each cytokine other than IL-6 obtained in the above manner were listed to form a vector of 145 samples, i.e., 145 dimensions. It is assumed that the vector obtained in this way represents the characteristics of each cytokine.

Therefore, the cosine similarity was then calculated for IL-6 and each of the 45 cytokines other than IL-6 using the following formula. The results are shown in Figure 2.

In the above formula, x represents the IL-6 content, y represents other cytokines, and n represents the number of data (145 samples).

Cosine similarity is equivalent to cos0 when the angle between the vectors of x and y is θ. In other words, the closer the cosine similarity is to 1, the closer the vectors of x (representing IL-6 levels) and y (representing other cytokines) are to having the same orientation. That is, vector similarity indicates the similarity in behavior between IL-6 and each of the other cytokines.

==Classification of Cytokines Based on Cosine Similarity==

Figure 3 shows a histogram of the cosine similarity of IL-6 with each of the other cytokines obtained as described above.

Based on Figure 3, cytokines were classified into two groups: those with a cosine similarity of less than 0.5 that were not associated with IL-6 (“Independent” in Figure 2) and those with a cosine similarity of more than 0.5 that were considered associated with IL-6 (“Near” and “Far” in Figure 2).

For the cytokine group considered to be associated with IL-6, cytokines with a cosine similarity of 0.6 or higher (“near” in Figure 2) were further selected as the first biomarker candidates for the IL-6 analog group.

The IL-6 analogue group contains 19 cytokines other than IL-6, namely IL-1α/IL-1F1, IL-1β/IL-1F2, IL-4, IL-18/IL-1F4, IL-1RA/IL-1F3, TNF-α, IFN-γ, GDF-15, CCL3/MIP-1α, CCL4/MIP-1β, G-CSF, CXCL9/MIG, CXCL10/IP-10/CRG-2, CCL27/CTACK, SCF/c-kitLigand, SCGF/CLEC11a, CD25/IL-2Rα, HGF, and CCL11/Eotaxin (eosinophil activation chemokine).

It should be noted that, for each cytokine included in the IL-6 analogue, a network diagram based on the cosine similarity with IL-6 is shown in Figure 4. In Figure 4, the thickness of the edges of the diagram corresponds to the magnitude of the cosine similarity.

==Clustering using the K-medoids method==

Based on the levels of cytokines contained in the IL-6 analogue, 145 specimens were clustered into 3 clusters using the K-medoids method.

Through this clustering, as shown in Figure 5, the samples were clustered into cluster 0, cluster 1, and cluster 2. Figures 6 (A, B) and 7 (A, B) are plots showing the distribution of the three clusters for each cytokine. In each plot, the horizontal axis 0 (zero) represents cluster 0, the horizontal axis 1 represents cluster 1, and the horizontal axis 2 represents cluster 2.

Figure 6 (A, B) shows the clustering results of cytokines located relatively close to IL-6 in the network diagram of Figure 4. Figure 7 (A, B) shows the clustering results of cytokines located relatively close to IL-1β in the network diagram of Figure 4.

[Example 3] Selection of biomarker candidates based on reliability (AUC)

For each cytokine contained in the IL-6 analogue group, an arbitrary series of candidate threshold values (e.g., equivalent to the values in the leftmost column of Figures 8 and 9) are set to distinguish the specimen groups of clusters 1 and 2 from the specimen group of cluster 0.

For each cytokine, when distinguishing using each of the threshold candidate values, the sensitivity and specificity are calculated based on whether it can be classified into a group including clusters 1 and 2 or cluster 0 using the above-mentioned K-medoids method.

As an example, the correspondence table of threshold, sensitivity, and specificity of IL-6 is shown in Figure 8, and the correspondence table of threshold, sensitivity, and specificity of IL-1β is shown in Figure 9.

Using the same sensitivity and specificity obtained for each cytokine contained in the IL-6 analogue group, ROC (Receiver Operating Curve) curves were plotted, and the area under the curve (AUC) was obtained.

AUC is an indicator of the reliability of the function in classifying the specimens from clusters 1 and 2 from the specimens from cluster 0 for each cytokine. Figure 10 shows the AUC of each cytokine included in the IL-6 analogue group.

Generally speaking, if the AUC is about 0.8 or higher, it is considered suitable for labeling.

Based on the above information, CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1 are cytokines that behave similarly to IL-6, i.e., they serve as indicators of IL-6 behavior in patients.

The thresholds for using these cytokines as markers to distinguish between groups with high IL-6 levels and groups with low IL-6 levels can be determined based on sensitivity and specificity.

Referring to Figures 8 and 9, for example, for IL-6, thresholds are appropriately set considering how patients will be identified, from thresholds corresponding to relatively high sensitivity and specificity of approximately 1.4 to approximately 1.6. In particular, thresholds for serum IL-6 levels with sensitivity and specificity around 1.5 are 2.12 (pg/mL), 2.29 (pg/mL), 2.43 (pg/mL), 2.54 (pg/mL), 2.73 (pg/mL), or 2.79 (pg/mL).

Additionally, for example, with IL-1β, thresholds are appropriately set considering how patients should be identified, from thresholds corresponding to relatively high sensitivity and specificity of approximately 1.4 to approximately 1.6. Specifically, the thresholds for serum IL-1β levels with sensitivity and specificity above approximately 1.5 are 4.49 (pg/mL), 4.50 (pg/mL), 5.04 (pg/mL), 5.13 (pg/mL), 5.59 (pg/mL), or 5.76 (pg/mL).

As described in the background section, there have been reports of elevated serum IL-6 and high expression of IL-6 receptors in diseased organs during PAH, as well as the effectiveness of IL-6 signaling pathway-targeted therapies for pulmonary hypertension. Serum IL-6 and/or organ-based IL-6 receptors in patients are direct targets for therapies based on IL-6 signaling pathway inhibitors, such as anti-IL-6 antibodies or anti-IL-6 receptor antibodies. That is, as mentioned above, pulmonary hypertension can be treated by inhibiting IL-6 action, and the IL-6 level in patients with pulmonary hypertension is considered an indicator of responsiveness to treatment based on IL-6 signaling pathway inhibitors.

Therefore, IL-6, CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1 are indicators for predicting the responsiveness to treatment based on IL-6 signaling pathway inhibitors in IL-6-related diseases, especially in patients with PAH.

SEQUENCE LISTING

<110> Chugai Pharmaceutical Co., Ltd.

International University of Health and Welfare

<120> Selection methods and biomarkers for patients with pulmonary hypertension

<130> JP214527

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<170> PatentIn version 3.5

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Claims (21)

1. A biomarker for predicting IL-6 levels in individuals with PAH. The biomarker comprises one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1. 2. A biomarker for predicting the response of individuals with PAH to treatment based on substances that block the IL-6 signaling pathway. The biomarker comprises one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, IL-6, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1. 3. A biomarker for selecting patients with PAH who are more likely to respond to treatment based on substances that block the IL-6 signaling pathway. The biomarker comprises one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, IL-6, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1. 4. Use of a biomarker for predicting IL-6 levels in subjects with PAH, said biomarker comprising one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1. 5. Use of a biomarker for predicting the response of a PAH subject to treatment based on an IL-6 signaling pathway inhibitor, said biomarker comprising one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, IL-6, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1. 6. Use of a biomarker for selecting patients with PAH who are more likely to respond to treatment based on an IL-6 signaling pathway inhibitor, said biomarker comprising one or more cytokines selected from CXCL9/MIG, CCL4/MIP-1β, CCL27/CTACK, IL-1β/IL-1F2, GDF-15, IL-4, G-CSF, IL-6, CXCL10/IP-10/CRG-2, and IL-1α/IL-1F1. 7. A method for predicting IL-6 levels in subjects with PAH, comprising: The content of the biomarker of claim 1 is determined in a sample taken from the object. 8. A method for predicting the response of a subject with PAH to treatment based on an IL-6 signaling pathway inhibitor, comprising: The content of the biomarker of claim 2 was determined in a sample taken from the object; Based on the levels of biomarkers, we can determine whether a subject is highly likely to respond to treatments based on substances that block the IL-6 signaling pathway. 9. A method for selecting patients with PAH who are highly likely to respond to treatment based on an IL-6 signaling pathway inhibitor, comprising: The content of the biomarker of claim 3 was determined in a sample taken from the object; Based on the levels of biomarkers, we can determine whether a subject is highly likely to respond to treatments based on substances that block the IL-6 signaling pathway. 10. A kit for predicting IL-6 levels in subjects with PAH, said kit comprising components for determining the biomarker of claim 1. 11. A kit for predicting the responsiveness of subjects with PAH to treatment based on an IL-6 signaling pathway inhibitor, said kit comprising a component for measuring the biomarker of claim 2. 12. A kit for selecting PAH patients who are more likely to respond to treatment based on an IL-6 signaling pathway inhibitor, said kit comprising a component for measuring the biomarker of claim 3. 13. A medicament for treating PAH comprising an IL-6 signaling pathway inhibitor, said medicament being administered to a PAH patient who is selected by the selection method of claim 9 as a patient with a high probability of responding to treatment based on the IL-6 signaling pathway inhibitor. 14. A method for selecting novel biomarkers, comprising: (a) Selecting biomarker candidates based on the similarity of vectors obtained using the content of reference biomarkers and the content of biomarker candidate substances in samples taken from the object group; and (b) Cluster the object groups based on the content of the biomarker candidates selected in (a). 15. A method for selecting a biomarker, said biomarker being used to predict IL-6 levels in subjects with PAH, said selection method comprising: (a) Biomarker candidates are selected based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from subjects with PAH; and (b) Cluster the object groups based on the content of the biomarker candidates selected in (a). 16. A method for selecting a biomarker for predicting the responsiveness of a PAH subject to treatment based on an IL-6 signaling pathway inhibitor, the selection method comprising: (a) Biomarker candidates are selected based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from subjects with PAH; and (b) Cluster the object groups based on the content of the biomarker candidates selected in (a). 17. A method for selecting a biomarker, said biomarker being used to select patients with PAH who are highly likely to respond to treatment based on an IL-6 signaling pathway inhibitor, said selection method comprising: (a) Biomarker candidates are selected based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from subjects with PAH; and (b) Cluster the object groups based on the content of the biomarker candidates selected in (a). 18. A novel biomarker selection device, comprising: (a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the content of a reference biomarker and the content of biomarker candidate substances in samples taken from the object group; and (b) Clustering Unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a). 19. A biomarker selection device for predicting IL-6 levels in subjects with PAH, the selection device comprising: (a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from a PAH-affected group; and (b) Clustering Unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a). 20. A biomarker selection device for predicting the response of a PAH patient to treatment based on an IL-6 signaling pathway inhibitor, the selection device comprising: (a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from a PAH-affected group; and (b) Clustering Unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a). 21. A biomarker selection device, the biomarker being used to select patients with PAH who are highly likely to respond to treatment based on an IL-6 signaling pathway inhibitor, the selection device comprising: (a) A selection unit that selects biomarker candidates based on the similarity of vectors obtained using the levels of IL-6 and biomarker candidate substances in samples taken from a PAH-affected group; and (b) Clustering Unit, which clusters the object group based on the content of the biomarker candidate substances selected in (a).