Methods for treating hepcidin-mediated disorders

Abstract

To provide methods for treating hepcidin-mediated disorders.SOLUTION: A method of treating a hepcidin-mediated disorder comprises administering a therapeutically effective amount of an IL-6 antagonist to a patient with a hepcidin-mediated disorder.SELECTED DRAWING: None

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 199,434, filed July 31, 2015, and U.S. Provisional Application No. 62 / 268,788, filed December 17, 2015, each of which is incorporated by reference in its entirety. [Background technology]

[0002] 2. Background The peptide hormone hepcidin plays a central role in whole-body iron homeostasis. Hentze et al., Cell 142:24-38 (2010). Hepcidin expression is known to be influenced by the TMPRSS6 gene, matriptase-2, the product of a type II transmembrane serine protease. Common variants in the TMPRSS6 gene have been shown to correlate with iron status. Benyamin et al., Nature Genetics 41(11):1173-1175 (2009). The rs855791 SNP (2321G→A, A736V) has been shown to correlate with naturally occurring variation in hepcidin expression and blood hemoglobin levels.

[0003] Hepcidin expression has also been implicated in human iron disorders, Pietrangelo, J. Hepatology 54:173-181 (2011), and anemia of chronic disease (ACD), also known as anemia of inflammation (AI). ACD is common in patients with chronic infection, autoimmune disease, cancer, and chronic kidney disease (CKD). Sun et al., Am. J. Hematol. 87(4):392-400 (2012). Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need in the art for methods for treating hepcidin-mediated disorders. [Means for solving the problem]

[0005] 3. Overview The inventors demonstrate that reducing IL-6 signaling confers clinical benefit in patients with hepcidin-mediated disorders, including anemia of chronic disease and hepcidin-mediated cytotoxicity, but that this benefit is conferred only in patients with at least one copy of the TMPRSS6 rs855791 major allele and is most effective in patients with elevated levels of IL-6.

[0006] Thus, in a first aspect, a method for treating a hepcidin-mediated disorder is provided. The method comprises administering a therapeutically effective amount of an IL-6 antagonist to a patient with a hepcidin-mediated disorder who has been determined to have at least one copy of the major allele at the TMPRSS6 rs855791 SNP. In a first series of embodiments, the patient has previously been determined to have at least one copy of the TMPRSS6 rs855791 major allele. In another series of embodiments, the method further comprises the initial step of determining that the patient has at least one copy of the TMPRSS6 rs855791 major allele. Typically, the patient has elevated pre-treatment serum levels of IL-6. In some embodiments, the patient has elevated pre-treatment serum levels of CRP.

[0007] In various embodiments, the hepcidin-mediated disorder is anemia of chronic disease.

[0008] In some anemia embodiments, the patient is male and has a pre-treatment hemoglobin (Hb) level of less than 14 g / dl, a pre-treatment Hb level of less than 13 g / dl, a pre-treatment Hb level of less than 12 g / dl, or a pre-treatment Hb level of less than 11 g / dl. In some anemia embodiments, the patient is female and has a pre-treatment Hb level of less than 12 g / dl, a pre-treatment Hb level of less than 11 g / dl, a pre-treatment Hb level of less than 10 g / dl, or a pre-treatment Hb level of less than 9 g / dl.

[0009] In some anemia embodiments, the patient is male and has a pre-treatment hematocrit of less than 40%, less than 35%, or 30-34%. In some embodiments, the patient is female and has a pre-treatment hematocrit of less than 36%, less than 35%, less than 34%, less than 33%, less than 32%, or less than 31%. In some embodiments, the female patient has a pre-treatment hematocrit of 26-29%.

[0010] In various anemia embodiments, the patient has received at least one pre-treatment administration of an erythropoiesis stimulating agent (ESA). In certain embodiments, the patient has received at least one pre-treatment administration of an ESA and has a normal Hb level or normal hematocrit. In various embodiments, the patient has received at least one pre-treatment administration of an iron supplement. In certain embodiments, the patient has received at least one pre-treatment administration of an iron supplement and has a normal Hb level or normal hematocrit. In various embodiments, the patient has received at least one pre-treatment transfusion of blood or packed red blood cells. In certain embodiments, the patient has received at least one pre-treatment transfusion of blood or packed red blood cells and has a normal Hb level or normal hematocrit.

[0011] In various anemia embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase the patient's Hb level above pre-treatment levels. In various embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase the patient's hematocrit above pre-treatment levels. In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow a reduction in the patient's ESA dose without reducing the patient's Hb level below the level present immediately prior to treatment. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow a reduction in the patient's ESA dose without reducing the patient's hematocrit below the level present immediately prior to treatment.

[0012] In various embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow at least a 10% reduction in the patient's ESA dose compared to the pre-treatment ESA dose, at least a 20% reduction in the patient's ESA dose compared to the pre-treatment ESA dose, at least a 30% reduction in the patient's ESA dose compared to the pre-treatment ESA dose, at least a 40% reduction in the patient's ESA dose compared to the pre-treatment ESA dose, or at least a 50% reduction in the patient's ESA dose compared to the pre-treatment ESA dose.

[0013] In some embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to restore functional iron deficiency.

[0014] In one series of embodiments, the hepcidin-mediated disorder is anemia of chronic disease, where the chronic disease is chronic kidney disease (CKD).

[0015] In some CKD embodiments, the patient has KDOQI stage 1 chronic kidney disease, KDOQI stage 2 chronic kidney disease, KDOQI stage 3 chronic kidney disease, KDOQI stage 4 chronic kidney disease, or KDOQI stage 5 chronic kidney disease. In specific embodiments, the patient has KDOQI stage 5 chronic kidney disease.

[0016] In some CKD embodiments, the patient has cardiorenal syndrome (CRS). In specific embodiments, the patient has CRS type 4. In certain embodiments, the patient has undergone at least one pre-treatment dialysis therapy.

[0017] In some CKD embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce cardiovascular (CV) mortality compared to age-matched, disease-matched historical controls.

[0018] In various embodiments, the hepcidin-mediated disorder is anemia of chronic disease where the chronic disease is a chronic inflammatory disease.

[0019] In some embodiments, the chronic inflammatory disease is rheumatoid arthritis (RA). In certain embodiments, the patient has a pre-treatment DAS28 score of greater than 5.1. In some embodiments, the patient has a pre-treatment DAS28 score of 3.2 to 5.1. In specific embodiments, the patient has a pre-treatment DAS28 score of less than 2.6. In selected embodiments, the patient's pre-treatment RA is moderately active to severely active.

[0020] In some RA embodiments, the patient has received at least one pretreatment dose of methotrexate. In some embodiments, the patient has received at least one pretreatment dose of a TNFα antagonist. In selected embodiments, the TNFα antagonist is selected from the group consisting of etanercept, adalimumab, infliximab, certolizumab, and golimumab.

[0021] In some RA embodiments, the patient has received at least one pre-treatment dose of an IL-6 antagonist, hi certain embodiments, the pre-treatment IL-6 antagonist is tocilizumab or tofacitinib.

[0022] In a preferred set of embodiments, the therapeutic IL-6 antagonist is MEDI5117.

[0023] In various embodiments, the hepcidin-mediated disorder is anemia of chronic disease, wherein the chronic disease is selected from the group consisting of juvenile idiopathic arthritis, ankylosing spondylitis, plaque psoriasis, psoriatic arthritis, inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

[0024] In some embodiments, the hepcidin-mediated disorder is anemia of chronic disease, wherein the chronic disease is cancer. In certain embodiments, the cancer is selected from the group consisting of solid tumors, small cell lung cancer, non-small cell lung cancer, hematological cancer, multiple myeloma, leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), lymphoma, Hodgkin's lymphoma, and hepatic adenoma.

[0025] In some embodiments, the hepcidin-mediated disorder is anemia of chronic disease, where the chronic disease is a chronic infection.

[0026] In some embodiments, the hepcidin-mediated disorder is anemia of chronic disease, where the chronic disease is congestive heart failure (CHF).

[0027] In some embodiments, the hepcidin-mediated disorder is iron-refractory iron deficiency anemia (IRIDA).

[0028] In some embodiments, the hepcidin-mediated disorder is acute coronary syndrome. In certain embodiments, the patient has suffered a myocardial infarction (MI) within 60 days, 30 days, 48 ​​hours, or 24 hours prior to the first administration of the IL-6 antagonist.

[0029] In some acute coronary syndrome embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to improve myocardial contractility compared to pre-treatment levels. In some acute coronary syndrome embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to improve cardiac ejection fraction compared to pre-treatment levels. In some acute coronary syndrome embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to reduce cardiac fibrosis compared to pre-treatment levels.

[0030] In some embodiments, the hepcidin-mediated disorder is Castleman's disease.

[0031] In another embodiment, a method for improving the treatment of a hepcidin-mediated disorder is provided, comprising discontinuing administration of an IL-6 antagonist to a patient with a hepcidin-mediated disorder who is determined to be homozygous for the TMPRSS6 rs855791 minor allele.

[0032] In another aspect, a method is provided for improving the treatment of a hepcidin-mediated disorder by discontinuing ineffective therapy, thereby reducing side effects and costs without losing therapeutic efficacy. The method includes discontinuing administration of an IL-6 antagonist to a patient with a hepcidin-mediated disorder who has been determined to be homozygous for the TMPRSS6 rs855791 minor allele. In one series of embodiments, the patient has previously been determined to be homozygous for the TMPRSS6 rs855791 minor allele. In another series of embodiments, the method further includes the initial step of determining that the patient is homozygous for the TMPRSS6 rs855791 minor allele. In typical embodiments, the patient has an elevated pre-treatment serum level of IL-6. In various embodiments, the patient has an elevated pre-treatment serum level of CRP. In various embodiments, the patient has a hepcidin-mediated disorder selected from those described in Section 5.2.1 herein. In certain embodiments, the patient has anemia of chronic disease.

[0033] The data presented in Examples 2, 3, and 5 below demonstrate that IL-6 antagonists confer therapeutic benefit in subjects who have elevated pretreatment IL-6 levels and who have at least one copy of the TMPRSS6 major allele, even in the absence of anemia. Accordingly, in another aspect, methods are provided for treating an IL-6-mediated inflammatory disorder in patients without anemia of chronic inflammation. The methods comprise administering a therapeutically effective amount of an IL-6 antagonist to a subject, typically a human patient, with an IL-6-mediated inflammatory disorder, wherein the patient is not anemic and the subject has been determined to have at least one copy of the TMPRSS6 rs855791 major allele. In a first series of embodiments, the subject has previously been determined to have at least one copy of the TMPRSS6 rs855791 major allele. In another series of embodiments, the methods further comprise the initial step of determining that the subject has at least one copy of the TMPRSS6 rs855791 major allele. Typically, the method positively excludes treatment of subjects who are homozygous for the TMPRSS6 rs855791 major allele. Typically, the patient has elevated pre-treatment serum levels of IL-6.

[0034] In specific embodiments of any of the methods of treatment, the patient has elevated pre-treatment serum levels of IL-6, hi certain embodiments, the patient has a pre-treatment serum IL-6 level greater than 2.5 pg / ml, greater than 5 pg / ml, greater than 7.5 pg / ml, greater than 10 pg / ml, or greater than 12.5 pg / ml.

[0035] In various embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce free IL-6 levels in the patient's serum below pre-treatment levels. In specific embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce free IL-6 levels by at least 10% compared to pre-treatment levels, by at least 20% compared to pre-treatment levels, or by at least 50% compared to pre-treatment levels.

[0036] In certain embodiments of any of the methods of treatment, the patient has elevated pre-treatment levels of C-reactive protein (CRP). In certain embodiments, the patient has a pre-treatment CRP level greater than 2 mg / ml, greater than 3 mg / ml, greater than 5 mg / ml, greater than 7.5 mg / ml, or greater than 10 mg / ml.

[0037] In various embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce the patient's CRP levels below pre-treatment levels. In a specific embodiment, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce the patient's CRP levels by at least 50% compared to pre-treatment levels.

[0038] In specific embodiments of any of the methods of treatment, the patient has been determined to have at least one copy of the TMPRSS6 rs855791 major allele using a TaqMan® real-time PCR assay.

[0039] In any embodiment of the methods of treatment, the IL-6 antagonist is an anti-IL-6 antibody or an antigen-binding fragment or derivative thereof.

[0040] In certain embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative has a K of less than 100 nM, less than 50 nM, less than 10 nM, or less than 1 nM for binding to human IL-6. D In certain embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative has an elimination half-life after intravenous administration of at least 7 days, at least 14 days, at least 21 days, or at least 30 days.

[0041] In various antibody embodiments, the IL-6 antagonist is a full-length monoclonal anti-IL-6 antibody, such as an IgG1 or IgG4 antibody.

[0042] In selected embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative is fully human. In some embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative is humanized.

[0043] In currently preferred embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable region CDRs of MED5117. In some of these embodiments, the antibody comprises the VH and VL of MED5117. Moreover, in certain embodiments, the antibody is MED5117.

[0044] In various embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable region CDRs of an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, elsilimomab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).

[0045] In some embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative comprises a heavy chain V region and a light chain V region derived from an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In a specific embodiment, the anti-IL-6 antibody is an antibody selected from the group consisting of siltuximab, gerilimu- zumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).

[0046] In some embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative is an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In a specific embodiment, the anti-IL-6 antibody is an antibody selected from the group consisting of siltuximab, gerilimu- zumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).

[0047] In various embodiments, the IL-6 antagonist is a single domain antibody, a VHH nanobody, a Fab, or an scFv.

[0048] In various embodiments, the IL-6 antagonist is an anti-IL-6R antibody, or an antigen-binding fragment or derivative thereof. In certain embodiments, the anti-IL-6R antibody, antigen-binding fragment or derivative is tocilizumab or bovalilizumab.

[0049] In various embodiments, the IL-6 antagonist is a JAK inhibitor. In specific embodiments, the JAK inhibitor is selected from the group consisting of tofacitinib (Xeljanz), decernotinib, ruxolitinib, upadacitinib, baricitinib, filgotinib, lestaurtinib, pacritinib, peficitinib, INCB-039110, ABT-494, INCB-047986, and AC-410.

[0050] In various embodiments, the IL-6 antagonist is a STAT3 inhibitor.

[0051] In some embodiments where the IL-6 antagonist is an antibody or antigen-binding fragment or derivative, the IL-6 antagonist is administered parenterally. In a specific embodiment, the IL-6 antagonist is administered subcutaneously.

[0052] In some embodiments where the IL-6 antagonist is a JAK inhibitor or a STAT3 inhibitor, the IL-6 antagonist is administered orally. 4. Brief description of the drawings [Brief explanation of the drawings]

[0053] [Figure 1]1A and 1B provide box plots showing that increased amounts of erythropoietin ("EPO") were required for treatment in chronic kidney disease patients (CKD stage 5 dialysis subjects) who had elevated serum IL-6 levels and at least one copy of the major allele at a known SNP in the TMPRSS6 gene, rs855791 (encoding a TMPRSS6 polypeptide containing a G or C at nucleotide position 2321 and an alanine (736A) at amino acid position 736), but were not required for treatment in chronic kidney disease patients homozygous for the rs855791 TMPRSS6 minor allele (encoding a TMPRSS6 polypeptide containing a T or A at nucleotide position 2321 and a valine (736V) at position 736). Data from patients homozygous for the minor allele (A / A) are shown in FIG. 1A. Data from patients with at least one copy of the major allele (homozygous G / G and heterozygous G / A) were pooled and are shown in FIG. 1B. Each of the two patient populations was further stratified into groups based on tertiles of serum IL-6 levels: "low" tertile (IL-6 < 5 pg / ml), "middle" tertile (IL-6 = 5-15 pg / ml), and "highest" tertile (IL-6 > 15 μg / ml). Box plots with error bars are overlaid on the raw data. Each box plot represents a patient group based on both IL-6 level and genotype. Details are described in Example 1. [Figure 2A] Figures 2A and 2B provide survival curves demonstrating that the TMPRSS6 rs855791 major allele confers higher all-cause mortality in response to elevated IL-6 levels in chronic kidney disease stage 5 dialysis subjects. Figure 2A shows data from patients homozygous for the minor allele (A / A). Each group was separated into tertiles of serum IL-6 levels using the IL-6 levels used in Figure 1. Details are described in Example 1. [Figure 2B]Figure 2A provides survival curves demonstrating that the TMPRSS6 rs855791 major allele confers higher all-cause mortality in response to elevated IL-6 levels in chronic kidney disease stage 5 dialysis subjects. Figure 2B shows data from patients with at least one copy of the major allele (homozygous G / G and heterozygous G / A). Each group was separated into tertiles of serum IL-6 levels using the IL-6 levels used in Figure 1. Details are described in Example 1. [Figure 3] 1 is a graph showing that increased EPO doses were required for therapy in chronic kidney disease patients (CKD stage 5 dialysis subjects) who had elevated serum levels of the acute phase reactant CRP and had at least one copy of the TMPRSS6 rs855791 major allele, but not in chronic kidney disease patients who had elevated serum levels of the acute phase reactant CRP and were homozygous for the rs855791 minor allele. Genotype groups were separated into serum CRP levels <2 mg / L vs. >2 mg / L. Details are described in Example 1. [Figure 4] Figures 4A and 4B are graphical representations demonstrating that the TMPRSS6 rs855791 major allele confers higher all-cause mortality in response to elevated IL-6 levels in patients after myocardial infarction ("MI"). Figure 4A plots the cumulative probability of a fatal event over time (y-axis) against the number of days after MI (x-axis) for a population homozygous for the TMPRSS6 rs855791 minor allele. Figure 4B plots the cumulative probability of a fatal event over time for a population with at least one copy of the TMPRSS6 rs855791 major allele. Each group was separated into tertiles of serum IL-6 levels as indicated. IL-6 levels were measured 1 month after MI. Mortality was measured 1 to 12 months after MI. Details are described in Example 2. [Figure 5]Figures 5A and 5B are graphical representations demonstrating that the TMPRSS6 rs855791 major allele confers a higher risk of heart failure ("HF") in response to elevated IL-6 levels in post-MI patients. Figure 5A plots the cumulative probability of HF (y-axis) over time against the number of days post-MI (x-axis) for a population homozygous for the TMPRSS6 rs855791 minor allele. Figure 5B plots the cumulative probability of HF events over time for a population with at least one copy of the TMPRSS6 rs855791 major allele. Each group was separated into tertiles of serum IL-6 levels as indicated. IL-6 levels were measured 1 month after myocardial infarction. HF was measured 1 to 12 months after myocardial infarction. Details are described in Example 2. [Figure 6A] Figure 6 shows the results of an assay of human iPS cells differentiated into cardiomyocytes when transfected with constructs constitutively expressing either the TMPRSS6 rs855791 minor or major allele and exposed to BMP2 plus IL-6 or BMP2 alone in vitro, demonstrating that the TMPRSS6 rs855791 major allele confers a higher risk of cell death (Trypan Blue positive) in response to IL-6. Figure 6A shows the results in a normoxic environment. The data imply that reduced IL-6 exposure should improve cardiomyocyte survival in patients with the TMPRSS6 rs855791 major allele, but not in patients with the TMPRSS6 rs855791 minor allele. Details are described in Example 3. [Figure 6B]Figure 6A shows the results of an assay of human iPS cells differentiated into cardiomyocytes when transfected with constructs constitutively expressing either the TMPRSS6 rs855791 minor or major allele and exposed to BMP2 plus IL-6 or BMP2 alone in vitro, demonstrating that the TMPRSS6 rs855791 major allele confers a higher risk of cell death (Trypan Blue positive) in response to IL-6. Figure 6B shows the results after exposure to hypoxic conditions and reoxygenation. The data imply that reduced IL-6 exposure should improve cardiomyocyte survival in patients with the TMPRSS6 rs855791 major allele, but not in patients with the TMPRSS6 rs855791 minor allele. Details are described in Example 3. [Figure 7] Figure 1 shows the experimental design of the cardiorenal syndrome study described in Example 4. CRS4 was induced in rats genotypically similar to humans, homozygous for the TMPRSS6 rs855791 major allele. This diagram illustrates the various events of the study along a timeline. In this study, myocardial infarction ("MI") was induced in rats at week 0. At week 2, each subject underwent a single nephrectomy ("Nx"). Starting on day 1 (D1) after nephrectomy, anti-IL-6 antibody (Ab9770, Abcam Plc, UK) (Rx) or an isotype control antibody ("IgG"; ab171516, Abcam Plc, UK) was administered once every three days until the end of the study. Standard of care therapy (ACE inhibitor - perindopril) was administered daily from day 1 after Nx until the end of the study. Rodents were sacrificed at week 6. MI and Nx were not performed in the control "sham" subject group. Various assessments of the rodents were performed at the time points indicated by the arrows. [Figure 8A]Figures 8A and 8B show cardiac ejection fractions in rats treated with an anti-IL-6 antibody ("IL-6 antibody"), a standard of care ACE inhibitor (perindopril or "Peri"), versus a control ("isotype") treatment group and sham-operated animals in a cardiorenal syndrome model summarized in Figure 7 and described in detail in Example 4. Figure 8A is a plot showing baseline ejection fraction levels for all groups 2 weeks after myocardial infarction but before nephrectomy. Results are expressed as mean ± SEM and demonstrate that anti-IL-6 therapy has therapeutic efficacy in a cardiorenal syndrome model comparable to standard of care therapy, as measured by changes in cardiac ejection fraction. [Figure 8B] Figure 8B shows cardiac ejection fractions of rats treated with an anti-IL-6 antibody ("IL-6 antibody"), a standard of care ACE inhibitor (perindopril or "Peri"), relative to a control ("isotype") treatment group and sham-operated animals in a cardiorenal syndrome model summarized in Figure 7 and described in detail in Example 4. Figure 8B is a plot showing ejection fraction levels for all groups after one week of treatment, one week after nephrectomy. Results are expressed as mean ± SEM and demonstrate that anti-IL-6 therapy has therapeutic efficacy in a cardiorenal syndrome model comparable to standard of care therapy, as measured by changes in cardiac ejection fraction. [Figure 8C] Figure 8C shows cardiac ejection fractions of rats treated with an anti-IL-6 antibody ("IL-6 antibody"), a standard of care ACE inhibitor (perindopril or "Peri"), relative to a control ("isotype") treatment group and sham-operated animals in a cardiorenal syndrome model summarized in Figure 7 and described in detail in Example 4. Figure 8C is a plot showing ejection fractions for all groups after 2 weeks of treatment, 2 weeks after nephrectomy. Results are expressed as mean ± SEM and demonstrate that anti-IL-6 therapy has therapeutic efficacy in a cardiorenal syndrome model comparable to standard of care therapy, as measured by changes in cardiac ejection fraction. [Figure 8D]Figure 8D shows cardiac ejection fractions of rats treated with an anti-IL-6 antibody ("IL-6 antibody"), a standard of care ACE inhibitor (perindopril or "Peri"), versus a control ("isotype") treatment group and sham-operated animals in a cardiorenal syndrome model summarized in Figure 7 and described in detail in Example 4. Figure 8D is a plot showing ejection fractions for all groups after 4 weeks of treatment, 4 weeks after nephrectomy. Results are expressed as mean ± SEM and demonstrate that anti-IL-6 therapy has therapeutic efficacy in a cardiorenal syndrome model comparable to standard of care therapy, as measured by changes in cardiac ejection fraction. [Figure 9] Figure 7 shows plots of cardiac contractility in rats treated with anti-IL-6 antibody ("IL-6 antibody"), standard of care (perindopril or "Peri"), versus a control ("isotype") treatment group in the cardiorenal syndrome model summarized in Figure 7 and described in detail in Example 4. Cardiac contractility was assessed at the end of the study by measuring dP / dtmax (mmHb / ms), a measure of pressure within the heart. Measurements are shown for all groups after 4 weeks of treatment and 4 weeks after nephrectomy. Results are expressed as mean ± SEM and demonstrate that anti-IL-6 therapy has a therapeutic effect comparable to standard of care therapy, as shown by the increased cardiac contractility in the anti-IL-6-treated rodent group. [Figure 10A] Figure 10A shows that anti-IL-6 therapy has an anti-cardiorenal syndrome effect comparable to standard of care therapy, as measured by the level of fibrosis in cardiac tissue from groups of rodents treated with anti-IL-6 therapy ("IL-6 Ab"), standard of care (perindopril or "Peri"), and control ("IgG"). Figure 10A is a photomicrograph showing a histological section of cardiac tissue stained with picrosirius red. Two regions of tissue were analyzed: a "normal" region and a "perifibrosis" region. An example of the "normal" region is shown by the delineated portion of the tissue section. The inset of the photomicrograph shows a magnified view of the "normal" region, demonstrating that a small portion of the "normal" region has fibrotic tissue. The "perifibrosis" region is the region of tissue in the "normal" region surrounding the fibrotic tissue. [Figure 10B] Figure 10A shows that anti-IL-6 therapy has a comparable anti-cardiorenal syndrome effect to standard of care therapy, as measured by the level of fibrosis in cardiac tissue from rodent groups treated with anti-IL-6 therapy ("IL-6 Ab"), standard of care (perindopril or "Peri"), and control ("IgG"). Figure 10B is a plot showing the percentage of area of ​​"normal" areas, indicated as fibrotic tissue (i.e., stained / dark areas), in tissue samples from all groups. Results are expressed as mean ± SEM. Details are provided in Example 4. [Figure 10C] Figure 10 shows that anti-IL-6 therapy has a comparable anti-cardiorenal syndrome effect to standard of care therapy, as measured by the level of fibrosis in cardiac tissue from groups of rodents treated with anti-IL-6 therapy ("IL-6 Ab"), standard of care (perindopril or "Peri"), and control ("IgG"). Figure 10C is a plot showing the percentage of the area of ​​the "perifibrotic" region, designated as fibrotic tissue, in tissue samples from all groups. Results are expressed as mean ± SEM. Details are provided in Example 4. [Figure 11A] Figure 11A shows data from an in vivo model in which myocardial infarction was induced in mice genotypically similar to humans homozygous for the TMPRSS6 rs855791 major allele. The control group received no therapy. The experimental group was treated with an anti-mouse IL-6 antibody. Figure 11B shows that treatment with anti-IL-6 provides a statistically significant improvement in ejection fraction. The data demonstrate that anti-IL-6 therapy given immediately after myocardial infarction improves left ventricular functional recovery in rodents mimicking human patients with the TMPRSS6 rs855791 major allele. Details are described in Example 5. [Figure 11B]Figure 11B shows data from an in vivo model in which myocardial infarction was induced in mice genotypically similar to humans homozygous for the TMPRSS6 rs855791 major allele. The control group received no therapy. The experimental group was treated with an anti-mouse IL-6 antibody. Figure 11B shows that treatment with anti-IL-6 confers a statistically significant improvement in cardiac contractility, measured as left ventricular fractional shortening. The data demonstrate that anti-IL-6 therapy given immediately after myocardial infarction improves left ventricular functional recovery in rodents mimicking human patients with the TMPRSS6 rs855791 major allele. Details are described in Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0054] The drawings depict various embodiments of the present invention for purposes of illustration only. Those skilled in the art will readily recognize that alternative embodiments of the structures and methods illustrated herein may be employed without departing from the principles of the present invention as described herein.

[0055] 5. Detailed Description 5.1. Summary of experimental results The peptide hormone hepcidin plays a central role in whole-body iron homeostasis. Hentze et al., Cell 142:24-38 (2010). Hepcidin expression is known to be influenced by the product of the TMPRSS6 gene, matriptase-2, a type II transmembrane serine protease. Common variants in the TMPRSS6 gene have been shown to correlate with iron status (Benyamin et al., Nature Genetics 41(11):1173-1175 (2009)). The rs855791 SNP (2321G→A, A736V) has been shown to correlate with naturally occurring variations in hepcidin expression and blood hemoglobin levels. Hepcidin expression has also been implicated in human iron disorders (Pietrangelo, J. Hepatology 54:173-181 (2011)) and anemia of chronic disease (ACD), also known as anemia of inflammation (AI). ACD is common in patients with chronic infection, autoimmune disease, cancer, and chronic kidney disease (CKD). Sun et al., Am. J. Hematol. 87(4):392-400 (2012).

[0056] To determine whether genotypes at the TMPRSS6 rs855791 SNP predict the degree of anemia in end-stage renal disease, we analyzed data previously collected in a clinical study of patients with chronic kidney disease, along with newly determined SNP genotyping. Because hepcidin expression is also regulated by IL-6 (Casanovas et al., PLOS Computational Biol. 10(1):e1003421 (2014)), we further analyzed the data to determine whether serum IL-6 levels could predict the degree of anemia in end-stage renal disease.

[0057] As described in Example 1 and shown in Figure 1, the degree of underlying anemia, measured as clinically titrated EPO dose, correlated with IL-6 levels only in patients with at least one copy of the major allele at the TMPRSS6 rs855791 SNP. In these patients, higher serum IL-6 levels correlated with higher required EPO doses (Figure 1B). In contrast, the degree of anemia in patients with two copies of the minor allele did not correlate with serum IL-6 levels (Figure 1A).

[0058] Similarly, overall survival correlated with IL-6 levels only in patients with at least one copy of the major allele at TMPRSS6 SNP rs855791. In subjects with at least one copy of the TMPRSS6 rs855791 major allele, survival was inversely correlated with serum IL-6 levels, with patients in the highest tertile of serum IL-6 levels statistically significantly worse than patients in the lowest tertile of IL-6 levels (Figure 2B). In contrast, overall survival in patients homozygous for the minor allele at rs855791 was not affected by IL-6 levels (Figure 2A).

[0059] Without wishing to be bound by theory, it is possible that in patients with at least one copy of the TMPRSS6 major allele, increased serum IL-6 causes increased hepcidin expression, thereby increasing anemia. The increased risk of death is the result of dysregulated iron metabolism, resulting anemia, and / or increased doses of erythropoiesis-stimulating agents, such as EPO, administered for treatment. These correlations suggest that reducing IL-6 levels or IL-6 signaling increases the likelihood of reducing anemia, reducing the required EPO dose, and improving survival in patients with chronic kidney disease, but only in those patients with at least one copy of the TMPRSS6 rs855791 major allele, and is most effective in those patients with elevated serum IL-6 levels.

[0060] In Example 2, to determine whether the TMPRSS6 rs855791 genotype influences IL-6 sensitivity in patients with acute rather than chronic disease, we analyzed data previously collected in a clinical study of patients hospitalized for acute coronary syndromes in conjunction with newly determined SNP genotyping.

[0061] Mortality in subjects homozygous for the TMPRSS6 rs855791 SNP minor allele (A) did not correlate with IL-6 variation (Figure 4A). However, one or two copies of the major allele (G) increased all-cause mortality in response to elevated IL-6 levels in subjects after myocardial infarction (Figure 4B). Thus, TMPRSS6 regulated IL-6-mediated mortality risk after myocardial infarction.

[0062] The effect of TMPRSS6 genotype on the risk of IL-6-mediated heart failure was also assessed. Heart failure in subjects homozygous for the minor allele (A) was not correlated with changes in IL-6 (Figure 5A). However, the G allele of TMPRSS6 conferred a higher rate of heart failure in response to elevated IL-6 levels in subjects after myocardial infarction (Figure 5B). Thus, TMPRSS6 regulated the risk of IL-6-mediated heart failure after myocardial infarction.

[0063] The data from Example 2 demonstrate that the correlation between TMPRSS6 genotype, IL-6 levels, and adverse clinical outcomes is not limited to patients with chronic kidney disease. Without wishing to be bound by theory, increased serum IL-6 increases hepcidin expression in patients with at least one copy of the TMPRSS6 major allele, resulting in increased iron sequestration in cardiomyocytes and subsequent iron-mediated cytotoxicity. These correlations raise the possibility that reducing IL-6 levels or IL-6 signaling may reduce heart failure and mortality in patients with acute coronary syndromes, but only in those patients with at least one copy of the TMPRSS6 rs855791 major allele, with the greatest effect in those patients with elevated serum IL-6 levels.

[0064] Although the correlations observed in Examples 1 and 2 strongly suggest that reduced IL-6-mediated signaling should confer clinical benefit in patients with at least one copy of the TMPRSS6 rs855791 major allele, elevated IL-6 levels, and anemia or hepcidin-mediated cytotoxicity, the observed correlations are insufficient to prove causality. Therefore, in Example 3, human induced pluripotent stem (iPS) cell cardiomyocytes were genetically engineered to express only the TMPRSS6 rs855791 major allele or only the minor allele and tested in vitro.

[0065] Hepcidin expression is regulated by both the BMP6 / SMAD and IL-6 / STAT signaling pathways, and both BMP and IL-6 act through their respective receptors to promote increased hepcidin expression. Casanovas et al., PLOS Comp. Biol. 10(1):e1003421 (2014). iPS cardiomyocytes bearing major and minor alleles were treated in vivo with recombinant BMP2 and IL-6, agonists of both signaling pathways, or with BMP2 alone to model clinical interventions in which IL-6 levels (or signaling) are reduced. Control iPS cells were not treated with either agonist. Cell mortality was measured under normoxia (normoxia) and under conditions of simulated hypoxia followed by reoxygenation (reperfusion).

[0066] Figure 6A shows the results when cells were treated at normoxia levels. iPS cardiomyocytes expressing only the TMPRSS6 rs855791 minor allele ("736V minor allele") are not significantly affected by the elimination of IL-6 signaling ("ns"). Cell mortality, measured as the percentage of trypan blue-positive cells, is not significantly reduced when cells are treated with BMP2 alone compared to treatment with BMP2 + IL-6. In contrast, iPS cardiomyocytes expressing the TMPRSS6 rs855791 major allele exhibit statistically significantly lower cell death when IL-6 signaling is eliminated.

[0067] Figure 6B shows the results when cells were subjected to hypoxia followed by reoxygenation. Compared to normoxic conditions, hypoxia / reoxygenation was toxic to iPS cardiomyocytes, killing approximately 40 percent of control cells of the major and minor alleles, compared with approximately 20% of control cells under normoxic conditions (compare Figure 6A). In contrast to this increased background toxicity, iPS cardiomyocytes of the minor allele were not significantly affected by elimination of IL-6 signaling. Cell mortality was not significantly reduced when cells were treated with BMP2 alone compared with treatment with BMP2 plus IL-6. In contrast, iPS cardiomyocytes expressing the TMPRSS6 rs855791 major allele exhibited statistically significantly lower cell death when IL-6 signaling was eliminated.

[0068] These data strengthen the inferences drawn from the post-hoc analysis of clinical trial data in Examples 1 and 2. Reduced IL-6 signaling is effective in reducing IL-6-mediated toxicity in cardiomyocytes expressing the TMPRSS6 rs855791 major allele, but not in cardiomyocytes expressing only the minor allele. Without wishing to be bound by theory, increased IL-6-induced toxicity in major allele iPS cardiomyocytes may result from an IL-6-mediated increase in hepcidin expression, resulting in increased intracellular sequestration of iron and subsequent iron-mediated cytotoxicity.

[0069] Patients with chronic kidney disease, such as those enrolled in the MIMICK study analyzed in Example 1, often develop cardiac dysfunction, which is a major cause of overall mortality. This secondary cardiac damage following primary chronic kidney disease is referred to as cardiorenal syndrome type 4 (CRS type 4). To directly test whether anti-IL-6 therapy is effective as a treatment for CRS4 patients who carry at least one copy of the TMPRSS6 rs855791 major allele, as suggested by the data in Examples 1 and 3, we used a model of CRS4 in rats that are genotypically similar to humans homozygous for the TMPRSS6 rs855791 major allele.

[0070] After 4 weeks of treatment, both treatment groups, i.e., the group treated with anti-IL-6 antibody and the group treated with perindopril, the standard of care for ACE inhibitor therapy, showed statistically significantly increased ejection fraction levels compared to the isotype control group (Figure 8D) (p < 0.001). The similar ejection fraction levels in the anti-IL-6 and standard of care groups measured after 4 weeks of treatment indicated that anti-IL-6 therapy was as effective as ACE inhibitors. Figure 9 shows that anti-IL-6 therapy was also as effective as ACE inhibitors in preserving cardiac contractility. Figures 10A-10C demonstrate that anti-IL-6 therapy was equally effective in reducing cardiac fibrosis.

[0071] These data demonstrate that treatment with anti-IL-6 agents is effective in reducing cardiac injury and restoring function in an in vivo model of cardiorenal syndrome in animals genotypically similar to humans who are homozygous for the TMPRSS6 rs855791 major allele.

[0072] Similarly, the data in Examples 2 and 3 suggest that reducing IL-6 levels or IL-6 signaling reduces heart failure and mortality in patients with acute coronary syndromes who have at least one copy of the TMPRSS6 rs855791 major allele, with the greatest benefit in those patients with elevated serum levels of IL-6.

[0073] A study was conducted to determine the effect of anti-IL-6 therapy after acute myocardial infarction in mice genotypically similar to humans homozygous for the TMPRSS6 rs855791 major allele.

[0074] Figures 11A and 11B show data from an in vivo model in which myocardial infarction was induced in mice genotypically similar to humans homozygous for the TMPRSS6 rs855791 major allele. The control group received no therapy. The experimental group was treated with an anti-mouse IL-6 antibody. Figure 11A shows that treatment with anti-IL-6 resulted in a statistically significant improvement in ejection fraction compared to controls. Figure 11B shows that treatment with anti-IL-6 resulted in a statistically significant improvement in contractility, measured as cardiac fractional shortening, compared to controls. These data demonstrate that anti-IL-6 therapy given immediately after myocardial infarction improves left ventricular functional recovery in rodents genotypically similar to human patients carrying the TMPRSS6 rs855791 major allele.

[0075] Taken together, the experimental data demonstrate that therapeutic interventions that reduce IL-6 signaling provide clinical benefit in patients with hepcidin-mediated disorders, such as anemia or hepcidin-mediated cytotoxicity, but only in those patients who carry at least one copy of the TMPRSS6 rs855791 major allele, with the greatest effect in patients with elevated levels of IL-6.

[0076] Thus, as further described below, in a first aspect, a method for treating a hepcidin-mediated disorder is provided. The method comprises administering a therapeutically effective amount of an IL-6 antagonist to a patient with a hepcidin-mediated disorder who has been determined to have at least one copy of the major allele at the TMPRSS6 rs855791 SNP. In a second aspect, a method for improving the treatment of a hepcidin-mediated disorder is provided, the method comprises discontinuing the administration of an IL-6 antagonist to a patient with a hepcidin-mediated disorder, wherein the patient has been determined to be homozygous for the TMPRSS6 rs855791 minor allele. Treatment is improved by discontinuing ineffective therapy, thereby reducing side effects and costs without losing therapeutic effectiveness. In a further aspect, a method is provided for treating an IL-6-mediated inflammatory disorder in a patient without anemia of chronic inflammation, the method comprising administering a therapeutically effective amount of an IL-6 antagonist to a patient with an IL-6-mediated inflammatory disorder and without anemia, wherein the subject has been determined to have at least one copy of the TMPRSS6 rs855791 major allele.

[0077] 5.2. Definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. As used herein, the following terms have the meanings ascribed to them below.

[0078] "Hepcidin" refers to a polypeptide or biologically active fragment thereof having at least about 85% or greater amino acid identity to the amino acid sequence provided in NCBI Accession No. NP_066998 ("hepcidin preprotein"). Exemplary hepcidin biological activities include binding to and reducing levels of the iron transport channel ferroportin, inhibiting iron transport, inhibiting intestinal iron absorption, and inhibiting iron release from macrophages and the liver. The amino acid sequence of an exemplary hepcidin preprotein is provided below:

[0079] 1 MALSSQIWAA CLLLLLLLAS LTSGSVFPQQ TGQLAELQPQ DRAGARASWM PMFQRRRRRD 61 THFPICIFCC GCCHRSKCGM CCKT (SEQ ID NO: 1)

[0080] With respect to the sequence above, hepcidin exists in various forms, including the preprohormone (amino acids 25-84), the prohormone (amino acids 25-84), and mature forms called hepcidin-25 (amino acids 60-84), hepcidin-22 (amino acids 63-84), and hepcidin-20 (amino acids 65-84).

[0081] A "hepcidin-mediated disorder" is any disorder in which hepcidin expression contributes to either the pathogenesis of the disorder or its symptoms. The contribution of hepcidin to the pathogenesis may be known, suspected, or inferred from the observation that administration of an IL-6 antagonist provides greater therapeutic benefit in patients with the disorder who have at least one copy of the TMPRSS6 rs855791 SNP major allele compared to patients with the disorder who are homozygous for the TMPRSS6 rs855791 SNP minor allele. Hepcidin-mediated disorders are further described in Section 5.2.1 below.

[0082] "Transmembrane protease serine 6 (TMPRSS6) polypeptide" refers to a polypeptide or fragment thereof having at least about 85% or more amino acid identity to the amino acid sequence provided in NCBI Accession No. NP_001275929 and having serine proteinase activity. TMPRSS6 polypeptide, also known as matriptase-2 (MT2), cleaves hemoduyelin and inhibits bone morphogenetic protein signaling. An exemplary TMPRSS6 amino acid sequence having an alanine at position 736 (736A) is provided below:

[0083] 1 MPVAEAPQVA GGQGDGGDGE EAEPEGMFKA CEDSKRKARG YLRLVPLFVL LALLVLASAG 61 VLLWYFLGYK AEVMVSQVYS GSLRVLNRHF SQDLTRRESS AFRSETAKAQ KMLKELITST 121 RLGTYYNSSS VYSFGEGPLT CFFWFILQIP EHRRLMLSPE VVQALLVEEL LSTVNSSAAV 181 PYRAEYEVDP EGLVILEASV KDIAALNSTL GCYRYSYVGQ GQVLRLKGPD HLASSCLWHL 241 QGPKDLMLKL RLEWTLAECR DRLAMYDVAG PLEKRLITSV YGCSRQEPVV EVLASGAIMA 301 VVWKKGLHSY YDPFVLSVQP VVFQACEVNL TLDNRLDSQG VLSTPYFPSY YSPQTHCSWH 361 LTVPSLDYGL ALWFDAYALR RQKYDLPCTQ GQWTIQNRRL CGLRILQPYA ERIPVVATAG 421 ITINFTSQIS LTGPGVRVHY GLYNQSDPCP GEFLCSVNGL CVPACDGVKD CPNGLDERNC 481 VCRATFQCKE DSTCISLPKV CDGQPDCLNG SDEEQCQEGV PCGTFTFQCE DRSCVKKPNP 541 QCDGRPDCRD GSDEEHCDCG LQGPSSRIVG GAVSSEGEWP WQASLQVRGR HICGGALIAD 601 RWVITAAHCF QEDSMASTVL WTVFLGKVWQ NSRWPGEVSF KVSRLLLHPY HEEDSHDYDV 661 ALLQLDHPVV RSAAVRPVCL PARSHFFEPG LHCWITGWGA LREGALRADA VALFYGWRNQ 721 GSETCCCPIS NALQKADVQL IPQDLCSEVY RYQVTPRMLC AGYRKGKKDA CQGDSGGPLV 781 CKALSGRWFL AGLVSWGLGC GRPNYFGVYT RITGVISWIQ QVVT (SEQ ID NO: 2)

[0084] An exemplary TMPRSS6 amino acid sequence with a valine at position 736 (736V) is provided below: 1 MPVAEAPQVA GGQGDGGDGE EAEPEGMFKA CEDSKRKARG YLRLVPLFVL LALLVLASAG 61 VLLWYFLGYK AEMVVSQVYS GSLRVLNRHF SQDLTRRESS AFRSETAKAQ KMLKELITST 121 RLGTYYNSSS VYSFGEGPLT CFFWFILQIP EHRRLMLSPE VVQALLVEEL LSTVNSSAAV 181 PYRAEYEVDP EGLVILEASV KDIAALNSTL GCYRYSYVGQ GQVLRLKGPD HLASSCLWHL 24 QGPKDLMLKL RLEWTLAECR DRLAMYDVAG PLEKRLITSV YGCSRQEPVV EVLASGAIMA 30 VVWKKGLHSY YDPFVLSVQP VVFQACEVNL TLDNRLDSQG VLSTPYFPSY YSPQTHCSWH 36 LTVPSLDYGL ALWFDAYALR RQKYDLPCTQ GQWTIQNRRL CGLRILQPYA ERIPVVATAG 42 ITINFTSQIS LTGPGVRVHY GLYNQSDPCP GEFLCSVNGL CVPACDGVKD CPNGLDERNC 48 VCRATFQCKE DSTCISLPKV CDGQPDCLNG SDEEQCQEGV PCGTFTFQCE DRSCVKKPNP 54 QCDGRPDCRD GSDEEHCDCG LQGPSSRIVG GAVSSEGEWP WQASLQVRGR HICGGALIAD 60 RWVITAAHCF QEDSMASTVL WTVFLGKVWQ NSRWPGEVSF KVSRLLLHPY HEEDSHDYDV 66 ALLQLDHPVV RSAAVRPVCL PARSHFFEPG LHCWITGWGA LREGALRADA VALFYGWRNQ 72 GSETCCCPIS NALQKVDVQL IPQDLCSEVY RYQVTPRMLC AGYRKGKKDA CQGDSGGPLV 78 CKALSGRWFL AGLVSWGLGC GRPNYFGVYT RITGVISWIQ QVVT (SEQ ID NO: 3)

[0085] By "TMPRSS6 nucleic acid molecule" is meant a polynucleotide encoding a TMPRSS6 polypeptide (matriptase-2, MT2). ​​An exemplary TMPRSS6 nucleic acid molecule sequence is provided in NCBI Accession No. NM_001289000. A TMPRSS6 nucleic acid sequence having a G at nucleotide position 2321 (the "G allele," "major allele") is provided below:

[0086] 1 GGACAAACAG AGGCTCCTGA GGCCTGTGTG CAGGCCCGGC ACCTATCTGC CGCTCCCAAA 61 GGATGCCCGT GGCCGAGGCC CCCCAGGTGG CTGGCGGGCA GGGGGACGGA GGTGATGGCG 121 AGGAAGCGGA GCCGGAGGGG ATGTTCAAGG CCTGTGAGGA CTCCAAGAGA AAAGCCCGGG 181 GCTACCTCCG CCTGGTGCCC CTGTTTGTGC TGCTGGCCCT GCTCGTGCTG GCTTCGGCGG 241 GGGTGCTACT CTGGTATTTC CTAGGGTACA AGGCGGAGGT GATGGTCAGC CAGGTGTACT 301 CAGGCAGTCT GCGTGTACTC AATCGCCACT TCTCCCAGGA TCTTACCCGC CGGGAATCTA 361 GTGCCTTCCG CAGTGAAACC GCCAAAGCCC AGAAGATGCT CAAGGAGCTC ATCACCAGCA 421 CCCGCCTGGG AACTTACTAC AACTCCAGCT CCGTCTATTC CTTTGGGGAG GGACCCCTCA 481 CCTGCTTCTT CTGGTTCATT CTCCAAATCC CCGAGCACCG CCGGCTGATG CTGAGCCCCG 541 AGGTGGTGCA GGCACTGCTG GTGGAGGAGC TGCTGTCCAC AGTCAACAGC TCGGCTGCCG 601 TCCCCTACAG GGCCGAGTAC GAAGTGGACC CCGAGGGCCT AGTGATCCTG GAAGCCAGTG 661 TGAAAGACAT AGCTGCATTG AATTCCACGC TGGGTTGTTA CCGCTACAGC TACGTGGGCC 721 AGGGCCAGGT CCTCCGGCTG AAGGGGCCTG ACCACCTGGC CTCCAGCTGC CTGTGGCACC 781 TGCAGGGCCC CAAGGACCTC ATGCTCAAAC TCCGGCTGGA GTGGACGCTG GCAGAGTGCC 841 GGGACCGACT GGCCATGTAT GACGTGGCCG GGCCCCTGGA GAAGAGGCTC ATCACCTCGG 901 TGTACGGCTG CAGCCGCCAG GAGCCCGTGG TGGAGGTTCT GGCGTCGGGG GCCATCATGG 961 CGGTCGTCTG GAAGAAGGC CTGCACAGCT ACTACGACCC CTTCGTGCTC TCCGTGCAGC 1021 CGGTGGTCTT CCAGGCCTGT GAAGTGAACC TGACGCTGGA CAACAGGCTC GACTCCCAGG 1081 GCGTCCTCAG CACCCCGTAC TTCCCCAGCT ACTACTCGCC CCAAACCCAC TGCTCCTGGC 1141 ACCTCACGGT GCCCTCTCTG GACTACGGCT TGGCCCTCTG GTTTGATGCC TATGCACTGA 1201 GGAGGCAGAA GTATGATTTG CCGTGCACCC AGGGCCAGTG GACGATCCAG AACAGGAGGC 1261 TGTGTGGCTT GCGCATCCTG CAGCCCTACG CCGAGAGGAT CCCCGTGGTG GCCACGGCCG 1321 GGATCACCAT CAACTTCACC TCCCAGATCT CCCTCACCGG GCCCGGTGTG CGGGTGCACT 1381 ATGGCTTGTA CAACCAGTCG GACCCCTGCC CTGGAGAGTT CCTCTGTTCT GTGAATGGAC 1441 TCGTTGTCCC TGCCTGTGAT GGGGTCAAGG ACTGCCCCAA CGGCCTGGAT GAGAGAAACT 1501 GCGTTTGCAG AGCCACATTC CAGTGCAAAG AGGACAGCAC ATGCATCTCA CTGCCCAAGG 1561 TCTGTGATGG GCAGCCTGAT TGTCTCAACG GCAGCGACGA AGAGCAGTGC CAGGAAGGGG 1621 TGCCATGTGG GACATTCACC TTCCAGTGTG AGGACCGGAG CTGCGTGAAG AAGCCCAACC 1681 CGCAGTGTGA TGGGCGGCCC GACTGCAGGG ACGGCTCGGA TGAGGAGCAC TGTGACTGTG 1741 GCCTCCAGGG CCCCTCCAGC CGCATTGTTG GTGGAGCTGT GTCCTCCGAG GGTGAGTGGC 1801 CATGGCAGGC CAGCCTCCAG GTTCGGGGTC GACACATCTG TGGGGGGGCC CTCATCGCTG 1861 ACCGCTGGGT GATAACAGCT GCCCACTGCT TCCAGGAGGA CAGCATGGCC TCCACGGTGC 1921 TGTGGACCGT GTTCCTGGGC AAGGTGTGGC AGAACTCGCG CTGGCCTGGA GAGGTGTCCT 1981 TCAAGGTGAG CCGCCTGCTC CTGCACCCGT ACCACGAAGA GGACAGCCAT GACTACGACG 2041 TGGCGCTGCT GCAGCTCGAC CACCCGGTGG TGCGCTCGGC CGCCGTGCGC CCCGTCTGCC 2101 TGCCCGCGCG CTCCCACTTC TTCGAGCCCG GCCTGCACTG CTGGATTACG GGCTGGGGCG 2161 CCTTGCGCGA GGGCGCCCTA CGGGCGGATG CTGTGGCCCT ATTTTATGGA TGGAGAAACC 2221 AAGGCTCAGA GACATGTTGC TGCCCCATCA GCAACGCTCT GCAGAAAGTG GATGTGCAGT 2281 TGATCCCACA GGACCTGTGC AGCGAGGTCT ATCGCTACCA GGTGACGCCA CGCATGCTGT 2341 GTGCCGGCTA CCGCAAGGGC AAGAAGGATG CCTGTCAGGG TGACTCAGGT GGTCCGCTGG 2401 TGTGCAAGGC ACTCAGTGGC CGCTGGTTCC TGGCGGGGCT GGTCAGCTGG GGCCTGGGCT 2461 GTGGCCGGCC TAACTACTTC GGCGTCTACA CCCGCATCAC AGGTGTGATC AGCTGGATCC 2521 AGCAAGTGGT GACCTGAGGA ACTGCCCCCC TGCAAAGCAG GGCCCACCTC CTGGACTCAG 2581 AGAGCCCAGG GCAACTGCCA AGCAGGGGGA CAAGTATTCT GGCGGGGGGT GGGGGAGAGA 2641 GCAGGCCCTG TGGTGGCAGG AGGTGGCATC TTGTCTCGTC CCTGATGTCT GCTCCAGTGA 2701 TGGCAGGAGG ATGGAGAAGT GCCAGCAGCT GGGGGTCAAG ACGTCCCCTG AGGACCCAGG 2761 CCCACACCCA GCCCTTCTGC CTCCCAATTC TCTCTCCTCC GTCCCCTTCC TCCACTGCTG 2821 CCTAATGCAA GGCAGTGGCT CAGCAGCAAG AATGCTGGTT CTACATCCCG AGGAGTGTCT 2881 GAGGTGCGCC CCACTCTGTA CAGAGGCTGT TTGGGCAGCC TTGCCTCCAG AGAGCAGATT 2941 CCAGCTTCGG AAGCCCCTGG TCTAACTTGG GATCTGGGAA TGGAAGGTGC TCCCATCGGA 3001 GGGGACCCTC AGAGCCCTGG AGACTGCCAG GTGGGCCTGC TGCCACTGTA AGCCAAAAGG 3061 TGGGGAAGTC CTGACTCCAG GGTCCTTGCC CCACCCCTGC CTGCCACCTG GGCCCTCACA 3121 GCCCAGACCC TCACTGGGAG GTGAGCTCAG CTGCCCTTTG GAATAAAGCT GCCTGATCCA 3181 AAAAAAAAAA AAAAAA (SEQ ID NO: 4)

[0087] The TMPRSS6 nucleic acid sequence having an A at nucleotide position 2321 is provided below: 1 GGACAAACAG AGGCTCCTGA GGCCTGTGTG CAGGCCCGGC ACCTATCTGC CGCTCCCAAA 61 GGATGCCCGT GGCCGAGGCC CCCCAGGTGG CTGGCGGGCA GGGGGACGGA GGTGATGGCG 121 AGGAAGCGGA GCCGGAGGGG ATGTTCAAGG CCTGTGAGGA CTCCAAGAGA AAAGCCCGGG 181 GCTACCTCCG CCTGGTGCCC CTGTTTGTGC TGCTGGCCCT GCTCGTGCTG GCTTCGGCGG 241 GGGTGCTACT CTGGTATTTC CTAGGGTACA AGGCGGAGGT GATGGTCAGC CAGGTGTACT 301 CAGGCAGTCT GCGTGTACTC AATCGCCACT TCTCCCAGGA TCTTACCCGC CGGGAATCTA 361 GTGCCTTCCG CAGTGAAACC GCCAAAGCCC AGAAGATGCT CAAGGAGCTC ATCACCAGCA 421 CCCGCCTGGG AACTTACTAC AACTCCAGCT CCGTCTATTC CTTTGGGGAG GGACCCCTCA 481 CCTGCTTCTT CTGGTTCATT CTCCAAATCC CCGAGCACCG CCGGCTGATG CTGAGCCCCG 541 AGGTGGTGCA GGCACTGCTG GTGGAGGAGC TGCTGTCCAC AGTCAACAGC TCGGCTGCCG 601 TCCCCTACAG GGCCGAGTAC GAAGTGGACC CCGAGGGCCT AGTGATCCTG GAAGCCAGTG 661 TGAAAGACAT AGCTGCATTG AATTCCACGC TGGGTTGTTA CCGCTACAGC TACGTGGGCC 721 AGGGCCAGGT CCTCCGGCTG AAGGGGCCTG ACCACCTGGC CTCCAGCTGC CTGTGGCACC 781 TGCAGGGCCC CAAGGACCTC ATGCTCAAAC TCCGGCTGGA GTGGACGCTG GCAGAGTGCC 841 GGGACCGACT GGCCATGTAT GACGTGGCCG GGCCCCTGGA GAAGAGGCTC ATCACCTCGG 901 TGTACGGCTG CAGCCGCCAG GAGCCCGTGG TGGAGGTTCT GGCGTCGGGG GCCATCATGG 961 CGGTCGTCTG GAAGAAGGC CTGCACAGCT ACTACGACCC CTTCGTGCTC TCCGTGCAGC 1021 CGGTGGTCTT CCAGGCCTGT GAAGTGAACC TGACGCTGGA CAACAGGCTC GACTCCCAGG 1081 GCGTCCTCAG CACCCCGTAC TTCCCCAGCT ACTACTCGCC CCAAACCCAC TGCTCCTGGC 1141 ACCTCACGGT GCCCTCTCTG GACTACGGCT TGGCCCTCTG GTTTGATGCC TATGCACTGA 1201 GGAGGCAGAA GTATGATTTG CCGTGCACCC AGGGCCAGTG GACGATCCAG AACAGGAGGC 1261 TGTGTGGCTT GCGCATCCTG CAGCCCTACG CCGAGAGGAT CCCCGTGGTG GCCACGGCCG 1321 GGATCACCAT CAACTTCACC TCCCAGATCT CCCTCACCGG GCCCGGTGTG CGGGTGCACT 1381 ATGGCTTGTA CAACCAGTCG GACCCCTGCC CTGGAGAGTT CCTCTGTTCT GTGAATGGAC 1441 TCTGTGTCCC TGCCTGTGAT GGGGTCAAGG ACTGCCCCAA CGGCCTGGAT GAGAGAAACT 1501 GCGTTTGCAG AGCCACATTC CAGTGCAAAG AGGACAGCAC ATGCATCTCA CTGCCCAAGG 1561 TCTGTGATGG GCAGCCTGAT TGTCTCAACG GCAGCGACGA AGAGCAGTGC CAGGAAGGGG 1621 TGCCATGTGG GACATTCACC TTCCAGTGTG AGGACCGGAG CTGCGTGAAG AAGCCCAACC 1681 CGCAGTGTGA TGGGCGGCCC GACTGCAGGG ACGGCTCGGA TGAGGAGCAC TGTGACTGTG 1741 GCCTCCAGGG CCCCTCCAGC CGCATTGTTG GTGGAGCTGT GTCCTCCGAG GGTGAGTGGC 1801 CATGGCAGGC CAGCCTCCAG GTTCGGGGTC GACACATCTG TGGGGGGGCC CTCATCGCTG 1861 ACCGCTGGGT GATAACAGCT GCCCACTGCT TCCAGGAGGA CAGCATGGCC TCCACGGTGC 1921 TGTGGACCGT GTTCCTGGGC AAGGTGTGGC AGAACTCGCG CTGGCCTGGA GAGGTGTCCT 1981 TCAAGGTGAG CCGCCTGCTC CTGCACCCGT ACCACGAAGA GGACAGCCAT GACTACGACG 2041 TGGCGCTGCT GCAGCTCGAC CACCCGGTGG TGCGCTCGGC CGCCGTGCGC CCCGTCTGCC 2101 TGCCCGCGCG CTCCCACTTC TTCGAGCCCG GCCTGCACTG CTGGATTACG GGCTGGGGCG 2161 CCTTGCGCGA GGGCGCCCTA CGGGCGGATG CTGTGGCCCT ATTTTATGGA TGGAGAAACC 2221 AAGGCTCAGA GACATGTTGC TGCCCCATCA GCAACGCTCT GCAGAAAGTG GATGTGCAGT 2281 TGATCCCACA GGACCTGTGC AGCGAGGTCT ATCGCTACCA AGTGACGCCA CGCATGCTGT 2341 GTGCCGGCTA CCGCAAGGGC AAGAAGGATG CCTGTCAGGG TGACTCAGGT GGTCCGCTGG 2401 TGTGCAAGGC ACTCAGTGGC CGCTGGTTCC TGGCGGGGCT GGTCAGCTGG GGCCTGGGCT 2461 GTGGCCGGCC TAACTACTTC GGCGTCTACA CCCGCATCAC AGGTGTGATC AGCTGGATCC 2521 AGCAAGTGGT GACCTGAGGA ACTGCCCCCC TGCAAAGCAG GGCCCACCTC CTGGACTCAG 2581 AGAGCCCAGG GCAACTGCCA AGCAGGGGGA CAAGTATTCT GGCGGGGGGT GGGGGAGAGA 2641 GCAGGCCCTG TGGTGGCAGG AGGTGGCATC TTGTCTCGTC CCTGATGTCT GCTCCAGTGA 2701 TGGCAGGAGG ATGGAGAAGT GCCAGCAGCT GGGGGTCAAG ACGTCCCCTG AGGACCCAGG 2761 CCCACACCCA GCCCTTCTGC CTCCCAATTC TCTCTCCTCC GTCCCCTTCC TCCACTGCTG 2821 CCTAATGCAA GGCAGTGGCT CAGCAGCAAG AATGCTGGTT CTACATCCCG AGGAGTGTCT 2881 GAGGTGCGCC CCACTCTGTA CAGAGGCTGT TTGGGCAGCC TTGCCTCCAG AGAGCAGATT 2941 CCAGCTTCGG AAGCCCCTGG TCTAACTTGG GATCTGGGAA TGGAAGGTGC TCCCATCGGA 3001 GGGGACCCTC AGAGCCCTGG AGACTGCCAG GTGGGCCTGC TGCCACTGTA AGCCAAAAGG 3061 TGGGGAAGTC CTGACTCCAG GGTCCTTGCC CCACCCCTGC CTGCCACCTG GGCCCTCACA 3121 GCCCAGACCC TCACTGGGAG GTGAGCTCAG CTGCCCTTTG GAATAAAGCT GCCTGATCCA 3181 AAAAAAAAAA AAAAAA (SEQ ID NO: 5)

[0088] "Variant" means a polynucleotide or polypeptide sequence that differs from a reference sequence by one or more nucleotides or one or more amino acids. An exemplary TMPRSS6 variant is TMPRSS6(A736V), resulting from SNP rs855791 (G→A).

[0089] "Single nucleotide polymorphism" or "SNP" refers to a naturally occurring DNA sequence variant in which a single nucleotide in the genome differs between members of a species or between paired chromosomes in an individual. SNPs can be used as genetic markers for variant alleles. In one embodiment, the TMPRSS6 SNP is rs855791.

[0090] "rs855791" refers to a single nucleotide polymorphism (SNP) in the human TMPRSS6 gene, 2321G→A, which results in an alanine to valine substitution (A736V) in the catalytic domain of matriptase-2 (MT2), encoded by the TMPRSS6 gene. The allele with the highest frequency in the human population (major allele) is 2321G, which encodes 736A. The allele with the lowest frequency in the human population (minor allele) is 2321A, which encodes 736V.

[0091] "Heterozygous" means that a chromosomal locus has two different alleles. In one embodiment of the methods described herein, heterozygous refers to a genotype in which one allele has a TMPRSS6 nucleic acid sequence encoding a TMPRSS6 polypeptide having an alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 major allele), and the other allele includes a variant TMPRSS6 nucleic acid sequence encoding a TMPRSS6 polypeptide including a valine at amino acid position 736 (e.g., having an A or T at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 minor allele).

[0092] "Homozygous" means that a chromosomal locus has two identical alleles. In certain embodiments of the methods described herein, homozygous refers to a genotype in which both alleles have a TMPRSS6 nucleic acid sequence that encodes a TMPRSS6 polypeptide containing an alanine at amino acid position 736 (e.g., a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 homozygous major allele). In some embodiments, homozygous refers to a genotype in which both alleles have a TMPRSS6 nucleic acid sequence that encodes a TMPRSS6 polypeptide containing a valine at amino acid position 736 (e.g., an A or T at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 homozygous minor allele).

[0093] "Determining that a patient has at least one copy of the TMPRSS6 rs855791 major allele" includes, but is not limited to, performing an assay to determine that a patient has at least one copy of the TMPRSS6 rs855791 major allele, ordering an assay to determine that a patient has at least one copy of the TMPRSS6 rs855791 major allele, prescribing an assay to determine that a patient has at least one copy of the TMPRSS6 rs855791 major allele, or otherwise directing or controlling the performance of an assay to determine that a patient has at least one copy of the TMPRSS6 rs855791 major allele, and reviewing TMRSS6 genotype assay data or protein or nucleic acid sequence data to determine that a patient has at least one copy of the TMPRSS6 rs855791 major allele.

[0094] "Interleukin 6 (IL-6)" or "IL-6 polypeptide" refers to a polypeptide or fragment thereof having at least about 85% or more amino acid identity to the amino acid sequence provided in NCBI Accession No. NP_000591 and having IL-6 biological activity. IL-6 is a covalent cytokine with multiple biological functions. Exemplary IL-6 biological activities include immunostimulatory and proinflammatory activities. An exemplary IL-6 amino acid sequence is provided below:

[0095] 1 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ 61 NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 121 PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ 181 GPPARPITVQ PTEAWPRTSQ GPSTRPVEVVP GGRAVAAILG LGLVLGLLGP LAILLALYLL 241 RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI (SEQ ID NO: 6)

[0096] "Interleukin 6 (IL-6) nucleic acid" means a polynucleotide that encodes an interleukin 6 (IL-6) polypeptide. An exemplary interleukin 6 (IL-6) nucleic acid sequence is provided under NCBI Accession No. NM_000600. An exemplary sequence under NCBI Accession No. NM_000600 is provided below:

[0097] 1 AATATTAGAG TCTCAACCCC CAATAAATAT AGGACTGGAG ATGTCTGAGG CTCATTCTGC 61 CCTCGAGCCC ACCGGGAACG AAAGAGAAGC TCTATCTCCC CTCCAGGAGC CCAGCTATGA 121 ACTCCTTCTC CACAAGCGCC TTCGGTCCAG TTGCCTTCTC CCTGGGGCTG CTCCTGGTGT 181 TGCCTGCTGC CTTCCCTGCC CCAGTACCCC TTCCAAAGAT GTAGCCGCCC 241 CACACAGACA GCCACTCACC TCTTCAGAAC GAATTGACAA ACAAATTCGG TACATCCTCG 301 ACGGCATCTC AGCCCTGAGA AAGGAGACAT GTAACAAGAG TAACATGTGT GAAAGCAGCA 361 AAGAGGCACT GGCAGAAAAC AACCTGAACC TTCCAAAGAT GGCTGAAAAA GATGGATGCT 421 TCCAATCTGG ATTCAATGAG GAGACTTGCC TGGTGAAAAT CATCACTGGT CTTTTGGAGT 481 TTGAGGTATA CCTAGAGTAC CTCCAGAACA GATTTGAGAG TAGTGAGGAA CAAGCCAGAG 541 CTGTGCAGAT GAGTACAAAA GTCCTGATCC AGTTCCTGCA GAAAAAGGCA AAGAATCTAG 601 ATGCAATAAC CACCCCTGAC CCAACCACAA ATGCCAGCCT GCTGACGAAG CTGCAGGCAC 661 AGAACCAGTG GCTGCAGGAC ATGACAACTC ATCTCATTCT GCGCAGCTTT AAGGAGTTCC 721 TGCAGTCCAG CCTGAGGGCT CTTCGGCAAA TGTAGCATGG GCACCTCAGA TTGTTGTTGT 781 TAATGGGCAT TCCTTCTTCT GGTCAGAAAC CTGTCCACTG GGCACAGAAC TTATGTTGTT 841 CTCTATGGAG AACTAAAAGT ATGAGCGTTA GGACACTATT TTAATTATTT TTAATTTATT 901 AATATTTAAA TATGTGAAGC TGAGTTAATT TATGTAAGTC ATATTTATAT TTTTAAGAAG 961 TACCACTTGA AACATTTTAT GTATTAGTTT TGAAATAATA ATGGAAAGTG GCTATGCAGT 1021 TTGAATATCC TTTGTTTCAG AGCCAGATCA TTTCTTGGAA AGTGTAGGCT TACCTCAAAT 1081 AAATGGCTAA CTTATACATA TTTTTAAAGA AATATTTATA TTGTATTTAT ATAATGTATA 1141 AATGGTTTTT ATACCAATAA ATGGCATTTT AAAAAATTCA GCAAAAAAAA AAAAAAAAAA 1201 A (SEQ ID NO: 7)

[0098] "Interleukin-6 receptor (IL-6R) complex" refers to a protein complex that includes IL-6 receptor subunit alpha (IL-6Rα) and interleukin-6 signal transducer glycoprotein 130, also known as interleukin-6 receptor subunit beta (IL-6Rβ).

[0099] "Interleukin-6 receptor subunit alpha (IL-6Rα) polypeptide" refers to a polypeptide or fragment thereof having at least about 85% amino acid identity with the amino acid sequence provided in NCBI Accession No. NP_000556 or NP_852004 and having the biological activity of an IL-6 receptor. Exemplary IL-6Rα biological activities include binding to IL-6, binding to glycoprotein 130 (gp130), and regulating cell proliferation and differentiation. An exemplary IL-6R sequence is provided below:

[0100] 1 MLAVGCALLA ALLAAPGAAL APRRCPAQEV ARGVLTSLPG DSVTLTCPGV EPEDNATVHW 61 VLRKPAAGSH PSRWAGMGRR LLLRSVQLHD SGNYSCYRAG RPAGTVHLLV DVPPEEPQLS 121 CFRKSPLSNV VCEWGPRSTP SLTTKAVLLV RKFQNSPAED FQEPCQYSQE SQKFSCQLAV 181 PEGDSSFYIV SMCVASSVGS KFSKTQTFQG CGILQPDPPA NITVTAVARN PRWLSVTWQD 241 PHSWNSSFYR LRFELRYRAE RSKTFTTWMV KDLQHHCVIH DAWSGLRHVV QLRAQEEFGQ 301 GEWSEWSPEA MGTPWTESRS PPAENEVSTP MQALTTNKDD DNILFRDSAN ATSLPVQDSS 361 SVPLPTFLVA GGSLAFGTLL CIAIVLRFKK TWKLRALKEG KTSMHPPYSL GQLVPERPRP 421 TPVLVPLISP PVSPSSLGSD NTSSHNRPDA RDPRSPYDIS NTDYFFPR (SEQ ID NO: 8)

[0101] "Interleukin-6 receptor subunit beta (IL-6Rβ) polypeptide" refers to a polypeptide or fragment thereof having at least about 85% amino acid identity with the amino acid sequence provided under NCBI Accession No. NP_002175, NP_786943, or NP_001177910, and having the biological activity of an IL-6 receptor. Exemplary IL-6Rβ biological activities include binding to IL-6Rα, IL-6 receptor signaling activity, and regulation of cell proliferation, differentiation, hepcidin expression, and the like. An exemplary IL-6Rβ sequence is provided below:

[0102] 1 MLTLQTWLVQ ALFIFLTTES TGELLDPCGY ISPESPVVQL HSNFTAVCVL KEKCMDYFHV 61 NANYIVWKTN HFTIPKEQYT IINRTASSVT FTDIASLNIQ LTCNILTFGQ LEQNVYGITI 121 ISGLPPEKPK NLSCIVNEGK KMRCEWDGGR ETHLETNFTL KSEWATHKFA DCKAKRDTPT 181 SCTVDYSTVY FVNIEVWVEA ENALGKVTSD HINFDPVYKV KPNPPHNLSV INSEELSSIL 241 KLTWTNPSIK SVIILKYNIQ YRTKDASTWS QIPPEDTAST RSSFTVQDLK PFTEYVFRIR 301 CMKEDGKGYW SDWSEEASGI TYEDRPSKAP SFWYKIDPSH TQGYRTVQLV WKTLPPFEAN 361 GKILDYEVTL TRWKSHLQNY TVNATKLTVN LTNDRYLATL TVRNLVGKSD AAVLTIPACD 421 FQATHPVMDL KAFPKDNMLW VEWTTPRESV KKYILEWCVL SDKAPCITDW QQEDGTVHRT 481 YLRGNLAESK CYLITVTPVY ADGPGSPESI KAYLKQAPPS KGPTVRTKKV GKNEAVLEWD 541 QLPVDVQNGF IRNYTIFYRT IIGNETAVNV DSSHTEYTLS SLTSDTLYMV RMAAYTDEGG 601 KDGPEFTFTT PKFAQGEIEA IVVPVCLAFL LTTLLGVLFC FNKRDLIKKH IWPNVPDPSK 661 SHIAQWSPHT PPRHNFNSKD QMYSDGNFTD VSVVEIEAND KKPFPEDLKS LDLFKKEKIN 721 TEGHSSGIGG SSCMSSSRPS ISSSDENESS QNTSSTVQYS TVVHSGYRHQ VPSVQVFSRS 781 ESTQPLLDSE ERPEDLQLVD HVDGGDGILP RQQYFKQNCS QHESSPDISH FERSKQVSSV 841 NEEDFVRLKQ QISDHISQSC GSGQMKMFQE VSAADAFGPG TEGQVERFET VGMEAATDEG 901 MPKSYLPQTV RQGGYMPQ (SEQ ID NO: 9)

[0103] The term "IL-6 antagonist" refers to an agent capable of reducing the biological activity of IL-6. IL-6 antagonists include agents that reduce serum IL-6 polypeptide levels; agents that reduce expression of IL-6 polypeptides or nucleic acids; agents that reduce the ability of IL-6 to bind to IL-6R; agents that reduce expression of IL-6R; and agents that reduce signaling by the IL-6R receptor upon binding to IL-6. In preferred embodiments, the IL-6 antagonist reduces the biological activity of IL-6 by at least about 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%. As further described in Section 5.7 below, IL-6 antagonists include IL-6-binding polypeptides, e.g., anti-IL-6 antibodies and antigen-binding fragments or derivatives thereof; IL-6R-binding polypeptides, e.g., anti-IL-6R antibodies and antigen-binding fragments or derivatives thereof; and synthetic chemical molecules, e.g., JAK1 and JAK3 inhibitors.

[0104] "IL-6 antibody" or "anti-IL-6 antibody" means an antibody that specifically binds to IL-6. Anti-IL-6 antibodies include monoclonal and polyclonal antibodies specific for IL-6, as well as antigen-binding fragments or derivatives thereof. IL-6 antibodies are described in more detail in Section 5.7.1 below.

[0105] By "IL-6-mediated inflammatory disorder" is meant any disorder in which IL-6 is known or suspected to contribute to either the pathogenesis of the disease or its symptoms.

[0106] "Erythropoietin (EPO)" refers to a polypeptide or fragment thereof having at least about 85% or more amino acid identity with the amino acid sequence provided in NCBI Accession No. NP_000790 and having EPO biological activity. Exemplary EPO biological activities include binding to the erythropoietin receptor and the resulting increase in proliferation and terminal differentiation of erythroid progenitor cells and / or erythropoiesis (red blood cell production). Exemplary EPO amino acid sequences are provided below:

[0107] 1 MGVHECPAWL WLLLSLLSLP LGLPVLGAPP RLICDSRVLE RYLLEAKEAE NITTGCAEHC 61 SLNENITVPD TKVNFYAWKR MEVGQQAVEV WQGLALLSEA VLRGQALLVN SSQPWEPLQL 121 HVDKAVSGLR SLTTLLRALG AQKEAISPPD AASAAPLRTI TADTFRKLFR VYSNFLRGKL 181 KLYTGEACRT GDR (SEQ ID NO: 10)

[0108] "Erythropoiesis-stimulating agent (ESA)" means an agent that stimulates erythropoiesis. ESAs include, but are not limited to, EPO, darbepoetin (Aranesp), epoetin beta (NeoRecormon), epoetin delta (Dynepo), epoetin omega (Epomax), and epoetin zeta.

[0109] "Erythropoietic factor" refers to an agent that increases the growth or proliferation of red blood cells or their precursor cells (e.g., hematopoietic stem cells) and / or reduces cell death in red blood cells or their precursor cells. In various embodiments, erythropoietic factors include erythropoiesis stimulators, HIF stabilizers, and supplemental iron.

[0110] "C-reactive protein (CRP) polypeptide" refers to a polypeptide or fragment thereof having at least about 85% or more amino acid identity with the amino acid sequence provided in NCBI Accession No. NP_000558 and having complement activation activity. CRP levels are elevated in response to inflammation. Exemplary CRP sequences are provided below:

[0111] 1 MEKLLCFLVL TSLSHAFGQT DMSRKAFVFP KESDTSYVSL KAPLTKPLKA FTVCLHFYTE 61 LSSTRGYSIF SYATKRQDNE ILIFWSKDIG YSFTVGGSEI LFEVPEVTVA PVHICTSWES 121 ASGIVEFWVD GKPRVRKSLK KGYTVGAEAS IILGQEQDSF GGNFEGSQSL VGDIGNVNMW 181 DFVLSPDEIN TIYLGGPFSP NVLNWRALKY EVQGEVFTKP QLWP (SEQ ID NO: 11)

[0112] By "drug" is meant any compound or composition suitable for administration in therapy, and expressly includes chemical compounds, proteins, including antibodies or antigen-binding fragments thereof, peptides; and nucleic acid molecules.

[0113] "Subject" means a human or non-human mammal, including, but not limited to, bovine, equine, canine, ovine, feline, and rodent subjects such as mice and rats. A "patient" is a human subject.

[0114] As used herein, the terms "treat," "treating," "treatment," and the like refer to the reduction or amelioration of a disorder and / or its associated signs or symptoms, or the slowing or halting of its progression. It is understood that, although not excluded, treating a disorder or condition does not require that the associated disorder, condition, or symptoms be completely eliminated.

[0115] "Pre-treatment" means before the first administration of an IL-6 antagonist according to the methods described herein. Pre-treatment does not exclude, and often includes, prior administration of a therapy other than an IL-6 antagonist.

[0116] In this disclosure, the words "comprise," "comprising," "containing," "having," "includes," "including," and linguistic variations thereof have the meaning ascribed to them in U.S. patent law and acknowledge the presence of additional components beyond those expressly listed.

[0117] By "biological sample" is meant any tissue, cell, fluid, or other substance derived from an organism (e.g., a human subject). In certain embodiments, the biological sample is serum or blood.

[0118] "Angiotensin-converting enzyme (ACE) inhibitor" refers to an agent that inhibits the biological function of angiotensin-converting enzyme, which converts angiotensin I to angiotensin II. ACE inhibitors include, but are not limited to, quinapril, perindopril, ramipril, captopril, benazepril, trandolapril, fosinopril, lisinopril, moexipril, and enalapril. In various embodiments, the ACE inhibitor is perindopril.

[0119] 5.1. Other Interpretation Conventions Unless otherwise specified, residue numbering in antibody constant regions is according to the EU index as set forth in Kabat.

[0120] Ranges provided herein are understood to be abbreviations for all values ​​within the range, inclusive of the recited endpoints. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0121] As used herein, the term "or" is understood to be inclusive unless otherwise stated or clear from context. As used herein, the terms "a," "an," and "the" are understood to be singular or plural unless otherwise stated or clear from context.

[0122] Unless otherwise stated or otherwise clear from the context, as used herein, the term "about" is understood to mean within normal tolerances in the art, e.g., within two standard deviations of the mean. About can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0123] 5.2. Methods for Treating Hepcidin-Mediated Disorders In a first aspect, a method for treating a hepcidin-mediated disorder is provided.

[0124] The method comprises administering a therapeutically effective amount of an IL-6 antagonist to a subject, typically a human patient, with a hepcidin-mediated disorder, wherein the subject has been determined to have at least one copy of the TMPRSS6 rs855791 major allele. In a first series of embodiments, the subject has previously been determined to have at least one copy of the TMPRSS6 rs855791 major allele. In another series of embodiments, the method further comprises the initial step of determining that the subject has at least one copy of the TMPRSS6 rs855791 major allele. Typically, the method affirmatively excludes treatment of subjects who are homozygous for the TMPRSS6 rs855791 minor allele. Typically, the patient has elevated pretreatment serum levels of IL-6.

[0125] 5.2.1. Hepcidin-mediated disorders 5.2.1.1. Anemia of chronic disease / chronic inflammation In various embodiments, the hepcidin-mediated disorder treated by the methods described herein is anemia of chronic disease, also known as anemia of chronic inflammation.

[0126] In various embodiments, the patient is male and has a pre-treatment hemoglobin (Hb) content of less than 14 g / dl. In some embodiments, the male patient has a pre-treatment Hb level of 13.0-13.9 g / dl, 12.0-12.9 g / dl, 11.0-11.9 g / dl, 10.0-10.9 g / dl, or less than 10 g / dl. In various embodiments, the patient is female and has a pre-treatment Hb content of less than 12 g / dl. In some embodiments, the female patient has a pre-treatment Hb level of 11.0-11.9 g / dl, 10.0-10.9 g / dl, 9.0-9.9 g / dl, 8.0-8.9 g / dl, or less than 8 g / dl. In some of these embodiments, the patient has previously been treated with an ESA. In some embodiments, the patient has been treated with iron supplementation. In some embodiments, the patient has been treated with transfusions of blood or packed red blood cells.

[0127] In various embodiments, the patient is male and has a pre-treatment hematocrit of less than 40%. In some embodiments, the male patient has a pre-treatment hematocrit of less than 39%, less than 38%, less than 37%, less than 36%, or less than 35%. In certain embodiments, the male patient has a pre-treatment hematocrit of 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, or 30%. In various embodiments, the patient is female and has a pre-treatment hematocrit of less than 36%. In some embodiments, the female patient has a pre-treatment hematocrit of less than 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, or 26%. In certain embodiments, the female patient has a pre-treatment hematocrit of 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, or 26%. In some of these embodiments, the patient is being treated with an ESA. In some embodiments, the patient is being treated with iron supplements. In some embodiments, the patient is being treated with a transfusion of blood or packed red blood cells.

[0128] In some embodiments, the patient is treated with an ESA and has a normal pre-treatment Hb content and / or normal pre-treatment hematocrit. In certain embodiments, the patient is male and has a pre-treatment hemoglobin (Hb) content of at least 14 g / dL and / or a pre-treatment hematocrit of at least 40%. In certain embodiments, the patient is female and has a pre-treatment Hb content of at least 12 g / dL and / or a hematocrit of at least 36%. In certain embodiments, the ESA is EPO. In certain embodiments, the ESA is darbepoetin alfa.

[0129] In some embodiments, the patient is being treated with iron supplementation and has a normal pre-treatment Hb content and / or normal pre-treatment hematocrit. In certain embodiments, the patient is male and has a pre-treatment hemoglobin (Hb) content of at least 14 g / dL and / or a pre-treatment hematocrit of at least 40%. In certain embodiments, the patient is female and has a pre-treatment Hb content of at least 12 g / dL and / or a hematocrit of at least 36%.

[0130] In some embodiments, the patient has been treated with a transfusion of whole blood or packed red blood cells and has a normal pre-treatment Hb content and / or normal pre-treatment hematocrit. In certain embodiments, the patient is male and has a pre-treatment hemoglobin (Hb) content of at least 14 g / dL and / or a pre-treatment hematocrit of at least 40%. In certain embodiments, the patient is female and has a pre-treatment Hb content of at least 12 g / dL and / or a hematocrit of at least 36%.

[0131] In some embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase the patient's Hb level above pre-treatment levels. In some embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase the patient's hematocrit above pre-treatment levels. In some embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase both the Hb level and the hematocrit above pre-treatment levels.

[0132] In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow a reduction in the patient's ESA dose without reducing the patient's Hb level below pre-treatment levels. In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow a reduction in the patient's ESA dose without reducing the patient's hematocrit below pre-treatment levels. In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow a reduction in the patient's ESA dose without reducing the patient's Hb level and hematocrit.

[0133] In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow at least a 10% reduction in the patient's ESA dose compared to the ESA dose before treatment. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow at least a 20%, 30%, 40%, or 50% reduction in the patient's ESA dose compared to the ESA dose before treatment. In specific embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow at least a 60%, or even at least a 75% reduction in the patient's ESA dose compared to the ESA dose before treatment.

[0134] In some embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to restore functional iron deficiency.

[0135] 5.2.1.1.1. Chronic kidney disease In various embodiments, the chronic disease is chronic kidney disease (CKD).

[0136] In some embodiments, the patient has KDOQI stage 1 chronic kidney disease. In certain embodiments, the patient has KDOQI stage 2 chronic kidney disease, KDOQI stage 3 chronic kidney disease, KDOQI stage 4 chronic kidney disease, or KDOQI stage 5 chronic kidney disease.

[0137] In some embodiments, the patient has cardiorenal syndrome (CRS). In certain embodiments, the patient has CRS type 4.

[0138] In some embodiments, the patient is being treated with dialysis.

[0139] In some embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce cardiovascular (CV) mortality compared to an age-matched, disease-matched historical cohort.

[0140] 5.2.1.1.2. Chronic inflammatory diseases In various embodiments, the chronic disease is a chronic inflammatory disease.

[0141] In some embodiments, the chronic inflammatory disease is rheumatoid arthritis (RA).

[0142] In certain embodiments, the patient has a pre-treatment DAS28 score of greater than 5.1. In some embodiments, the patient has a pre-treatment DAS28 score of 3.2 to 5.1. In some embodiments, the patient has a pre-treatment DAS28 score of less than 2.6. In various embodiments, the patient's pre-treatment RA is severely active. In some embodiments, the patient's pre-treatment RA is moderately active.

[0143] In certain embodiments, the patient is being treated with methotrexate. In some embodiments, methotrexate is discontinued when treatment with an IL-6 antagonist is initiated. In some embodiments, methotrexate treatment is continued when treatment with an IL-6 antagonist is initiated.

[0144] In certain embodiments, the patient is being treated with an anti-TNFα agent. In specific embodiments, the anti-TNFα agent is selected from etanercept, adalimumab, infliximab, certolizumab, and golimumab. In specific embodiments, the anti-TNFα agent is discontinued when treatment with an IL-6 antagonist is initiated.

[0145] In certain embodiments, the patient is being treated with an IL-1 receptor antagonist. In a specific embodiment, the IL-1 receptor antagonist is anakinra. In a specific embodiment, the IL-1 receptor antagonist is discontinued when treatment with an IL-6 antagonist is initiated.

[0146] In certain embodiments, the patient is being treated with abatacept. In a specific embodiment, the abatacept is discontinued when treatment with an IL-6 antagonist is initiated.

[0147] In certain embodiments, the patient is being treated with an IL-6 antagonist, and the method further comprises continuing to administer the IL-6 antagonist only to those patients who are newly determined to have at least one copy of the TMPRSS6 rs855791 major allele. In certain embodiments, the IL-6 antagonist is tocilizumab. In certain embodiments, the IL-6 antagonist is tofacitinib.

[0148] In various embodiments, the chronic inflammatory disease is selected from the group consisting of juvenile idiopathic arthritis, ankylosing spondylitis, plaque psoriasis, psoriatic arthritis, inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

[0149] 5.2.1.1.3. Cancer In various embodiments, the chronic disease is cancer.

[0150] In some embodiments, the cancer is selected from the group consisting of a solid tumor, small cell lung cancer, non-small cell lung cancer, a hematological cancer, multiple myeloma, leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), lymphoma, and Hodgkin's lymphoma.

[0151] 5.2.1.1.4. Chronic infections In various embodiments, the chronic disease is a chronic infectious disease.

[0152] Congestive heart failure In various embodiments, the chronic disease is congestive heart failure (CHF).

[0153] 5.2.1.2 Iron-refractory iron deficiency anemia (IRIDA) In various embodiments, the hepcidin-mediated disorder is iron-refractory iron deficiency anemia (IRIDA).

[0154] 5.2.1.3. Anemia associated with hepcidin-producing hepatic adenoma In various embodiments, the hepcidin-mediated disorder is anemia associated with hepcidin-producing hepatic adenoma.

[0155] 5.2.1.4. Acute coronary syndrome The data presented in Examples 2, 3, and 5 below demonstrate that IL-6 antagonists are effective in reducing the risk of heart failure and death, improving cardiac function, and reducing fibrosis after acute myocardial infarction. Thus, in various embodiments, the hepcidin-mediated disorder is acute coronary syndrome.

[0156] In certain embodiments, the patient suffered a myocardial infarction 60 days prior to the first administration of the IL-6 antagonist, hi specific embodiments, the patient suffered a myocardial infarction within 30 days, 14 days, 7 days, 48 ​​hours, or 24 hours prior to the first administration of the IL-6 antagonist.

[0157] In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to improve myocardial contractility compared to pre-treatment levels. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to improve cardiac ejection fraction compared to pre-treatment levels. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to reduce cardiac fibrosis compared to pre-treatment levels.

[0158] 5.2.1.5. Castleman disease In various embodiments, the hepcidin-mediated disorder is Castleman's disease.

[0159] 5.3. Methods for Improving the Treatment of Hepcidin-Mediated Disorders In another aspect, a method is provided for improving the treatment of hepcidin-mediated disorders by discontinuing ineffective therapy, thereby reducing side effects and costs without losing therapeutic efficacy. The method comprises discontinuing administration of an IL-6 antagonist to a patient with a hepcidin-mediated disorder, wherein the patient has been determined to be homozygous for the TMPRSS6 rs855791 minor allele. In one series of embodiments, the patient has previously been determined to be homozygous for the TMPRSS6 rs855791 minor allele. In another series of embodiments, the method further comprises an initial step of determining that the patient is homozygous for the TMPRSS6 rs855791 minor allele. In a typical embodiment, the patient has elevated pre-treatment serum levels of IL-6. In various embodiments, the patient has elevated pre-treatment serum levels of CRP.

[0160] In various embodiments, the patient has a hepcidin-mediated disorder selected from those described above in Section 5.2.1. In certain embodiments, the patient has anemia of chronic disease.

[0161] 5.4. Methods for Treating IL-6-Mediated Inflammatory Disorders The data presented in Examples 2, 3, and 5 below demonstrate that IL-6 antagonists provide therapeutic benefit in subjects with elevated pretreatment IL-6 levels and at least one copy of the TMPRSS6 major allele, even in the absence of anemia. Thus, in another aspect, methods are provided for treating IL-6-mediated inflammatory disorders in patients without anemia of chronic inflammation.

[0162] The method comprises administering a therapeutically effective amount of an IL-6 antagonist to a subject, typically a human patient, with an IL-6-mediated inflammatory disorder, wherein the patient does not have anemia and the subject has been determined to have at least one copy of the TMPRSS6 rs855791 major allele. In a first series of embodiments, the subject has previously been determined to have at least one copy of the TMPRSS6 rs855791 major allele. In another series of embodiments, the method further comprises the initial step of determining that the subject has at least one copy of the TMPRSS6 rs855791 major allele. Typically, the method affirmatively excludes treatment of subjects who are homozygous for the TMPRSS6 rs855791 minor allele. Typically, the patient has elevated pretreatment serum levels of IL-6.

[0163] In some embodiments, the IL-6 mediated disorder is rheumatoid arthritis (RA).

[0164] In certain embodiments, the patient has a pre-treatment DAS28 score of greater than 5.1. In some embodiments, the patient has a pre-treatment DAS28 score of 3.2 to 5.1. In some embodiments, the patient has a pre-treatment DAS28 score of less than 2.6. In various embodiments, the patient's pre-treatment RA is severely active. In some embodiments, the patient's pre-treatment RA is moderately active.

[0165] In certain embodiments, the patient is being treated with methotrexate. In some embodiments, methotrexate is discontinued when treatment with an IL-6 antagonist is initiated. In some embodiments, methotrexate is continued when treatment with an IL-6 antagonist is initiated.

[0166] In certain embodiments, the patient is being treated with an anti-TNFα agent. In specific embodiments, the anti-TNFα agent is selected from etanercept, adalimumab, infliximab, certolizumab, and golimumab. In specific embodiments, the anti-TNFα agent is discontinued when treatment with the IL-6 antagonist is initiated.

[0167] In certain embodiments, the patient is being treated with an IL-1 receptor antagonist. In a specific embodiment, the IL-1 receptor antagonist is anakinra. In a specific embodiment, the IL-1 receptor antagonist is discontinued when treatment with the IL-6 antagonist is initiated.

[0168] In certain embodiments, the patient is being treated with abatacept, hi a specific embodiment, the abatacept is discontinued when treatment with the IL-6 antagonist is initiated.

[0169] In various embodiments, the IL-6 mediated disorder is selected from the group consisting of juvenile idiopathic arthritis, ankylosing spondylitis, plaque psoriasis, psoriatic arthritis, inflammatory bowel disease, Crohn's disease, and ulcerative colitis.

[0170] 5.5. Pre-treatment serum IL-6 and CRP levels In an exemplary embodiment of the methods described herein, the patient has elevated pre-treatment serum levels of IL-6.

[0171] In some embodiments, the patient has a pre-treatment serum IL-6 level greater than 2.5 pg / ml. In various embodiments, the patient has a pre-treatment serum IL-6 level greater than 5 pg / ml, greater than 7.5 pg / ml, greater than 10 pg / ml, greater than 12.5 pg / ml, or greater than 15 pg / ml.

[0172] In some embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce the patient's serum IL-6 levels below pre-treatment levels, hi certain embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce the patient's serum IL-6 levels by at least 10%, 20%, 30%, 40%, or 50%.

[0173] In various embodiments, the patient has elevated pre-treatment levels of C-reactive protein (CRP). In some embodiments, the patient has a pre-treatment CRP level greater than 2 mg / ml, 2.5 mg / ml, 3 mg / ml, 3.5 mg / ml, 4 mg / ml, 4.5 mg / ml, or 5 mg / ml. In some embodiments, the patient has a pre-treatment CRP level greater than 7.5 mg / ml, 10 mg / ml, 12.5 mg / ml, or 15 mg / ml.

[0174] In some embodiments, the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce the patient's CRP levels below pre-treatment levels, hi certain embodiments, the IL-6 antagonist is administered at a dose, on a schedule, or for a duration sufficient to reduce the patient's CRP levels by at least 10%, 20%, 30%, 40%, or 50% compared to pre-treatment levels.

[0175] TMPRSS6 rs855791 genotyping The methods described herein involve administering a therapeutically effective amount of an IL-6 antagonist to a subject determined to have at least one copy of the TMPRSS6 rs855791 major allele. Preferably, both alleles corresponding to the gene of interest are identified, thereby enabling identification and differentiation of patients who are homozygous for the TMPRSS6 rs855791 major allele, heterozygous for both the major and minor TMPRSS6 rs855791 alleles, and homozygous for the TMPRSS6 rs855791 minor allele.

[0176] The absence (major allele) or presence (minor allele) of SNP rs855791 (2321G→A) in the TMPRSS6 gene is determined using standard techniques.

[0177] Typically, PCR is used to amplify a biological sample obtained from a patient.

[0178] In some embodiments, the absence or presence of a polymorphism is detected simultaneously with amplification using real-time PCR (RT-PCR). In certain embodiments, RT-PCR assays use 5' nucleases (TaqMan® probes), molecular beacons, and / or FRET hybridization probes. These are reviewed in Espy et al., Clin. Microbiol. Rev. 2006 Jan; 19(1):165-256, incorporated herein by reference in its entirety. In typical embodiments, commercially available assays are used. In selected embodiments, the commercially available assays are selected from the group consisting of TaqMan™ SNP genotyping assays (ThermoFisher), PCR SNP genotyping assays (Qiagen), Novallele genotyping assays (Canon), and SNP Type™ assays (formerly SNPtype) (Fluidigm).

[0179] In some embodiments, the absence or presence of the polymorphism is detected after amplification by hybridization using a probe specific for SNP rs855791, restriction endonuclease digestion, nucleic acid sequencing, primer extension, microarray or gene chip analysis, mass spectrometry, and / or DNAse protection assay. In some embodiments, the allelic variant is determined by sequencing. In certain embodiments, Sanger sequencing is used. In certain embodiments, one of a variety of next-generation sequencing technologies is used, including, for example, a sequencing technology selected from the group consisting of microarray sequencing, Solexa sequencing (Illumina), Ion Torrent (Life Technologies), SOliD (Applied Biosystems), pyrosequencing, single-molecule real-time sequencing (Pacific Bio), nanopore sequencing, and tunneling current sequencing.

[0180] 5.7. IL-6 Antagonists The IL-6 antagonists used in the methods described herein are capable of reducing the biological activity of IL-6.

[0181] 5.7.1. Anti-IL-6 antibodies In various embodiments, the IL-6 antagonist is an anti-IL-6 antibody or an antigen-binding fragment or derivative thereof.

[0182] In some embodiments, the IL-6 antagonist is a full-length anti-IL-6 monoclonal antibody. In a specific embodiment, the full-length monoclonal antibody is an IgG antibody. In certain embodiments, the full-length monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the IL-6 antagonist is a polyclonal composition comprising multiple full-length anti-IL-6 antibodies, each of which has a unique CDR. In some embodiments, the IL-6 antagonist is an antibody fragment selected from Fab, Fab', and F(ab')2 fragments. In some embodiments, the IL-6 antagonist is a single-domain antibody, such as an scFv, a disulfide-linked Fv (dsFv), or a camelid-derived VHH single-domain nanobody. In some embodiments, the IL-6 antagonist is an immunoconjugate or fusion comprising an IL-6 antigen-binding fragment. In some embodiments, the antibody is bispecific or multispecific, and at least one of the antigen-binding moieties has specificity for IL-6.

[0183] In some embodiments, the antibody is fully human. In some embodiments, the antibody is humanized. In some embodiments, the antibody is chimeric, having non-human V region and human C region domains. In some embodiments, the antibody is murine.

[0184] In an exemplary embodiment, the anti-IL-6 antibody has a K of less than 100 nM for binding to human IL-6. D For some embodiments, the anti-IL-6 antibody has a K of less than 75 nM, 50 nM, 25 nM, 20 nM, 15 nM, or 10 nM for binding to human IL-6. D In specific embodiments, the anti-IL-6 antibody has a K of less than 5 nM, 4 nM, 3 nM, or 2 nM for binding to human IL-6. D In selected embodiments, the anti-IL-6 antibody has a K of less than 1 nM, 750 pM, or 500 pM for binding to human IL-6. DIn certain embodiments, the anti-IL-6 antibody has a K of 500 pM, 400 pM, 300 pM, 200 pM, or 100 pM or less for binding to human IL-6. D It has.

[0185] In typical embodiments, the anti-IL-6 antibody neutralizes the biological activity of IL-6. In some embodiments, the neutralizing antibody prevents IL-6 from binding to the IL-6 receptor.

[0186] In typical embodiments, the anti-IL-6 antibody has an elimination half-life after intravenous administration of at least 7 days, hi certain embodiments, the anti-IL-6 antibody has an elimination half-life of at least 14 days, at least 21 days, or at least 30 days.

[0187] In some embodiments, the anti-IL-6 antibody has a human IgG constant region with at least one amino acid substitution that extends serum half-life compared to an unsubstituted human IgG constant domain.

[0188] In certain practical forms, the IgG constant domain comprises substitutions at residues 252, 254, and 256, where the amino acid substitution at amino acid residue 252 is with tyrosine, the amino acid substitution at amino acid residue 254 is with threonine, and the amino acid substitution at amino acid residue 256 is with glutamic acid ("YTE"). See U.S. Patent No. 7,083,784, which is incorporated herein by reference in its entirety. In certain extended half-life embodiments, the IgG constant domain comprises substitutions selected from T250Q / M428L (Hinton et al., J. Immunology 176:346-356 (2006)), N434A (Yeung et al., J. Immunology 182:7663-7671 (2009)), or T307A / E380A / N434A (Petkova et al., International Immunology, 18:1759-1769 (2006)).

[0189] In some embodiments, the excretion half-life of an anti-IL-6 antibody is increased by utilizing the FcRN binding property of human serum albumin. In certain embodiments, the antibody is conjugated to albumin (Smith et al., Bioconjug. Chem., 12: 750-756 (2001)). In some embodiments, the anti-IL-6 antibody is fused to a bacterial albumin-binding domain (Stork et al., Prot. Eng. Design Science, 20: 569-76 (2007)). In some embodiments, the anti-IL-6 antibody is fused to an albumin-binding peptide. ((Nguygen et al., Prot Eng Design Sel 19: 291-297 (2006)). In some embodiments, the anti-IL-1 antibody is bispecific, with one specificity directed against IL-6R and one specificity directed against human serum albumin (Ablynx, WO2006 / 122825 (bispecific nanobody)).

[0190] In some embodiments, the elimination half-life of an anti-IL-6 antibody is increased by PEGylation (Melmed et al., Nature Reviews Drug Discovery 7: 641-642 (2008)), HPMA copolymer conjugation (Lu et al., Nature Biotechnology 17: 1101-1104 (1999)), dextran conjugation (Nuclear Medicine Communications, 16: 362-369 (1995)), conjugation with homoamino acid polymers (HAPs, Hapylation) (Schlapschy et al., Prot Eng Design Sel 20: 273-284 (2007)), or polysialylation (Constantinou et al., Bioconjug. Chem. 20: 924-931 (2009)).

[0191] 5.7.1.1.1. MED5117 and derivatives In certain embodiments, the anti-IL-6 antibody, or antigen-binding portion thereof, comprises all six CDRs of MEDI5117. In specific embodiments, the antibody, or antigen-binding portion thereof, comprises the MEDI5117 heavy chain V region and light chain V region. In particular embodiments, the antibody is a full-length MEDI5117 antibody. The MEDI5117 antibody is described in WO2010 / 088444 and US 2012 / 0034212, the disclosures of which are incorporated herein by reference in their entireties. The MEDI5117 antibody has the following CDRs and heavy and light chain sequences:

[0192] MEDI5117 VH CDR1 SNYMI (SEQ ID NO: 12) MEDI5117 VH CDR2 DLYYYAGDTYYADSVKG (SEQ ID NO: 13) MEDI5117 VH CDR3 WADDHPPWIDL (SEQ ID NO: 14) MEDI5117 VL CDR1 RASQGISSWLA (SEQ ID NO: 15) MEDI5117 VL CDR2 KASTLES (SEQ ID NO: 16) MEDI5117 VL CDR3 QQSWLGGS (SEQ ID NO: 17)

[0193] MEDI5117 heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTISSNYMIWVRQAPGKGLEWVSDLYYYAGDTYY ADSVKGRFTMSRDISKNTVYLQMNSLRAEDTAVYYCARWADDHPPWIDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18)

[0194] MEDI5117 light chain DIQMTQSPSTLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKVLIYKASTLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQSWLGGSFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19)

[0195] In various embodiments, the anti-IL-6 antibody is a derivative of MED5117.

[0196] In some embodiments, MED5117 derivatives contain one or more amino acid substitutions in the MED5117 heavy and / or light chain V regions.

[0197] In certain embodiments, the derivative has the same structure as the original V of the MEDI5117 anti-IL-6 antibody. H and / or V L Compared to the above, it contains fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, fewer than 2 amino acid substitutions, or fewer than 1 amino acid substitution while retaining specificity for human IL-6.

[0198] In certain embodiments, a MED5117 derivative comprises an amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the VH and VL domains of MEDI5117. Percent sequence identity is determined using the BLAST algorithm using default parameters.

[0199] In certain embodiments, a MED5117 derivative comprises an amino acid sequence whose CDRs are at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the respective CDR of MEDI5117. Percent sequence identity is determined using the BLAST algorithm using default parameters.

[0200] In certain embodiments, V H and / or V L The CDR derivatives contain conservative amino acid substitutions at one or more predicted non-essential amino acid residues (ie, amino acid residues that are not important for the antibody to specifically bind human IL-6).

[0201] 5.7.1.1.2. Other Anti-IL-6 Antibodies In various embodiments, the anti-IL-6 antibody comprises six CDRs from an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, elsilimomab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (Argen-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In certain embodiments, the anti-IL-6 antibody comprises a heavy chain V region and a light chain V region derived from an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In specific embodiments, the anti-IL-6 antibody is an antibody selected from the group consisting of siltuximab, gerilimu- zumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).

[0202] In some embodiments, the anti-IL-6 antibody is described in US 2016 / 0168243, US 2016 / 0130340, 2015 / 0337036, US 2015 / 0203574, US 2015 / 0140011, US 2015 / 0125468, US 2014 / 0302058, US 2014 / 0141013, US 2013 / 0280266, US 2013 / 0017575, US 2010 / 0215654, US 2008 / 0075726, U.S. Pat. No. 5,856,135, US 2006 / 0240012, US 2006 / 0257407, or U.S. Patent No. 7,291,721.

[0203] 5.7.2. Anti-IL-6 Receptor Antibodies In various embodiments, the IL-6 antagonist is an anti-IL-6 receptor antibody or an antigen-binding fragment or derivative thereof.

[0204] In some embodiments, the IL-6 antagonist is a full-length anti-IL-6 receptor monoclonal antibody. In specific embodiments, the full-length monoclonal antibody is an IgG antibody. In certain embodiments, the full-length monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the IL-6 antagonist is a polyclonal composition comprising multiple species of full-length anti-IL-6 receptor antibodies, each of which has a unique CDR. In some embodiments, the IL-6 antagonist is an antibody fragment selected from Fab and Fab' fragments. In some embodiments, the IL-6 antagonist is a single-domain antibody, an scFv, including a camelid-derived VHH single-domain nanobody. In some embodiments, the antibody is bispecific or multispecific, and at least one of the antigen-binding portions has specificity for IL-6R.

[0205] In some embodiments, the antibody is fully human. In some embodiments, the antibody is humanized. In some embodiments, the antibody is chimeric, having non-human V region and human C region domains. In some embodiments, the antibody is of murine origin.

[0206] In an exemplary embodiment, the anti-IL-6 receptor antibody has a K of less than 100 nM for binding to human IL-6R. D In some embodiments, the anti-IL-6R antibody has a K of less than 75 nM, 50 nM, 25 nM, 20 nM, 15 nM, or 10 nM for binding to human IL-6R. D In specific embodiments, the anti-IL-6 receptor antibody has a K of less than 5 nM, 4 nM, 3 nM, or 2 nM for binding to human IL-6R. D In selected embodiments, the anti-IL-6 receptor antibody has a K of less than 1 nM, 750 pM, or 500 pM for binding to human IL-6R. D In specific embodiments, the anti-IL-6 receptor antibody has a K of 500 pM, 400 pM, 300 pM, 200 pM, or 100 pM or less for binding to human IL-6R. D It has.

[0207] In an exemplary embodiment, the anti-IL-6R reduces the biological activity of IL-6.

[0208] In typical embodiments, the anti-IL-6R antibody has an elimination half-life after intravenous administration of at least 7 days, hi certain embodiments, the anti-IL-6R antibody has an elimination half-life of at least 14 days, at least 21 days, or at least 30 days.

[0209] In some embodiments, the anti-IL-6R antibody has a human IgG constant region with at least one amino acid substitution that extends serum half-life compared to an unsubstituted human IgG constant domain.

[0210] In certain practical forms, the IgG constant domain comprises substitutions at residues 252, 254, and 256, where the amino acid substitution at amino acid residue 252 is with tyrosine, the amino acid substitution at amino acid residue 254 is with threonine, and the amino acid substitution at amino acid residue 256 is with glutamic acid ("YTE"). See U.S. Patent No. 7,083,784, which is incorporated herein by reference in its entirety. In certain extended half-life embodiments, the IgG constant domain comprises substitutions selected from T250Q / M428L (Hinton et al., J. Immunology 176:346-356 (2006)), N434A (Yeung et al., J. Immunology 182:7663-7671 (2009)), or T307A / E380A / N434A (Petkova et al., International Immunology, 18:1759-1769 (2006)).

[0211] In some embodiments, the excretion half-life of an anti-IL-6R antibody is increased by utilizing the FcRN-binding properties of human serum albumin. In certain embodiments, the antibody is conjugated to albumin (Smith et al., Bioconjug. Chem., 12: 750-756 (2001)). In some embodiments, the anti-IL-6R antibody is fused to a bacterial albumin-binding domain (Stork et al., Prot. Eng. Design Science, 20: 569-76 (2007)). In some embodiments, the anti-IL-6 antibody is fused to an albumin-binding peptide (Nguygen et al., Prot Eng Design Sel, 19: 291-297 (2006)). In some embodiments, the anti-IL-1 antibody is bispecific, with one specificity directed against IL-6R and one specificity directed against human serum albumin (Ablynx, WO2006 / 122825 (bispecific nanobody)).

[0212] In some embodiments, the elimination half-life of an anti-IL-6R antibody is increased by PEGylation (Melmed et al., Nature Reviews Drug Discovery 7: 641-642 (2008)), HPMA copolymer conjugation (Lu et al., Nature Biotechnology 17: 1101-1104 (1999)), dextran conjugation (Nuclear Medicine Communications, 16: 362-369 (1995)), conjugation with homoamino acid polymers (HAPs, Hapylation) (Schlapschy et al., Prot Eng Design Sel 20: 273-284 (2007)), or polysialylation (Constantinou et al., Bioconjug. Chem. 20: 924-931 (2009)).

[0213] In certain embodiments, the anti-IL-6R antibody or antigen-binding portion thereof comprises all six CDRs of tocilizumab. In specific embodiments, the antibody or antigen-binding portion thereof comprises the heavy chain V region and light chain V region of tocilizumab. In particular embodiments, the antibody is a full-length tocilizumab antibody.

[0214] In certain embodiments, the anti-IL-6R antibody or antigen-binding portion thereof comprises all six CDRs of sarilumab. In specific embodiments, the antibody or antigen-binding portion thereof comprises the heavy chain V region and light chain V region of sarilumab. In particular embodiments, the antibody is a full-length sarilumab antibody.

[0215] In certain embodiments, the anti-IL-6R antibody, or antigen-binding portion thereof, comprises all six CDRs of VX30 (Vaccinex), ARGX-109 (arGEN-X), FM101 (Formatech), SA237 (Roche), NI-1201 (NovImmune), or an antibody described in US 2012 / 0225060.

[0216] In certain embodiments, the anti-IL-6R antibody, or antigen-binding portion thereof, is a single-domain antibody. In a specific embodiment, the single-domain antibody is a camelid VHH single-domain antibody. In a specific embodiment, the antibody is bovalilizumab (ALX-0061) (Ablynx NV).

[0217] 5.7.3. Anti-IL-6:IL-6R complex antibody In various embodiments, the IL-6 antagonist is an antibody specific for the complex of IL-6 and IL-6R, hi certain embodiments, the antibody has six CDRs of an antibody selected from those described in US 2011 / 0002936, the entire contents of which are incorporated herein by reference.

[0218] JAK and STAT Inhibitors IL-6 is known to signal through the JAK-STAT pathway.

[0219] In various embodiments, the IL-6 antagonist is an inhibitor of the JAK signaling pathway. In some embodiments, the JAK inhibitor is a JAK1-specific inhibitor. In some embodiments, the JAK inhibitor is a JAK3-specific inhibitor. In some embodiments, the JAK inhibitor is a pan-JAK inhibitor.

[0220] In certain embodiments, the JAK inhibitor is selected from the group consisting of tofacitinib (Xeljanz), decernotinib, ruxolitinib, upadacitinib, baricitinib, filgotinib, lestaurtib, pacritinib, peficitinib, INCB-039110, ABT-494, INCB-047986, and AC-410.

[0221] In various embodiments, the IL-6 antagonist is a STAT3 inhibitor. In certain embodiments, the inhibitor is AZD9150 (AstraZeneca, Isis Pharmaceuticals), a STAT3 antisense molecule.

[0222] 5.7.5. Additional IL-6 Antagonists In various embodiments, the IL-6 antagonist is an antagonist peptide.

[0223] In certain embodiments, the IL-6 antagonist is C326 (an IL-6 inhibitor by Avidia, also known as AMG220) or FE301, a recombinant protein inhibitor of IL-6 (Ferring International Center SA, Conaris Research Institute AG). In some embodiments, the anti-IL-6 antagonist comprises soluble gp130, FE301 (Conaris / Ferring).

[0224] 5.8. Medication Regimen 5.8.1. Antibodies, Antigen-Binding Fragments, and Peptides In an exemplary embodiment, the antibody, antigen-binding fragment, and peptide IL-6 antagonists are administered parenterally.

[0225] In some parenteral embodiments, the IL-6 antagonist is administered intravenously. In certain intravenous embodiments, the IL-6 antagonist is administered as a bolus. In certain intravenous embodiments, the IL-6 antagonist is administered as an infusion. In certain intravenous embodiments, the IL-6 antagonist is administered as a bolus followed by an infusion. In some parenteral embodiments, the IL-6 antagonist is administered subcutaneously.

[0226] In various embodiments, the antibody, antigen-binding fragment, or peptide IL-6 antagonist is administered at a dose that is independent of the patient's weight or surface area (flat dose).

[0227] In some embodiments, the intravenous flat dose is 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In some embodiments, the intravenous flat dose is 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg. In some embodiments, the intravenous flat dose is 25 mg, 30 mg, 40 mg, or 50 mg. In some embodiments, the intravenous flat dose is 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg. In some embodiments, the intravenous flat dose is 1 to 10 mg, 10 to 15 mg, 15 to 20 mg, 20 to 30 mg, 30 to 40 mg, or 40 to 50 mg. In some embodiments, the intravenous flat dose is 1 to 40 mg or 50 to 100 mg.

[0228] In some embodiments, the subcutaneous flat dose is 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg. In some embodiments, the subcutaneous flat dose is 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, or 200 mg. In some embodiments, the subcutaneous flat dose is 210 mg, 220 mg, 230 mg, 240 mg, or 250 mg. In some embodiments, the subcutaneous flat dose is 10-100 mg, 100-200 mg, or 200-250 mg. In some embodiments, the subcutaneous flat dose is 10-20 mg, 20-30 mg, 30-40 mg, 40-50 mg, 50-60 mg, 60-70 mg, 70-80 mg, 80-90 mg, or 90-100 mg. In some embodiments, the subcutaneous flat dose is 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, or 200-250 mg.

[0229] In various embodiments, the antibody, antigen-binding fragment, or peptide IL-6 antagonist is administered as a patient weight-based dose.

[0230] In some embodiments, the antagonist is administered at an intravenous dose of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1.0 mg / kg. In some embodiments, the antagonist is administered at a dose of 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, or 5 mg / kg.

[0231] In some embodiments, the subcutaneous weight-based dose is 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1.0 mg / kg. In some embodiments, the antagonist is administered at a dose of 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, or 5 mg / kg.

[0232] In various intravenous embodiments, the IL-6 antagonist is administered once every 7 days, once every 14 days, once every 21 days, once every 28 days, or once a month. In various subcutaneous embodiments, the IL-6 antagonist is administered once every 14 days, once every 28 days, once a month, once every two months (every other month), or once every three months.

[0233] In certain preferred embodiments, the IL-6 antagonist is a MEDI5117 antibody. In various embodiments, MEDI5117 is administered IV once weekly at a flat dose of 1-30 mg. In certain embodiments, MEDI5117 antibody is administered IV once weekly at a flat dose of 1, 2, 3, 4, 5, 7.5, 10, 15, 20, 25, or 30 mg. In some embodiments, MEDI5117 antibody is administered sc once monthly to once every three months at a flat dose of 25-250 mg. In specific embodiments, MEDI5117 is administered sc once monthly, every two months, or every three months at a dose of 30 mg, 45 mg, 60 mg, 75 mg, 100 mg, 120 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, or 250 mg.

[0234] In some embodiments, the IL-6 antagonist is tocilizumab. In various embodiments, tocilizumab is administered sc once weekly to patients weighing 100 kg or more at a starting dose of 162 mg. In some embodiments, tocilizumab is administered intravenously at a dose of 4 mg / kg once every four weeks, then increased to 8 mg / kg every four weeks based on clinical response.

[0235] JAK and STAT Inhibitors In an exemplary embodiment, the small molecule JAK inhibitor and STAT inhibitor are administered orally.

[0236] In various embodiments, the inhibitor is administered at an oral dose of 1-10 mg, 10-20 mg, 20-30 mg, 30-40 mg, or 40-50 mg once or twice daily. In some embodiments, the inhibitor is administered at a dose of 50-60 mg, 60-70 mg, 70-80 mg, 80-90 mg, or 90-100 mg once or twice daily. In some embodiments, the inhibitor is administered at a dose of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg PO once or twice daily. In some embodiments, the inhibitor is administered at a dose of 75 mg PO QD or BID, 100 mg PO QD or BID.

[0237] In certain embodiments, the JAK inhibitor is tofacitinib and is administered at a dose of 5 mg PO BID or 11 mg PO qDay.

[0238] In certain embodiments, the JAK inhibitor is decernotinib and is administered at a dose of 25 mg, 50 mg, 100 mg, or 150 mg PO BID.

[0239] In certain embodiments, the inhibitor is ruxolitinib and is administered at a dose of 25 mg PO BID, 20 mg PO BID, 15 mg PO BID, 10 mg PO BID, or 5 mg PO BID.

[0240] 5.9. Additional Therapeutic Agents In various embodiments of the methods described herein, the methods further comprise administering an additional therapeutic agent to the IL-6 antagonist, wherein the second therapeutic agent is also capable of decreasing hepcidin expression.

[0241] In some embodiments, the second therapeutic agent is a BMP antagonist. In certain embodiments, the BMP antagonist is an anti-BMP6 antibody. In certain embodiments, the anti-BMP6 antibody has six CDRs of the antibodies described in US 2016 / 0176956 or US 2016 / 0159896, the disclosures of which are incorporated herein by reference in their entireties.

[0242] In certain embodiments, the second therapeutic agent is a hemojuvelin antagonist. In particular embodiments, the hemojuvelin antagonist is an anti-hemojuvelin antibody. In particular embodiments, the anti-hemojuvelin antibody has six CDRs of the antibody disclosed in Kovac et al., Haematologica (2016) doi:10.3324 / haematol.2015.140772 [ePub ahead of print].

[0243] In certain embodiments, the second therapeutic agent is a hepcidin antagonist. In certain embodiments, the hepcidin antagonist is an anti-hepcidin antibody. In certain embodiments, the antibody has six CDRs derived from the antibody described in US 2016 / 0017032, the disclosure of which is incorporated herein by reference in its entirety.

[0244] Kit In another aspect, a kit is provided.

[0245] In an exemplary embodiment, the kit provides reagents for determining a patient's genotype at the TMPRSS6 SNP rs855791 position from a biological sample obtained from the patient.

[0246] 5.11. Further Aspects and Embodiments 5.11.1. Methods for Treating Inflammation in Chronic Kidney Disease or Cardiovascular Disease In other aspects and embodiments, methods for characterizing and treating inflammation in chronic kidney disease or cardiovascular disease with IL-6 antagonists, as well as compositions and methods for characterizing patient responsiveness to treatment, are provided.

[0247] These aspects and embodiments are based, at least in part, on the discovery that inflammation in patients with chronic kidney disease and cardiovascular disease who have one or more alleles of TMPRSS6 containing a G or C at nucleotide position 2321 (encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736) places these patients at a higher risk of mortality, and that such subjects could be treated with an IL-6 antagonist to reduce this risk. As reported in more detail below, patients with chronic kidney disease were genotyped and their serum levels of IL-6 and CRP were assayed, and these diagnostic data were compared with the administered EPO dose and risk of mortality. Patients who have one or more alleles of TMPRSS6 containing a G or C at nucleotide position 2321 (encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736) and who had elevated IL-6 and / or CRP levels required higher EPO doses for treatment and had higher mortality rates. The nucleotide at this position has been shown to be important in identifying patients with iron deficiency anemia (see Finberg, Nat. Genet. 2008, 40(5):569-571, which is incorporated herein by reference in its entirety for all it teaches, as well as sequences, variants, nomenclature, etc.). These data strongly support the identification of a subset of patients based on TMPRSS6 genotype who require higher EPO doses and / or have a higher risk of mortality, and who may respond to IL-6 inhibition with or without standard therapy for the treatment of anemia (e.g., associated with chronic kidney disease). By inhibiting inflammation, EPO doses can be reduced, thereby avoiding the adverse side effects of EPO (e.g., cardiovascular risk).

[0248] These aspects and embodiments are further based on the discovery that patients with one or more alleles of TMPRSS6 containing a G or C at nucleotide position 2321 (encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736) are at higher risk of myocardial infarction or cardiovascular disease-related mortality. These patients may also benefit from IL-6 inhibition, which reduces inflammation and increased risk.

[0249] Thus, therapeutic methods are provided for treating inflammation associated with cardiovascular disease or chronic kidney disease, including anemia of chronic kidney disease, and / or reducing the risk of mortality associated with such conditions by inhibiting the biological activity of IL-6, e.g., by blocking the binding of IL-6 or its receptor (gp80) to each other, or by blocking their signaling or expression (e.g., by anti-IL-6 antibodies or anti-IL-6R antibodies or JAK1 / STAT3 inhibition), in patients selected by genotyping TMPRSS6 at SNP rs855791. In one embodiment, the treatment of chronic kidney disease is performed with or without standard treatment for the anemia, and methods are performed to characterize the responsiveness of patients with chronic kidney disease to treatment for the anemia, e.g., by genotyping TMPRSS6 at SNP rs855791 and detecting levels of inflammatory markers (e.g., elevated serum levels of IL-6 and / or CRP).

[0250] Administering an agent that inhibits IL-6 biological activity or expression provides a method for treating anemia and / or reducing mortality in cardiovascular disease or chronic kidney disease associated with chronic inflammation in such patients.

[0251] In some aspects and embodiments, compositions and methods are provided for treating chronic inflammation, which contributes to mortality in subjects with chronic kidney disease or cardiovascular disease, and for characterizing patient responsiveness to such therapy. In specific embodiments, methods are provided for characterizing and treating chronic inflammatory anemia and mortality (e.g., in chronic kidney disease), and for characterizing patient responsiveness to treatment of anemia (e.g., administration of erythropoietin or an erythropoiesis-stimulating agent). In one aspect, a method is provided for treating chronic inflammation in a selected subject, the method comprising administering to the subject an IL-6 antagonist, wherein the subject is selected for treatment by having one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736.

[0252] In another aspect, a method is provided for treating inflammation or chronic inflammation in a selected subject with cardiovascular disease or chronic kidney disease, the method involving administering an IL-6 antagonist (e.g., an anti-IL-6 antibody) to the subject, wherein the subject is selected for treatment by having one or more alleles encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736. In one embodiment, the method reduces the subject's risk of death. In one embodiment, the subject has a history of myocardial infarction or heart failure.

[0253] In another aspect, a method is provided for reducing inflammation and risk of mortality in a selected subject with cardiovascular or renal disease, the method comprising administering an IL-6 antagonist (e.g., an anti-IL-6 antibody) to the subject, wherein the subject is selected as having one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736 and experiencing increased inflammation compared to a reference. In one embodiment, the subject has a history of myocardial infarction or heart failure.

[0254] In another aspect, a method is provided for reducing the risk of death in a subject with chronic kidney disease or heart failure, the method comprising administering an IL-6 antagonist to the subject, wherein the subject is identified as having one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736 and has increased inflammation compared to a reference.

[0255] In another aspect, a method for treating anemia in a subject is provided, the method involving administering an IL-6 antagonist, alone or in combination with an anemia therapy, to the subject, wherein the subject is identified as having one or more alleles encoding a TMPRSS6 polypeptide (also called matriptase-2, MT2) containing an alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) and has increased inflammation compared to a reference.

[0256] In another aspect, a method is provided for treating anemia in a subject with increased inflammation, the method involving administering an IL-6 antagonist (e.g., an IL-6 antibody) alone or in combination with an erythropoietic factor in an amount effective to neutralize inflammation in a subject having one or more alleles encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule).

[0257] In yet another aspect, a method is provided for enhancing responsiveness to EPO in a subject identified as being in need thereof, the method comprising administering an IL-6 antagonist (e.g., an IL-6 antibody) in an amount effective to neutralize inflammation in a subject having one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule), thereby reducing the EPO dose.

[0258] In another aspect, a method for reducing mortality in a subject with increased inflammation is provided, the method involving administering an IL-6 antagonist in an amount effective to neutralize inflammation in a subject with one or more alleles encoding a TMPRSS6 polypeptide including an alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule).

[0259] In yet another aspect, a method is provided for selecting therapy for a subject identified as being in need thereof, the method involving characterizing subjects having one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) and detecting levels of one or more inflammatory markers, IL-6 or CRP, wherein the characterization indicates that an IL-6 antagonist should be administered alone or in combination with treatment for anemia.

[0260] In yet another aspect, a method is provided for increasing the proliferation or survival of red blood cells or their precursor cells (e.g., hematopoietic stem cells, proerythroblasts, erythroblasts, or reticulocytes) in a subject identified as being in need thereof, the method comprising administering to the subject an IL-6 antagonist and an erythropoietic factor, wherein the subject is identified as having one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) and experiencing increased inflammation compared to a reference.

[0261] In various embodiments of any of the aspects described herein, the subject has or is identified as having anemia, including anemia of cancer, anemia in chronic autoimmune disease, anemia in chronic inflammatory disease, anemia in cardiovascular disease, anemia in metabolic syndrome, etc. In various embodiments of any of the aspects described herein, the subject has or is identified as having chronic kidney disease. In various embodiments of any of the aspects described herein, the subject has or is identified as having inflammation. In various embodiments of any of the aspects described herein, the subject has or is identified as having an increased risk of mortality associated with chronic inflammation, chronic kidney disease, or cardiovascular disease. In various embodiments of any of the aspects described herein, the subject is identified as being in need of treatment. In various embodiments of any of the aspects described herein, the subject has or is identified as having increased inflammation. In various embodiments of any of the aspects described herein, the subject has, or is identified as having, one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736 (e.g., a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) and has increased inflammation compared to a reference. In various embodiments of any of the aspects described herein, the method comprises administering to the subject an IL-6 antagonist. In various embodiments of any of the aspects described herein, the method comprises administering to the subject an IL-6 antagonist and an anemia therapy. In various embodiments of any of the aspects described herein, the subject is human.

[0262] In various embodiments of any of the aspects described herein, the anemia therapy comprises administering an erythropoietic factor, hi various embodiments, the erythropoietic factor is one or more of erythropoietin, an erythropoietic stimulant, a HIF stabilizer, and supplemental iron.

[0263] In various embodiments, increased inflammation is characterized by increased levels of IL-6 and / or CRP compared to a reference (e.g., as measured by a conventional CRP assay or a high-sensitivity assay (hsCRP), both of which detect CRP but have different analytical performance). In various embodiments, increased inflammation is characterized as IL-6 greater than about 5 pg / ml. In various embodiments, increased inflammation is characterized as CRP greater than about 2 mg / L.

[0264] In various embodiments of any of the aspects described herein, the IL-6 antagonist is administered in an amount effective to neutralize inflammation. In various embodiments, an amount effective to neutralize inflammation reduces IL-6 to less than about 15 pg / ml, less than about 10 pg / ml, or less than about 5 pg / ml. In various embodiments, an amount effective to neutralize inflammation reduces CRP to less than about 2 mg / L or less than about 0.2 mg / L.

[0265] In various embodiments of any of the aspects delineated herein, administering an IL-6 antagonist or anti-IL-6 antibody reduces the dose of EPO. In certain embodiments, the dose of EPO is reduced by about 40 IU / kg / week, about 50 IU / kg / week, about 80 IU / kg / week, about 100 IU / kg / week, or more. In various embodiments, administering an IL-6 antagonist or anti-IL-6 antibody reduces the side effects of increased EPO doses.

[0266] In one embodiment, patients with chronic kidney disease are treated with or without standard anemia treatment. In particular, an agent that inhibits IL-6 biological activity or expression is provided to a subject with anemia associated with chronic kidney disease, with or without anemia treatment (e.g., EPO, ESA, HIF stabilizer, supplemental iron, or red blood cell transfusion). Anemia treatment works by stimulating erythropoiesis or erythropoiesis. Thus, an agent that increases the growth or proliferation of red blood cells or their precursor cells and / or reduces cell death of red blood cells or their precursor cells. Erythroid precursor cells include, for example, hematopoietic stem cells, general myeloid precursor cells, proerythroblasts, erythroblasts, reticulocytes, or any cells that can differentiate or mature into red blood cells.

[0267] An agent that inhibits IL-6 biological activity by blocking the binding of IL-6 or its receptor (gp80) to each other, or by blocking its signal transduction or expression, can be provided to a subject with anemia associated with chronic kidney disease in a pharmaceutical composition, wherein the pharmaceutical composition comprises an effective amount of the agent, an agent for treating anemia (e.g., EPO, ESA, HIF prolyl-hydroxylase inhibitor, supplemental iron), and a suitable excipient. In one embodiment, the agent is an IL-6 antagonist or anti-IL-6 antibody that reduces the level or activity of an IL-6 polypeptide or nucleic acid molecule in a subject or inhibits intracellular signaling induced by IL-6 receptor activation. An anti-IL-6 antibody (e.g., MEDI5117) can be administered in combination with anemia treatment (e.g., administration of EPO, ESA, HIF stabilizer, supplemental iron). Methods for treating anemia vary depending on the patient's TMPRSS6 genotype and the patient's inflammatory state. Patients who are homozygous or heterozygous for the major allele of TMPRSS6 containing a G or C at nucleotide position 2321 (encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736) and who have elevated levels of inflammatory markers (e.g., IL-6 and / or CRP) are administered an IL-6 antagonist or anti-IL-6 antibody that reduces the level or activity of the IL-6 polypeptide in the context of treatment for anemia (e.g., administration of EPO, ESA, HIF stabilizers, or supplemental iron). Patients who are homozygous for the minor allele of TMPRSS6 containing an A or T at nucleotide position 2321 (encoding a TMPRSS6 polypeptide containing a valine at amino acid position 736) do not require anti-IL-6 therapy to supplement their treatment for anemia. Methods for treating anemia can vary depending on the stage of chronic kidney disease and the patient's age, health, and physical condition.

[0268] In another aspect, an assay useful for characterizing a subject with anemia associated with chronic inflammation (e.g., in chronic kidney disease) is provided. The inflammatory markers IL-6 and CRP can be detected by any suitable method. The methods described herein can be used individually or in combination to detect IL-6 or CRP biomarkers and / or inflammatory conditions. In one embodiment, inflammation is characterized by detecting the level of IL-6 and / or CRP polypeptides in a subject's biological sample (e.g., serum) compared to expression in a reference (e.g., serum from a healthy control subject), where increased IL-6 and / or CRP expression is indicative of inflammation. In another embodiment, increased IL-6 and / or CRP expression indicates that the subject with anemia associated with chronic kidney disease will not respond to treatment for the anemia and / or will respond to treatment for the anemia when administered in combination with an IL-6 antagonist (e.g., an anti-IL-6 antibody).

[0269] In one embodiment, IL-6 and / or CRP polypeptide levels are measured by immunoassay. Immunoassays typically utilize antibodies (or other agents that specifically bind to the marker) to detect the presence or level of the biomarker in a sample. Antibodies can be produced by methods well known in the art, for example, by immunizing an animal with the biomarker or a fragment thereof. Biomarkers can be isolated from samples based on their binding properties. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.

[0270] In various embodiments, traditional immunoassays are used, including, for example, Western blots, sandwich immunoassays, including ELISA and other enzyme immunoassays, fluorescence-based immunoassays, and chemiluminescence. Nephelometry is a liquid-phase assay in which the antibody is present in solution. Binding of the antigen to the antibody results in a change in absorbance, which is measured. Other forms of immunoassays include magnetic immunoassays, radioimmunoassays, and real-time immunoquantitative PCR (iqPCR). Other detection methods include liquid chromatography and mass spectrometry.

[0271] Immunoassays can be performed on solid substrates (e.g., chips, beads, microfluidic platforms, membranes) or on any other format that supports binding of antibodies to markers and subsequent detection. A single marker may be detected at once, or a multiplexed format may be used. Multiplexed immunoassays can include planar microarrays (protein chips) and bead-based microarrays (suspension arrays).

[0272] Chronic kidney disease patients with anemia identified as having elevated IL-6 and / or CRP polypeptide levels are selected for treatment with an agent that reduces IL-6 expression or activity (e.g., an anti-IL-6 antibody) in combination with treatment for anemia. Patients treated by the methods of the invention can be monitored by detecting changes in hemoglobin, hematocrit, erythropoietin dose, IL-6 and / or CRP expression after treatment. Patients showing decreased expression of IL-6 and / or CRP and / or decreased inflammation are identified as responsive to IL-6 inhibition.

[0273] Other aspects and embodiments are provided in the following numbered sections. 1. A method for treating chronic inflammation in a selected subject, comprising administering an IL-6 antagonist to the subject, wherein the subject is selected for treatment by having one or more alleles encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736. 2. A method for treating inflammation in a selected subject with cardiovascular disease, heart failure, and / or chronic kidney disease, comprising administering an IL-6 antagonist to the subject, wherein the subject is selected for treatment by having one or more alleles encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736. 3. A method for reducing the risk of inflammation and death in a selected subject with cardiovascular disease, heart failure and / or chronic kidney disease, comprising administering an IL-6 antagonist to the subject, wherein the subject is selected as having one or more alleles encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736 and has increased inflammation compared to a reference. 4. A method for treating anemia in a subject with chronic kidney disease, comprising administering an IL-6 antagonist to the subject, wherein the subject is identified as having one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736 and has increased inflammation compared to a reference. 5. The method according to any one of items 1 to 4, wherein the IL-6 antagonist is administered in an amount effective to neutralize inflammation. 6. The method according to any one of items 1 to 4, wherein the IL-6 antagonist is an anti-IL-6 antibody. 7. The method of item 5, further comprising administering an erythropoietic factor to the subject. 8. The method of any one of items 1 to 4, wherein the method reduces the subject's risk of mortality. 9. A method for reducing the risk of death in a subject with chronic kidney disease or heart failure, comprising administering an IL-6 antagonist to the subject, wherein the subject is identified as having one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736 and has increased inflammation compared to a reference.

[0274] 10. A method for treating anemia in a subject with increased inflammation, comprising administering an erythropoietic factor and an anti-IL-6 antibody in amounts effective to neutralize inflammation in a subject having one or more alleles encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736. 11. The method according to any one of items 1 to 10, wherein increased inflammation is characterized by increased IL-6 and / or CRP levels compared to a reference. 12. The method of item 11, wherein the increased inflammation is characterized as IL-6 greater than about 5 pg / ml, about 10 pg / ml, or about 15 pg / ml. 13. The method of item 10, wherein increased inflammation is characterized as a CRP greater than about 2 mg / L. 14. The method of claim 10, wherein the erythropoietic factor is one or more of erythropoietin, an erythropoiesis promoter, a HIF stabilizer, and supplemental iron. 15. A method for enhancing responsiveness to EPO in a subject identified as one in need thereof, comprising administering an IL-6 antagonist and an anti-IL-6 antibody in amounts effective to neutralize inflammation in a subject having one or more alleles encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736, thereby enhancing the subject's responsiveness to EPO. 16. The method of item 15, wherein the amount of anti-IL-6 antibody effective to neutralize inflammation reduces IL-6 to less than about 15 pg / ml, less than about 10 pg / ml, or less than about 5 pg / ml. 17. The method of item 16, wherein the amount of IL-6 antagonist or anti-IL-6 antibody effective to neutralize inflammation reduces CRP to less than about 2 mg / L. 18. The method of item 15, wherein administering an IL-6 antagonist or an anti-IL-6 antibody reduces the dose of EPO. 19. The method of item 17, wherein the dose of EPO is reduced by about 40 IU / kg / week, about 50 IU / kg / week, about 80 IU / kg / week, about 100 IU / kg / week or more. 20. The method of item 15, wherein administering an IL-6 antagonist or an anti-IL-6 antibody reduces the side effects of increased EPO.

[0275] 21. A method for selecting a treatment for a subject identified as being in need thereof, comprising: a) characterizing the subject as having one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736; and b) detecting levels of one or more inflammatory markers, IL-6 and CRP, wherein the characterization indicates that an IL-6 antagonist should be administered in combination with therapy for anemia. 22. The method of item 21, further comprising administering to the subject an IL-6 antagonist and an anemia therapy. 23. The method of item 21, wherein the therapy for anemia comprises administering an erythropoietic factor. 24. A method for increasing the proliferation or survival of red blood cells or their precursor cells in a subject identified as being in need thereof, comprising administering to the subject an IL-6 antagonist and an erythropoietic factor, wherein the subject is identified as having one or more alleles encoding a TMPRSS6 polypeptide comprising an alanine at amino acid position 736, and the subject has increased inflammation compared to a reference. 25. The method according to item 24, wherein cell death in erythrocytes or their precursor cells is reduced. 26. The method according to item 24, wherein the progenitor cells are hematopoietic stem cells, proerythroblasts, erythroblasts, or reticulocytes. 27. The method of any one of items 15 to 24, wherein the subject has chronic kidney disease. 28. The method of any one of items 15 to 24, wherein the subject has anemia. 29. The method according to item 28, wherein the anemia is cancer anemia, anemia in a chronic autoimmune disease, anemia in a chronic inflammatory disease, or anemia in metabolic syndrome.

[0276] 30. The method of any one of items 15 to 24, wherein the IL-6 antagonist is administered in an amount effective to neutralize inflammation. 31. The method of any one of items 15 to 24, wherein the IL-6 antagonist is an anti-IL-6 antibody. 32. The method of any one of claims 15 to 24, wherein the increased inflammation is characterized by increased levels of IL-6 and / or CRP compared to a reference. 33. The method of any one of items 15 to 24, wherein the increased inflammation is characterized as IL-6 greater than about 5 pg / ml, about 10 pg / ml, or about 15 pg / ml. 34. The method of any one of items 15 to 24, wherein increased inflammation is characterized as a CRP greater than about 2 mg / L. 35. The method of any one of items 15 to 24, wherein the amount effective to neutralize inflammation reduces IL-6 to less than about 10 pg / ml or less than about 5 pg / ml. 36. The method of any one of items 15 to 24, wherein the amount effective to neutralize inflammation reduces CRP to less than about 2 mg / L. 37. The method of any one of items 15 to 24, wherein the erythropoietic factor is one or more of erythropoietin, an erythropoiesis-stimulating agent, a HIF stabilizer, and supplemental iron. 38. The method of item 24, wherein administering the IL-6 antagonist reduces the dose of EPO. 39. The method of item 38, wherein the IL-6 antagonist is an anti-IL-6 antibody.

[0277] 40. The method of item 38, wherein the dose of EPO is reduced by about 40 IU / kg / week, about 50 IU / kg / week, about 80 IU / kg / week, about 100 IU / kg / week or more. 41. The method of item 23, wherein administering an IL-6 antagonist reduces the side effects of increased EPO. 42. The method of any one of items 1 to 40, wherein the allele comprises G at position 2321 of the TMPRSS6 polynucleotide. 43. The IL-6 antagonist comprises the following nucleic acid sequence: SNYMI (SEQ ID NO: 12), DLYYYAGDTYYADSVKG (SEQ ID NO: 13), WADDHPPWIDL (SEQ ID NO: 14), RASQGISSWLA (SEQ ID NO: 15) KASTLES (SEQ ID NO: 16), and QQSWLGGS (SEQ ID NO: 17) 43. The method according to any one of items 1 to 42, wherein the anti-IL-6 antibody has one or more CDRs selected from the group consisting of: 44. The method of item 42, wherein the anti-IL-6 antibody has a heavy chain CDR1 comprising the sequence SNYMI (sequence number 12), a heavy chain CDR2 comprising the sequence DLYYYAGDTYYADSVKG (sequence number 13), a heavy chain CDR3 comprising the sequence WADDHPPWIDL (sequence number 14), a light chain CDR1 comprising the sequence RASQGISSWLA (sequence number 15), a light chain CDR2 comprising the sequence KASTLES (sequence number 16), and a light chain CDR3 comprising the sequence QQSWLGGS (sequence number 17). 45. An anti-IL-6 antibody having the following sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTISSNYMIWVRQAPGKGLEWVSDLYYYAGDTYY ADSVKGRFTMSRDISKNTVYLQMNSLRAEDTAVYYCARWADDHPPWIDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18) 43. The method of claim 42, wherein the heavy chain comprises: 46. ​​An anti-IL-6 antibody having the following sequence: DIQMTQSPSTLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKVLIYKASTLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQSWLGGSFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19) 43. The method of claim 42, wherein the light chain comprises: 47. The method of item 42, wherein the anti-IL-6 antibody is MEDI5117. 48. The method of any one of items 1 to 47, wherein the subject is a human.

[0278] 5.11.2. Methods for Treating Cardio-Renal Syndrome In other aspects and embodiments, compositions and methods for treating cardiorenal syndrome are provided. will be done.

[0279] These aspects and embodiments are based, at least in part, on the discovery that anti-IL-6 treatment of cardiac injury in a rodent model of cardiorenal syndrome is as effective as standard of care therapy. As reported in more detail below, rodent models of cardiorenal syndrome were treated with anti-IL-6 or standard of care therapy (ACE inhibitor, perindopril) after myocardial infarction. After treatment, ejection fraction, myocardial contractility, and the percentage of fibrotic tissue in cardiac tissue were measured. The ejection fraction levels in both the anti-IL-6-treated group and the standard of care-treated group were increased compared to those in the control-treated group. Cardiac contractility in both the anti-IL-6-treated group and the standard of care-treated group was increased compared to those in the control-treated group. The amount of fibrotic tissue in both the anti-IL-6-treated group and the standard of care-treated group was reduced compared to those in the control-treated group. Furthermore, the levels of ejection fraction and fibrotic tissue burden were similar in subjects treated with anti-IL-6 and standard of care therapy. The results demonstrate that anti-IL-6 therapy is as effective as standard of care therapy in treating cardiorenal syndrome in rodent models.

[0280] These aspects and embodiments are further based, at least in part, on the discovery that patients identified as having cardiorenal syndrome after a myocardial infarction and as having elevated levels of IL-6 have a particularly elevated risk of cardiovascular death, including heart failure. Without being bound by theory, IL-6 may play a causal role in the development and / or progression of cardiorenal syndrome. Thus, patients with elevated IL-6 levels after a myocardial infarction, or patients with cardiorenal syndrome and elevated IL-6 levels, are likely to benefit from IL-6 inhibition.

[0281] Thus, therapeutic methods for treating cardiac and / or renal damage in a subject with cardiorenal syndrome are provided, involving administering an IL-6 antagonist to the subject. In some embodiments, the treatment of cardiac and / or renal damage in a subject with cardiorenal syndrome is carried out with or without standard treatment for cardiorenal syndrome. Methods for characterizing the risk of cardiovascular death in patients after myocardial infarction are provided, involving detecting increased IL-6 levels in a biological sample obtained from the patient.

[0282] In one aspect, a method is provided for treating cardiac / kidney damage in a subject with cardiorenal syndrome, the method involving administering to the subject an IL-6 antagonist.

[0283] In another embodiment, a method is provided for increasing cardiac function in a subject with cardiorenal syndrome, the method involving administering to the subject an IL-6 antagonist.

[0284] In yet another embodiment, a method of reducing fibrosis in a subject with cardiorenal syndrome is provided, the method involving administering to the subject an IL-6 antagonist.

[0285] In various embodiments of any of the aspects delineated herein, the method further includes administering to the subject a standard of care regimen. In various embodiments, the standard of care regimen is an angiotensin-converting enzyme (ACE) inhibitor.

[0286] In various embodiments of any of the aspects delineated herein, the increased cardiac function is characterized by an increase in the subject's ejection fraction and / or myocardial contractility compared to a reference. In various embodiments of any of the aspects delineated herein, the decreased fibrosis is characterized by a decrease in the proportion of fibrotic tissue in a tissue sample from the subject compared to a reference. In various embodiments, the fibrosis is in cardiac tissue.

[0287] In various embodiments of any of the aspects delineated herein, the subject has cardiac and / or renal damage. In various embodiments of any of the aspects delineated herein, the subject has renal damage secondary to cardiac damage.

[0288] In another aspect, the present invention provides a method for identifying an increased risk of cardiovascular death (e.g., heart failure) in a subject following a myocardial infarction in the subject, the method involving measuring the level of one or more IL-6 polynucleotides or polypeptides in a sample from the subject compared to a reference, wherein an increased level of one or more IL-6 polynucleotides or polypeptides indicates an increased risk of cardiovascular death.

[0289] In yet another aspect, the present invention provides a method for characterizing the risk of cardiovascular death (e.g., heart failure) in a subject following a myocardial infarction, the method involving measuring the level of one or more IL-6 polynucleotides or polypeptides in a sample from the subject compared to a reference, wherein an increased level of one or more IL-6 polynucleotides or polypeptides indicates an increased risk of cardiovascular death.

[0290] In various embodiments of any of the aspects delineated herein, the subject has cardiorenal syndrome, heart failure, chronic kidney disease, or is free of cardiorenal pathology. In various embodiments of any of the aspects delineated herein, the subject is identified as having cardiorenal syndrome, heart failure, chronic kidney disease, or is free of cardiorenal pathology about one month after myocardial infarction.

[0291] In another aspect, the present invention provides a method for treating cardiac and / or renal injury in a selected subject having a cardiorenal syndrome, the method involving administering an IL-6 antagonist to the subject, wherein the subject is selected for treatment by detecting an increased level of one or more IL-6 polynucleotides or polypeptides in a biological sample from the subject compared to a reference.

[0292] In yet another aspect, the invention provides a method of reducing the risk of cardiovascular death (e.g., heart failure) in a selected subject with cardiorenal syndrome, the method involving administering an IL-6 antagonist to the subject, wherein the subject is selected by detecting an increased level of one or more IL-6 polynucleotides or polypeptides in a biological sample from the subject compared to a reference. In various embodiments of any of the aspects delineated herein, the subject has had a myocardial infarction.

[0293] In various embodiments of any of the aspects delineated herein, the IL-6 antagonist is an anti-IL-6 antibody. In various embodiments, the anti-IL-6 antibody is MEDI5117.

[0294] In various embodiments of any of the aspects delineated herein, the biological sample is a plasma sample or a serum sample. In various embodiments of any of the aspects delineated herein, the subject is a human.

[0295] In another aspect, methods are provided for treating cardiorenal syndrome in a patient and / or reducing the risk of death or heart failure in such a patient by administering an agent that inhibits IL-6 biological activity or expression. In one embodiment, a patient with cardiorenal syndrome is treated with or without standard treatment for cardiorenal syndrome (e.g., angiotensin-converting enzyme (ACE) inhibitors). In particular, an agent that inhibits IL-6 biological activity or expression is provided to a subject with cardiorenal syndrome (e.g., administration of an anti-IL-6 antibody).

[0296] In another aspect, methods for increasing cardiac function and reducing fibrosis in a subject with cardiorenal syndrome are provided. The methods include administering to the subject an agent that inhibits the biological activity or expression of IL-6. In some embodiments, increased cardiac function is measured by an increase in the subject's ejection fraction compared to a reference (e.g., the ejection fraction of a healthy control subject), or an increase in myocardial contractility (e.g., dP / dtmax In some embodiments, reduced fibrosis is characterized by a decrease in the percentage of fibrotic tissue in a tissue sample from a subject compared to a reference (e.g., a tissue sample obtained from a healthy control subject). In one embodiment, the fibrosis is in cardiac tissue.

[0297] A pharmaceutical composition containing an effective amount of an agent that inhibits the biological activity of IL-6 by blocking the binding of IL-6 or its receptor (gp80) to each other, or by blocking their signal transduction or expression, can be provided to a subject with cardiorenal syndrome, where the pharmaceutical composition comprises a suitable excipient. In one embodiment, the agent is an IL-6 antagonist or an anti-IL-6 antibody that reduces the level or activity of IL-6 polypeptide or polynucleotide in the subject, or inhibits intracellular signal transduction induced by IL-6 receptor activation. An anti-IL-6 antibody (e.g., MEDI5117) can be administered. The method of treating cardiorenal syndrome may vary depending on the stage of cardiorenal syndrome and the patient's age, health, and physical condition.

[0298] In various embodiments, subjects with cardiorenal syndrome are treated with an IL-6 antagonist. Furthermore, subjects at increased risk of cardiovascular death and / or heart failure after myocardial infarction can be identified by characterizing the subject's plasma level of IL-6. Subjects with elevated IL-6 levels are at increased risk of cardiovascular death and / or heart failure. Such subjects can be selected for treatment with an IL-6 antagonist. Additionally, subjects with cardiorenal syndrome and elevated IL-6 levels, including those who have suffered a myocardial infarction, can be selected for treatment. Once selected for treatment, such subjects can be administered virtually any IL-6 antagonist known in the art. Suitable IL-6 antagonists include, for example, known IL-6 antagonists, commercially available IL-6 antagonists, IL-6 antagonists developed using methods well known in the art, and antagonists of the IL-6R-related intracellular signaling system.

[0299] In another aspect, an assay for characterizing cardiovascular death, risk of heart failure, and / or mortality in a subject after myocardial infarction is provided. The assay features detection of IL-6 in a biological sample obtained from the subject. IL-6 can be detected by any suitable method. In one embodiment, the risk of cardiovascular death or heart failure is characterized by detecting the level of IL-6 polypeptide in the subject's biological sample (e.g., serum or plasma) compared to expression in a reference (e.g., serum or plasma from a healthy control subject or a control subject without cardiac-renal pathology), where increased IL-6 indicates an increased risk of cardiovascular death or heart failure. Subjects identified as having an increased risk of cardiovascular death, heart failure, or mortality can be selected for treatment. In another embodiment, subjects with cardiorenal syndrome and increased IL-6 levels are selected for treatment with an IL-6 antagonist (e.g., an anti-IL-6 antibody).

[0300] In one embodiment, IL-6 polynucleotide levels are measured. IL-6 polynucleotide levels can be measured by standard methods such as quantitative PCR, Northern blot, microarray, mass spectrometry, and in situ hybridization.

[0301] In one embodiment, IL-6 polypeptide levels are measured. IL-6 polypeptide levels can be measured by standard methods, such as immunoassays. Immunoassays typically utilize antibodies (or other agents that specifically bind to markers) to detect the presence or level of biomarkers in a sample. Antibodies can be produced by methods well known in the art, for example, by immunizing animals with the biomarkers or fragments thereof. Biomarkers can be isolated from samples based on their binding properties. Alternatively, if the amino acid sequence of a polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies by methods well known in the art.

[0302] In various embodiments, the assay uses traditional immunoassays, including Western blots, sandwich immunoassays, including ELISA and other enzyme immunoassays, fluorescence-based immunoassays, and chemiluminescence. Nephelometry is a liquid-phase assay in which the antibody is present in solution. Binding of the antigen to the antibody results in a change in absorbance, which is measured. Other forms of immunoassays include magnetic immunoassays, radioimmunoassays, and real-time immunoquantitative PCR (iqPCR). Other detection methods include liquid chromatography and mass spectrometry.

[0303] Immunoassays can be performed on solid substrates (e.g., chips, beads, microfluidic platforms, membranes) or on any other format that supports binding of antibodies to markers and subsequent detection. A single marker may be detected at once, or a multiplexed format may be used. Multiplexed immunoassays can include planar microarrays (protein chips) and bead-based microarrays (suspension arrays).

[0304] Patients with cardiorenal syndrome identified as having increased IL-6 polypeptide levels are selected for treatment with an agent that reduces IL-6 expression or activity (e.g., an anti-IL-6 antibody). Treatment may be administered in combination with standard treatments for cardiorenal syndrome (e.g., an ACE inhibitor). Patients treated with the methods of the present invention can be monitored by detecting changes in IL-6 levels after treatment.

[0305] Other aspects and embodiments are provided in the following numbered sections. 1. A method for treating cardiac and / or renal damage in a subject with cardiorenal syndrome, comprising administering to the subject an IL-6 antagonist. 2. A method for increasing cardiac function in a subject with cardiorenal syndrome, comprising administering to the subject an IL-6 antagonist. 3. A method for reducing fibrosis in a subject with cardiorenal syndrome, comprising administering to the subject an IL-6 antagonist. 4. The method of item 2, wherein the increased cardiac function is characterized by an increase in the subject's ejection fraction compared to a reference. 5. The method according to item 3, wherein the fibrosis is in cardiac tissue. 6. The method of item 3 or 5, wherein the reduction in fibrosis is characterized by a decrease in the proportion of fibrous tissue in a tissue sample from the subject compared to a reference. 7. The method according to any one of items 1 to 6, wherein the subject has cardiac and / or renal damage. 8. The method of any one of items 1 to 7, wherein the subject has kidney damage secondary to cardiac damage. 9. The method of any one of items 1 to 8, further comprising administering a standard of care regimen to the subject. 10. The method of any one of items 1 to 9, wherein the standard of care therapy is an angiotensin-converting enzyme (ACE) inhibitor.

[0306] 11. A method for identifying an increased risk of cardiovascular death in a subject following a myocardial infarction in the subject, the method comprising measuring the level of one or more IL-6 polynucleotides or polypeptides in a sample from the subject compared to a reference, wherein an increased level of one or more IL-6 polynucleotides or polypeptides indicates an increased risk of cardiovascular death. 12. A method for characterizing the risk of cardiovascular death in a subject following a myocardial infarction, comprising measuring the level of one or more IL-6 polynucleotides or polypeptides in a sample from the subject compared to a reference, wherein an increased level of one or more IL-6 polynucleotides or polypeptides indicates an increased risk of cardiovascular death. 13. The method of item 11 or 12, wherein the subject has cardiorenal syndrome, heart failure, chronic kidney disease, or no cardiorenal pathology. 14. The method of any one of items 11 to 13, wherein the subject is identified as having cardiorenal syndrome, heart failure, chronic kidney disease, or no cardiorenal pathology about one month after myocardial infarction. 15. A method for treating cardiac and / or renal damage in a selected subject with a cardiorenal syndrome, comprising administering an IL-6 antagonist to the subject, wherein the subject is selected for treatment by detecting an increase in the level of one or more IL-6 polynucleotides or polypeptides in a biological sample from the subject compared to a reference. 16. A method for reducing the risk of cardiovascular death in a selected subject with cardiorenal syndrome, comprising administering an IL-6 antagonist to the subject, wherein the subject is selected by detecting an increased level of one or more IL-6 polynucleotides or polypeptides in a biological sample from the subject compared to a reference. 17. The method according to item 15 or 16, wherein the subject has had a myocardial infarction. 18. The method of any one of items 1 to 10 or 15 to 17, wherein the IL-6 antagonist is an anti-IL-6 antibody. 19. The method of item 18, wherein the anti-IL-6 antibody is MEDI5117. 20. The method according to any one of items 11 to 19, wherein the biological sample is a plasma sample. 21. The method according to any one of items 1 to 20, wherein the subject is a human.

[0307] [Example] 5.12 Working Example The following examples are offered by way of illustration and not by way of limitation.

[0308] [Example 1] 5.12.1. EPO Dose and Overall Survival in Patients with Chronic Kidney Disease Correlate with Serum IL-6 and CRP Levels Only in Patients with at Least One Copy of the TMPRSS6 SNP rs855791 Major Allele The peptide hormone hepcidin plays a central role in whole-body iron homeostasis (Hentze et al., Cell 142:24-38 (2010)). Hepcidin expression is known to be influenced by the product of the TMPRSS6 gene, matriptase-2, a type II transmembrane serine protease. Common variants in the TMPRSS6 gene have been shown to correlate with iron status (Benyamin et al., Nature Genetics 41(11):1173-1175 (2009)), and specific mutations in the TMPRSS6 gene have been shown to cause iron-refractory iron deficiency anemia (IRIDA) (Finberg et al., Nature Genetics 40(5):569-571 (2008)). SNP rs855791 (2321G→A; A736V) is a naturally occurring variation in the TMPRSS6 gene that is associated with naturally occurring variations in hepcidin expression and blood hemoglobin levels.

[0309] To determine whether the TMPRSS6 rs855791 SNP genotype predicts the degree of anemia in end-stage renal disease, we analyzed previously collected data from a clinical trial of patients with chronic kidney disease, along with newly determined SNP genotyping. Because hepcidin expression is also regulated by IL-6 (Casanovas et al., PLOS Computational Biol. 10(1):e1003421 (2014)), we further analyzed the data to determine whether serum IL-6 levels could predict the degree of anemia in end-stage renal disease.

[0310] method Data from N = 257 patients recruited between October 2003 and September 2004 in six dialysis departments in the Stockholm-Uppsala (Sweden) region and enrolled in the MIMICK1, MIMICK2 (Mapping Inflammatory Markers in Chronic Kidney Disease) and MIA (Malnutrition, Inflammation and Atherosclerosis) cohorts were aggregated to N = 208 based on the general dialysis criteria of ferritin > 100 ng / ml and Hb > 10 mg / dL to select patients who were stable on hemodialysis and not iron deficient or significantly anemic, thereby excluding patients with factors that could decouple iron processing from hemoglobin levels.

[0311] Clinical data of all patients, including erythropoietin (EPO) dose (IU / kg / week), IL-6 serum level (pg / ml), CRP serum level (mg / L), survival rate (months), and TMPRSS6 genotype at SNP rs855791, were collated and analyzed using statistical analysis software (SPSS Statistics Desktop, IBM). The tested TMPRSS6 alleles and their nucleotide and amino acid sequences are shown in Table 1.

[0312] [Table 1]

[0313] The cohort was separated into rs855791 subgroups (homozygous AA, heterozygous AG, and homozygous GG), and each genotype group was separated into tertiles or quartiles of serum IL-6 levels (e.g., IL-6 <5 pg / ml vs. >10 pg / ml and IL-6 <5 pg / ml vs. >15 pg / ml) or serum CRP levels (CRP <2 mg / L vs. >2 mg / L). Comparisons were made between EPO doses in the upper and lower tertiles and quartiles. Statistical analysis was performed within genotype groups by Student's t-test and between groups by ANOVA.

[0314] result Because each patient's EPO dose was titrated by the treating physician to achieve normal hemoglobin levels, EPO dose could be used as a surrogate for the degree of underlying anemia. EPO dose in subjects homozygous for the minor allele (A / A) was found to be relatively insensitive to IL-6 fluctuations (Figure 1A; left panel). However, EPO dose in subjects with at least one copy of the major allele, i.e., patients heterozygous (A / G) or homozygous (G / G) for the major allele (G), was sensitive to their IL-6 levels (Figure 1B; right panel). In these latter subjects, increased serum IL-6 levels (e.g., >5 pg / ml) were associated with increased EPO dose.

[0315] Without wishing to be bound by any particular theory, homozygosity for the minor allele eliminated the effect of IL-6 on iron handling, and therefore, EPO doses in these patients (A / A) were similar regardless of IL-6 levels.

[0316] Subjects homozygous for the TMPRSS6 rs855791 minor allele (A) showed similar mortality rates regardless of IL-6 levels (Figure 2A). However, survival in subjects with at least one copy of the major allele, i.e., patients heterozygous or homozygous for the major allele (G), varied according to IL-6 levels (Figure 2B). Indeed, the TMPRSS6 G allele conferred higher all-cause mortality in response to elevated IL-6 levels in dialysis subjects with stage 5 chronic kidney disease. In subjects with at least one copy of the major allele (G), IL-6 levels ≥ 5 pg / ml (i.e., middle and highest tertiles of IL-6) were associated with increased mortality compared with IL-6 levels < 5 pg / ml (i.e., lower tertiles of IL-6) (Figure 2B).

[0317] In addition, levels of the acute phase reactant CRP (a marker of inflammation) also correlated with increasing EPO dose in subjects heterozygous or homozygous for the major allele (G), but not in patients homozygous for the minor allele (Figure 3).

[0318] Consider As shown in Figure 1, the degree of underlying anemia, measured as clinically titrated EPO dose, correlated with IL-6 levels only in patients with at least one copy of the major allele at the TMPRSS6 rs855791 SNP. In these patients, the higher the serum IL-6 level, the higher the EPO dose required (Figure 1B). In contrast, the degree of anemia in patients with two copies of the minor allele did not correlate with serum IL-6 levels (Figure 1A).

[0319] Similarly, overall survival correlated with IL-6 levels only in patients with at least one copy of the major allele of TMPRSS6 SNP rs855791. In subjects with at least one copy of the TMPRSS6 rs855791 major allele, survival was inversely correlated with serum IL-6 levels, with patients in the highest tertile of serum IL-6 levels exhibiting statistically significantly poorer survival than those in the lowest tertile of IL-6 levels (Figure 2B). In contrast, overall survival in patients homozygous for the minor allele at rs855791 was not affected by IL-6 levels (Figure 2A).

[0320] Without wishing to be bound by theory, it is possible that increased serum IL-6 in patients with at least one copy of the TMPRSS6 major allele leads to increased hepcidin expression, thereby increasing anemia. The increased risk of death is the result of dysregulated iron metabolism, resulting anemia, and / or increased doses of erythropoiesis-stimulating agents such as EPO. If these correlations reflect a causal relationship, it raises the possibility that reduced IL-6 levels or IL-6 signaling may reduce anemia, reduce the required EPO dose, and increase survival in patients with chronic kidney disease, but only in those patients with at least one copy of the TMPRSS6 rs855791 major allele, and be most effective in those patients with elevated serum IL-6 levels.

[0321] [Example 2] 5.12.2. Risk of Death and Heart Failure After Acute Myocardial Infarction Correlates with IL-6 Serum Levels Only in Patients with at Least One Copy of the TMPRSS6 SNP rs855791 Major Allele To determine whether the TMPRSS6 rs855791 genotype influences IL-6 sensitivity in patients with acute rather than chronic illness, we analyzed previously collected data in a clinical trial of patients hospitalized for acute coronary syndromes along with newly determined SNP genotyping.

[0322] method Data from subjects previously enrolled in the multicenter Platelet Inhibition and Patient Outcomes (PLATO) study were analyzed. Patients were eligible for PLATO enrollment if they had been hospitalized for acute coronary syndrome with symptoms presenting in the previous 24 hours. Mortality and the presence of heart failure were measured in these subjects beginning 30 days after myocardial infarction.

[0323] result Mortality in subjects homozygous for the TMPRSS6 rs855791 SNP minor allele (A) did not correlate with IL-6 changes (Figure 4A). However, one or two copies of the major allele (G) conferred higher all-cause mortality in response to elevated IL-6 levels in subjects after myocardial infarction (Figure 4B). Thus, TMPRSS6 regulated IL-6-mediated mortality risk after myocardial infarction.

[0324] The effect of TMPRSS6 genotype on IL-6-mediated heart failure risk was also measured in subjects enrolled in PLATO, starting 30 days after myocardial infarction. Heart failure in subjects homozygous for the minor allele (A) did not correlate with changes in IL-6 (Figure 5A). However, the G allele of TMPRSS6 conferred a higher rate of heart failure in response to elevated IL-6 levels in subjects after myocardial infarction (Figure 5B). Thus, TMPRSS6 regulated the IL-6-mediated risk of heart failure after myocardial infarction.

[0325] Consider These data demonstrate that the correlation between TMPRSS6 genotype, IL-6 levels, and adverse clinical outcomes is not limited to patients with chronic kidney disease. Without wishing to be bound by theory, increased serum IL-6 in patients with at least one copy of the TMPRSS6 major allele leads to increased hepcidin expression, resulting in increased iron sequestration in cardiomyocytes and subsequent iron-mediated cytotoxicity. If these correlations reflect a causal relationship, it raises the possibility that reducing IL-6 levels or IL-6 signaling may reduce heart failure and mortality in patients with acute coronary syndromes, but only in those patients with at least one copy of the TMPRSS6 rs855791 major allele, and may be most effective in those patients with elevated serum IL-6 levels.

[0326] [Example 3] 5.12.3. In Vitro Studies of iPS-Derived Human Cardiomyocytes Confirm a Causal Relationship between TMPRSS6 Genotype and IL-6-Mediated Cytotoxicity Although the correlations observed in Examples 1 and 2 suggest that reduced IL-6-mediated signaling may confer clinical benefit in patients with at least one copy of the TMPRSS6 rs855791 major allele, elevated IL-6 levels, and either anemia or hepcidin-mediated cytotoxicity, the observed correlations are insufficient to prove causality. Therefore, to examine the effects of BMP and BMP + IL-6 on hepcidin expression and cellular susceptibility to ischemic injury, experiments were performed in human induced pluripotent cell-derived cardiomyocytes (iPS-CMs) transfected with mutant forms of TMPRSS6.

[0327] 5.12.3.1. Method Cultivation of human iPS-derived cardiomyocytes iCell Cardiomyocytes (Cellular Dynamics International, CDI Inc.) were seeded onto 0.1% gelatin-coated 6-well or 96-well cell culture plates containing iCell Cardiomyocyte Plating Medium (CDI Inc.). 48 hours after plating, the plating medium was replaced with Maintenance Medium (CDI Inc.). The Maintenance Medium was replaced every other day until the day of the experiment.

[0328] Simulated ischemia / reoxygenation protocoliPS cardiomyocytes were subjected to simulated ischemia (SI) for 90 min by replacing the cell culture medium with "ischemic buffer" containing 118 mM NaCl, 24 mM NaHCO3, 1.0 mM NaH2PO4, 2.5 mM CaCl2-2H2O, 1.2 mM MgCl2, 20 mM sodium lactate, 16 mM KCl, 10 mM 2-deoxyglucose (pH adjusted to 6.2), as previously reported (Das, A., Xi, L., and Kukreja, KC (2005) J. Biol. Chem. 280: 12944-12955; Das A, Smolenski A, Lohmann SM, Kukreja RC. (2006) J. Biol. Chem. 281(50):38644-52). Cells were incubated at 37°C in a trigger gas incubator regulated with 1-2% O and 5% CO throughout the SI period. Reoxygenation (RO) was achieved by replacing the ischemic buffer with normal cell culture medium under normoxic conditions. Cell necrosis occurred after 2 or 18 hours of reoxygenation, respectively. iCells were subjected to 4 hours of SI and 24 hours of RO as described above.

[0329] Assessment of cell viability and apoptosis Trypan blue exclusion assay was performed to assess cell necrosis as previously reported (Das, A., Xi, L., and Kukreja, KC (2005) J. Biol. Chem. 280, 12944-12955; Das A, Smolenski A, Lohmann SM, Kukreja RC. (2006) J. Biol. Chem. 281(50):38644-52).

[0330] Transfection of iCell CardiomyocytesOn day 8 after plating, the medium was replaced with fresh maintenance medium and the cells were incubated for 4 hours. Cells were transfected with pCMV6-XL5 TMPRSS6(K523) or pCMV6-XL5 TMPRSS6(K523)V763A using ViaFect™ transfection reagent according to the manufacturer's instructions (Promega Corp., Madison, WI). 48 hours after transfection, the cells were subjected to further experiments.

[0331] Western blot analysisWestern blots were performed as previously reported (Das, A., Xi, L., and Kukreja, KC (2005) J. Biol. Chem. 280, pp. 12944-12955; Das A, Smolenski A, Lohmann SM, Kukreja RC. (2006) J. Biol. Chem. 281(50):38644-52). Total soluble proteins were extracted from cells using lysis buffer (Cell Signaling, MA). The homogenate was centrifuged at 10,000 × g for 5 minutes at 4°C, and the supernatant was collected. Proteins (50 μg from each sample) were separated on a 12% acrylamide gel, transferred to a nitrocellulose membrane, and then blocked for 1 h with 5% nonfat dry milk in TBST (10 m m Tris-HCl, pH 7.4, 100 m m NaCl, 0.1% Tween 20). The membrane was then incubated overnight with rabbit monoclonal / polyclonal or goat polyclonal primary antibodies diluted 1:000 for each protein. The antibodies were: phospho-Beclin-1 (Ser93) (D9A5G) rabbit mAb, Beclin-1, SQSTM1 / p62, LC3A / B (D3U4C) XP® rabbit mAb, phospho-Akt (Ser473) (D9E) XP® rabbit mAb, Akt(pan) (C67E7) rabbit mAb, phospho-S6 ribosomal protein (Ser240 / 244) (D68F8) XP® rabbit mAb, S6 ribosomal protein (5G10) rabbit mAb (Cell Signaling, MA), anti-matriptase 2 (TMPRSS6) and anti-SLC40A1 (ferropurtin) (Abcam Company, MA), and goat polyclonal actin-HRP (Santa Cruz Biotechnology, TX). The membrane was then incubated with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:2000 dilution, Amersham Biosciences) for 2 hours. The blot was developed using a chemiluminescence system, and the bands were scanned and quantified by densitometry analysis.

[0332] Real-time PCR-Taqman assayTotal RNA, including small RNAs, was isolated using the miRNeasy Mini Kit according to the manufacturer's protocol (QIAGEN Sciences, MD, USA). The concentration and purity of the isolated RNA were measured using a Nanodrop ND-1000 spectrophotometer (Agilent Technologies, CA, USA). Briefly, 1 μg of total RNA was converted to cDNA with random hexamers using a High-Capacity cDNA Synthesis Kit (Applied Biosystems, CA, USA). Reverse transcription was performed using the following PCR conditions: 25°C for 10 minutes, 37°C for 120 minutes, and 85°C for 5 minutes. Real-time PCR was performed using the following PCR cycle conditions: 95°C for 10 minutes, 95°C for 15 seconds, and 60°C for 60 seconds, using a Taqman amplicon-specific probe (Applied Biosystems, CA, USA) Hamp (CGGCTCTGCAGCCTTG) (SEQ ID NO: 20). Hamp expression was normalized to the GAPDH (CTTCCAGGAGCGAGATCCCGCTAA) (SEQ ID NO: 21) housekeeping gene. Relative gene expression was analyzed using the 2-ΔΔCt method.

[0333] TMPRSS6 mutagenesis and transfection of iPS cells- pCMV6-XL5 TMPRSS6 was purchased from Origene Technologies (Rockville, MD), catalog number SC306623, corresponding to GenBank accession number NM_153609. This clone contains the mutation K253A, resulting in an amino acid change. Site-directed mutagenesis was performed to revert the amino acid at position 253 to the canonical lysine (K). Once reversion was confirmed, site-directed mutagenesis was performed to introduce the V736A mutation. All mutagenesis reactions were performed using the Agilent Technologies QuikChange II XL Site-Directed Mutagenesis Kit (Santa Clara, CA, catalog number 200521). All vectors were sequenced and verified. The primer sequences used were as follows: antisense (as) TMPRSS6 E253K GCATGAGGTCCTTGGGGCCCTGCAG (SEQ ID NO: 22), sense (s) TMPRSS6 E253K CTGCAGGGCCCCAAGGACCTCATGC (SEQ ID NO: 23), antisense (as) TMPRSS6 V736A CCTGGTAGCGATAGGCCTCGCTGCACAGG (SEQ ID NO: 24), sense (s) TMPRSS6 V736A CCTGTGCAGCGAGGCCTATCGCTACCAGG (SEQ ID NO: 25).

[0334] 5.12.3.2. Results Human iPS-CMs minimally express matriptase-2 at baseline. Cells were transfected with constructs driving constitutive expression of matriptase-2 736A, encoded by the TMPRSS6 rs855791 SNP major allele, or matriptase-2 736V, encoded by its minor allele, to mimic cardiomyocytes homozygous for the major and minor alleles, respectively.

[0335] Hepcidin expression is regulated by both the BMP6 / SMAD and IL-6 / STAT signaling pathways, and both BMP and IL-6 act through their respective receptors to promote increased hepcidin expression. Casanovas et al., PLOS Comp. Biol. 10(1):e1003421 (2014). iPS cardiomyocytes bearing major and minor alleles were treated in vitro with agonists of both signaling pathways—recombinant BMP2 and IL-6—or with BMP2 alone to model clinical interventions in which IL-6 levels (or signaling) are reduced. Control iPS cells were not treated with either agonist. Cell mortality was measured under normoxia (normoxia) and under conditions of simulated hypoxia followed by reoxygenation (reperfusion).

[0336] Figure 6A shows the results when cells are treated at normoxia levels. iPS cardiomyocytes expressing only the TMPRSS6 rs855791 minor allele ("736V minor allele") are not significantly affected ("ns") by the elimination of IL-6 signaling: cell death, measured as the percentage of trypan blue-positive cells, is significantly reduced when cells are treated with BMP2 alone compared to treatment with BMP2 + IL-6. In contrast, iPS cardiomyocytes expressing the TMPRSS6 rs855791 major allele exhibit statistically significantly lower cell death when IL-6 signaling is eliminated.

[0337] Figure 6B shows the results when cells were subjected to hypoxia followed by reoxygenation. Compared to normoxic conditions, hypoxia / reoxygenation was significantly toxic to iPS cardiomyocytes, with approximately 40% of control cells of the major and minor alleles dying compared to approximately 20% of control cells under normoxic conditions (compare Figures 6B to 6A). In contrast to this increased background toxicity, iPS cardiomyocytes of the minor allele were not significantly affected by elimination of IL-6 signaling: cell mortality was slightly reduced when cells were treated with BMP2 alone compared to treatment with BMP2 + IL-6. In contrast, iPS cardiomyocytes expressing the TMPRSS6 rs855791 major allele exhibited statistically significantly lower cell death when IL-6 signaling was eliminated.

[0338] Consideration These data strengthen the inferences drawn from the post-hoc analysis of clinical trial data in Examples 1 and 2: reduced IL-6 signaling may be effective in reducing IL-6-mediated toxicity in cardiomyocytes expressing the TMPRSS6 rs855791 major allele, but not the minor allele. Without wishing to be bound by theory, increased IL-6-induced toxicity in major allele iPS cardiomyocytes may result from an IL-6-mediated increase in hepcidin expression, resulting in increased intracellular iron sequestration and subsequent iron-mediated cytotoxicity.

[0339] [Example 4] 5.12.4. Anti-IL-6 Therapy Is As Effective as the Current Standard of Care in a Model of Cardiorenal Syndrome in Rats Genotypically Similar to Humans Homozygous for the TMPRSS6 rs855791 Major Allele Patients with chronic kidney disease, such as those enrolled in the MIMICK study analyzed in Example 1, often develop impaired cardiac function, which is a major cause of mortality. This secondary cardiac damage following primary chronic kidney disease is called cardiorenal syndrome type 4 (CRS type 4).

[0340] To test whether anti-IL-6 therapy is effective as a treatment in CRS4 patients who carry at least one copy of the TMPRSS6 rs855791 major allele, as suggested by the data in Examples 1 and 3, we used a model of CRS4 that is genotypically similar to humans who are homozygous for the TMPRSS6 rs855791 major allele.

[0341] Figure 7 shows an overview of the study design.

[0342] At week 0, myocardial infarction was induced in CRS animals. At week 2, nephrectomy was performed. The control group underwent a sham operation instead. Prior to nephrectomy, subjects underwent various assessments. These included serum creatinine, glomerular filtration rate, 24-hour urinary protein levels, echocardiography, tail cuff blood pressure, and measurements of plasma and urinary biomarkers.

[0343] Treatment began on day 1 after nephrectomy. Animals were divided into three groups: (i) control treatment, (ii) anti-IL-6 therapy, and (iii) standard of care therapy. Anti-IL-6 therapy was an anti-IL-6 antibody suitable for use in rodents. Standard of care therapy was administration of perindopril, an angiotensin-converting enzyme (ACE) inhibitor. At the start of treatment, subjects in all groups were assessed. Assessments included measurement of serum creatinine, glomerular filtration rate, 24-hour protein levels, and plasma biomarkers.

[0344] Subjects in all groups were assessed on days 3 and 7 after nephrectomy. Assessments included measurements of serum creatinine and plasma biomarkers on day 3, and serum creatinine, glomerular filtration rate, 24-hour protein levels, echocardiography, blood pressure, and plasma biomarkers on day 7.

[0345] Subjects were sacrificed at week 6. Prior to sacrifice, subjects in all groups underwent various assessments. Assessments included measurements of serum creatinine, glomerular filtration rate, 24-hour protein levels, blood pressure, plasma biomarkers, echocardiography, and pressure-volume loop analysis. After sacrifice, tissues were collected from subjects in all groups for histological evaluation (i.e., Sirius Red staining of cardiac tissue).

[0346] Figures 8A-8D show cardiac ejection fractions of CRS-free rats ("Sham"), CRS animals treated with a pharmacologically irrelevant isotype control antibody ("Isotype"), CRS animals treated with an anti-IL-6 antibody ("IL-6 ab"), and CRS animals treated with a standard of care ACE inhibitor ("Peri") in the cardiorenal syndrome model summarized in Figure 7.

[0347] Figure 8A shows the baseline ejection fraction levels of all groups two weeks after myocardial infarction, but before nephrectomy and treatment, demonstrating that experimentally induced myocardial infarction causes a significant decrease in cardiac ejection fraction. Figure 8B is a plot showing the ejection fraction levels of all groups one week after nephrectomy and one week after treatment. Figure 8C is a plot showing the ejection fraction levels of all groups two weeks after nephrectomy and two weeks after treatment. Figure 8D is a plot showing the ejection fraction levels of all groups four weeks after nephrectomy and four weeks after treatment. Results are expressed as mean + / - SEM.

[0348] After 4 weeks of treatment, both treatment groups, i.e., the anti-IL-6 treated group and the standard of care ACE inhibitor therapy group, showed statistically significantly increased ejection fraction levels compared to the isotype control group (Figure 8D) (p<0.001). Similar ejection fraction levels in the anti-IL-6 and standard of care groups measured after 4 weeks of treatment indicated that anti-IL-6 therapy had comparable efficacy to the ACE inhibitor perindopril (standard of care therapy), demonstrating that anti-IL-6 therapy has therapeutic efficacy in preserving cardiac function in this cardiorenal syndrome model, as measured by changes in cardiac ejection fraction, comparable to standard of care therapy.

[0349] Cardiac contractility measurements (Figure 9) showed that anti-IL-6 therapy was also as effective as standard of care therapy with an ACE inhibitor. After 4 weeks of treatment, cardiac contractility in the group treated with anti-IL-6 and standard of care therapy was significantly increased compared to that of the isotype control group. Similar cardiac contractility in the anti-IL-6 and standard of care groups demonstrates that anti-IL-6 therapy is as effective in preserving cardiac function in this cardiorenal syndrome model as the ACE inhibitor perindopril (standard of care therapy), as measured by contractility.

[0350] Measurement of fibrosis in cardiac tissue harvested from animals in all groups also demonstrated that anti-IL-6 therapy was as effective as standard of care therapy (Figures 10A-10C). Fibrosis in cardiac tissue was quantified by measuring the percentage of fibrotic tissue in two regions: a "normal" region and a "perifibrosis" region. An example of a "normal" region is shown by the delineated portion of the tissue section shown in the photomicrograph in Figure 10A. The inset of the photomicrograph shows a magnified view of the "normal" region, demonstrating that a small portion of the "normal" region has fibrotic tissue. The "perifibrosis" region is the region of tissue in the "normal" region that is surrounding the fibrotic tissue.

[0351] The plots in Figures 10B and 10C show that cardiac tissue from subjects treated with anti-IL-6 or standard of care therapy had significantly reduced percent fibrotic tissue area compared to the isotype control group, both measured in the "normal" region (Figure 10B) or the "perifibrotic" region (Figure 10C). Furthermore, the percent fibrotic tissue area measured in the anti-IL-6 and standard of care therapy groups was similar (in both the "normal" and "perifibrotic" regions), indicating that anti-IL-6 has an antifibrotic effect comparable to that of the ACE inhibitor perindopril (standard of care therapy).

[0352] These data demonstrate that treatment with anti-IL-6 agents is effective in reducing cardiac injury and recovery function in an in vivo model of cardiorenal syndrome in animals genotypically similar to humans who are homozygous for the TMPRSS6 rs855791 major allele.

[0353] [Example 5] 5.12.5. Anti-IL-6 Therapy Is Effective in Preserving Cardiac Function in a Model of Acute Myocardial Infarction in Mice Genotypically Similar to Human TMPRSS6 rs855791 Major Allele Homozygotes The data in Examples 2 and 3 suggest that reducing IL-6 levels or IL-6 signaling can reduce heart failure and mortality in patients with acute coronary syndromes, but only in those patients who have at least one copy of the TMPRSS6 rs855791 major allele, and is most effective in those patients with elevated serum levels of IL-6.

[0354] Rodent studies were conducted to determine the effects of anti-IL-6 therapy after acute myocardial infarction in mice genotypically similar to humans that are homozygous for the TMPRSS6 rs855791 major allele.

[0355] Figures 11A and 11B show data from an in vivo model in which myocardial infarction was induced in mice genotypically similar to humans homozygous for the TMPRSS6 rs855791 major allele. The control group received no treatment. The experimental group was treated with an anti-mouse IL-6 antibody. Figure 11A shows that treatment with anti-IL-6 resulted in a statistically significant improvement in ejection fraction. Figure 11B shows that treatment with anti-IL-6 resulted in a statistically significant improvement in contractility, measured as cardiac fractional shortening. The data demonstrate that anti-IL-6 therapy given immediately after myocardial infarction improves left ventricular functional recovery in rodents genotypically similar to human patients carrying the TMPRSS6 rs855791 major allele.

[0356] The present invention encompasses, for example, the following embodiments: [Embodiment 1] A method for treating a hepcidin-mediated disorder, comprising: administering a therapeutically effective amount of an IL-6 antagonist to a patient having a hepcidin-mediated disorder; wherein the patient is determined to have at least one copy of the TMPRSS6 rs855791 major allele, [Embodiment 2] The method of embodiment 1, wherein the patient has previously been determined to have at least one copy of the TMPRSS6 rs855791 major allele. [Embodiment 3] The method of embodiment 1, further comprising the initial step of determining that the patient has at least one copy of the TMPRSS6 rs855791 major allele. [Embodiment 4] The method of any one of embodiments 1 to 3, wherein the patient has elevated pre-treatment serum levels of IL-6. [Embodiment 5] The method of any one of embodiments 1 to 4, wherein the patient has elevated pre-treatment serum levels of CRP. [Embodiment 6] The method of any one of embodiments 1 to 5, wherein the hepcidin-mediated disorder is anemia of chronic disease. [Embodiment 7] The method of embodiment 6, wherein the patient is male and has a pre-treatment hemoglobin (Hb) level of less than 14 g / dl. [Embodiment 8] The method of embodiment 7, wherein the patient has a pre-treatment Hb level of less than 13 g / dl. [Embodiment 9] The method of embodiment 8, wherein the patient has a pre-treatment Hb level of less than 12 g / dl. [Embodiment 10] The method of embodiment 9, wherein the patient has a pre-treatment Hb level of less than 11 g / dl. [Embodiment 11] The method of embodiment 6, wherein the patient is female and has a pre-treatment Hb level of less than 12 g / dl. [Embodiment 12] The method of embodiment 11, wherein the patient has a pre-treatment Hb level of less than 11 g / dl. [Embodiment 13] The method of embodiment 12, wherein the patient has a pre-treatment Hb level of less than 10 g / dl. [Embodiment 14] The method of embodiment 13, wherein the patient has a pre-treatment Hb level of less than 9 g / dl. [Embodiment 15] The method of any one of embodiments 6 to 10, wherein the patient is male and has a pre-treatment hematocrit of less than 40%. [Embodiment 16] The method of embodiment 15, wherein the patient has a pre-treatment hematocrit of less than 35%. [Embodiment 17] The method of embodiment 16, wherein the patient has a pre-treatment hematocrit of 30-34%. [Embodiment 18] The method of any one of embodiments 6 and 11 to 14, wherein the patient has a pre-treatment hematocrit of less than 36%. [Embodiment 19] The method of embodiment 18, wherein the patient has a pre-treatment hematocrit of less than 30%. [Embodiment 20] The method of embodiment 19, wherein the patient has a pre-treatment hematocrit of 26-29%. [Embodiment 21] The method of any one of embodiments 6 to 20, wherein the patient has received at least one pretreatment administration of an ESA. [Embodiment 22] The method of embodiment 6, wherein the patient has received at least one pretreatment dose of an ESA and has a normal Hb level or normal hematocrit. [Embodiment 23] The method of any one of embodiments 6 to 20, wherein the patient has received at least one pretreatment dose of iron supplementation. [Embodiment 24] The method of embodiment 6, wherein the patient has received at least one pretreatment dose of iron supplementation and has a normal Hb level or normal hematocrit. [Embodiment 25] The method of any one of embodiments 6 to 20, wherein the patient has received at least one pre-treatment transfusion of blood or packed red blood cells. [Embodiment 26] The method of embodiment 6, wherein the patient has received at least one pre-treatment transfusion of blood or packed red blood cells and has a normal Hb level or normal hematocrit. [Embodiment 27] The method of any of Embodiments 6 to 26, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase the patient's Hb level above pre-treatment levels. The method according to any one of the preceding claims. [Embodiment 28] The method of any one of embodiments 6 to 27, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase the patient's hematocrit above pre-treatment levels. [Embodiment 29] The method of embodiment 21 or 22, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to allow a reduction in the patient's ESA dose without reducing the patient's Hb level below the level present immediately before treatment. [Embodiment 30] The method of embodiment 21 or 22, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to allow a reduction in the patient's ESA dose without reducing the patient's hematocrit below the level present immediately before treatment. [Embodiment 31] The method of any one of embodiments 21, 22, 29, or 30, wherein the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow at least a 10% reduction in the patient's ESA dose compared to the ESA dose before treatment. [Embodiment 32] The method of embodiment 31, wherein the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow at least a 20% reduction in the patient's ESA dose compared to the ESA dose before treatment. [Embodiment 33] The method of embodiment 32, wherein the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to allow at least a 50% reduction in the patient's ESA dose compared to the ESA dose before treatment. [Embodiment 34] The method of any one of embodiments 6 to 33, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to restore functional iron deficiency. [Embodiment 35] The method described in any one of embodiments 6 to 34, wherein the chronic disease is chronic kidney disease (CKD). [Embodiment 36] The method of embodiment 35, wherein the patient has KDOQI stage 1 chronic kidney disease, KDOQI stage 2 chronic kidney disease, KDOQI stage 3 chronic kidney disease, KDOQI stage 4 chronic kidney disease, or KDOQI stage 5 chronic kidney disease. [Embodiment 37] The method of embodiment 36, wherein the patient has KDOQI stage 5 chronic kidney disease. [Embodiment 38] The method of embodiment 35, wherein the patient has cardiorenal syndrome (CRS). [Embodiment 39] The method of embodiment 38, wherein the patient has CRS type 4. [Embodiment 40] The method described in any one of embodiments 35 to 39, wherein the patient has undergone at least one pre-treatment dialysis treatment. [Embodiment 41] The method of any one of embodiments 35 to 40, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce cardiovascular (CV) mortality compared to age-matched, disease-matched historical controls. [Embodiment 42] The method of any one of embodiments 6 to 34, wherein the chronic disease is a chronic inflammatory disease. [Embodiment 43] The method of embodiment 42, wherein the chronic inflammatory disease is rheumatoid arthritis (RA). [Embodiment 44] The method of embodiment 43, wherein the patient has a pre-treatment DAS28 score of greater than 5.1. [Embodiment 45] The method described in embodiment 43, wherein the patient has a pre-treatment DAS28 score of 3.2 to 5.1. [Embodiment 46] The method of embodiment 43, wherein the patient has a pre-treatment DAS28 score of less than 2.6. [Embodiment 47] The method of embodiment 43, wherein the patient's pre-treatment RA is moderately active to severely active. [Embodiment 48] The method of any one of embodiments 43 to 47, wherein the patient has received at least one pretreatment dose of methotrexate. [Embodiment 49] The method of any one of embodiments 43 to 48, wherein the patient has received at least one pretreatment administration of a TNFα antagonist. [Embodiment 50] The method of embodiment 49, wherein the TNFα antagonist is selected from the group consisting of etanercept, adalimumab, infliximab, certolizumab, and golimumab. [Embodiment 51] The method described in any one of embodiments 43 to 47, wherein the patient has received at least one pretreatment administration of an IL-6 antagonist. [Embodiment 52] The method of embodiment 51, wherein the pre-treatment IL-6 antagonist is tocilizumab. [Embodiment 53] The method of embodiment 51, wherein the pretreatment IL-6 antagonist is tofacitinib. [Embodiment 54] The method of any one of embodiments 51 to 53, wherein the treating IL-6 antagonist is MEDI5117. [Embodiment 55] The method of embodiment 42, wherein the chronic inflammatory disease is selected from the group consisting of juvenile idiopathic arthritis, ankylosing spondylitis, plaque psoriasis, psoriatic arthritis, inflammatory bowel disease, Crohn's disease, and ulcerative colitis. [Embodiment 56] The method described in any one of embodiments 6 to 34, wherein the chronic disease is cancer. [Embodiment 57] The method described in embodiment 56, wherein the cancer is selected from the group consisting of solid tumors, small cell lung cancer, non-small cell lung cancer, blood cancer, multiple myeloma, leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), lymphoma, Hodgkin's lymphoma, and liver adenoma. [Embodiment 58] The method described in any one of embodiments 6 to 34, wherein the chronic disease is a chronic infectious disease. [Embodiment 59] The method of any one of embodiments 6 to 34, wherein the chronic disease is congestive heart failure (CHF). [Embodiment 60] The method of any one of embodiments 1 to 5, wherein the hepcidin-mediated disorder is iron-refractory iron deficiency anemia (IRIDA). [Embodiment 61] The method of any one of embodiments 1 to 5, wherein the hepcidin-mediated disorder is acute coronary syndrome. [Embodiment 62] The method of embodiment 61, wherein the patient has suffered a myocardial infarction (MI) within 60 days prior to the first administration of the IL-6 antagonist. [Embodiment 63] The method described in embodiment 62, wherein the patient has suffered an MI within 30 days prior to the first administration of the IL-6 antagonist. [Embodiment 64] The method of embodiment 63, wherein the patient has suffered an MI within 48 hours prior to the first administration of the IL-6 antagonist. [Embodiment 65] The method of embodiment 64, wherein the patient has suffered an MI within 24 hours prior to the first administration of the IL-6 antagonist. [Embodiment 66] A method according to any one of embodiments 61 to 65, wherein the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to improve myocardial contractility compared to pre-treatment levels. [Embodiment 67] The method described in any one of embodiments 61 to 66, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to improve cardiac ejection fraction compared to pre-treatment levels. [Embodiment 68] The method described in any one of embodiments 61 to 67, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce cardiac fibrosis compared to pre-treatment levels. [Embodiment 69] The method of any one of embodiments 1 to 5, wherein the hepcidin-mediated disorder is Castleman's disease.

[0077] [Embodiment 70] A method for improving treatment of a hepcidin-mediated disorder, comprising: discontinuing administration of an IL-6 antagonist to a patient with a hepcidin-mediated disorder; wherein the patient is determined to be homozygous for the TMPRSS6 rs855791 minor allele. [Embodiment 71] The method of embodiment 70, wherein the patient has previously been determined to be homozygous for the TMPRSS6 rs855791 minor allele. [Embodiment 72] The method of embodiment 70, further comprising the initial step of determining that the patient is homozygous for the TMPRSS6 rs855791 minor allele. [Embodiment 73] A method for treating an IL-6-mediated inflammatory disorder in a patient without anemia of chronic inflammation, comprising: administering a therapeutically effective amount of an IL-6 antagonist to a patient with an IL-6-mediated inflammatory disease without anemia; wherein the patient is determined to have at least one copy of the TMPRSS6 rs855791 major allele, [Embodiment 74] The method of embodiment 73, wherein the patient has previously been determined to have at least one copy of the TMPRSS6 rs855791 major allele. [Embodiment 75] The method of embodiment 73, further comprising the initial step of determining that the patient has at least one copy of the TMPRSS6 rs855791 major allele. [Embodiment 76] The method of any one of embodiments 1 to 75, wherein the patient has elevated pre-treatment serum levels of IL-6. [Embodiment 77] The method of embodiment 76, wherein the patient has a pre-treatment serum IL-6 level greater than 2.5 pg / ml. [Embodiment 78] The method of embodiment 77, wherein the patient has a pre-treatment serum IL-6 level greater than 5 pg / ml. [Embodiment 79] The method of embodiment 78, wherein the patient has a pre-treatment serum IL-6 level greater than 7.5 pg / ml. [Embodiment 80] The method of embodiment 79, wherein the patient has a pre-treatment serum IL-6 level greater than 10 pg / ml. [Embodiment 81] The method of embodiment 80, wherein the patient has a pre-treatment serum IL-6 level greater than 12.5 pg / ml. [Embodiment 82] The method described in any one of embodiments 76 to 81, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce the free IL-6 level in the patient's serum below the pre-treatment level. [Embodiment 83] The method of embodiment 82, wherein the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to reduce free IL-6 levels by at least 10% compared to pre-treatment levels. [Embodiment 84] The method of embodiment 83, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce the level of free IL-6 in the patient's serum by at least 20% compared to pre-treatment levels. [Embodiment 85] The method of embodiment 84, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce the level of free IL-6 in the patient's serum by at least 50% compared to pre-treatment levels. [Embodiment 86] The method of any one of embodiments 1 to 85, wherein the patient has elevated pre-treatment levels of C-reactive protein (CRP). [Embodiment 87] The method of embodiment 86, wherein the patient has a pre-treatment CRP level greater than 2 mg / ml. [Embodiment 88] The method of embodiment 87, wherein the patient has a pre-treatment CRP level greater than 3 mg / ml. [Embodiment 89] The method of embodiment 88, wherein the patient has a pre-treatment CRP level greater than 5 mg / ml. [Embodiment 90] The method of embodiment 89, wherein the patient has a pre-treatment CRP level greater than 7.5 mg / ml. [Embodiment 91] The method of embodiment 90, wherein the patient has a pre-treatment CRP level greater than 10 mg / ml. [Embodiment 92] The method of any one of embodiments 86 to 91, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce the patient's CRP level below pre-treatment levels. [Embodiment 93] The method of embodiment 92, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce the patient's CRP levels by at least 50% compared to pre-treatment levels. [Embodiment 94] The method of any one of embodiments 1 to 93, wherein the patient has been determined to have at least one copy of the TMPRSS6 rs855791 major allele using a TaqMan® real-time PCR assay. [Embodiment 95] The method described in any one of embodiments 1 to 94, wherein the IL-6 antagonist is an anti-IL-6 antibody or an antigen-binding fragment or derivative thereof. [Embodiment 96] The anti-IL-6 antibody, or antigen-binding fragment or derivative thereof, has a K of less than 100 nM for binding to human IL-6. D 96. The method of embodiment 95, comprising:

[0099] [Embodiment 97] The antibody or antigen-binding fragment or derivative has a K of less than 50 nM for binding to human IL-6. D 97. The method of embodiment 96, comprising:

[0099] [Embodiment 98] The antibody or antigen-binding fragment or derivative has a K D 98. The method of embodiment 97, comprising: [Embodiment 99] The antibody or antigen-binding fragment or derivative has a K of less than 1 nM for binding to human IL-6. D 99. The method of embodiment 98, comprising: [Embodiment 100] The method described in any one of embodiments 95 to 99, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative has an elimination half-life after intravenous administration of at least 7 days. [Embodiment 101] The method of embodiment 100, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative has an elimination half-life after intravenous administration of at least 14 days. [Embodiment 102] The method of embodiment 101, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative has an elimination half-life after intravenous administration of at least 21 days. [Embodiment 103] The method of embodiment 102, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative has an elimination half-life after intravenous administration of at least 30 days. [Embodiment 104] The method of any one of embodiments 95 to 103, wherein the IL-6 antagonist is a full-length monoclonal anti-IL-6 antibody. [Embodiment 105] The method described in embodiment 104, wherein the antibody is an IgG1 or IgG4 antibody. [Embodiment 106] The method described in embodiment 105, wherein the antibody is an IgG1 antibody. [Embodiment 107] The method of any one of embodiments 95 to 106, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is fully human. [Embodiment 108] The method of any one of embodiments 95 to 106, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is humanized. [Embodiment 109] The method described in any one of embodiments 95 to 108, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable region CDRs of MED5117. [Embodiment 110] The method described in embodiment 109, wherein the antibody comprises the VH and VL of MED5117. [Embodiment 111] The method described in embodiment 110, wherein the antibody is MED5117. [Embodiment 112] The anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable region CDRs of an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, elsilimomab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). [Embodiment 9] A method according to any one of claims 5 to 108. [Embodiment 113] The method described in embodiment 112, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises a heavy chain V region and a light chain V region derived from an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In a specific embodiment, the anti-IL-6 antibody is an antibody selected from the group consisting of siltuximab, gerilimu- zumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). [Embodiment 114] The method described in embodiment 113, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In a specific embodiment, the anti-IL-6 antibody is an antibody selected from the group consisting of siltuximab, gerilimu- zumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). [Embodiment 115] The method described in any one of embodiments 95 to 103, wherein the IL-6 antagonist is a single domain antibody, a VHH nanobody, a Fab, or an scFv. [Embodiment 116] The method of any one of embodiments 1 to 94, wherein the IL-6 antagonist is an anti-IL-6R antibody, or an antigen-binding fragment or derivative thereof. [Embodiment 117] The method described in embodiment 116, wherein the anti-IL-6R antibody, antigen-binding fragment or derivative is tocilizumab. [Embodiment 118] The method of embodiment 116, wherein the anti-IL-6R antibody, antigen-binding fragment or derivative is bovalilizumab. [Embodiment 119] The method of any one of embodiments 1 to 94, wherein the IL-6 antagonist is a JAK inhibitor. [Embodiment 120] The method of embodiment 119, wherein the JAK inhibitor is selected from the group consisting of tofacitinib (Xeljanz), decernotinib, ruxolitinib, upadacitinib, baricitinib, filgotinib, lestaurtinib, pacritinib, peficitinib, INCB-039110, ABT-494, INCB-047986, and AC-410. [Embodiment 121] The method of any one of embodiments 1 to 94, wherein the IL-6 antagonist is a STAT3 inhibitor. [Embodiment 122] The method of any one of embodiments 95 to 118, wherein the IL-6 antagonist is administered parenterally. [Embodiment 123] The method of embodiment 122, wherein the IL-6 antagonist is administered subcutaneously. [Embodiment 124] The method of embodiment 119 or 120, wherein the IL-6 antagonist is administered orally. 6. Incorporation by Reference All publications, patents, patent applications, and other documents cited in this application are herein incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference for all purposes.

[0357] 7. Equivalents While various specific embodiments have been illustrated and described, the above specification is not intended to be limiting. It will be understood that various modifications can be made without departing from the spirit and scope of the invention. Numerous variations will become apparent to those skilled in the art upon review of this specification.

Claims

1. 1. A composition for use in a method of treating a hepcidin-mediated disorder, comprising an IL-6 antagonist, The method comprises administering a therapeutically effective amount of an IL-6 antagonist to a patient having a hepcidin-mediated disorder; The composition, wherein the patient is determined to have at least one copy of the TMPRSS6 rs855791 major allele.

2. 10. The composition of claim 1, wherein the hepcidin-mediated disorder is anemia of chronic disease.

3. 3. The composition of claim 2, wherein the patient is male and has a pre-treatment hemoglobin (Hb) level of less than 14 g / dl.

4. 4. The composition of claim 2 or 3, wherein the patient has a pre-treatment hematocrit of less than 35%.

5. The composition according to any one of claims 2 to 4, wherein the chronic disease is chronic kidney disease (CKD).

6. 6. The composition of claim 5, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to reduce cardiovascular (CV) mortality compared to age-matched, disease-matched historical controls.

7. The composition according to any one of claims 2 to 4, wherein the chronic disease is a chronic inflammatory disease.

8. 8. The composition of claim 7, wherein the chronic inflammatory disease is rheumatoid arthritis (RA).

9. The composition of any one of claims 1 to 8, wherein the therapeutic IL-6 antagonist is MEDI5117.

10. The composition according to any one of claims 2 to 4, wherein the chronic disease is cancer.

11. The composition according to any one of claims 2 to 4, wherein the chronic disease is a chronic infectious disease.

12. The composition according to any one of claims 2 to 4, wherein the chronic disease is congestive heart failure (CHF).

13. 2. The composition of claim 1, wherein the hepcidin-mediated disorder is iron-refractory iron deficiency anemia (IRIDA).

14. The composition of claim 1, wherein the hepcidin-mediated disorder is acute coronary syndrome.

15. The composition of claim 14, wherein the patient has suffered a myocardial infarction (MI) within 60 days prior to the first administration of the IL-6 antagonist.

16. 1. A composition for use in a method of treating a hepcidin-mediated disorder, comprising an IL-6 antagonist, The method comprises discontinuing administration of an IL-6 antagonist to a patient having a hepcidin-mediated disorder; The composition, wherein the patient is determined to be homozygous for the TMPRSS6 rs855791 minor allele.

17. 1. A composition for use in a method for treating an IL-6-mediated inflammatory disorder in a patient without anemia of chronic inflammation, comprising an IL-6 antagonist, The method comprises administering a therapeutically effective amount of an IL-6 antagonist to a patient having an IL-6-mediated inflammatory disorder without anemia; The composition, wherein the patient is determined to have at least one copy of the TMPRSS6 rs855791 major allele.

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