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 Jul. 31, 2015, and U.S. Provisional Application No. 62 / 268,788, filed Dec. 17, 2015, each of which is incorporated herein by reference in its entirety.

Background Art

[0002] 2. Background Hepcidin, a peptide hormone, plays a central role in systemic iron homeostasis. Hentze et al., Cell 142:24-38 (2010). Hepcidin expression is known to be affected by the product of the TMPRSS6 gene, matriptase-2, a type II transmembrane serine protease. Common variants of 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 natural variation in hepcidin expression and blood hemoglobin levels.

[0003] Hepcidin expression is also involved 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 infections, autoimmune diseases, cancer, and chronic kidney disease (CKD). Sun et al., Am. J. Hematol. 87(4):392-400 (2012).

Summary of the Invention

Problems 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. Summary The inventors have demonstrated that a decrease in IL-6 signaling provides a clinical benefit in patients with hepcidin-mediated disorders, including anemia of chronic disease and hepcidin-mediated cytotoxicity, but this benefit is provided only in patients having at least one copy of the TMPRSS6 rs855791 major allele and is most effective in patients having elevated levels of IL-6.

[0006] Accordingly, 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 having a hepcidin-mediated disorder 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 an 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 embodiments of anemia, the patient is male and has a pre-treatment hemoglobin (Hb) level of less than 14 g / dl, less than 13 g / dl, less than 12 g / dl, or less than 11 g / dl. In some embodiments of anemia, the patient is female and has a pre-treatment Hb level of less than 12 g / dl, less than 11 g / dl, less than 10 g / dl, or less than 9 g / dl.

[0009] In some embodiments of anemia, 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 embodiments of anemia, 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 a normal hematocrit. In various embodiments, the patient has received at least one pre-treatment administration of iron supplementation. In certain embodiments, the patient has received at least one pre-treatment administration of iron supplementation and has a normal Hb level or a 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 a normal hematocrit.

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

[0012] In various embodiments, the IL-6 antagonist is administered in a dose, schedule, and duration sufficient to allow for 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 in a dose, schedule, and duration sufficient to restore functional iron deficiency.

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

[0015] In some embodiments of CKD, 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 a specific embodiment, the patient has KDOQI stage 5 chronic kidney disease.

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

[0017] In some embodiments of CKD, the IL-6 antagonist is administered in a dose, schedule, and duration sufficient to reduce cardiovascular (CV) mortality compared to age- and 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 greater than 5.1. In some embodiments, the patient has a pre-treatment DAS28 score of 3.2 - 5.1. In a specific embodiment, the patient has a pre-treatment DAS28 score less than 2.6. In selected embodiments, the patient's pre-treatment RA is moderately to severely active.

[0020] In some embodiments of RA, the patient has received at least one pre-treatment administration of methotrexate. In some embodiments, the patient has received at least one pre-treatment administration 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 embodiments of RA, the patient has received at least one pre-treatment dose of an IL-6 antagonist. In certain embodiments, the pre-treatment IL-6 antagonist is tocilizumab or tofacitinib.

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

[0023] In various embodiments, the hepcidin-mediated disorder is anemia of a 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 a 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, blood cancers, multiple myeloma, leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), lymphoma, Hodgkin lymphoma, and hepatic adenoma.

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

[0026] In some embodiments, the hepcidin-mediated disorder is anemia of a chronic disease, wherein 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 had a myocardial infarction (MI) within 60 days prior to the first administration of the IL-6 antagonist, within 30 days prior to the first administration of the IL-6 antagonist, within 48 hours prior to the first administration of the IL-6 antagonist, or within 24 hours prior to the first administration of the IL-6 antagonist.

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

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

[0031] In another aspect, a method for improving the treatment of hepcidin-mediated disorders is provided. The method includes discontinuing administration of an IL-6 antagonist to a patient having a hepcidin-mediated disorder who has been determined to be homozygous for the TMPRSS6 rs855791 minor allele.

[0032] In another aspect, a method for improving the treatment of hepcidin-mediated disorders is provided by discontinuing ineffective therapies, thereby reducing side effects and reducing costs without loss of treatment efficacy. The method includes discontinuing the administration of an IL-6 antagonist to a patient having a hepcidin-mediated disorder who has been determined to be homozygous for the TMPRSS6 rs855791 minor allele. In a 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 an initial step of determining that the patient is homozygous for the TMPRSS6 rs855791 minor allele. In typical embodiments, the patient has elevated pre-treatment serum levels of IL-6. In various embodiments, the patient has elevated pre-treatment serum levels 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 shown in Examples 2, 3, and 5 below demonstrate that an IL-6 antagonist confers a therapeutic benefit in subjects with elevated pre-treatment IL-6 levels and having at least one copy of the TMPRSS6 major allele, even in the absence of anemia. Accordingly, in another aspect, a method for treating an IL-6 mediated inflammatory disorder in a patient without chronic inflammatory anemia is provided. The method comprises administering to a subject, typically a human patient, having an IL-6 mediated inflammatory disorder, a therapeutically effective amount of an IL-6 antagonist, 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 method further comprises an 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 that are homozygous for the TMPRSS6 rs855791 major allele. Typically, the patient has elevated pre-treatment serum levels of IL-6.

[0034] In certain specific embodiments of any of the treatment methods, the patient has elevated pre-treatment serum levels of IL-6. In certain embodiments, the patient has a pre-treatment serum IL-6 level that is 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 in a dosage, schedule, and duration sufficient to reduce the free IL-6 level in the patient's serum below the pre-treatment level. In specific embodiments, the IL-6 antagonist is administered in a dosage, schedule, and duration sufficient to reduce the free IL-6 level by at least 10% compared to the pre-treatment level, at least 20% compared to the pre-treatment level, or at least 50% compared to the pre-treatment level.

[0036] In certain embodiments of any of the treatment methods, the patient has an elevated pre-treatment level of C-reactive protein (CRP). In certain embodiments, the patient has a pre-treatment CRP level that is 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 in a dose, schedule, and duration sufficient to reduce the patient's CRP level below the pre-treatment level. In specific embodiments, the IL-6 antagonist is administered in a dose, schedule, and duration sufficient to reduce the patient's CRP level by at least 50% compared to the pre-treatment level.

[0038] In certain specific embodiments of any of the treatment methods, 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 certain embodiments of any of the treatment methods, 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 D less than 100 nM, less than 50 nM, less than 10 nM, or less than 1 nM for binding to human IL-6. 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 certain 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 presently 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. Further, 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, golimumab, 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, golimumab, 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, golimumab, 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, golimumab, 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, golimumab, 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, VHH nanobody, Fab, or scFv.

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

[0049] In various embodiments, the IL-6 antagonist is a JAK inhibitor. In a specific embodiment, 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 DESCRIPTION OF THE DRAWINGS

[0053]

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Mode for Carrying Out the Invention

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

[0055] 5. Detailed description 5.1. Summary of experimental results Hepcidin, a peptide hormone, plays a central role in systemic iron homeostasis. Hentze et al., Cell 142:24-38 (2010). Hepcidin expression is known to be affected by the product of the TMPRSS6 gene, matriptase-2, a type II transmembrane serine protease. Common variants of 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 natural variation in hepcidin expression and blood hemoglobin levels. Hepcidin expression is also involved 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 infections, autoimmune diseases, cancer, and chronic kidney disease (CKD). Sun et al., Am. J. Hematol. 87(4):392-400 (2012).

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

[0057] As described in Example 1 and as shown in FIG. 1, the underlying degree of anemia, measured as the clinically titrated EPO dose, correlated with IL-6 levels only in patients having 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 (FIG. 1B). In contrast, the degree of anemia in patients having two copies of the minor allele did not correlate with serum IL-6 levels (FIG. 1A).

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

[0059] Without wishing to be bound by theory, in patients having at least one copy of the TMPRSS6 major allele, an increase in serum IL-6 may cause an increase in 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 a decrease in IL-6 levels or IL-6 signaling may reduce anemia, reduce the required EPO dose, and increase the likelihood of increased survival only in those patients with chronic kidney disease who have at least one copy of the TMPRSS6 rs855791 major allele, and was most effective in those patients with elevated serum levels of IL-6.

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

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

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

[0063] 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 being bound by theory, in patients having at least one copy of the TMPRSS6 major allele, increased serum IL-6 increases hepcidin expression, which in turn increases sequestration of iron in cardiomyocytes, subsequently resulting in iron-mediated cytotoxicity. These correlations raise the possibility that a decrease in IL-6 levels or IL-6 signaling can reduce heart failure and mortality only in those patients who are acute coronary syndrome patients but have at least one copy of the TMPRSS6 rs855791 major allele, and that the greatest effect is seen in those patients with elevated serum levels of IL-6.

[0064] The correlations observed in Examples 1 and 2 strongly suggest that a decrease in IL-6-mediated signaling should confer a clinical benefit in patients having at least one copy of the TMPRSS6 rs855791 major allele, elevated IL-6 levels, and anemia or hepcidin-mediated cytotoxicity, although the correlations observed are insufficient to prove causation. Thus, in Example 3, human induced pluripotent stem (iPS) cell cardiomyocytes were genetically engineered to express only the TMPRSS6 rs855791 major or 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 with the major and minor alleles were treated in vivo with recombinant BMP2 and IL-6, which are agonists of both signaling pathways, or with BMP2 alone, to model clinical interventions in which IL-6 levels (or signaling) are decreased. Control iPS cells were not treated with any agonist. Cell death rate was measured under normoxic (normoxia) conditions and after simulating hypoxia and then under reoxygenation (reperfusion) conditions.

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

[0067] Figure 6B shows the results when cells are subjected to hypoxia followed by reoxygenation. Compared to normal oxygen conditions, hypoxia / reoxygenation is toxic to iPS cardiomyocytes, with approximately 40 percent of control cells for both the major and minor alleles dying, compared to approximately 20% of control cells dying under normal oxygen conditions (compare with Figure 6A). In response to this increased background toxicity, iPS cardiomyocytes of the minor allele are not significantly affected by elimination of IL-6 signaling. The cell death rate does not decrease significantly when cells are treated with BMP2 alone compared to treatment with BMP2 + IL-6. In contrast, iPS cardiomyocytes expressing the TMPRSS6 rs855791 major allele show statistically significantly lower cell death when IL-6 signaling is eliminated.

[0068] These data reinforce the inferences drawn from the post hoc analysis of the clinical trial data in Examples 1 and 2. Reduction of 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 being bound by theory, the increased IL-6-induced toxicity in major allele iPS cardiomyocytes may result from an IL6-mediated increase in hepcidin expression, which results in increased intracellular sequestration of iron and subsequently 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 injury following primary chronic kidney disease is called cardiorenal syndrome type 4 (CRS type 4). As suggested by the data in Examples 1 and 3, to directly test whether anti-IL-6 therapy is effective as a treatment for CRS4 patients having at least one copy of the TMPRSS6 rs855791 major allele, the inventors used a model of CRS4 in rats that are genotypically similar to humans that are homozygous for the TMPRSS6 rs855791 major allele.

[0070] After 4 weeks of treatment, both treatment groups, i.e., the group treated with the anti-IL-6 antibody and the group treated with perindopril, which is 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). Similar ejection fraction levels in the anti-IL-6 group and the standard of care group measured after the 4th week of treatment indicated that anti-IL-6 therapy had equivalent efficacy to the ACE inhibitor. Figure 9 shows that anti-IL-6 therapy was also equally effective as the ACE inhibitor in maintaining 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 an anti-IL-6 agent is effective in reducing cardiac injury and restoring function in an in vivo model of cardiorenal syndrome in animals that are genotypically similar to humans that are homozygous for the TMPRSS6 rs855791 major allele.

[0072] Similarly, the data from Examples 2 and 3 suggest that a decrease in IL-6 levels or IL-6 signaling reduces heart failure and mortality in patients with acute coronary syndrome who have at least one copy of the TMPRSS6 rs855791 major allele, and suggest that it has the greatest effect 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 that are 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 that are 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 the control. Figure 11B shows that treatment with anti-IL-6 resulted in a statistically significant improvement in contractility measured as shortening of cardiac fractional compared to the control. These data demonstrate that anti-IL-6 therapy administered immediately after myocardial infarction improves left ventricular function recovery in rodents that are genotypically similar to human patients with the TMPRSS6 rs855791 major allele.

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

[0076] Accordingly, in a first aspect, as further described below, a method for treating hepcidin-mediated disorders is provided. The method includes administering a therapeutically effective amount of an IL-6 antagonist to a patient having a hepcidin-mediated disorder 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 hepcidin-mediated disorders is provided, the method including discontinuing administration of an IL-6 antagonist to a patient having a hepcidin-mediated disorder, where 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 treatment efficacy. In a further aspect, a method for treating an IL-6-mediated inflammatory disorder in a patient without chronic inflammatory anemia is provided, the method including administering a therapeutically effective amount of an IL-6 antagonist to a patient having an IL-6-mediated inflammatory disorder and not having anemia, where the subject has been determined to have at least one copy of the TMPRSS6 rs855791 major allele.

[0077] 5.2. Definitions 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" means a polypeptide having at least about 85% amino acid identity with the amino acid sequence provided by NCBI accession number NP_066998 ("hepcidin preprotein") or a biologically active fragment thereof. Exemplary hepcidin biological activities include binding to the iron transport channel ferroportin and reduction of its levels, inhibition of iron transport, inhibition of intestinal iron absorption, and inhibition of iron release from macrophages and the liver. An exemplary amino acid sequence of the prepeptide protein is provided below:

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

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

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

[0082] "Transmembrane protease serine 6 (TMPRSS6) polypeptide" means a polypeptide or a fragment thereof that has at least about 85% or more amino acid identity with the amino acid sequence provided by NCBI accession number NP_001275929 and has serine protease activity. The TMPRSS6 polypeptide, also known as matriptase-2 (MT2), cleaves hemojuvelin and inhibits the signaling of bone morphogenetic proteins. An exemplary TMPRSS6 amino acid sequence having alanine (736A) at position 736 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 having valine (736V) at position 736 is provided below: 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 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] The "TMPRSS6 nucleic acid molecule" means a polynucleotide encoding the TMPRSS6 polypeptide (matriptase-2, MT2). An exemplary TMPRSS6 nucleic acid molecule sequence is provided at NCBI accession number NM_001289000. The TMPRSS6 nucleic acid sequence having a G at nucleotide position 2321 ("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 GAAGAAGGGC 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 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 GAAGAAGGGC 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] The term "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" means a naturally occurring DNA sequence variant in which a single nucleotide in the genome differs between members of a biological species or between paired chromosomes in an individual. SNPs can be used as genetic markers for mutant alleles. In one embodiment, the TMPRSS6 SNP is rs855791.

[0090] "rs855791" means a single nucleotide polymorphism (SNP) of 2321G→A in the human TMPRSS6 gene, which results in a substitution of alanine to valine (A736V) in the catalytic domain of matriptase-2 (MT2) encoded by the TMPRSS6 gene. The allele with the highest frequency (major allele) in the human population is 2321G, which encodes 736A. The allele with the lowest frequency (minor allele) in the human population 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 alanine at amino acid position 736 (e.g., having G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 major allele), and the other allele contains a mutant TMPRSS6 nucleic acid sequence encoding a TMPRSS6 polypeptide containing valine at amino acid position 736 (e.g., having 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 encoding a TMPRSS6 polypeptide that includes alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of a 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 encoding a TMPRSS6 polypeptide that includes valine at amino acid position 736 (e.g., having an A or T at nucleotide position 2321 of a 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, otherwise instructing or controlling the performance of an assay to determine that a patient has at least one copy of the TMPRSS6 rs855791 major allele, and examining 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" means a polypeptide or a fragment thereof that has at least about 85% or more amino acid identity with the amino acid sequence provided by NCBI accession number NP_000591 and has IL-6 biological activity. IL-6 is a pleiotropic cytokine with multiple biological functions. Exemplary biological activities of IL-6 include immunostimulatory and pro-inflammatory 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 GPSTRPVEVP GGRAVAAILG LGLVLGLLGP LAILLALYLL 241 RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI (SEQ ID NO: 6)

[0096] "Interleukin-6 (IL-6) nucleic acid" means a polynucleotide encoding the interleukin-6 (IL-6) polypeptide. An exemplary interleukin-6 (IL-6) nucleic acid sequence is provided by NCBI accession number NM_000600. The exemplary sequence in NCBI accession number 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 CAGGAGAAGA 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] The "interleukin-6 receptor (IL-6R) complex" means a protein complex containing the interleukin-6 receptor subunit alpha (IL-6Rα) and the interleukin-6 signal transducer glycoprotein 130, also called interleukin-6 receptor subunit beta (IL-6Rβ).

[0099] The "Interleukin-6 receptor subunit alpha (IL-6Rα) polypeptide" refers to a polypeptide or a fragment thereof that has at least about 85% or more amino acid identity with the amino acid sequence provided by NCBI accession number NP_000556 or NP_852004 and has the biological activity of the IL-6 receptor. Exemplary IL-6Rα biological activities include binding to IL-6, binding to glycoprotein 130 (gp130), and regulation of cell growth and differentiation. Exemplary IL-6R sequences are 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] The "interleukin-6 receptor subunit beta (IL-6Rβ) polypeptide" refers to a polypeptide or a fragment thereof that has at least about 85% or more amino acid identity with the amino acid sequences provided by NCBI accession numbers NP_002175, NP_786943, or NP_001177910 and has the biological activity of the 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, etc. Exemplary IL-6Rβ sequences are 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] "IL-6 antagonist" refers to an agent that can reduce the biological activity of IL-6. IL-6 antagonists include agents that reduce the level of IL-6 polypeptide in serum; agents that reduce the expression of IL-6 polypeptide or nucleic acid, agents that reduce the ability of IL-6 to bind to IL-6R, agents that reduce the expression of IL-6R, and agents that reduce signal transduction by the IL-6R receptor when bound to IL-6. In a preferred embodiment, 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, such as anti-IL-6 antibodies and antigen-binding fragments or derivatives thereof; IL-6R binding polypeptides, such as anti-IL-6R antibodies and antigen-binding fragments or derivatives thereof; and synthetic chemical molecules, such as 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] "IL-6-mediated inflammatory disorder" means any disorder in which IL-6 is known or suspected to contribute to either the etiology of the disease or its symptoms.

[0106] "Erythropoietin (EPO)" means a polypeptide or fragment thereof that has at least about 85% amino acid identity with the amino acid sequence provided by NCBI accession number NP_000790 and has EPO biological activity. Exemplary EPO biological activities include binding to the erythropoietin receptor and the resulting increase in the proliferation and terminal differentiation of erythroid progenitor cells and / or erythropoiesis (erythrocyte production). An exemplary EPO amino acid sequence is 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" means an agent that increases the growth or proliferation of erythrocytes or their progenitor cells (e.g., hematopoietic stem cells) and / or decreases cell death in erythrocytes or their progenitor cells. In various embodiments, the erythropoietic factor includes an erythropoiesis-stimulating agent, an HIF stabilizer, and supplemental iron.

[0110] "C-reactive protein (CRP) polypeptide" means a polypeptide or fragment thereof having at least about 85% or more amino acid identity with the amino acid sequence provided by NCBI accession number NP_000558 and having complement activation activity. CRP levels increase 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] "Agent" means any compound or composition suitable for administration in a therapy, and explicitly includes chemical compounds, antibodies or proteins including antigen-binding fragments thereof, peptides; and nucleic acid molecules.

[0113] "Subject" means a human or non-human mammal, and includes, without limitation, bovine, equine, canine, ovine, feline, and rodent subjects such as mice and rats. "Patient" is a human subject.

[0114] As used herein, the terms "treat", "treating", "treatment", etc. mean the reduction or alleviation of a disorder and / or associated symptoms or signs, or the delay or arrest of its progression. It is understood that treatment of a disorder or condition, without being excluded, does not require the complete elimination of the disorder, condition or symptoms associated therewith.

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

[0116] In the present disclosure, "comprise", "comprising", "containing", "having", "includes", "including", and their linguistic variants have the meaning ascribed to them in the United States Patent Law and recognize the presence of additional components beyond those explicitly recited.

[0117] "Biological sample" means any tissue, cell, liquid, or other substance derived from a living organism (e.g., a human subject). In certain embodiments, the biological sample is serum or blood.

[0118] "Angiotensin-converting enzyme (ACE) inhibitor" means a drug that inhibits the biological function of angiotensin-converting enzyme that 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 interpretive conventions Unless otherwise specified, the residue numbering of the antibody constant region follows the EU index shown in Kabat.

[0120] The ranges provided herein are to be understood as shorthand for all values within the range including the recited endpoints. For example, the range 1-50 is to be understood as including any number, combination of numbers, or sub-range 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] Unless otherwise indicated or apparent from the context, as used herein, the term "or" is to be understood as inclusive. Unless otherwise indicated or apparent from the context, as used herein, the terms "a", "an", and "the" are to be understood as singular or plural.

[0122] Unless otherwise specified or otherwise apparent from the context, as used herein, the term "about" is understood to be within the normal tolerance in the art, e.g., within 2 standard deviations of the mean. About can be understood to be 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 apparent 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 hepcidin-mediated disorders is provided.

[0124] The method comprises administering to a subject, typically a human patient having a hepcidin-mediated disorder, a therapeutically effective amount of an IL-6 antagonist, 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 an 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 that are homozygous for the TMPRSS6 rs855791 minor allele. Typically, the patient has elevated pre-treatment serum levels of IL-6.

[0125] 5.2.1. Hepcidin-mediated disorders 5.2.1.1. Chronic disease / chronic inflammatory anemia 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 been previously treated with an ESA. In some embodiments, the patient is being treated with iron supplementation. In some embodiments, the patient is being treated with a blood or packed red blood cell transfusion.

[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 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, or less than 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 has been previously treated with an ESA. In some embodiments, the patient is being treated with iron supplementation. In some embodiments, the patient is being treated with a blood or packed red blood cell transfusion.

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

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

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

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

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

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

[0134] In some embodiments, the IL-6 antagonist is administered in a dosage, schedule, and 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 chronic kidney disease at KDOQI stage 1. In certain embodiments, the patient has chronic kidney disease at KDOQI stage 2, KDOQI stage 3, KDOQI stage 4, or KDOQI stage 5.

[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 in a dosage, schedule, and duration sufficient to reduce cardiovascular (CV) mortality as compared to a historical cohort matched by age and disease.

[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 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 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, treatment with methotrexate 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 particular embodiment, the IL-1 receptor antagonist is anakinra. In specific embodiments, 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 specific embodiments, abatacept is discontinued when treatment with an IL-6 antagonist is initiated.

[0147] In certain embodiments, the patient has been treated with an IL-6 antagonist, and the method further comprises continuing to administer the IL-6 antagonist only to those patients 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 solid tumors, small cell lung cancer, non-small cell lung cancer, blood cancers, multiple myeloma, leukemia, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), lymphoma, and Hodgkin lymphoma.

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

[0152] 5.2.1.1.5. 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 shown in Examples 2, 3, and 5 below demonstrate that an IL-6 antagonist is effective in reducing the risk of heart failure and death, enhancing 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 has suffered a myocardial infarction 60 days prior to the first administration of the IL-6 antagonist. In specific embodiments, the patient has 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 in a dose, schedule, and duration sufficient to improve myocardial contractility compared to pre-treatment levels. In certain embodiments, the IL-6 antagonist is administered in 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 in 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 disease.

[0159] 5.3. Methods for improving the treatment of hepcidin-mediated disorders In another aspect, a method for improving the treatment of hepcidin-mediated disorders is provided by discontinuing ineffective therapies, thereby reducing side effects and reducing costs without loss of treatment efficacy. The method includes discontinuing administration of an IL-6 antagonist to a patient having a hepcidin-mediated disorder, where the patient has been determined to be homozygous for the TMPRSS6 rs855791 minor allele. In a 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 an initial step of determining that the patient is homozygous for the TMPRSS6 rs855791 minor allele. In exemplary embodiments, 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 in Session 5.2.1 above. In certain embodiments, the patient has anemia of chronic disease.

[0161] 5.4. Methods for treating IL-6-mediated inflammatory disorders The data shown in Examples 2, 3 and 5 below demonstrate that IL-6 antagonists provide a therapeutic benefit in subjects with elevated pre-treatment IL-6 levels and having at least one copy of the TMPRSS6 major allele, even in the absence of anemia. Accordingly, in another aspect, a method for treating IL-6-mediated inflammatory disorders in patients without anemia of chronic inflammation is provided.

[0162] The method comprises administering to a subject, typically a human patient, having an IL-6 mediated inflammatory disorder, a therapeutically effective amount of an IL-6 antagonist, 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 set of embodiments, the subject has previously been determined to have at least one copy of the TMPRSS6 rs855791 major allele. In another set of embodiments, the method further comprises an 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 that are homozygous for the TMPRSS6 rs855791 minor allele. Typically, the patient has elevated pre-treatment 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 greater than 5.1. In some embodiments, the patient has a pre-treatment DAS28 score of 3.2 - 5.1. In some embodiments, the patient has a pre-treatment DAS28 score 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 the IL-6 antagonist is initiated. In some embodiments, methotrexate is continued when treatment with the IL-6 antagonist is initiated.

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

[0167] In certain embodiments, the patient is being treated with an IL-1 receptor antagonist. In a particular 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.

[0168] In certain embodiments, the patient is being treated with abatacept. In a specific embodiment, abatacept is discontinued when treatment with an 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. Pretreatment 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 in a dose, schedule, and duration sufficient to reduce the patient's serum IL-6 level below the pre-treatment level. In certain embodiments, the IL-6 antagonist is administered in a dose, schedule, and duration sufficient to reduce the patient's serum IL-6 level by at least 10%, 20%, 30%, 40%, or 50%.

[0173] In various embodiments, the patient has a pre-treatment level of C-reactive protein (CRP) that is elevated. 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 in a dose, schedule, and duration sufficient to reduce the patient's CRP level below the pre-treatment level. In certain embodiments, the IL-6 antagonist is administered in a dose, schedule, or duration sufficient to reduce the patient's CRP level by at least 10%, 20%, 30%, 40%, or 50% compared to the pre-treatment level.

[0175] 5.6. TMPRSS6 rs855791 genotyping The methods described herein include 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, and thus, it is possible to identify and distinguish patients who are homozygous for the TMPRSS6 rs855791 major allele, heterozygous for 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 biological samples obtained from a patient.

[0178] In some embodiments, the absence or presence of the polymorphism is detected simultaneously with amplification using real-time PCR (RT-PCR). In certain embodiments, the RT-PCR assay uses 5' nuclease (TaqMan® probe), molecular beacons, and / or FRET hybridization probes. It is reviewed in Espy et al., Clin. Microbiol. Rev. January 2006, 19(1): 165-256, which is hereby incorporated by reference in its entirety. In typical embodiments, a commercially available assay is used. In selected embodiments, the commercially available assay is selected from the group consisting of TaqMan® SNP Genotyping Assay (ThermoFisher), PCR SNP Genotyping Assay (Qiagen), Novallele Genotyping Assay (Canon), and SNP Type® Assay (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 called by sequencing. In certain embodiments, Sanger sequencing is used. In certain embodiments, one of the various next-generation sequencing technologies is used, for example, sequencing technologies 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 antagonist used in the methods described herein can reduce 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 species of full-length anti-IL-6 antibodies, each of the multiple species having 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 scFv, disulfide-bonded Fv (dsFv), or VHH single-domain nanobody derived from camel. 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 portions 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 and has 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 D of less than 100 nM for binding to human IL-6. For some embodiments, the anti-IL-6 antibody has a K D of less than 75 nM, 50 nM, 25 nM, 20 nM, 15 nM, or 10 nM for binding to human IL-6. In a specific embodiment, the anti-IL-6 antibody has a K D of less than 5 nM, 4 nM, 3 nM, or 2 nM for binding to human IL-6. In a selected embodiment, the anti-IL-6 antibody has a K DIt has. In 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 inhibits the binding of IL-6 to the IL-6 receptor.

[0186] In typical embodiments, the anti-IL-6 antibody has an elimination half-life after intravenous administration for at least 7 days. In 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 the serum half-life compared to the unsubstituted human IgG constant domain.

[0188] In certain practical embodiments, the IgG constant domain contains substitutions at residues 252, 254, and 256, where the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, the amino acid substitution at amino acid residue 254 is a substitution with threonine, and the amino acid substitution at amino acid residue 256 is a substitution with glutamic acid (“YTE”). See U.S. Patent No. 7,083,784, which is hereby incorporated by reference in its entirety. In certain extended half-life embodiments, the IgG constant domain contains 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 elimination half-life of the anti-IL-6 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-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, one specificity being for the IL-6R and one specificity being for human serum albumin (Ablynx, WO2006 / 122825 (bispecific nanobody)).

[0190] In some embodiments, the elimination half-life of the anti-IL-6 antibody is increased by PEGylation (Melmed et al., Nature Reviews Drug Discovery 7: 641-642 (2008)), by HPMA copolymer conjugation (Lu et al., Nature Biotechnology 17: 1101-1104 (1999)), by dextran conjugation (Nuclear Medicine Communications, 16: 362-369 (1995)), by conjugation with homoamino acid polymers (HAPs, Hapylation) (Schlapschy et al., Prot Eng Design Sel 20: 273-284 (2007)), or by 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 a specific embodiment, the antibody or antigen-binding portion thereof comprises the MEDI5117 heavy chain V region and the light chain V region. In a particular embodiment, the antibody is the 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 entirety. The MEDI5117 antibody has the following CDRs as well as 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 STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE 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, the MED5117 derivative comprises one or more amino acid substitutions in the MED5117 heavy chain and / or light chain V regions.

[0197] In certain embodiments, the derivative retains specificity for human IL-6 while comprising less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, less than 2 amino acid substitutions, or less than 1 amino acid substitution, as compared to the original V H and / or V L of the MEDI5117 anti-IL-6 antibody.

[0198] In certain embodiments, the 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 sequences of the VH and VL domains of MEDI5117. The percent sequence identity is determined using the BLAST algorithm with default parameters.

[0199] In certain embodiments, the MED5117 derivative comprises an amino acid sequence in which the 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 sequences of the respective CDRs of MEDI5117. The percent sequence identity is determined using the BLAST algorithm with default parameters.

[0200] In certain embodiments, V H and / or V L CDR derivatives contain conservative amino acid substitutions at one or more predicted non-essential amino acid residues (i.e., amino acid residues that are not important for the antibody to specifically bind to 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 derived from an antibody selected from the group consisting of siltuximab, golimumab, 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, golimumab, 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, golimumab, 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 comprises six CDRs derived from an antibody selected from those 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. Patent No. 5,856,135, US 2006 / 0240012, US 2006 / 0257407, or U.S. Patent No. 7,291,721, the disclosures of which are incorporated herein by reference in their entireties.

[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 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 species of full-length anti-IL-6 receptor antibodies, each of the multiple species having unique CDRs. 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 such as an scFv comprising a VHH single-domain nanobody derived from camel. In some embodiments, the antibody is bispecific or multispecific and at least one of the antigen-binding portions has specificity for the 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 and has non-human V region and human C region domains. In some embodiments, the antibody is of murine origin.

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

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

[0208] In exemplary embodiments, the anti-IL-6R antibody has an elimination half-life after intravenous administration for at least 7 days. In 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 the serum half-life compared to the unmodified human IgG constant domain.

[0210] ​In certain embodiments, the IgG constant domain comprises substitutions at residues 252, 254, and 256, where the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, the amino acid substitution at amino acid residue 254 is a substitution with threonine, and the amino acid substitution at amino acid residue 256 is a substitution with glutamic acid (“YTE”). See U.S. Patent No. 7,083,784, which is hereby incorporated 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 elimination half-life of the 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 for IL-6R and one specificity for human serum albumin (Ablynx, WO2006 / 122825 (bispecific nanobody)).

[0212] In some embodiments, the elimination half-life of the anti-IL-6R antibody is increased by PEGylation (Melmed et al., Nature Reviews Drug Discovery 7: 641-642 (2008)), by HPMA copolymer conjugation (Lu et al., Nature Biotechnology 17: 1101-1104 (1999)), by dextran conjugation (Nuclear Medicine Communications, 16: 362-369 (1995)), by conjugation with homoamino acid polymers (HAPs, Hapylation) (Schlapschy et al., Prot Eng Design Sel 20: 273-284 (2007)), or by 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 a specific embodiment, the antibody or antigen-binding portion thereof comprises the heavy chain V region and the light chain V region of tocilizumab. In a particular embodiment, the antibody is the 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 a specific embodiment, the antibody or antigen-binding portion thereof comprises the heavy chain V region and the light chain V region of sarilumab. In a particular embodiment, the antibody is the 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 the 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 particular embodiment, the antibody is bapineuzumab (ALX-0061) (Ablynx NV).

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

[0218] 5.7.4. 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, lestaurtinib, pacritinib, peficitinib, INCB-039110, ABT-494, INCB-047986, and AC-410.

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

[0222] 5.7.5. Further 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 S.A., Conaris Research Institute AG). In some embodiments, the anti-IL-6 antagonist includes soluble gp130, FE301 (Conaris / Ferring).

[0224] 5.8. Dosage regimens 5.8.1. Antibodies, antigen-binding fragments, peptides In exemplary embodiments, the antibody, antigen-binding fragment, and peptide IL-6 antagonist 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 by infusion. In certain intravenous embodiments, the IL-6 antagonist is administered as a bolus followed by 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 (flat dose) independent of the patient's weight or surface area.

[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 - 10 mg, 10 - 15 mg, 15 - 20 mg, 20 - 30 mg, 30 - 40 mg, or 40 - 50 mg. In some embodiments, the intravenous flat dose is 1 - 40 mg or 50 - 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 (bi-monthly), or once every three months.

[0233] In certain preferred embodiments, the IL-6 antagonist is the MEDI5117 antibody. In various embodiments, MEDI5117 is administered IV at a flat dose of 1-30 mg once a week. In certain embodiments, the MEDI5117 antibody is administered IV at a flat dose of 1, 2, 3, 4, 5, 7.5, 10, 15, 20, 25, or 30 mg once a week. In some embodiments, the MEDI5117 antibody is administered s.c. at a flat dose of 25-250 mg once a month to once every three months. In certain embodiments, MEDI5117 is administered s.c. 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 once a month, once every two months, or once every three months.

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

[0235] 5.8.2. JAK and STAT inhibitors In typical embodiments, the small molecule JAK inhibitors and STAT inhibitors are administered orally.

[0236] In various embodiments, the inhibitor is administered once or twice a day at an oral dose of 1-10 mg, 10-20 mg, 20-30 mg, 30-40 mg, or 40-50 mg. In some embodiments, the inhibitor is administered once or twice a day at a dose of 50-60 mg, 60-70 mg, 70-80 mg, 80-90 mg, or 90-100 mg. In some embodiments, the inhibitor is administered once or twice a day at a PO dose of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg. 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 method further comprises administration of a therapeutic agent in addition to the IL-6 antagonist, wherein the second therapeutic agent can also decrease 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 the 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 entirety.

[0242] In certain embodiments, the second therapeutic agent is a hemojuvelin antagonist. In certain embodiments, the hemojuvelin antagonist is an anti-hemojuvelin antibody. In certain embodiments, the anti-hemojuvelin antibody has the six CDRs of the antibodies 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 specific embodiments, the hepcidin antagonist is an anti-hepcidin antibody. In certain embodiments, the antibody has six CDRs derived from the antibodies described in US 2016 / 0017032, the disclosure of which is incorporated herein by reference in its entirety.

[0244] 5.10. Kits In another aspect, a kit is provided.

[0245] In an exemplary embodiment, the kit provides reagents for determining the genotype of a patient at the position of the TMPRSS6 SNP rs855791 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 an IL-6 antagonist, as well as compositions and methods for characterizing responsiveness to treatment of a patient, are provided.

[0247] These aspects and embodiments are based at least in part on the finding that inflammation in patients with chronic kidney disease and patients with cardiovascular disease having one or more alleles of TMPRSS6 that include a G or a C at nucleotide position 2321 (encoding a TMPRSS6 polypeptide that includes an alanine at amino acid position 736) makes these patients at higher risk of death and that such subjects could have been treated with an IL-6 antagonist to reduce this risk. As reported in more detail below, patients with chronic kidney disease were genotyped, serum levels of IL-6 and CRP were assayed, and these diagnostic data were compared with the administered EPO dose and risk of death. Patients having one or more alleles of TMPRSS6 that include a G or a C at nucleotide position 2321 (encoding a TMPRSS6 polypeptide that includes an alanine at amino acid position 736) and having elevated IL-6 and / or CRP levels required higher EPO doses for treatment and had a higher mortality rate. Nucleotides at this position have been shown to be important in the identification of patients with iron deficiency anemia (see Finberg, Nat. Genet. 2008, 40(5): 569-571, which is hereby incorporated by reference in its entirety, including all that it teaches, and 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 are at higher risk of death and who may respond to IL-6 inhibition regardless of the presence or absence of standard therapies for the treatment of anemia (e.g., associated with chronic kidney disease). By inhibiting inflammation, the EPO dose can be reduced, thereby avoiding harmful side effects of EPO (e.g., cardiovascular risk).

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

[0249] Accordingly, by inhibiting the biological activity of IL-6, for example, by determining the genotype of TMPRSS6 at SNP rs855791, in patients selected thereby, by blocking the binding of IL-6 or its receptor (gp80) to each other, or by blocking its signaling or expression (e.g., by an anti-IL-6 antibody or by an anti-IL-6R antibody or JAK1 / STAT3 inhibition), a method for treating inflammation associated with chronic kidney disease, including anemia of chronic kidney disease or cardiovascular disease, and / or for reducing the risk of death associated with such conditions is provided. In one embodiment, the treatment of chronic kidney disease is carried out regardless of the presence or absence of standard treatment for anemia, and the responsiveness of patients suffering from chronic kidney disease is characterized by treatment for anemia. For example, methods for determining the genotype of TMPRSS6 at SNP rs855791 and for detecting levels of inflammatory markers (e.g., elevation of IL-6 and / or CRP serum levels) are carried out.

[0250] A method is provided for treating anemia of cardiovascular disease or chronic kidney disease associated with chronic inflammation and / or reducing death in such patients by administering an agent that inhibits IL-6 biological activity or expression.

[0251] In some aspects and embodiments, compositions and methods are provided for treating chronic inflammation contributing to mortality in subjects having chronic kidney disease or cardiovascular disease and for characterizing patient responsiveness to such therapies. 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 erythropoiesis-stimulating agents). In one aspect, a method for treating chronic inflammation in a selected subject is provided, the method 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 that includes alanine at amino acid position 736.

[0252] In another aspect, a method for treating inflammation or chronic inflammation in a selected subject having cardiovascular disease or chronic kidney disease is provided, 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 that includes 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 for reducing inflammation and the risk of death in a selected subject having cardiovascular disease or kidney disease is provided, 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 that includes alanine at amino acid position 736 and having increased inflammation as compared to a reference. In one embodiment, the subject has a history of myocardial infarction or heart failure.

[0254] In another aspect, a method for reducing the risk of death in a subject having chronic kidney disease or heart failure is provided, the method comprising administering an IL-6 antagonist to the subject, where the subject is identified as having one or more alleles encoding a TMPRSS6 polypeptide comprising alanine at amino acid position 736 and has increased inflammation as 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 to the subject either alone or in combination with a therapy for anemia, where the subject is identified as having one or more alleles (e.g., having a G or C at nucleotide position 2321 of a TMPRSS6 nucleic acid molecule) encoding a TMPRSS6 polypeptide (also called matriptase-2, MT2) comprising alanine at amino acid position 736 and has increased inflammation as compared to a reference.

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

[0257] In yet another aspect, a method for enhancing responsiveness to EPO in a subject identified as in need thereof is provided, 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 (e.g., having a G or C at nucleotide position 2321 of a TMPRSS6 nucleic acid molecule) encoding a TMPRSS6 polypeptide comprising alanine at amino acid position 736, whereby the EPO dose is reduced.

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

[0259] In yet another aspect, a method for selecting a therapy for a subject identified as in need thereof is provided, the method comprising characterizing a subject having one or more alleles encoding a TMPRSS6 polypeptide comprising alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of a TMPRSS6 nucleic acid molecule), and detecting the 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 of anemia.

[0260] In yet another aspect, a method for increasing the proliferation or survival of erythrocytes or their progenitor cells (e.g., hematopoietic stem cells, proerythroblasts, erythroblasts or reticulocytes) in a subject identified as in need thereof is provided, 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 alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of a TMPRSS6 nucleic acid molecule) and having increased inflammation as compared to a reference.

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

[0263] In various embodiments, the increased inflammation is characterized by an increase in the levels of IL-6 and / or CRP as compared to a reference (e.g., as measured by conventional CRP assays or high-sensitivity assays (hsCRP) that both detect CRP but have different analytical performance). In various embodiments, the increased inflammation is characterized by IL-6 greater than about 5 pg / ml. In various embodiments, the increased inflammation is characterized by 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, the 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, the 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 depicted herein, administration of an IL-6 antagonist or an anti-IL-6 antibody results in a decrease in the dose of EPO. In certain embodiments, the dose of EPO is decreased 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, administration of an IL-6 antagonist or an anti-IL-6 antibody reduces the side effects of the increased EPO dose.

[0266] In one embodiment, patients with chronic kidney disease are treated regardless of the presence or absence of standard treatment for anemia. In particular, agents that inhibit IL-6 biological activity or expression are provided to subjects with anemia associated with chronic kidney disease regardless of the presence or absence of treatment for anemia (e.g., EPO, ESA, HIF stabilizers, adjunctive iron, or red blood cell transfusions). Treatment for anemia acts by stimulating erythropoiesis or red blood cell production. Thus, agents that increase the growth or proliferation of red blood cells or their progenitor cells and / or decrease the cell death of red blood cells or their progenitor cells. Red blood cell progenitor cells include, for example, hematopoietic stem cells, common myeloid progenitor cells, proerythroblasts, erythroblasts, reticulocytes, or any cell that can differentiate or mature into a red blood cell.

[0267] Agents that inhibit 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 in a pharmaceutical composition to a subject having anemia associated with chronic kidney disease, 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 IL-6 polypeptide or nucleic acid molecule in a subject, or inhibits intracellular signal transduction induced by activation of the IL-6 receptor. Anti-IL-6 antibodies (e.g., MEDI5117) can be administered in combination with treatment of anemia (e.g., administration of EPO, ESA, HIF stabilizer, supplemental iron). The method for treating anemia varies depending on the patient's TMPRSS6 genotype and the patient's inflammatory status. Patients who are homozygous or heterozygous for the major allele of TMPRSS6 containing G or C at nucleotide 2321 (encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736) and 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 of anemia (e.g., administration of EPO, ESA, HIF stabilizer, supplemental iron). Patients who are homozygous for the minor allele of TMPRSS6 containing A or T at nucleotide 2321 (encoding a TMPRSS6 polypeptide containing valine at amino acid position 736) do not require anti-IL-6 therapy to supplement the treatment of anemia. The method for treating anemia can vary depending on the stage of chronic kidney disease, the patient's age, health status, and physical condition.

[0268] In another aspect, an assay useful for characterizing a subject having 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 for the detection of IL-6 or CRP biomarkers and / or inflammatory conditions. In one embodiment, the inflammation is characterized by detecting the levels of IL-6 and / or CRP polypeptides in a biological sample (e.g., serum) of the subject as compared to their expression in a reference (e.g., serum from a healthy control subject), wherein an increase in IL-6 and / or CRP expression is an indicator of inflammation. In another embodiment, an increase in IL-6 and / or CRP expression indicates that a subject having anemia associated with chronic kidney disease does not respond to treatment of the anemia and / or is responsive to treatment of the anemia when administered in combination with an IL-6 antagonist (e.g., an anti-IL-6 antibody).

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

[0270] In various embodiments, traditional immunoassays are used, including, for example, Western blot, ELISA and other enzyme immunoassays such as sandwich immunoassays, fluorescence-based immunoassays, and chemiluminescence. Nephelometry is an assay performed in the liquid phase where antibodies are present in solution. The binding of antigen to the antibody results in a change in the measured absorbance. Other forms of immunoassays include magnetic immunoassays, radioimmunoassays, and real-time immuniquantitative 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 form that supports the binding of the antibody to the marker and subsequent detection. A single marker can be detected at a time, or a multiplex format can be used. Multiplex 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., anti-IL-6 antibody) in combination with treatment of the 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 a decrease in the expression of IL-6 and / or CRP and / or a decrease in inflammation are identified as responders to IL-6 inhibition.

[0273] Other aspects and embodiments are provided in the following numbered items. 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 comprising alanine at amino acid position 736. 2. A method for treating inflammation in a selected subject having 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 comprising alanine at amino acid position 736. 3. A method for reducing the risk of inflammation and death in a selected subject having 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 comprising alanine at amino acid position 736 and having increased inflammation compared to a reference. 4. A method for treating anemia in a subject having 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 alanine at amino acid position 736 and having 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 according to item 5, further comprising administering an erythropoietic factor to the subject. 8. The method according to any one of items 1 to 4, wherein the method reduces the risk of death of the subject. 9. A method for reducing the risk of death in a subject having 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 alanine at amino acid position 736 and having increased inflammation as compared to a reference.

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

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

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

[0277] 40. The method according to 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 according to item 23, wherein administering an IL-6 antagonist reduces the increased side effects of EPO. 42. The method according to any one of items 1 to 40, wherein the allele contains a G at position 2321 of the TMPRSS6 polynucleotide. 43. The IL-6 antagonist is 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) The method according to any one of items 1 to 42, which is an anti-IL-6 antibody having one or more CDRs selected from the following: 44. The method according to item 42, wherein the anti-IL-6 antibody has a heavy chain CDR1 containing the sequence SNYMI (SEQ ID NO: 12), a heavy chain CDR2 containing the sequence DLYYYAGDTYYADSVKG (SEQ ID NO: 13), a heavy chain CDR3 containing the sequence WADDHPPWIDL (SEQ ID NO: 14), a light chain CDR1 containing the sequence RASQGISSWLA (SEQ ID NO: 15), a light chain CDR2 containing the sequence KASTLES (SEQ ID NO: 16), and a light chain CDR3 containing the sequence QQSWLGGS (SEQ ID NO: 17). 45. The anti-IL-6 antibody has the following sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTISSNYMIWVRQAPGKGLEWVSDLYYYAGDTYY ADSVKGRFTMSRDISKNTVYLQMNSLRAEDTAVYYCARWADDHPPWIDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18) The method according to item 42, having a heavy chain comprising: 46. The anti-IL-6 antibody has the following sequence: DIQMTQSPSTLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKVLIYKASTLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQSWLGGSFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19) The method according to item 42, having a light chain comprising: 47. The method according to item 42, wherein the anti-IL-6 antibody is MEDI5117. 48. The method according to any one of items 1 to 47, wherein the subject is human.

[0278] 5.11.2. Methods for treating cardiorenal syndrome In other aspects and embodiments, compositions and methods for treating cardiorenal syndrome are provided thereof.

[0279] These aspects and embodiments are based at least in part on the finding that anti-IL-6 treatment of cardiac injury in a rodent model of cardiorenal syndrome has an effect equivalent to that of the standard of care. As reported in more detail below, a rodent model of cardiorenal syndrome was treated with anti-IL-6 or the standard of care (ACE inhibitor, perindopril) after myocardial infarction. After treatment, the ejection fraction, myocardial contractility, and the proportion of fibrous tissue in the heart tissue were measured. The levels of ejection fraction in both the group treated with anti-IL-6 and the group treated with the standard of care were increased compared to the levels in the group treated with control treatment. The cardiac contractility in both the group treated with anti-IL-6 and the group treated with the standard of care was increased compared to the levels in the group treated with control treatment. The amount of fibrous tissue in both the group treated with anti-IL-6 and the group treated with the standard of care was decreased compared to the amount in the group treated with control treatment. Furthermore, the levels of ejection fraction and the amount of fibrotic tissue were similar in the group treated with anti-IL-6 and the group treated with the standard of care. The results demonstrate that anti-IL-6 therapy has efficacy equivalent to that of the standard of care in the treatment of cardiorenal syndrome in a rodent model.

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

[0281] Accordingly, there is provided a method of treating heart and / or kidney injury in a subject having cardio-renal syndrome, which involves administering an IL-6 antagonist to the subject. In some embodiments, the treatment of heart and / or kidney injury in a subject having cardio-renal syndrome is carried out with or without using standard treatment for cardio-renal syndrome. There is provided a method for characterizing the risk of cardiovascular death in a patient after myocardial infarction, the method involving detecting an increase in IL-6 level in a biological sample obtained from the patient.

[0282] In one aspect, there is provided a method for treating heart / kidney injury in a subject having cardio-renal syndrome, the method involving administering an IL-6 antagonist to the subject.

[0283] In another aspect, there is provided a method for increasing cardiac function in a subject having cardio-renal syndrome, the method involving administering an IL-6 antagonist to the subject.

[0284] In yet another aspect, there is provided a method for reducing fibrosis in a subject having cardio-renal syndrome, the method involving administering an IL-6 antagonist to the subject.

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

[0286] In various embodiments of any of the aspects described herein, the increase in cardiac function is characterized by an increase in the ejection fraction and / or myocardial contractility of the subject as compared to a reference. In various embodiments of any of the aspects described herein, the reduction in fibrosis is characterized by a decrease in the proportion of fibrous tissue in a tissue sample derived from the subject as compared to a reference. In various embodiments, the fibrosis is in the heart tissue.

[0287] In various embodiments of any of the aspects described herein, the subject has heart and / or kidney damage. In various embodiments of any of the aspects described herein, the subject has kidney damage following heart damage.

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

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

[0290] In various embodiments of any of the aspects described herein, the subject has cardio-renal syndrome, heart failure, chronic kidney disease, or has no cardio-renal condition. In various embodiments of any of the aspects described herein, the subject is identified as having or not having cardio-renal syndrome, heart failure, chronic kidney disease about 1 month after myocardial infarction.

[0291] In another aspect, the invention provides a method for treating heart and / or kidney damage in a selected subject having cardio-renal syndrome, the method involving 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 as compared to a reference.

[0292] In yet another aspect, the present invention provides a method of reducing the risk of cardiovascular death (e.g., heart failure) in a selected subject having cardiorenal syndrome, the method involving administering an IL-6 antagonist to the subject, where the subject is selected by detecting an increase in one or more levels of an IL-6 polynucleotide or polypeptide in a biological sample derived from the subject as compared to a reference. In various embodiments of any of the aspects described herein, the subject had a myocardial infarction.

[0293] In various embodiments of any of the aspects described 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 described herein, the biological sample is a plasma sample or a serum sample. In various embodiments of any of the aspects described herein, the subject is human.

[0295] In another aspect, a method is provided for treating a patient's cardiorenal syndrome 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 having cardiorenal syndrome is treated with or without standard treatment for cardiorenal syndrome (e.g., an angiotensin-converting enzyme (ACE) inhibitor). In particular, an agent that inhibits IL-6 biological activity or expression is provided to a subject having cardiorenal syndrome (e.g., administration of an anti-IL-6 antibody).

[0296] In another aspect, a method of increasing cardiac function and a method of reducing fibrosis in a subject having cardiorenal syndrome are provided. The method includes administering to the subject an agent that inhibits IL-6 biological activity or expression. In some embodiments, the increase in cardiac function is an increase in the ejection fraction of the subject as compared to a reference (e.g., the ejection fraction of a healthy control subject), or an increase in myocardial contractility (e.g., dP / dt) as compared to a reference (e.g., the myocardial contractility of a healthy control subject).max ) is characterized by an increase. In some embodiments, the decrease in fibrosis is characterized by a decrease in the proportion of fibrotic tissue in a tissue sample from a subject as compared to a reference (e.g., a tissue sample obtained from a healthy control subject). In one embodiment, the fibrosis is in the heart tissue.

[0297] In a pharmaceutical composition, 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 its signal transduction or expression, can be provided to a subject having cardiorenal syndrome, wherein the pharmaceutical composition comprises an effective amount of the agent and a suitable excipient. In one embodiment, the agent is an IL-6 antagonist or an anti-IL-6 antibody that decreases the level or activity of IL-6 polypeptide or polynucleotide in the subject, or inhibits the intracellular signal transduction induced by IL-6 receptor activation. An anti-IL-6 antibody (e.g., MEDI5117) can be administered. The treatment method of cardiorenal syndrome can vary depending on the stage of cardiorenal syndrome, the age, health status, and physical condition of the patient.

[0298] In various embodiments, a subject having cardiorenal syndrome is treated with an IL-6 antagonist. Further, a subject having an increased risk of cardiovascular death and / or heart failure after myocardial infarction can be identified by characterizing the plasma level of IL-6 in the subject. A subject with elevated IL-6 levels has an increased risk of cardiovascular death and / or heart failure. Such a subject can be selected for treatment with an IL-6 antagonist. In addition, a subject having cardiorenal syndrome and having increased IL-6 levels, including a subject having suffered myocardial infarction, can be selected for treatment. When selected for treatment, such a subject can be substantially administered 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 to the intracellular signaling system related to IL-6R.

[0299] In another aspect, an assay for characterizing cardiovascular death, risk of heart failure, and / or death in a subject after myocardial infarction is provided. This assay features the 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 a biological sample (e.g., serum or plasma) of the subject as compared to the expression in a reference (e.g., serum or plasma from a healthy control subject or a control subject without cardiorenal pathology), where an increase in IL-6 indicates an increased risk of cardiovascular death or heart failure. A subject identified as having an increased risk of cardiovascular death, heart failure, or mortality can be selected for treatment. In another embodiment, a subject having cardiorenal syndrome and having increased IL-6 levels is selected for treatment with an IL-6 antagonist (e.g., an anti-IL-6 antibody).

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

[0301] In one embodiment, the IL-6 polypeptide level is measured. The level of the IL-6 polypeptide can be measured by standard methods such as immunoassay. Immunoassays typically utilize an antibody (or other agent that specifically binds to the marker) to detect the presence or level of a biomarker in a sample. Antibodies can be produced by methods well known in the art, for example, by immunizing an animal with a biomarker or a fragment thereof. Biomarkers can be isolated from a sample 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 an antibody by methods well known in the art.

[0302] In various embodiments, the assay uses traditional immunoassays, including, for example, Western blot, ELISA and other enzyme immunoassays, sandwich immunoassays, fluorescence-based immunoassays, and chemiluminescence. Nephelometry is an assay performed in the liquid phase where the antibody is present in solution. The binding of an antigen to the antibody results in a change in absorbance that is measured. Other forms of immunoassays include magnetic immunoassay, radioimmunoassay, and real-time immunometric PCR (iqPCR). Other detection methods include liquid chromatography and mass spectrometry.

[0303] Immunoassays can be performed on a solid substrate (e.g., a chip, beads, a microfluidic platform, a membrane), or in any other format that supports the binding of an antibody to a marker and subsequent detection. A single marker may be detected at a time, or a multiplex format may be used. Multiplex immunoassays can include planar microarrays (protein chips) and bead-based microarrays (suspension arrays).

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

[0305] Other aspects and embodiments are provided in the following numbered items. 1. A method for treating cardiac and / or renal injury in a subject having cardiorenal syndrome, the method comprising administering an IL-6 antagonist to the subject. 2. A method for increasing cardiac function in a subject having cardiorenal syndrome, the method comprising administering an IL-6 antagonist to the subject. 3. A method for reducing fibrosis in a subject having cardiorenal syndrome, the method comprising administering an IL-6 antagonist to the subject. 4. The method of item 2, wherein the increase in cardiac function is characterized by an increase in the ejection fraction of the subject compared to a reference. 5. The method of item 3, wherein the fibrosis is in the heart tissue. 6. The method of item 3 or 5, wherein the reduction in fibrosis is characterized by a decrease in the percentage of fibrotic tissue in a tissue sample from the subject compared to a reference. 7. The method according to any one of items 1-6, wherein the subject has cardiac and / or renal injury. 8. The method according to any one of items 1 to 7, wherein the subject has kidney injury following heart injury. 9. The method according to any one of items 1 to 8, further comprising administering a standard of care therapy to the subject. 10. The method according to 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 myocardial infarction, comprising measuring the level of one or more IL-6 polynucleotides or polypeptides in a sample derived from the subject, compared to a reference, wherein an increase in the level of one or more of the 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 myocardial infarction, comprising measuring the level of one or more IL-6 polynucleotides or polypeptides in a sample derived from the subject, compared to a reference, wherein an increase in the level of one or more of the IL-6 polynucleotides or polypeptides indicates an increased risk of cardiovascular death. 13. The method according to item 11 or 12, wherein the subject has cardiorenal syndrome, heart failure, chronic kidney disease, or has no cardiorenal condition. 14. The method according to any one of items 11 to 13, wherein the subject is identified as having or not having cardiorenal syndrome, heart failure, chronic kidney disease about 1 month after myocardial infarction. 15. A method for treating heart and / or kidney injury in a selected subject having 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 derived from the subject, compared to a reference. 16. A method for reducing the risk of cardiovascular death in a selected subject having cardiorenal syndrome, comprising administering an IL-6 antagonist to the subject, wherein the subject is selected by detecting an increase in one or more levels of IL-6 polynucleotide or polypeptide in a biological sample derived from the subject as compared to a reference. 17. The method according to item 15 or 16, wherein the subject had myocardial infarction. 18. The method according to 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 according to 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 human.

[0307] [Examples] 5.12. Examples The following examples are provided for illustrative purposes and are not limiting.

[0308] [Example 1] 5.12.1. In patients having at least one copy of the TMPRSS6 SNP rs855791 major allele, the EPO dosage and overall survival rate in patients with chronic kidney disease are correlated with serum IL-6 and CRP levels Hepcidin, a peptide hormone, plays a central role in systemic iron homeostasis. Hentze et al., Cell 142:24-38 (2010). Hepcidin expression is known to be affected by the product of the TMPRSS6 gene, matriptase-2, a type II transmembrane serine protease. Common variants of 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 natural variation in hepcidin expression and blood hemoglobin levels.

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

[0310] Methods Data from N = 257 patients recruited between October 2003 and September 2004 in six dialysis units in the Stockholm-Uppsala (Sweden) region and registered in the MIMICK1, MIMICK2 (mapping of inflammatory markers in chronic kidney disease), and MIA (malnutrition, inflammation, and atherosclerosis) cohorts were aggregated to N = 208 based on common dialysis criteria of ferritin > 100 ng / ml and Hb > 10 mg / dL to select patients who were not iron deficient, not severely anemic, and stable on hemodialysis, thereby excluding patients with factors that could dissociate iron treatment from hemoglobin levels.

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

[0312]

Table 1

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

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

[0315] Without wishing to be bound by a particular theory, homozygosity for the minor allele removed the influence of IL-6 on iron handling. Thus, the EPO dose in these patients (A / A) was nearly the same regardless of IL-6 levels.

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

[0317] Also, the level of CRP (a marker of inflammation), an acute-phase reactant, also correlated with an increase in EPO dose in subjects who were heterozygous or homozygous for the major allele (G), but not in patients who were homozygous for the minor allele (Figure 3).

[0318] Discussion As shown in Figure 1, the degree of underlying anemia measured as clinically titrated EPO dose correlated with IL-6 level only in patients who had 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 who had two copies of the minor allele did not correlate with serum IL-6 level (Figure 1A).

[0319] Similarly, overall survival correlated with IL-6 level only in patients who had at least one copy of the major allele of the TMPRSS6 SNP rs855791. In subjects who had at least one copy of the TMPRSS6 rs855791 major allele, survival was inversely correlated with serum IL-6 level, and patients in the highest tertile of serum IL-6 level showed statistically significantly inferior survival compared to those in the lowest tertile of IL-6 level (Figure 2B). In contrast, the overall survival of patients who were homozygous for the minor allele at rs855791 was not affected by IL-6 level (Figure 2A).

[0320] Although not intended to be bound by theory, in patients having at least one copy of the TMPRSS6 major allele, an increase in serum IL-6 may cause an increase in hepcidin expression, thereby potentially increasing anemia. The increased risk of death is a 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, a decrease in IL-6 levels or IL-6 signaling would decrease anemia, decrease the required EPO dose, increase survival rate, and potentially be most effective in those patients with elevated serum levels of IL-6, but only in those patients with chronic kidney disease who have at least one copy of the TMPRSS6 rs855791 major allele.

[0321] [Example 2] 5.12.2. The risks of death and heart failure after acute myocardial infarction correlate with IL-6 serum levels only in patients having at least one copy of the TMPRSS6 SNP rs855791 major allele To determine whether the TMPRSS6 rs855791 genotype affects IL-6 sensitivity in patients with acute rather than chronic disease, data previously collected in a clinical trial of patients admitted for acute coronary syndrome were analyzed together with newly determined SNP genotyping.

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

[0323] Results The mortality rate of subjects homozygous for the minor allele (A) of the TMPRSS6 rs855791 SNP was not correlated with the variation in IL-6 (Figure 4A). However, one or two copies of the major allele (G) gave a higher all-cause mortality rate in response to the increase in IL-6 levels in subjects after myocardial infarction (Figure 4B). Therefore, TMPRSS6 regulated the IL-6-mediated death risk after myocardial infarction.

[0324] The effect of the TMPRSS6 genotype on the IL-6-mediated heart failure risk was also measured starting from the 30th day after myocardial infarction in the subjects enrolled in PLATO. Heart failure in subjects homozygous for the minor allele (A) was not correlated with the variation in IL-6 (Figure 5A). However, the G allele of TMPRSS6 gave a higher heart failure rate in response to the increase in IL-6 levels in subjects after myocardial infarction (Figure 5B). Therefore, TMPRSS6 regulated the IL-6-mediated risk of heart failure after myocardial infarction.

[0325] Discussion These data demonstrate that the correlation between the TMPRSS6 genotype, IL-6 levels, and adverse clinical outcomes is not limited to patients with chronic kidney disease. Without being bound by theory, in patients having at least one copy of the TMPRSS6 major allele, an increase in serum IL-6 causes an increase in hepcidin expression, which results in an increase in iron sequestration in cardiomyocytes, subsequently leading to iron-mediated cytotoxicity. If these correlations reflect a causal relationship, a decrease in IL-6 levels or IL-6 signaling would reduce heart failure and mortality in acute coronary syndrome patients, but only in those patients having at least one copy of the TMPRSS6 rs855791 major allele, creating the possibility that it would be most effective in those patients with elevated serum levels of IL-6.

[0326] [Example 3] 5.12.3. In vitro studies of iPS-derived human cardiomyocytes confirm the causal relationship between TMPRSS6 genotype and IL-6-mediated cytotoxicity The correlations observed in Examples 1 and 2 suggest that a decrease in IL-6-mediated signaling should confer a clinical benefit in patients with at least one copy of the TMPRSS6 rs855791 major allele, an increase in IL-6 levels, and either anemia or hepcidin-mediated cytotoxicity, although the observed correlations are insufficient to prove causality. Therefore, to examine the effects of BMP and BMP+IL-6 on hepcidin expression and cellular sensitivity to ischemic injury, experiments were conducted in human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) transfected with variants of TMPRSS6.

[0327] 5.12.3.1. Methods Culture 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.). Forty-eight 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 protocol - iPS cardiomyocytes were prepared as previously reported (Das, A., Xi, L., and Kukreja, K. C. (2005) J. Biol. Chem. 280: 12944-12955; Das A, Smolenski A, Lohmann SM, Kukreja RC. (2006) J. Biol Chem. 281(50):38644-52), and the cell medium was 3 、1.0 mM NaH 2 PO 4 、2.5 mm CaCl 2 -2H 2 O、1.2 mM MgCl 2It was subjected to simulated ischemia (SI) for 90 minutes by replacing it with "ischemic buffer" containing 20 mM sodium lactate, 16 mM KCl, and 10 mM 2-deoxyglucose (pH adjusted to 6.2). The cells were incubated at 37 °C in a tri-gas incubator adjusted to 1~2% O 2 and 5% CO 2 throughout the SI period. Reoxygenation (RO) was achieved by replacing the ischemic buffer with normal cell culture medium under normoxic conditions. Cell death after 2 or 18 hours of reoxygenation, respectively. iCell was subjected to 4 hours of SI and 24 hours of RO as described above.

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

[0330] Transfection of iCell cardiomyocytes - On the 8th day 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). Forty-eight hours after transfection, the cells were used for further experiments.

[0331] Western blot analysis- Western blot was performed as previously reported (Das, A., Xi, L., and Kukreja, K. C. (2005) J. Biol. Chem. 280, 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 with 5% non-fat dry milk in TBST (10 mM Tris-HCl, pH 7.4, 100 mM NaCl, 0.1% Tween 20) for 1 hour. Next, the membrane was incubated overnight with rabbit monoclonal / polyclonal or goat polyclonal primary antibodies diluted 1:1000 for each protein. The antibodies were, namely, 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 (ferroportin) (Abcam Company, MA) and goat polyclonal actin-HRP (Santa Cruz Biotechnology, TX). Next, the membrane was 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, the bands were scanned, and quantified by densitometry analysis.

[0332] Real-time PCR-Taqman assay- Total RNA containing 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 using random hexamers with a high-capacity cDNA synthesis kit (Applied Biosystems, CA, USA). The reverse transcription reaction was carried out using the following PCR conditions: 10 min at 25 °C, 120 min at 37 °C, and 5 min at 85 °C. Real-time PCR was performed using the following PCR cycle conditions: 10 min at 95 °C, 15 s at 95 °C, and 60 s at 60 °C, using the Taqman amplicon-specific probe (Applied Biosystems, CA, USA) Hamp (CGGCTCTGCAGCCTTG) (SEQ ID NO: 20). The expression of Hamp 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), and catalog number SC306623 corresponded to GenBank accession number NM_153609. This clone contained the mutation K253A, which resulted in an amino acid change. Site-directed mutagenesis was performed to revert the amino acid at position 253 to the reference lysine (K). Once the 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 for confirmation. 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 the constitutive expression of matriptase-2 736A, encoded by the TMPRSS6 rs855791 SNP major allele, or matriptase-2 736V, encoded by its minor allele, mimicking 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 an increase in hepcidin expression. Casanovas et al., PLOS Comp. Biol. 10(1):e1003421 (2014). iPS cardiomyocytes with the 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 any agonist. Cell death rate was measured under normoxic conditions (normoxia), and after simulating hypoxia, under conditions of reoxygenation (reperfusion).

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

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

[0338] 5.12.3.3. Discussion These data reinforce the inference drawn from the post hoc analysis of the clinical trial data of Examples 1 and 2: a decrease in IL-6 signaling may be 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 being bound by theory, the increased toxicity induced by IL-6 in iPS cardiomyocytes with the major allele may result from an increase in IL-6-mediated hepcidin expression, which in turn leads to increased sequestration of iron within the cell and subsequently 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 homozygotes for the human TMPRSS6 rs855791 major allele Patients with chronic kidney disease, such as those enrolled in the MIMICK study analyzed in Example 1, often develop impairment of cardiac function, a major cause of mortality. This secondary cardiac injury following primary chronic kidney disease is called cardiorenal syndrome type 4 (CRS type 4).

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

[0341] The general outline of the test design is shown in FIG. 7.

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

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

[0344] On the third and seventh days after nephrectomy, assessments of the subjects in all groups were performed. The assessments included measurement of serum creatinine and plasma biomarkers on the third day, and serum creatinine, glomerular filtration rate, 24-hour protein levels, echocardiogram, blood pressure, and measurement of plasma biomarkers on the seventh day.

[0345] The subject was sacrificed at week 6. Prior to sacrifice, various assessments of the subject in all groups were performed. The assessments included measurements of serum creatinine, glomerular filtration rate, 24-hour protein level, blood pressure, plasma biomarkers, echocardiogram, and pressure-volume loop analysis. After sacrifice, tissues were harvested from the subjects in all groups for histological evaluation (i.e., Sirius red staining of heart tissue).

[0346] Figures 8A - 8D show the cardiac ejection fractions of rats without CRS ("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 the standard of care ACE inhibitor in the cardio-renal syndrome model summarized in Figure 7 ("Peri").

[0347] Figure 8A shows the baseline ejection fraction levels of all groups 2 weeks after myocardial infarction, but before nephrectomy and before 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 1 week after nephrectomy and 1 week after treatment. Figure 8C is a plot showing the ejection fraction levels of all groups 2 weeks after nephrectomy and 2 weeks after treatment. Figure 8D is a plot showing the ejection fraction levels of all groups 4 weeks after nephrectomy and 4 weeks of treatment. Results are expressed as mean + / - SEM.

[0348] Four weeks after treatment, both treatment groups, i.e., the group treated with anti-IL-6 and the group treated with the standard of care 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 4 weeks after treatment indicate that anti-IL-6 therapy has equivalent efficacy to the ACE inhibitor perindopril (standard of care therapy), demonstrating that anti-IL-6 therapy has therapeutic efficacy in the preservation of cardiac function in the cardio-renal syndrome model equivalent to the standard of care therapy as measured by changes in cardiac ejection fraction.

[0349] Measurement of cardiac contractility (Figure 9) showed that anti-IL-6 therapy also had an effect equivalent to the standard of care with an ACE inhibitor. Four weeks after treatment, cardiac contractility in the groups treated with anti-IL-6 and the standard of care was significantly increased compared to that in the control isotype group. Similar cardiac contractility in the anti-IL-6 and standard of care groups demonstrated that anti-IL-6 therapy is effective in preserving cardiac function in a cardio-renal syndrome model equivalent to the ACE inhibitor perindopril (standard of care) as measured by contractility.

[0350] Measurement of fibrosis in cardiac tissue harvested from animals in all groups also demonstrated that anti-IL-6 therapy had an effect equivalent to the standard of care (Figures 10A - 10C). Fibrosis in cardiac tissue was quantified by measuring the proportion of the area of fibrous tissue in two regions: the "normal" region and the "perifibrotic" region. An example of the "normal" region is shown by the depicted portion of the tissue section in the micrograph of Figure 10A. The inset in the micrograph shows a magnified view of the "normal" region and indicates that a small portion of the "normal" region has fibrotic tissue. The "perifibrotic" region is the area of tissue in the "normal" region surrounding the fibrous tissue.

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

[0352] These data demonstrate that treatment with an anti-IL-6 agent is effective in reducing heart injury and recovery function in an in vivo model of cardio-renal syndrome in animals that are genotypically similar to humans that 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 homozygotes for the human TMPRSS6 rs855791 major allele The data of Examples 2 and 3 suggest that reducing IL-6 levels or IL-6 signaling can reduce heart failure and mortality only in those patients who are patients with acute coronary syndrome but 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] A rodent study was conducted to determine the effect of anti-IL-6 therapy after acute myocardial infarction in mice that are 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 that are genotypically similar to humans that are homozygous for the TMPRSS6 rs855791 major allele. The control group received no treatment. The experimental group was treated with anti-mouse IL-6 antibody. Figure 11A shows that treatment with anti-IL-6 results in a statistically significant improvement in the ejection rate. Figure 11B shows that treatment with anti-IL-6 results in a statistically significant improvement in contractility, measured as a shortening of the cardiac fractional. The data demonstrate that anti-IL-6 therapy given immediately after myocardial infarction improves left ventricular functional recovery in rodents that are genotypically similar to human patients with the TMPRSS6 rs855791 major allele.

[0356] The present invention includes, for example, the following embodiments: [Embodiment 1] A method for treating hepcidin-mediated disorders, comprising administering to a patient having a hepcidin-mediated disorder a therapeutically effective amount of an IL-6 antagonist, wherein the patient has been determined to have at least one copy of the TMPRSS6 rs855791 major allele. A method as described above. [Embodiment 2] The method according to 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 according to Embodiment 1, further comprising an initial step of determining that the patient has at least one copy of the TMPRSS6 rs855791 major allele. [Embodiment 4] The method according to any one of Embodiments 1 to 3, wherein the patient has elevated pre-treatment serum levels of IL-6. [Embodiment 5] The method according to any one of Embodiments 1 to 4, wherein the patient has elevated pre-treatment serum levels of CRP. [Embodiment 6] The method according to any one of Embodiments 1 to 5, wherein the hepcidin-mediated disorder is anemia of chronic disease. [Embodiment 7] The method according to 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 according to Embodiment 7, wherein the patient has a pre-treatment Hb level of less than 13 g / dl. [Embodiment 9] The method according to Embodiment 8, wherein the patient has a pre-treatment Hb level of less than 12 g / dl. [Embodiment 10] The method according to Embodiment 9, wherein the patient has a pre-treatment Hb level of less than 11 g / dl. [Embodiment 11] The method according to Embodiment 6, wherein the patient is female and has a pre-treatment Hb level of less than 12 g / dl. [Embodiment 12] The method according to Embodiment 11, wherein the patient has a pre-treatment Hb level of less than 11 g / dl. [Embodiment 13] The method according to Embodiment 12, wherein the patient has a pre-treatment Hb level of less than 10 g / dl. [Embodiment 14] The method according to Embodiment 13, wherein the patient has a pre-treatment Hb level of less than 9 g / dl. [Embodiment 15] The method according to 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 according to Embodiment 15, wherein the patient has a pre-treatment hematocrit of less than 35%. [Embodiment 17] The method according to Embodiment 16, wherein the patient has a pre-treatment hematocrit of 30 to 34%. [Embodiment 18] The method according to 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 according to Embodiment 18, wherein the patient has a pre-treatment hematocrit of less than 30%. [Embodiment 20] The method according to Embodiment 19, wherein the patient has a pre-treatment hematocrit of 26 to 29%. [Embodiment 21] The method according to any one of Embodiments 6 to 20, wherein the patient has received at least one pre-treatment administration of an ESA. [Embodiment 22] The method according to Embodiment 6, wherein the patient has received at least one pre-treatment administration of an ESA and has a normal Hb level or a normal hematocrit. [Embodiment 23] The method according to any one of Embodiments 6 to 20, wherein the patient has received at least one pre-treatment administration of iron supplementation. [Embodiment 24] The method according to Embodiment 6, wherein the patient has received at least one pre-treatment administration of iron supplementation and has a normal Hb level or a normal hematocrit. [Embodiment 25] The method according to 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 according to 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 a normal hematocrit. [Embodiment 27] The IL-6 antagonist is administered in a dose, schedule, and duration sufficient to increase the patient's Hb level above the pre-treatment level, according to any one of Embodiments 6 to 26. The method described anywhere. [Embodiment 28] The IL-6 antagonist is administered in a dose, schedule, and period sufficient to increase the patient's hematocrit above the pre-treatment level, according to the method described in any of Embodiments 6 to 27. [Embodiment 29] The IL-6 antagonist is administered in a dose, schedule, and period 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, according to the method described in Embodiment 21 or 22. [Embodiment 30] The IL-6 antagonist is administered in a dose, schedule, and period 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, according to the method described in Embodiment 21 or 22. [Embodiment 31] The IL-6 antagonist is administered in a dose, schedule, and period sufficient to allow at least a 10% reduction in the patient's ESA dose compared to the pre-treatment ESA dose, according to the method described in any of Embodiments 21, 22, 29, or 30. [Embodiment 32] The IL-6 antagonist is administered in a dose, schedule, and period sufficient to allow at least a 20% reduction in the patient's ESA dose compared to the pre-treatment ESA dose, according to the method described in Embodiment 31. [Embodiment 33] The IL-6 antagonist is administered in a dose, schedule, and period sufficient to allow at least a 50% reduction in the patient's ESA dose compared to the pre-treatment ESA dose, according to the method described in Embodiment 32. [Embodiment 34] The IL-6 antagonist is administered in a dose, schedule, and period sufficient to restore functional iron deficiency, according to the method described in any of Embodiments 6 to 33. [Embodiment 35] The chronic disease is chronic kidney disease (CKD), according to the method described in any of Embodiments 6 to 34. [Embodiment 36] The method according to 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 according to Embodiment 36, wherein the patient has KDOQI stage 5 chronic kidney disease. [Embodiment 38] The method according to Embodiment 35, wherein the patient has cardiorenal syndrome (CRS). [Embodiment 39] The method according to Embodiment 38, wherein the patient has CRS type 4. [Embodiment 40] The method according to any one of Embodiments 35 to 39, wherein the patient has received at least one pre-treatment dialysis treatment. [Embodiment 41] The IL-6 antagonist is administered in a dose, schedule, and duration sufficient to reduce cardiovascular (CV) mortality compared to age- and disease-matched historical controls, according to any one of Embodiments 35 to 40. [Embodiment 42] The chronic disease is a chronic inflammatory disease, according to any one of Embodiments 6 to 34. [Embodiment 43] The chronic inflammatory disease is rheumatoid arthritis (RA), according to Embodiment 42. [Embodiment 44] The method according to Embodiment 43, wherein the patient has a pre-treatment DAS28 score greater than 5.1. [Embodiment 45] The method according to Embodiment 43, wherein the patient has a pre-treatment DAS28 score of 3.2 to 5.1. [Embodiment 46] The method according to Embodiment 43, wherein the patient has a pre-treatment DAS28 score less than 2.6. [Embodiment 47] The pre-treatment RA of the patient is moderately active to severely active, according to Embodiment 43. [Embodiment 48] The method according to any one of Embodiments 43 to 47, wherein the patient has received at least one pre-treatment administration of methotrexate. [Embodiment 49] The method according to any one of Embodiments 43 to 48, wherein the patient has received at least one pre-treatment administration of a TNFα antagonist. [Embodiment 50] The method according to Embodiment 49, wherein the TNFα antagonist is selected from the group consisting of etanercept, adalimumab, infliximab, certolizumab, and golimumab. [Embodiment 51] The method according to any one of Embodiments 43 to 47, wherein the patient has received at least one pretreatment dose of an IL-6 antagonist. [Embodiment 52] The method according to Embodiment 51, wherein the pretreatment IL-6 antagonist is tocilizumab. [Embodiment 53] The method according to Embodiment 51, wherein the pretreatment IL-6 antagonist is tofacitinib. [Embodiment 54] The method according to any one of Embodiments 51 to 53, wherein the treatment IL-6 antagonist is MEDI5117. [Embodiment 55] The method according to 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 according to any one of Embodiments 6 to 34, wherein the chronic disease is cancer. [Embodiment 57] The method according to 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 according to any one of Embodiments 6 to 34, wherein the chronic disease is a chronic infectious disease. [Embodiment 59] The method according to any one of Embodiments 6 to 34, wherein the chronic disease is congestive heart failure (CHF). [Embodiment 60] The method according to any one of Embodiments 1 to 5, wherein the hepcidin-mediated disorder is iron-refractory iron deficiency anemia (IRIDA). [Embodiment 61] The method according to any one of Embodiments 1 to 5, wherein the hepcidin-mediated disorder is acute coronary syndrome. [Embodiment 62] The method according to 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 according to embodiment 62, wherein the patient has suffered from MI within 30 days prior to the first administration of the IL-6 antagonist. [Embodiment 64] The method according to embodiment 63, wherein the patient has suffered from MI within 48 hours prior to the first administration of the IL-6 antagonist. [Embodiment 65] The method according to embodiment 64, wherein the patient has suffered from MI within 24 hours prior to the first administration of the IL-6 antagonist. [Embodiment 66] The method according to any one of embodiments 61 - 65, wherein the IL-6 antagonist is administered in a dose, schedule, and duration sufficient to improve myocardial contractility as compared to the pre-treatment level. [Embodiment 67] The method according to any one of embodiments 61 - 66, wherein the IL-6 antagonist is administered in a dose, schedule, and duration sufficient to improve cardiac ejection fraction as compared to the pre-treatment level. [Embodiment 68] The method according to any one of embodiments 61 - 67, wherein the IL-6 antagonist is administered in a dose, schedule, and duration sufficient to reduce cardiac fibrosis as compared to the pre-treatment level. [Embodiment 69] The method according to any one of embodiments 1 - 5, wherein the hepcidin-mediated disorder is Castleman's disease. [Embodiment 70] A method for improving the treatment of a hepcidin-mediated disorder, comprising discontinuing the administration of an IL-6 antagonist to a patient having a hepcidin-mediated disorder, wherein the patient has been determined to be homozygous for the TMPRSS6 rs855791 minor allele. [Embodiment 71] The method according to embodiment 70, wherein the patient has previously been determined to be homozygous for the TMPRSS6 rs855791 minor allele. [Embodiment 72] The method according to embodiment 70, further comprising an 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 chronic inflammatory anemia, comprising administering to a patient having an IL-6-mediated inflammatory disease without anemia a therapeutically effective amount of an IL-6 antagonist, wherein the patient has been determined to have at least one copy of the TMPRSS6 rs855791 major allele. [Embodiment 74] The method according to 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 according to Embodiment 73, further comprising an initial step of determining that the patient has at least one copy of the TMPRSS6 rs855791 major allele. [Embodiment 76] The method according to any one of Embodiments 1-75, wherein the patient has elevated pretreatment serum levels of IL-6. [Embodiment 77] The method according to Embodiment 76, wherein the patient has pretreatment serum IL-6 levels greater than 2.5 pg / ml. [Embodiment 78] The method according to Embodiment 77, wherein the patient has pretreatment serum IL-6 levels greater than 5 pg / ml. [Embodiment 79] The method according to Embodiment 78, wherein the patient has pretreatment serum IL-6 levels greater than 7.5 pg / ml. [Embodiment 80] The method according to Embodiment 79, wherein the patient has pretreatment serum IL-6 levels greater than 10 pg / ml. [Embodiment 81] The method according to Embodiment 80, wherein the patient has pretreatment serum IL-6 levels greater than 12.5 pg / ml. [Embodiment 82] The IL-6 antagonist is administered in a dose, schedule, and duration sufficient to reduce the free IL-6 level in the patient's serum below the pretreatment level. The method according to any one of Embodiments 76-81. [Embodiment 83] The method according to embodiment 82, wherein the IL-6 antagonist is administered in a dosage, schedule, and duration sufficient to reduce the free IL-6 level by at least 10% compared to the pre-treatment level. [Embodiment 84] The method according to embodiment 83, wherein the IL-6 antagonist is administered in a dosage, schedule, and duration sufficient to reduce the free IL-6 level in the patient's serum by at least 20% compared to the pre-treatment level. [Embodiment 85] The method according to embodiment 84, wherein the IL-6 antagonist is administered in a dosage, schedule, and duration sufficient to reduce the free IL-6 level in the patient's serum by at least 50% compared to the pre-treatment level. [Embodiment 86] The method according to any one of embodiments 1 to 85, wherein the patient has an elevated pre-treatment level of C-reactive protein (CRP). [Embodiment 87] The method according to embodiment 86, wherein the patient has a pre-treatment CRP level exceeding 2 mg / ml. [Embodiment 88] The method according to embodiment 87, wherein the patient has a pre-treatment CRP level exceeding 3 mg / ml. [Embodiment 89] The method according to embodiment 88, wherein the patient has a pre-treatment CRP level exceeding 5 mg / ml. [Embodiment 90] The method according to embodiment 89, wherein the patient has a pre-treatment CRP level exceeding 7.5 mg / ml. [Embodiment 91] The method according to embodiment 90, wherein the patient has a pre-treatment CRP level exceeding 10 mg / ml. [Embodiment 92] The method according to any one of embodiments 86 to 91, wherein the IL-6 antagonist is administered in a dosage, schedule, and duration sufficient to reduce the patient's CRP level below the pre-treatment level. [Embodiment 93] The method according to embodiment 92, wherein the IL-6 antagonist is administered in a dosage, schedule, and duration sufficient to reduce the patient's CRP level by at least 50% compared to the pre-treatment level. [Embodiment 94] The method according to 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 (registered trademark) real-time PCR assay. [Embodiment 95] The method according to 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 method according to Embodiment 95, wherein the anti-IL-6 antibody or an antigen-binding fragment or derivative thereof has a K D of less than 100 nM for binding to human IL-6. [Embodiment 97] The method according to Embodiment 96, wherein the antibody or antigen-binding fragment or derivative thereof has a K D of less than 50 nM for binding to human IL-6. [Embodiment 98] The method according to Embodiment 97, wherein the antibody or antigen-binding fragment or derivative thereof has a K D of less than 10 nM for binding to human IL-6. [Embodiment 99] The method according to Embodiment 98, wherein the antibody or antigen-binding fragment or derivative thereof has a K D of less than 1 nM for binding to human IL-6. [Embodiment 100] The method according to any one of Embodiments 95 to 99, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative thereof has an excretion half-life after intravenous administration for at least 7 days. [Embodiment 101] The method according to Embodiment 100, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative thereof has an excretion half-life after intravenous administration for at least 14 days. [Embodiment 102] The method according to Embodiment 101, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative thereof has an excretion half-life after intravenous administration for at least 21 days. [Embodiment 103] The method according to Embodiment 102, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative thereof has an excretion half-life after intravenous administration for at least 30 days. [Embodiment 104] The method according to 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 according to Embodiment 104, wherein the antibody is an IgG1 or IgG4 antibody. [Embodiment 106] The method according to Embodiment 105, wherein the antibody is an IgG1 antibody. [Embodiment 107] The method according to 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 according to 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 according to 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 according to Embodiment 109, wherein the antibody comprises the VH and VL of MED5117. [Embodiment 111] The method according to 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, golimumab, 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). The method according to any one of Embodiments 9 5 to 108. [Embodiment 113] The anti-IL-6 antibody or antigen-binding fragment or derivative is the method according to Embodiment 112, comprising the heavy chain V region and the light chain V region derived from an antibody selected from the group consisting of siltuximab, golimumab, 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, golimumab, 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 according to Embodiment 113, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is an antibody selected from the group consisting of siltuximab, golimumab, 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, golimumab, 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 according to any one of Embodiments 95 to 103, wherein the IL-6 antagonist is a single-domain antibody, VHH nanobody, Fab, or scFv. [Embodiment 116] The method according to 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 according to Embodiment 116, wherein the anti-IL-6R antibody, antigen-binding fragment or derivative is tocilizumab. [Embodiment 118] The method according to Embodiment 116, wherein the anti-IL-6R antibody, antigen-binding fragment or derivative is baricitinib. [Embodiment 119] The method according to any one of Embodiments 1 to 94, wherein the IL-6 antagonist is a JAK inhibitor. [Embodiment 120] The JAK inhibitor is the method according to Embodiment 119, 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 IL-6 antagonist is the method according to any one of Embodiments 1 to 94, which is a STAT3 inhibitor. [Embodiment 122] The IL-6 antagonist is the method according to any one of Embodiments 95 to 118, which is administered parenterally. [Embodiment 123] The IL-6 antagonist is the method according to Embodiment 122, which is administered subcutaneously. [Embodiment 124] The method according to Embodiment 119 or 120, wherein the IL-6 antagonist is administered orally. 6. Incorporation by reference All publications, patents, patent applications, and other writings cited in this application are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other writing were individually indicated to be incorporated by reference for all purposes.

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

Claims

1. A composition comprising an anti-IL-6 antibody for use in a method of treating cardiovascular disease in a subject, wherein the method comprises administering to the subject a therapeutically effective amount of the anti-IL-6 antibody, the subject has at least one copy of the TMPRSS6 rs855791 major allele, and the anti-IL-6 antibody comprises all six variable region CDRs of MEDI5117.

2. The composition according to claim 1, wherein the cardiovascular disease is atherosclerosis.

3. The composition according to claim 1, wherein the subject has a history of myocardial infarction or heart failure.

4. The composition according to claim 1, wherein the subject has a pre-treatment CRP level greater than 2 mg / L.

5. The composition according to claim 4, wherein the anti-IL-6 antibody is administered in a dose, schedule, and duration sufficient to reduce the subject's CRP level below the pre-treatment level.

6. The composition according to claim 5, wherein the anti-IL-6 antibody is administered in a dose, schedule, and duration sufficient to reduce the subject's CRP level by at least 50% compared to the pre-treatment level.

7. The composition according to claim 1, wherein the subject has a pre-treatment serum IL-6 level greater than 2.5 pg / ml.

8. The composition according to claim 1 for use in a method of treating heart failure after acute myocardial infarction in a subject.

9. The composition according to claim 8, wherein the subject has a history of myocardial infarction or heart failure.

10. The composition according to claim 8, wherein the subject has had a myocardial infarction within 60 days prior to the first administration of the anti-IL-6 antibody.

11. The composition according to claim 10, wherein the subject has had a myocardial infarction within 30 days prior to the first administration of the anti-IL-6 antibody, within 48 hours prior to the first administration of the anti-IL-6 antibody, or within 24 hours prior to the first administration of the anti-IL-6 antibody.

12. The composition according to claim 8, which, upon use, reduces the risk of heart failure in the subject.

13. The composition according to claim 1, wherein the anti-IL-6 antibody comprises the VH and VL of MEDI5117.

14. The composition according to claim 13, wherein the anti-IL-6 antibody is MEDI5117.

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