EA041768B1 - METHODS FOR THE TREATMENT OF HEPSIDIN-MEDIATED DISORDERS - Google Patents

Abstract

) The invention relates to methods for treating hepcidin-mediated disorders.

Description

1. Links to related applications

This application claims priority under U.S. Provisional Application No. 62/199434, filed July 31, 2015, and U.S. Provisional Application No. 62/268,788, filed December 17, 2015, each of which incorporated herein by reference in their entirety.

2. Sequence listing

The application for this invention contains a sequence listing, which was presented in electronic ASCII format and incorporated into the present description by reference in its entirety. The specified ASCII copy, created on September 12, 2016, is 34392PCT_CRF_sequencelisting.txt and is 51 KB in size.

3. Background of the present invention

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

Hepcidin expression has been implicated in human iron metabolism disorders, Pietrangelo, J. Hepatology, 54:173-181 (2011), and in the development of anemia of chronic disease (ACD) (also known as anemia of inflammation (AI) ). ACD most commonly occurs in patients with chronic infection, autoimmune disease, cancer, and chronic kidney disease (CKD). Sun et al., Am. J. Hematol. 87(4):392-400 (2012).

There is a need in the art for methods of treating hepcidin-mediated disorders.

4. The essence of the invention

The present inventors have demonstrated that a reduction in IL-6 signaling has a clinical benefit in patients with hepcidin-mediated disorder, including chronic disease anemia and hepcidin-mediated cellular toxicities, but only in those patients who have at least one a copy of the main rs855791 TMPRSS6 allele, with the greatest effect in patients with elevated IL-6 levels.

Therefore, in accordance with the first aspect, the present invention relates to methods of treatment mediated by hepcidin disorders. The methods include administering a therapeutically effective amount of an IL-6 antagonist to a patient with a hepcidin mediated disorder that has been determined to have at least one copy of the major allele with the rs855791 TMPRSS6 SNP. In accordance with the first series of embodiments, the patient was previously determined to have at least one copy of the core rs855791 TMPRSS6 allele. In accordance with another series of embodiments, the method further comprises an earlier step of determining that the patient has at least one copy of the TMPRSS6 core rs855791 allele. Typically, the patient has elevated serum levels of IL-6 before starting treatment. In accordance with some embodiments, the patient has elevated serum levels of CRP prior to treatment.

According to various embodiments, the hepcidin-mediated disorder is anemia of chronic disease.

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

In some anemia-related embodiments, the patient is a male patient and has a pre-treatment hematocrit of less than 40%, less than 35%, or 30-34%. In some embodiments, the patient is a female patient 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 accordance with some embodiments, the female patient has a pre-treatment hematocrit of 26-29%.

According to various anemia-related embodiments, the patient has received at least one administration of an erythropoiesis-stimulating agent (ESA) prior to treatment. In certain embodiments, the patient has received at least one administration of ESA prior to treatment and has a normal Hb level or normal hematocrit. In accordance with various embodiments, the patient has received at least one administration of an iron supplement prior to starting treatment. According to certain embodiments, the patient has received at least one iron supplement prior to treatment and has a normal Hb level or normal hematocrit. In accordance with various embodiments, the patient has received at least one blood or red blood cell transfusion prior to treatment. In accordance with certain embodiments, the patient has received at least one blood or packed red cell transfusion prior to treatment and has a normal Hb level or normal hematocrit.

According to various anemia-related embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to increase the patient's Hb above pre-treatment levels. According to various embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to increase the patient's hematocrit above pre-treatment levels. In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to allow the patient to reduce the dose of ESA without reducing the patient's Hb levels below levels present immediately before treatment. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to allow the patient to reduce the dose of ESA without reducing the patient's hematocrit below levels present immediately before treatment.

According to various embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to allow at least 10% reduction in the patient's ESA dose from pre-treatment ESA dose of at least 20 % reduce the patient's ESA dose from the pre-treatment ESA dose, reduce the patient's ESA dose by at least 30% from the pre-treatment ESA dose, reduce the patient's ESA dose by at least 40% from the pre-treatment dose ESA before treatment, or at least 50% reduce the patient's dose of ESA compared to the dose of ESA before treatment.

In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to reverse functional iron deficiency anemia.

According to a series of embodiments, the hepcidin-mediated disorder is anemia in chronic disease, the chronic disease being chronic kidney disease (CKD).

According to certain CKD-related embodiments, the patient has KDOQI chronic kidney disease stage 1, KDOQI chronic kidney disease stage 2, KDOQI chronic kidney disease stage 3, KDOQI chronic kidney disease stage 4, or 5- th stage of chronic kidney disease KDOQI. In accordance with certain variants of implementation, the patient has stage 5 chronic kidney disease KDOQI.

According to some CKD-related embodiments, the patient has cardiorenal syndrome (CRS). According to certain embodiments, the patient has type 4 CRS. In accordance with certain embodiments, the patient has received at least one dialysis treatment before starting treatment.

In some CKD-related embodiments, an IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to reduce cardiovascular (CV)-related mortality compared to age- and disease-matched historical controls. .

According to various embodiments, the hepcidin-mediated disorder is anemia of chronic disease, wherein the chronic disease is a chronic inflammatory disease.

According to some embodiments, the chronic inflammatory disease is rheumatoid arthritis (RA). In accordance with certain embodiments, the patient has a DAS28 score greater than 5.1 prior to treatment. In accordance with some embodiments, the patient has a DAS28 score of 3.2 to 5.1 prior to treatment. In certain embodiments, the patient has a DAS28 score of less than 2.6 prior to treatment. According to selected embodiments, the patient's RA is moderate to high prior to treatment.

In some RA-related embodiments, the patient has received at least one administration of methotrexate prior to starting treatment. In accordance with some embodiments, the patient has received at least one administration of a TNFa antagonist prior to starting treatment. According to the selected embodiments, the TNFa antagonist is selected from the group consisting of etanercept, adalimumab, infliximab, certolizumab, and golimumab.

In some RA-related embodiments, the patient has received at least one administration of an IL-6 antagonist prior to starting treatment. In certain embodiments, the IL-6 antagonist administered prior to treatment is tocilizumab or tofacitinib.

- 2 041768

According to a preferred series of embodiments, the IL-6 antagonist administered during treatment is MEDI5117.

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

In some embodiments, the hepcidin-mediated disorder is anemia of chronic disease, wherein the chronic disease is cancer. In accordance with certain embodiments, the cancer is selected from the group consisting of solid tumors, small cell lung cancer, non-small cell lung cancer, hematologic cancer, multiple myeloma, various forms of leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia ( CML), various forms of lymphoma, Hodgkin's lymphoma and liver adenoma.

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

In some embodiments, the hepcidin-mediated disorder is anemia of chronic disease, the chronic disease being chronic heart failure (CHF).

According to some embodiments, the hepcidin-mediated disorder is iron refractory and iron deficiency anemia (IRIDA).

In accordance with some embodiments, the hepcidin-mediated disorder is an acute coronary syndrome. In certain embodiments, the patient 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 -6 or within 24 hours preceding the first administration of the IL-6 antagonist.

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

In accordance with some embodiments, the hepcidin-mediated disorder is Castleman's disease.

According to another aspect, the present invention relates to methods for improving the treatment of a hepcidin-mediated disorder. The method involves stopping the administration of an IL-6 antagonist to a patient with a hepcidin-mediated disorder, the patient being determined to be homozygous for the rs855791 minor TMPRSS6 allele.

According to another aspect, the present invention relates to methods for improving the treatment of a hepcidin-mediated disorder by discontinuing therapy that is ineffective, thereby reducing side effects and reducing costs without loss of treatment efficacy. The methods involve stopping the administration of an IL-6 antagonist to a patient with a hepcidin mediated disorder, the patient being determined to be homozygous for the rs855791 minor TMPRSS6 allele. In accordance with a series of embodiments, the patient was previously determined to be homozygous for the rs855791 minor TMPRSS6 allele. In accordance with another series of embodiments, the method further comprises an earlier step of determining that the patient is homozygous for the minor rs855791 TMPRSS6 allele. According to exemplary embodiments, the patient has elevated serum levels of IL-6 prior to treatment. In accordance with various embodiments, the patient has elevated serum levels of CRP prior to treatment. In accordance with various embodiments, the patient has a hepcidin-mediated disorder selected from the disorders described in section 5.2.1 of this specification. In accordance with certain embodiments, the patient is anemic for a chronic disease.

From the data presented in Examples 2, 3, and 5 below, it can be seen that IL-6 antagonists are beneficial in subjects who have elevated pre-treatment IL-6 levels and at least one copy of the major TMPRSS6 allele, even in the absence of anemia. . Therefore, in another aspect, the present invention relates to methods for treating IL-6 mediated inflammatory disorders in non-anemic patients with chronic inflammation. The methods include administering a therapeutically effective amount of an IL-6 antagonist to a subject, typically a human patient, who has an IL-6 mediated inflammatory disease, the patient is not anemic, and the subject has been determined to have at least one copy of the major rs855791 TMPRSS6 allele. In accordance with the first series of embodiments, the subject has previously been determined to have at least one copy of the core rs855791 TMPRSS6 allele. In accordance with another series of embodiments, the method further comprises an earlier step of determining that the subject has at least one copy of the core rs855791 TMPRSS6 allele. Typically, the methods include excluding treatment of subjects if they are positively homozygous for the rs855791 minor allele of TMPRSS6. Usually the patient has elevated serum levels of IL6 before starting treatment.

In accordance with specific embodiments of any of the treatments, the patient has elevated serum levels of IL-6 prior to treatment. In accordance with certain embodiments, the patient has a pre-treatment serum IL-6 level of 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.

In various embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to reduce the patient's free IL-6 serum levels below pre-treatment levels. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to reduce free IL-6 levels by at least 10% from pre-treatment levels by at least 20% compared to pre-treatment levels or at least 50% compared to pre-treatment levels.

In accordance with specific embodiments of any of the treatments, the patient has elevated levels of C-reactive protein (CRP) prior to treatment. In certain embodiments, the patient has a C-reactive protein level of greater than 2 mg/l, greater than 3 mg/l, greater than 5 mg/l, greater than 7.5 mg/l, or even greater than 10 mg/l prior to treatment.

According to various embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to reduce the patient's CRP levels below pre-treatment levels. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to reduce the patient's CRP levels by at least 50% from pre-treatment levels.

In accordance with specific embodiments of any of the treatments, the patient was determined to have at least one copy of the core rs855791 TMPRSS6 allele by TaqMan® real-time PCR analysis.

According to embodiments of any of the treatments, the IL-6 antagonist is an anti-IL-6 antibody, or an antigen-binding fragment or derivative thereof.

In certain embodiments, the anti-IL-6 antibody, or antigen-binding fragment or derivative thereof, has a K _D for human IL-6 binding of less than 100 nM, less than 50 nM, less than 10 nM, or less than 1 nM. In certain embodiments, the anti-IL-6 antibody, or antigen-binding fragment or derivative thereof, has a half-life following intravenous administration of at least 7 days, at least 14 days, at least 21 days, or at least 30 days.

According to various embodiments, the IL-6 antagonist is a full-length anti-IL-6 monoclonal antibody, such as an IgG1 or IgG4 antibody.

In accordance with selected embodiments, the anti-IL-6 antibody, or antigen-binding fragment or derivative thereof, is fully human. In accordance with some embodiments, the anti-IL-6 antibody, or antigen-binding fragment or derivative thereof, is humanized.

According to presently preferred embodiments, the anti-IL-6 antibody, or antigen-binding fragment or derivative thereof, contains all six variable region CDRs from MED5117. According to some such embodiments, the antibody comprises VH and VL from MED5117. And in accordance with certain embodiments, the antibody is MED5117.

In various embodiments, an anti-IL-6 antibody, or antigen-binding fragment or derivative thereof, comprises all six CDRs of an antibody variable region selected from the group consisting of siltuximab, gerilimzumab, sirukumab, clasakizumab, 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 some embodiments, the anti-IL-6 antibody, or antigen-binding fragment or derivative thereof, comprises a heavy chain V region and a light chain V region from an antibody selected from the group consisting of siltuximab, gerilimzumab, sirukumab, clasakizumab,

- 4 041768 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 certain embodiments, the anti-IL-6 antibody is an antibody selected from the group consisting of siltuximab, gerilimzumab, sirukumab, clasakizumab, 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 some embodiments, the anti-IL-6 antibody, or antigen-binding fragment or derivative thereof, is an antibody selected from the group consisting of siltuximab, gerilimzumab, sirukumab, clasakizumab, olokizumab, VX30 (VOP-R003; Vaccinex), EB-007 ( EBI-029; Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518/BMS945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In certain embodiments, the anti-IL-6 antibody is an antibody selected from the group consisting of siltuximab, gerilimzumab, sirukumab, clasakizumab, 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).

According to various embodiments, the IL-6 antagonist is a single domain antibody, VHH nanobody, Fab, or scFv.

According to various embodiments, the IL-6 antagonist is an anti-IL-6R antibody, or an antigen-binding fragment or derivative thereof. In certain embodiments, the anti-IL-6R antibody, antigen-binding fragment, or derivative is tocilizumab or vobarilizumab.

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

According to various embodiments, the IL-6 antagonist is a STAT3 inhibitor.

In some embodiments, in which the IL-6 antagonist is an antibody, or antigen-binding fragment, or derivative, the IL-6 antagonist is administered parenterally. In certain embodiments, the IL-6 antagonist is administered subcutaneously.

In some embodiments, in which the IL-6 antagonist is a JAK inhibitor or a STAT3 inhibitor, the IL-6 antagonist is administered orally.

5. Brief description of the figures

In FIG. 1A and 1B are box plots showing that increased amounts of erythropoietin are needed to treat patients with chronic kidney disease (subjects on dialysis with CKD stage 5) (EPO) who had elevated serum levels of IL-6 and at least one copy of the major allele with a known SNP in the TMPRSS6 gene, rs855791 (G or C at nucleotide position 2321 encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736; 736A), but not in patients with chronic kidney disease who had elevated levels of IL-6 and were homozygous for the minor allele rs855791 TMPRSS6 (T or A at nucleotide position 2321 encoding a TMPRSS6 polypeptide having a valine at position 736; 736V). Data from patients homozygous for the minor allele (A/A) are shown in FIG. 1A; data from patients having at least one copy of the major allele (homozygous G/G and heterozygous G/A) were pooled and shown in FIG. 1B. Each of the two patient populations was further stratified into groups according to the tertiles of serum IL-6 levels: lower tertile (IL-6 <5 pg/mL); middle tertile (IL-6 = 5-15 pg/ml); highest tertile (IL-6 >15 pg/ml). Boxplots with whiskers superimposed on raw data. Each boxplot represents a group of patients by both IL-6 level and genotype. See example 1 for details.

In FIG. 2A and 2B are survival curves showing that the major rs855791 TMPRSS6 allele results in higher overall mortality in response to elevated levels of IL-6 in dialysis subjects with stage 5 chronic kidney disease.

In FIG. 2A shows data from patients homozygous for the minor allele (A/A).

In FIG. 2B shows data from patients having at least one copy of the major allele (homozygous G/G and heterozygous G/A).

Each group was divided into tertiles of serum IL-6 levels using the IL-6 levels used in FIG. 1. See example 1 for details.

In FIG. 3 is a graph showing that increased amounts of EPO are required for therapy in patients with chronic kidney disease (subjects on dialysis with stage 5 CKD) who had elevated serum levels of the acute inflammatory phase protein CRP and who had at least at least one copy of the core rs855791 TMPRSS6 allele, but not in patients with chronic

- 5 041768 kidney disease who had elevated serum levels of the acute phase inflammatory protein CRP and who were homozygous for the minor allele rs855791. Each genotype group was divided into serum CRP levels: <2 mg/l versus >2 mg/l. See example 1 for details.

In FIG. 4A and 4B are graphs showing that the major rs855791 TMPRSS6 allele results in higher overall mortality in response to elevated levels of IL-6 in post-myocardial infarction (MI) patients.

In FIG. 4A shows the cumulative probability of death over time (y-axis) versus days after MI (x-axis) for a population homozygous for the minor rs855791 TMPRSS6 allele.

In FIG. 4B shows the cumulative probability of death over time for a population having at least one copy of the major rs855791 TMPRSS6 allele.

Each group was divided into tertiles of serum IL-6 levels as previously indicated. The level of IL-6 was measured one month after myocardial infarction. Mortality was measured 1-12 months after myocardial infarction. Details are given in example 2.

In FIG. 5A and 5B are graphs showing that the major rs855791 TMPRSS6 allele confers a higher risk of heart failure (HF) in response to elevated levels of IL-6 in post-MI patients.

In FIG. 5A shows the cumulative probability of HF over time (y-axis) versus days after MI (x-axis) for a population homozygous for the minor rs855791 TMPRSS6 allele.

In FIG. 5B shows the cumulative probability of an HF event over time for a population having at least one copy of the core rs855791 TMRPSS6 allele.

Each group was divided into tertiles of serum IL-6 levels as previously indicated. The level of IL-6 was measured one month after myocardial infarction. HF was measured 1-12 months after myocardial infarction. Details are given in example 2.

In FIG. 6A and 6B show the results of analyzes of human iPS cells that were transfected with constructs, constitutively expressed either the minor or major rs855791 TMPRSS6 allele, and differentiated into cardiomyocytes when exposed to in vitro BMP2+IL-6 or BMP2 alone, indicating that the major rs855791 allele TMPRSS6 confers a higher risk of cell death (trypan blue positive) in response to IL-6.

In FIG. 6A shows results in a normal oxygen environment.

In FIG. 6B shows results after exposure to hypoxic conditions and reoxygenation.

The data suggest that reducing exposure to IL-6 should improve cardiomyocyte survival in patients with the major rs855791 TMPRSS6 allele, but not in patients with the minor rs855791 TMPRSS6 allele. Details are given in example 3.

In FIG. 7 is a diagram illustrating the design of the cardiorenal syndrome experiment described in Example 4. CRS4 was induced in rats genotypically similar to humans homozygous for the major rs855791 TMPRSS6 allele. The chart shows the different events in the study along the timeline. In the study, myocardial infarction (MI) was induced in rats at week 0. At week 2, a nephrectomy of one kidney (Nx) was performed in each subject. Anti-IL-6 antibody (ab9770, Abcam Plc, UK) (Rx) or isotype control antibody (IgG, ab171516, Abcam Plc, UK) was injected every 3 days, starting on day 1 (D1) after nephrectomy until the end research. The standard prophylactic agent (an ACE inhibitor called perindopril) was administered daily from day 1 after Nx until the end of the study. At week 6, the rodents were sacrificed. MI and Nx were not performed in the control group called the blank group. At the time points indicated by the arrows, various assessments were made in rodents.

In FIG. 8A-8D show cardiac ejection fraction in rats treated with an anti-IL-6 antibody (anti-IL-6 Ab), a standard prophylactic agent, i. an ACE inhibitor (perindopril or Peri), a relatively treated (isotype) control group, and sham surgery animals in the cardiorenal syndrome model that is briefly described in FIG. 7 and described in detail in example 4.

In FIG. 8A is a graph showing baseline ejection fraction levels for all groups two weeks after myocardial infarction but before nephrectomy.

In FIG. 8B is a graph showing ejection fraction levels for all groups one week after nephrectomy, after 1 week of treatment.

In FIG. 8C is a graph showing ejection fraction levels for all groups two weeks after nephrectomy, after 2 weeks of treatment.

In FIG. 8D is a graph showing ejection fraction levels for all groups four weeks after nephrectomy, after 4 weeks of treatment.

Results are expressed as mean ± SEM and indicate that anti-IL-6 antibody therapy had therapeutic efficacy in a model of cardiorenal syndrome equivalent to standard prophylactic therapy as measured by changes in cardiac ejection fraction.

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In FIG. 9 is a graph showing cardiac contractility in rats treated with anti-IL-6 antibody (anti-IL-6 Ab), standard prophylactic (perindopril or Peri), versus control (isotype) treated group in a model of cardiorenal syndrome, which is briefly described in Fig. 7 and described in detail in Example 4. At the end of the study, cardiac contractility was assessed by measuring dP/dt _max (mmHg/ms), which is an indicator of the pressure inside the heart. The measurement results are shown for all groups four weeks after nephrectomy, after 4 weeks of treatment. The results are expressed as mean ± SEM and indicate that anti-IL-6 antibody therapy had a therapeutic effect equivalent to standard prophylactic therapy, as evidenced by an increase in cardiac contractility in the anti-IL-6 antibody treated groups of rodents.

In FIG. 10A-10C show that anti-IL-6 antibody therapy had a beneficial effect on cardiorenal syndrome equivalent to standard prophylactic therapy as measured by levels of fibrosis in heart tissue from anti-IL-6 antibody (anti-IL-6 Ab) treated groups of rodents, standard preventive therapy (perindopril or Peri) and control (IgG).

In FIG. 10A is a photomicrograph showing a histological section of heart tissue stained with picrosirius red. Two areas of tissue were analyzed: a normal area and a fibrous edge area. An example of a normal area is indicated by the outlined portion of the tissue section. The micrograph inset shows an enlarged view of the normal area showing that small portions of the normal area had fibrous tissue. The fibrous margin site was an area of tissue in the normal area that was peripheral to the fibrous tissue.

In FIG. 10B is a graph showing area percentages of normal area designated as fibrous tissue (ie stained/dark areas) in tissue samples from all groups.

In FIG. 10C is a graph showing the area percentages of the fibrous rim area designated as fibrous tissue in tissue samples from all groups.

Results are expressed as mean ± SEM. Details are given in example 4.

In FIG. 11A and 11B show data from an in vivo model in which myocardial infarction was induced in mice genotypically similar to humans homozygous for the major rs855791 TMPRSS6 allele. The control group received no therapy. The experimental group was treated with anti-mouse IL-6 antibody.

In FIG. 11A shows that anti-IL-6 antibody treatment provided a statistically significant improvement in ejection fraction.

In FIG. 11B shows that anti-IL-6 antibody treatment provided a statistically significant improvement in contractility, measured as a shortening of the left ventricular cardiac fraction.

The data show that anti-IL-6 antibody therapy given immediately after myocardial infarction improved functional recovery of the left ventricle in rodents that mimic human patients with the major TMPRSS6 rs855791 allele. Details are given in example 5.

Various embodiments of the present invention are shown in the drawings for illustrative purposes only. From the following review, one skilled in the art will readily appreciate that alternative embodiments of the structures and methods illustrated herein can be used without departing from the teachings of the present invention described herein.

6. Detailed description of the invention

6.1. Review of experimental results.

The peptide hormone, hepcidin, plays a central role in systemic iron homeostasis. Hentze et al., Cell, 142:24-38 (2010). Hepcidin expression is known to be influenced by the product of the TMPRSS6 gene, matriptase-2, a type II transmembrane serine protease. Common variants of the TMPRSS6 gene have been shown to correlate with iron status, Benyamin et al., Nature Genetics, 41(11):1173-1175 (2009), while the SNP rs855791 (2321G^A; A736V) has been shown to correlate with natural fluctuations in the expression of hepcidin and hemoglobin levels in the blood. Hepcidin expression has been implicated in human iron metabolism disorders, Pietrangelo, J. Hepatology, 54:173-181 (2011), and in the development of anemia of chronic disease (ACD) (also known as anemia of inflammation (AI) ). ACD most commonly occurs in patients with chronic infection, autoimmune disease, cancer, and chronic kidney disease (CKD). Sun et al., Am. J. Hematol. 87(4):392-400 (2012).

To determine whether the rs855791 TMPRSS6 SNP genotype can predict the degree of anemia in end-stage kidney disease, data previously collected from clinical trials in patients with chronic kidney disease 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 could predict the degree of

- 7 041768 anemia in end-stage kidney disease.

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

Similarly, overall survival correlated with IL-6 levels only in patients with at least one copy of the major allele with the rs855791 TMPRSS6 SNP. In subjects with at least one copy of the major rs855791 TMPRSS6 allele, survival was inversely correlated with serum IL-6 levels, with patients in the highest tertile of serum IL-6 levels having statistically significantly worse survival than those in the lowest tertile of IL levels. -6 (Fig. 2B). In contrast, overall survival of patients homozygous for the minor allele in rs855791 was independent of IL-6 levels (Fig. 2A).

Without wishing to be bound by any theory, in patients with at least one copy of the major TMPRSS6 allele, an increase in serum IL-6 may lead to an increase in hepcidin expression, thereby increasing anemia. The increased risk of mortality is due to impaired iron metabolism resulting from anemia and/or an increased dose of an erythropoiesis-stimulating agent, such as EPO, administered for treatment. These correlations raise the possibility that lowering IL-6 levels or IL-6 signaling may reduce anemia, reduce the required dose of EPO, and increase survival in patients with chronic kidney disease, but only in those patients who have at least one copy. the main allele rs855791 TMPRSS6, and with the greatest effect in patients with elevated serum levels of IL-6.

To determine whether the TMPRSS6 rs855791 genotype affects IL-6 susceptibility in patients with acute but not chronic disease, data previously collected in clinical trials of patients hospitalized with acute coronary syndrome was analyzed in Example 2, in combination with newly determined genotyping. SNP.

Mortality of subjects homozygous for the minor allele (A) of the rs855791 TMPRSS6 SNP did not correlate with fluctuations in IL-6 (Fig. 4A). However, one or two copies of the major allele (G) gave higher overall mortality in response to elevated levels of IL-6 in subjects after myocardial infarction (Fig. 4B). Thus, TMPRSS6 modulated the IL-6 mediated risk of death after myocardial infarction.

The effect of the TMPRSS6 genotype on IL-6 mediated risk of heart failure was also assessed. Heart failure in subjects homozygous for the minor allele (A) did not correlate with fluctuations in IL-6 (FIG. 5A). However, the TMPRSS6 G allele conferred a higher incidence of heart failure in response to elevated levels of IL-6 in subjects after myocardial infarction (FIG. 5B). Thus, TMPRSS6 modulated the IL-6 mediated risk of heart failure after myocardial infarction.

From the data in Example 2, it appears that the correlation between TMPRSS6 genotype, IL-6 levels, and adverse clinical outcomes is not limited to patients with chronic kidney disease. Without wishing to be bound by any theory, in patients with at least one copy of the major TMPRSS6 allele, an increase in serum IL-6 may lead to an increase in hepcidin expression, followed by an increase in cardiomyocyte iron sequestration and subsequent iron-mediated cellular toxicity. Such correlations raise the possibility that lowering IL-6 levels or IL-6 signaling may reduce heart failure and mortality in patients with acute coronary syndrome, but only in those patients who have at least one copy of the major rs855791 TMPRSS6 allele. and with the greatest effect in patients with elevated serum levels of IL-6.

Although the correlations observed in Examples 1 and 2 strongly suggest that a reduction in IL-6 mediated signaling should be clinically beneficial in patients with at least one copy of the major rs855791 TMPRSS6 allele, elevated levels of IL- 6 and either anemia or hepcidin-mediated cellular toxicity, the observed correlations did not support a causal relationship. Accordingly, in Example 3, induced human pluripotent cell-derived cardiomyocytes (iPS) were constructed and tested in vitro to express only the major or minor allele of rs855791 TMPRSS6.

Hepcidin expression is regulated by both BMP6/SMAD and IL-6/STAT signaling pathways, with BMP and IL-6 acting through their respective receptors and driving hepcidin expression. Casanovas et al., PLOS Comp. Biol. 10(1):e1003421 (2014). Major and minor allele iPS cardiomyocytes were treated in vitro with agonists of both signaling pathways: recombinant BMP2 and IL-6, or BMP2 alone to model clinical interventions in which IL-6 levels (or signaling) are reduced. iPS control cells were not treated with any agonist at all. Cellular

- 8 041768 mortality was measured at normal oxygen pressure (normal oxygen conditions), as well as under conditions that simulate hypoxia followed by reoxygenation (reperfusion).

In FIG. 6A shows results when cells were treated at normal oxygen levels. iPS cardiomyocytes expressing only the minor rs855791 TMPRSS6 allele (minor allele 736V) were not significantly affected (n.s.) by abolishing IL-6 signaling: cell death, measured as the percentage of trypan blue positive cells, was not significantly reduced when cells were treated with only BMP2 versus BMP2+IL-6 treatment. In contrast, iPS cardiomyocytes expressing the major rs855791 TMPRSS6 allele showed statistically significant lower cell death when IL-6 signaling was abolished.

In FIG. 6B shows the results when the cells were subjected to hypoxia followed by reoxygenation. Compared to normal oxygen conditions, hypoxia/reoxygenation is toxic to iPS cardiomyocytes, with approximately 40% of major and minor allele control cells dying compared to approximately 20% of control cells dying under normal oxygen conditions (cf. FIG. .6A). With respect to this increased background toxicity, minor allele iPS cardiomyocytes were not significantly affected by abolishing IL-6 signaling: cell death was not significantly reduced when cells were treated with BMP2 alone compared with treatment with BMP2 + IL-6. In contrast, iPS cardiomyocytes expressing the major rs855791 TMPRSS6 allele showed statistically significant lower cell death when IL-6 signaling was abolished.

These data provide strong support for the conclusions drawn from the post hoc analysis of 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 major rs855791 TMPRSS6 allele, but not in cardiomyocytes. expressing only the minor allele. Without wishing to be bound by any theory, the IL-6-driven increase in toxicity in iPS cardiomyocytes with the major allele may be the result of an IL6-mediated increase in hepcidin expression, followed by an increase in cellular iron sequestration, and subsequent iron-mediated cellular toxicity.

Patients with chronic kidney disease, such as those who participated in the MIMICK studies analyzed in example 1, often develop impaired cardiac function, which is a major factor influencing overall mortality. This secondary myocardial injury after primary chronic kidney disease is called cardiorenal syndrome type 4 (CRS type 4). To directly test whether anti-IL-6 antibody therapy is effective as a treatment in CRS4 patients having at least one copy of the major rs855791 TMPRSS6 allele, as suggested by the data in Examples 1 and 3, we used a CRS4 model in rats that are genotypically similar to humans homozygous for the major rs855791 TMPRSS6 allele.

After 4 weeks of treatment, both treatment groups, ie. the anti-IL-6 antibody treated group and the standard ACE inhibitor prophylactic drug perindopril treated group had statistically significantly increased ejection fraction levels compared to the isotype control group (FIG. 8D) (p<0.001). Similar ejection fraction levels in the anti-IL-6 antibody group and the standard prophylactic therapy group measured after the 4th week of treatment show that the anti-IL-6 antibody therapy had equivalent efficacy to an ACE inhibitor. In FIG. 9 shows that anti-IL-6 antibody therapy was also effective, equivalent to an ACE inhibitor while maintaining cardiac contractility. In FIG. 10A-10C show that anti-IL-6 antibody therapy was equivalently effective in reducing cardiac fibrosis.

From these data, it appears that anti-IL-6 antibody treatment is effective in reducing myocardial injury and restoring function in an in vivo model of cardiorenal syndrome in animals that are genotypically similar to humans homozygous for the major rs855791 TMPRSS6 allele.

Similarly, the data in Examples 2 and 3 suggest that lowering IL-6 levels or IL-6 signaling may reduce heart failure and mortality in patients with acute coronary syndrome, but only in those patients who have at least one a copy of the main rs855791 TMPRSS6 allele, and with the greatest effect in patients with elevated serum levels of IL-6.

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

In FIG. 11A and 11B show data from an in vivo model in which myocardial infarction was induced in mice genotypically similar to humans homozygous for the major rs855791 TMPRSS6 allele. The control group received no therapy. The experimental group was treated with anti-mouse IL-6 antibody. In FIG. 11A shows that anti-IL-6 antibody treatment provided a statistically significant improvement in ejection fraction compared to controls. In FIG. 11B shows

- 9 041768 that treatment with antibody to IL-6 provided a statistically significant improvement in contractility, measured as a shortening of the cardiac fraction compared to controls. From these data, it appears that anti-IL-6 antibody therapy given immediately after myocardial infarction improved left ventricular functional recovery in rodents that are genetically similar to human patients with the major rs855791 TMPRSS6 allele.

Collectively, it appears from experimental data that therapeutic interventions that reduce IL-6 signaling are clinically beneficial in patients with a hepcidin-mediated disorder such as anemia or hepcidin-mediated cellular toxicity, but only in those who have at least one copy of the main rs855791 TMPRSS6 allele, with the greatest effect in patients with elevated IL-6 levels.

Therefore, as further described below, in accordance with the first aspect, the present invention relates to methods of treating a hepcidin-mediated disorder. The methods include administering a therapeutically effective amount of an IL-6 antagonist to a patient with a hepcidin mediated disorder that has been determined to have at least one copy of the major allele with the rs855791 TMPRSS6 SNP. According to a second aspect, the present invention relates to methods for improving the treatment of hepcidin-mediated disorders, the method comprising stopping the administration of an IL-6 antagonist to a patient with a hepcidin-mediated disorder, the patient having been determined to be homozygous for the rs855791 minor TMPRSS6 allele. Treatment is improved by discontinuing therapy that is ineffective, thereby reducing side effects and reducing costs without losing the effectiveness of the treatment. According to a further aspect, the present invention relates to methods for treating IL-6 mediated inflammatory disorders in patients without anemia of chronic inflammation, the methods comprising administering a therapeutically effective amount of an IL-6 antagonist to a patient who has an IL-6 mediated inflammatory disorder, does not have anemia, and the subject was determined to have at least one copy of the core rs855791 TMPRSS6 allele.

6.2. Definitions.

Unless otherwise defined, all technical and scientific terms used herein have the meaning that is generally understood by a person skilled in the art to which the present invention pertains. In the context of this document, the following terms have the meanings assigned to them below.

By hepcidin is meant a polypeptide having an amino acid sequence that is at least about 85% or more identical to the amino acid sequence listed in NCBI under accession number NP 066998 (hepcidin precursor protein), or a biologically active fragment thereof. Exemplary biological activities of hepcidin include binding to and lowering levels of an iron export channel called ferroportin, which inhibits iron transport, inhibits iron absorption in the intestine, and inhibits iron release from macrophages and the liver. The following is an illustrative amino acid sequence of the hepcidin precursor protein:

MALSSQIWAA CLLLLLLLAS LTSGSVFPQQ TGQLAELQPQ DRAGARASWM PMFQRRRRRD

THFPICIFCC GCCHRSKCGM SSCT (SEQ ID NO:1)

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

A hepcidin-mediated disorder is any disorder in which the expression of hepcidin contributes to the etiology of the disorder or any of its symptoms. The contribution of hepcidin to the etiology may be known, may be inferred, or may be drawn from the observation that administration of an IL-6 antagonist produces a greater therapeutic effect in patients with the disorder who have at least one copy of the major rs855791 TMPRSS6 SNP allele compared to patients with a disorder that are homozygous for the minor allele rs855791 TMPRSS6. Hepcidin-mediated disorders are further described in section 6.2.1.

The phrase transmembrane serine protease 6 polypeptide (TMPRSS6) is understood to mean a polypeptide or fragment thereof having an amino acid sequence that is at least about 85% or more identical to the amino acid sequence listed in NCBI under accession number NP 001275929 and possessing serine protease activity. The TMPRSS6 polypeptide, also known as matriptase-2 (MT2), cleaves hemojuvelin and inhibits signaling involving a bone morphogenetic protein. Below is an exemplary amino acid sequence of TMPRSS6 having an alanine at position 736 (736A):

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MPVAEAPQVA GGQGDGGDGE EAEPEGMFKA CEDSKRKARG YLRLVPLFVL LALLVLASAG

VLLWYFLGYK AEVMVSQVYS GSLRVLNRHF SQDLTRRESS AFRSETAKAQ KMLKELITST

121 RLGTYYNSSS VYSFGEGPLT CFFWFILQIP EHRRLMLSPE WQALLVEEL LSTVNSSAAV

181 PYRAEYEVDP EGLVILEASV KDIAALNSTL GCYRYSYVGQ GQVLRLKGPD HLASSCLWHL

241 QGPKDLMLKL RLEWTLAECR DRLAMYDVAG PLEKRLITSV YGCSRQEPW EVLASGAIMA

301 WWKKGLHSY YDPFVLSVQP WFQACEVNL TLDNRLDSQG VLSTPYFPSY YSPQTHCSWH

361 LTVPSLDYGL ALWFDAYALR RQKYDLPCTQ GQWTIQNRRL CGLRILQPYA ERIPWATAG

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 ALLQLDHPW RSAAVRPVCL PARSHFFEPG LHCWITGWGA LREGALRADA VALFYGWRNQ

721 GSETCCCPIS NALQKADVQL IPQDLCSEVY RYQVTPRMLC AGYRKGKKDA CQGDSGGPLV

781 CKALSGRWFL AGLVSWGLGC GRPNYFGVYT RITGVISWIQ QWT (SEQ ID NO: 2)

The following is an exemplary amino acid sequence of TMPRSS6 having a valine at position 736 (736V):

MPVAEAPQVA GGQGDGGDGE EAEPEGMFKA CEDSKRKARG YLRLVPLFVL LALLVLASAG

VLLWYFLGYK AEVMVSQVYS GSLRVLNRHF SQDLTRRESS AFRSETAKAQ KMLKELITST

121 RLGTYYNSSS VYSFGEGPLT CFFWFILQIP EHRRLMLSPE WQALLVEEL LSTVNSSAAV

181 PYRAEYEVDP EGLVILEASV KDIAALNSTL GCYRYSYVGQ GQVLRLKGPD HLASSCLWHL

QGPKDLMLKL RLEWTLAECR DRLAMYDVAG PLEKRLITSV YGCSRQEPW EVLASGAIMA

WWKKGLHSY YDPFVLSVQP WFQACEVNL TLDNRLDSQG VLSTPYFPSY YSPQTHCSWH

LTVPSLDYGL ALWFDAYALR RQKYDLPCTQ GQWTIQNRRL CGLRILQPYA ERIPWATAG

ITINFTSQIS LTGPGVRVHY GLYNQSDPCP GEFLCSVNGL CVPACDGVKD CPNGLDERNC

VCRATFQCKE DSTCISLPKV CDGQPDCLNG SDEEQCQEGV PCGTFTFQCE DRSCVKKPNP

QCDGRPDCRD GSDEEHCDCG LQGPSSRIVG GAVSSEGEWP WQASLQVRGR HICGGALIAD

RWVITAAHCF QEDSMASTVL WTVFLGKVWQ NSRWPGEVSF KVSRLLLHPY HEEDSHDYDV

ALLQLDHPW RSAAVRPVCL PARSHFFEPG LHCWITGWGA LREGALRADA VALFYGWRNQ

GSETCCCPIS NALQKVDVQL IPQDLCSEVY RYQVTPRMLC AGYRKGKKDA CQGDSGGPLV

CKALSGRWFL AGLVSWGLGC GRPNYFGVYT RITGVISWIQ QWT (SEQ ID NO: 3)

The phrase TMPRSS6 nucleic acid molecule is understood to mean a polynucleotide encoding a TMPRSS6 polypeptide (matriptase-2; MT2). An exemplary TMPRSS6 nucleic acid molecule sequence is available from the NCBI under accession number NM 001289000. The following is a TMPRSS6 nucleic acid sequence having a G at nucleotide position 2321 (G allele; main allele):

GGACAAAACAG AGGCTCCTGA GGCCTGTGTG CAGGCCCGGC ACCTATCTGC CGCTCCCAAA

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

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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 CAAGGAGCTC 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)

Below is the TMPRSS6 nucleic acid sequence having an A at nucleotide position 2321:

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GGACAAAACAG AGGCTCCTGA GGCCTGTGTG

GGATGCCCGT GGCCGAGGCC CCCCAGGTGG

121 AGGAAGCGGA GCCGGAGGGG ATGTTCAAGG

181 GCTACCTCCG CCTGGTGCCC CTGTTTGTGC

241 GGGTGCTACT CTGGTATTTC CTAGGGTACA

301 CAGGCAGTCT GCGTGTACTC AATCGCCACT

361 GTGCCTTCCG CAGTGAAACC GCCAAAGCCC

421 CCCGCCTGGG AACTTACTAC AACTCCAGCT

481 CCTGCTTCTT CTGGTTCATT CTCCAAATCC

541 AGGTGGTGCA GGCACTGCTG GTGGAGGAGC

601 TCCCCTACAG GGCCGAGTAC GAAGTGGACC

661 TGAAAGACAT AGCTGCATTG AATTCCACGC

721 AGGGCCAGGT CCTCCGGCTG AAGGGGCCTG

781 TGCAGGGCCC CAAGGACCTC ATGCTCAAAC

841 GGGACCGACT GGCCATGTAT GACGTGGCCG

901 TGTACGGCTG CAGCCGCCAG GAGCCCGTGG

961 CGGTCGTCTG GAAGAAGGGC CTGCACAGCT

1021 CGGTGGTCTT CCAGGCCTGT GAAGTGAACC

1081 GCGTCCTCAG CACCCCGTAC TTCCCCAGCT

1141 ACCTCACGGT GCCCTCTCTG GACTACGGCT

1201 GGAGGCAGAA GTATGATTTG CCGTGCACCC

1261 TGTGTGGCTT GCGCATCCTG CAGCCCTACG

1321 GGATCACCAT CAACTTCACC TCCCAGATCT

1381 ATGGCTTGTA CAACCAGTCG GACCCCTGCC

1441 TCTGTGTCCC TGCCTGTGAT GGGGTCAAGG

1501 GCGTTTCAG AGCCACATTC CAGTGCAAAG

1561 TCTGTGATGG GCAGCCTGAT TGTCTCAACG

1621 TGCCATGTGG GACATTCACC TTCCAGTGTG

CAGGCCCGGC ACCTATCTGC CGCTCCCAAA CTGGCGGGCA GGGGGACGGA GGTGATGGCG CCTGTGAGGA CTCCAAGAGA AAAGCCCGGG TGCTGGCCCT GCTCGTGCTG GCTTCGGCGG AGGCGGAGGT GATGGTCAGC CAGGTGTACT TCTCCCAGGA TCTTACCCGC CGGGAATCTA AGAAGATGCT CAAGGAGCTC ATCACCAGCA CCGTCTATTC CTTTGGGGAG GGACCCCTCA CCGAGCACCG CCGGCTGATG CTGAGCCCCG TGCTGTCCAC AGTCAACAGC TCGGCTGCCG CCGAGGGCCT AGTGATCCTG GAAGCCAGTG TGGGTTGTTA CCGCTACAGC TACGTGGGCC ACCACCTGGC CTCCAGCTGC CTGTGGCACC TCCGGCTGGA GTGGACGCTG GCAGAGTGCC GGCCCCTGGA GAAGAGGCTC ATCACCTCGG TGGAGGTTCT GGCGTCGGGG GCCATCATGG ACTACGACCC CTTCGTGCTC TCCGTGCAGC TGACGCTGGA GAAGAGGCTC GACTCCCAGG ACTACTCGCC CCAAACCCAC TGCTCCTGGC TGGCCCTCTG GTTTGATGCC TATGCACTGA AGGGCCAGTG GACGATCCAG AACAGGAGGC CCGAGAGGAT CCCCGTGGTG GCCACGGCCG CCCTCACCGG GCCCGGTGTG CGGGTGCACT CTGGAGAGTT CCTCTGTTCT GTGAATGGAC ACTGCCCCAA CGGCCTGGAT GAGAGAAACT AGGACAGCAC ATGCATCTCA CTGCCCAAGG GCAGCGACGA AGAGCAGTGC CAGGAAGGGG AGGACCGGAG CTGCGTGAAG AAGCCCAACC

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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)

By variant is meant a polynucleotide or polypeptide sequence that differs from the reference sequence in one or more nucleotides or in one or more amino acids. An exemplary variant of TMPRSS6 is TMPRSS6 (A736V) derived from SNP rs855791 (G>A).

By single nucleotide polymorphism or SNP is meant a naturally occurring variant of a DNA sequence in which one nucleotide in the genome differs among members of a species or paired chromosomes in an individual. SNPs can be used as genetic markers for variant alleles. According to one embodiment, the TMPRSS6 SNP is rs855791.

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

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

Homozygous means that a chromosomal locus has two identical alleles. According to specific 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 containing an alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of the nucleic acid molecule TMPRSS6) (homozygous main allele rs855791). In some embodiments, homozygous refers to a genotype in which both alleles have a TMPRSS6 nucleic acid sequence encoding a TMPRSS6 polypeptide containing valine at amino acid position 736 (e.g., having an A or a T at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule ) (homozygous minor allele rs855791).

Determining that a patient has at least one copy of the core rs855791 TMPRSS6 allele includes, without limitation, performing an assay to determine that the patient has at least one copy of the core rs855791 TMPRSS6 allele; ordering an assay to determine that the patient has at least one copy of the major rs855791 TMPRSS6 allele; prescribing an assay to determine that the patient has at least one copy of the major rs855791 TMPRSS6 allele; otherwise directing or monitoring that an assay is performed to determine that the patient has at least one copy of the major rs855791 TMPRSS6 allele; and examining the TMRSS6 genotype analysis data or protein or nucleic acid sequence data to determine that the patient has at least one copy of the TMPRSS6 core rs855791 allele.

By the term interleukin 6 (IL-6) or an IL-6 polypeptide is meant a polypeptide or fragment thereof having an amino acid sequence that is at least about 85% or more identical to the amino acid sequence listed in the NCBI under accession number NP_000591 and possessing biological activity IL-6. IL-6 is a pleiotropic cytokine with multiple biological functions. Exemplary biological activities of IL-6 include immunostimulatory and pro-inflammatory activities. The following is an exemplary amino acid sequence of IL-6:

MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ

NTVCRPCGPG FYNDWSSKP 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)

The term interleukin 6 (IL-6) nucleic acid is understood to mean a polynucleotide encoding an interleukin 6 (IL-6) polypeptide. An exemplary nucleic acid sequence for interleukin 6 (IL-6) is available from the NCBI under accession number NM 000600. Below is an exemplary sequence from the NCBI under accession number NM 000600:

AATATTAGAG TSTSAASSSS CAATAAATAT AGGACTGGAG ATGTCTGAGG CTCATTCTGC

CCTCGAGCCC ACCGGGAACG AAAGAGAAGC TSTATSTCSS CTCCAGGAGC CCAGCTATGA

121 ASTSSTTTS 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 CCAACCACAAA ATGCCAGCCT GCTGACGAAG CTGCAGGCAC

661 AGAACCAGTG GCTGCAGGAC ATGACAACTC ATTCSATTCT GCGCAGCTTT AAGGAGTTCC

721 TGCAGTCCAG CCTGAGGGCT CTTCGGCAAA TGTAGCATGG GCACCTCAGA TTGTTGTTGT

781 TAATGGGCAT TSSTTTTTST GGTCAGAAAC CTGTCCACTG GGCACAGAAC TTATGTTGTT

841 CTCTATTGGAG AACTAAAAAGT ATGAGCGTTA GGACACTATT TTAATTTTT TTAATTTTT

901 AATATTTAAA TATGTGAAGC TGAGTTAATT TATGTAAGTC ATATTTAAT TTTTAAGAAG

961 TACCACTTGA AASATTTAT GTATTAGTTT TGAAATAATA ATGGAAAGTG GCTATGCAGT

1021 TTGAATATCC TTTGTTTCAG AGCCAGATCA TTTCTTGGAA AGTGTAGGCT TASSTSAAAT

1081 AAATGGCTAA STTTATASATA TTTTTAAAGA AATATTTATA TTGTATTTAT ATAATGTATA

1141 AATGGTTTTTT ATASSAAAA ATGGCATTTTT AAAAAATTSA GCAAAAAAAA AAAAAAAAAAAA

1201A (SEQ ID NO:7)

Interleukin 6 receptor complex (IL-6R) is understood to be a protein complex comprising the alpha subunit of the IL-6 receptor (IL-6Ra) and the interleukin 6 signal transducer glycoprotein 130, also referred to as the P subunit of the interleukin 6 receptor (IL-6Re).

An interleukin 6 receptor α-subunit (IL-6Ra) polypeptide is understood to mean a polypeptide or fragment thereof having an amino acid sequence that is at least about 85% or more identical to the amino acid sequence listed in the NCBI under accession number NP 000556 or NP 852004, and having biological activity of the IL-6 receptor. Exemplary biological activities include binding to IL-6, binding to glycoprotein 130 (gp130), and regulation of cell growth and differentiation. The following is an exemplary IL-6R sequence:

MLAVGCALLA ALLAAPGAAL APRRCPAQEV ARGVLTSLPG DSVTLTCPGV EPEDNATVHW

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 DAWSGLRHW 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)

An interleukin 6 receptor β-subunit polypeptide (IL-6RP) is understood to mean a polypeptide or fragment thereof having an amino acid sequence that is at least about 85% or more identical to the amino acid sequence listed in the NCBI under accession number NP 002175, NP 786943, or NP 001177910 and having IL-6 receptor biological activity. Exemplary biological activities of IL-6Re include binding to IL-6Ra, IL-6 receptor signaling, and regulation of cell growth, cell differentiation, hepcidin expression, and so on. The following is an exemplary sequence of IL-6Re:

MLTLQTWLVQ ALFIFLTTES TGELLDPCGY ISPESPWQL HSNFTAVCVL KEKCMDYFHV

NANYIVWKTN HFTIPKEQYT IINRTASSVT FTDIASLNIQ LTCNILTFGQ LEQNVYGITI

121 ISGLPPEKPK NLSCIVNEGK KMRCEWDGGR ETHLETNFTL KSEWATHKFA DCKAKRDPTPT

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 IWPVCLAFL LTTLLGVLFC FNKRDLIKKH IWPNVPDPSK 661 SHIAQWSPHT PPRHNFNSKD QMYSDGNFTD VSWEIEAND KKPFPEDLKS LDLFKKEKIN 721 TEGHSSGIGG SSCMSSSRPS ISSSDENESS QNTSSTVQYS TWHSGYRHQ VPSVQVFSRS 781 ESTQPLLDSE ERPEDLQLVD HVDGGDGILP RQQYFKQNCS QHESSPDISH FERSKQVSSV 841 NEEDFVRLKQ QISDHISQSC GSGQMKMFQE VSAADAFGPG TEGQVERFET VGMEAATDEG 901 MPKSYLPQTV RQGGYMPQ (SEQ ID NO:9)

An antagonist of IL-6 is understood to mean an agent that is capable of reducing the biological activity of IL-6. IL-6 antagonists include agents that lower the serum level of an IL-6 polypeptide, including agents that decrease the expression of an IL-6 polypeptide or nucleic acid, agents that decrease the ability of IL-6 to bind to IL-6R, agents , which down-regulate IL-6R expression; and agents that down-regulate IL-6R receptor signaling when bound to IL-6. According to preferred embodiments, the IL-6 antagonist reduces the biological activity of IL-6 by at least about 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%. Section 6.7 below further describes that 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.

By IL-6 antibody or anti-IL-6 antibody is meant an antibody that specifically binds IL-6. Anti-IL-6 antibodies include monoclonal and polyclonal antibodies that are specific for IL-6 and antigen-binding fragments or derivatives thereof. IL-6 antibodies are described in more detail in section 6.7.1 below.

By IL-6 mediated inflammatory disorder is meant any disorder for which IL-6 is known or suspected to contribute to the etiology of a disease or any of its symptoms.

By erythropoietin (EPO) is meant a polypeptide or fragment thereof having an amino acid sequence that is at least about 85% or more identical to the amino acid sequence listed in the NCBI under accession number NP 000790 and possessing the biological activity of EPO. Exemplary biological activities of EPO include binding to the erythropoietin receptor and the resulting proliferation and eventual differentiation of erythroid progenitor cells and/or increased erythropoiesis (erythrocyte production).

The following is an exemplary amino acid sequence of EPO:

MGVHECPAWL WLLLSLLSLP LGLPVLGAPP RLICDSRVLE RYLLEAKEAE NITTGCAEHC

SLNENITVPD TKVNFYAWKR MEVGQQAVEV WQGLALLSEA VLRGQALLVN SSQPWEPLQL

121 HVDKAVSGLR SLTTLLRALG AQKEAISPPD AASAAPLRTI TADTFRKLFR VYSNFLRGKL

181 KLYTGEACRT GDR (SEQ ID NO:10)

An erythropoiesis-stimulating agent (ESA) is understood to mean an agent that stimulates erythropoiesis. ESAs include, without limitation, EPO, darbepoetin (Aranesp), epoetin beta (NeoRecormon), epoetin delta (Dynepo), epoetin omega (Epomax), epoetin zeta.

An erythropoietic factor is understood to mean an agent that increases the growth or proliferation of an erythrocyte or its progenitor cell (eg, hematopoietic stem cell) and/or reduces the cell death of an erythrocyte or its progenitor cell. According to various embodiments, erythropoietic factors include erythropoiesis-stimulating agents, HIF stabilizers, and supplemental iron.

A C-reactive protein (CRP) polypeptide is understood to mean a polypeptide or fragment thereof having an amino acid sequence that is at least about 85% or more identical to the amino acid sequence listed in NCBI accession number NP 000558 and possessing complement activating activity. CRP levels rise in response to inflammation. The following is an illustrative CRP sequence:

MEKLLCFLVL TSLSHAFGQT DMSRKAFVFP KESDTSYVSL KAPLTKPLKA FTVCLHFYTE

LSSTRGYSIF SYATKRQDNE ILIFWSKDIG YSFTVGGSEI LFEVPEVTVA PVHICTSWES

121 ASGIVEFWVD GKPRVRKSLK KGYTVGAEAS IILGQEQDSF GGNFEGSQSL VGDIGNVNMW

181 DFVLSPDEIN TIYLGGPFSP NVLNWRALKY EVQGEVFTKP QLWP (SEQ ID NO:11)

By agent is meant any compound or composition suitable for administration in therapy and specifically includes chemical compounds, proteins, including antibodies or antigen-binding fragments thereof, peptides and nucleic acid molecules.

A subject is understood to mean a human or non-human mammal, including, without limitation, bovine, equine, canine, ovine, feline, and rodent subjects, including murine and rat subjects. The patient is a human subject.

As used herein, the terms treat, treat, cure, and the like. refer to the reduction or amelioration of a disorder and/or associated signs or symptoms, or to slow or stop their progression. It is understood, although not excluded, that the treatment of a disorder or condition does not require complete elimination of the disorder, condition, or associated symptoms.

Before the start of treatment means before the first administration of the IL-6 antagonist in accordance with the methods described herein. Prior to initiation of treatment does not preclude, and often includes, prior administration of a treatment other than an IL-6 antagonist.

In the present disclosure, contains, containing, constituting, having, includes, including, and their linguistic variants have the meaning assigned to them in US patent law, allowing the presence of additional components in addition to those explicitly indicated.

A biological sample is understood to mean any tissue, cell, fluid, or other material derived from an organism (eg, a human subject). In certain embodiments, the biological sample is blood serum or blood.

An angiotensin-converting enzyme (ACE) inhibitor is understood to mean an agent that inhibits an angiotensin-converting enzyme's biological function of converting angiotensin I to angiotensin II. ACE inhibitors include, without limitation, quinapril, perindopril, ramipril, captopril, benazepril, trandolapril, fosinopril, lisinopril, moexipril, and enalapril. According to various embodiments, the ACE inhibitor is perindopril.

6.3. Other rules of interpretation.

Unless otherwise indicated, antibody constant region residue numbering follows Kabat's EU numbering system.

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

Unless specifically stated or obvious from the context, the term or used in the context of this document is understood to be inclusive. Unless specifically stated or obvious from the context, terms used herein in the singular are understood to be in the singular or in the plural.

- 17 041768

Unless specifically stated or otherwise apparent from the context, the term used herein is approximately understood to be within the range of normal error in the art, for example, within two standard deviations from the mean. Approximately can be understood as within 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05 or 0.01% of the indicated value. Unless otherwise clear from the context, all numerical values presented herein are modified by the term approximately.

6.4. Methods for the treatment of hepcidin-mediated disorders.

According to a first aspect, the present invention relates to methods for treating a hepcidin-mediated disorder.

The methods include administering a therapeutically effective amount of an IL-6 antagonist to a subject, typically a human patient, who has a hepcidin-mediated disorder, the subject being determined to have at least one copy of the core rs855791 TMPRSS6 allele. In accordance with the first series of embodiments, the subject has previously been determined to have at least one copy of the core rs855791 TMPRSS6 allele. In accordance with another series of embodiments, the method further comprises an earlier step of determining that the subject has at least one copy of the core rs855791 TMPRSS6 allele. Typically, the methods include excluding treatment of subjects if they are positively homozygous for the rs855791 minor allele of TMPRSS6. Usually the patient has elevated serum levels of IL6 before starting treatment.

6.4.1. Hepcidin mediated disorders.

6.4.1.1. Anemia in chronic disease/chronic inflammation.

In various embodiments, the hepcidin-mediated disorder being treated using the methods described herein is anemia of chronic disease, also known as anemia of chronic inflammation.

In accordance with various embodiments, the patient is a male patient and has a pre-treatment hemoglobin (Hb) content of less than 14 g/dL. In accordance with some embodiments, the male patient has a pretreatment Hb level of 13.0-13.9, 12.0-12.9, 11.0-11.9, 10.0-10.9, or less than 10 g/dl According to various embodiments, the patient is a female patient and has a pre-treatment Hb level of less than 12 g/dL. In accordance with some embodiments, the female patient has a pretreatment Hb level of 11.0-11.9, 10.0-10.9, 9.0-9.9, 8.0-8.9, or less than 8 g/dl In accordance with some such embodiments, the patient has previously been treated with an ESA. In accordance with some embodiments, the patient has been treated with an iron supplement. In accordance with some embodiments, the patient has been treated with a blood or red blood cell transfusion.

According to various embodiments, the patient is a male patient and has a pre-treatment hematocrit of less than 40%. In accordance with 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 accordance with certain embodiments, the male patient has a pre-treatment hematocrit of 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, or 30%. According to various embodiments, the patient is a female patient and has a pre-treatment hematocrit of less than 36%. In accordance with some embodiments, the female patient has a pre-treatment hematocrit of less than 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, or 26%. According to certain embodiments, the female patient has a pre-treatment hematocrit of 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, or 26%. In accordance with some such embodiments, the patient has been treated with an ESA. In accordance with some embodiments, the patient has been treated with an iron supplement. In accordance with some embodiments, the patient has been treated with a blood or red blood cell transfusion.

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

In some embodiments, the patient has been treated with an iron supplement and has normal pre-treatment Hb and/or normal pre-treatment hematocrit. In certain embodiments, the patient is a male patient and has a pre-treatment hemoglobin (Hb) of at least 14 g/dl and/or a pre-treatment hematocrit of at least

40%. In certain embodiments, the patient is a female patient and has a pre-treatment Hb of at least 12 g/dL and/or a hematocrit of at least 36%.

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

In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to increase the patient's Hb above pre-treatment levels. In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to increase the patient's hematocrit above pre-treatment levels. In some embodiments, the IL-6 antagonist is dosed, scheduled, and for a period sufficient to increase both Hb levels and hematocrit above pre-treatment levels.

In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to allow the patient to reduce the dose of ESA without reducing the patient's Hb levels below pre-treatment levels. In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to allow the patient to reduce the dose of ESA without reducing the patient's hematocrit below pre-treatment levels. In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to allow the patient to reduce the dose of ESA without decreasing the patient's Hb and hematocrit levels.

In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to allow at least 10% reduction in the patient's ESA dose compared to pre-treatment ESA dose. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to allow at least 20%, 30%, 40%, or 50% reduction in the patient's ESA dose compared to the pre-treatment ESA dose. . In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to allow at least 60% or even at least 75% reduction in the patient's ESA dose compared to the pre-ER dose of ESA. start of treatment.

In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to reverse functional iron deficiency anemia.

6.4.1.1.1. Chronic kidney disease.

According to various embodiments, the chronic disease is chronic kidney disease (CKD).

In accordance with some embodiments, the patient has stage 1 chronic kidney disease KDOQI. According to specific embodiments, the patient has KDOQI chronic kidney disease stage 2, KDOQI chronic kidney disease stage 3, KDOQI chronic kidney disease stage 4, or KDOQI chronic kidney disease stage 5.

In accordance with some embodiments, the patient has cardiorenal syndrome (CRS). In accordance with specific embodiments, the patient has type 4 CRS.

In accordance with some embodiments, the patient has been treated with dialysis.

In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to reduce cardiovascular (CV)-related mortality compared to age-matched and disease-matched historical cohorts.

6.4.1.1.2. Chronic inflammatory diseases.

According to various embodiments, the chronic disease is a chronic inflammatory disease.

According to some embodiments, the chronic inflammatory disease is rheumatoid arthritis (RA).

In accordance with specific embodiments, the patient has a DAS28 score greater than 5.1 prior to treatment. In accordance with some embodiments, the patient has a DAS28 score of 3.2 to 5.1 prior to treatment. In accordance with some embodiments, the patient has a DAS28 score of less than 2.6 prior to treatment. According to various embodiments, the patient's RA is highly active prior to treatment. According to some embodiments, the patient's RA is moderately active prior to treatment.

In accordance with certain embodiments, the patient has been treated with methotrexate. According to some embodiments, methotrexate is discontinued when treatment with the IL-6 antagonist is initiated. In some embodiments, methotrexate treatment is continued when treatment with the IL-6 antagonist is initiated.

In accordance with certain embodiments, the patient has been treated with an anti-TNFa agent. In certain embodiments, the anti-TNFa agent is selected from etanercept, adalimumab, infliximab, certolizumab, and golimumab. In certain embodiments, the anti-TNFa agent is discontinued when treatment with the IL-6 antagonist is initiated.

In accordance with certain embodiments, the patient has been treated with an IL-1 receptor antagonist. According to specific embodiments, the IL-1 receptor antagonist is anakinra. According to certain embodiments, the IL-1 receptor antagonist is interrupted when treatment with the IL-6 antagonist is initiated.

In accordance with certain embodiments, the patient has been treated with abatacept. In certain embodiments, abatacept is discontinued when treatment with an IL-6 antagonist is started.

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 who are again determined to have at least one copy of the major rs855791 TMPRSS6 allele. According to specific embodiments, the IL-6 antagonist is tocilizumab. According to specific embodiments, the IL-6 antagonist is tofacitinib.

According to 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.

6.4.1.1.3. Malignant tumor.

According to various embodiments, the chronic disease is a cancer.

In some embodiments, the cancer is selected from the group consisting of solid tumors, small cell lung cancer, non-small cell lung cancer, hematologic cancer, multiple myeloma, various forms of leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia ( CML), various forms of lymphoma and Hodgkin's lymphoma.

6.4.1.1.4. chronic infection.

According to various embodiments, a chronic disease is a chronic infection.

6.4.1.1.5. Chronic heart failure.

According to various embodiments, the chronic disease is chronic heart failure (CHF).

6.4.1.2. Iron refractory and iron deficiency anemia (IRIDA).

According to various embodiments, the hepcidin-mediated disorder is iron refractory and iron deficiency anemia (IRIDA).

6.4.1.3. Anemia associated with hepcidin-producing forms of liver adenoma.

According to various embodiments, the hepcidin-mediated disorder is anemia associated with a hepcidin-producing adenoma of the liver.

6.4.1.4. Acute coronary syndrome.

From the data presented in Examples 2, 3 and 5 below, it can be seen that IL-6 antagonists are effective in reducing the risk of heart failure and death and in increasing cardiac function and reducing fibrosis after acute myocardial infarction. Accordingly, according to various embodiments, the hepcidin-mediated disorder is an acute coronary syndrome.

In certain embodiments, the patient has had a myocardial infarction within 60 days prior to the first administration of the IL-6 antagonist. In certain embodiments, the patient has had 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.

In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to improve myocardial contractility from levels before

- 20 041768 start of treatment. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to improve cardiac ejection fraction from pre-treatment levels. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to reduce cardiac fibrosis from pre-treatment levels.

6.4.1.5. Castleman disease.

According to various embodiments, the hepcidin-mediated disorder is Castleman's disease.

6.5. Methods for improving the treatment of hepcidin-mediated disorders.

According to another aspect, the present invention relates to methods for improving the treatment of a hepcidin-mediated disorder by discontinuing therapy that is ineffective, thereby reducing side effects and reducing costs without loss of treatment efficacy. The methods involve stopping the administration of an IL-6 antagonist to a patient with a hepcidin mediated disorder, the patient being determined to be homozygous for the rs855791 minor TMPRSS6 allele. In accordance with a series of embodiments, the patient was previously determined to be homozygous for the rs855791 minor TMPRSS6 allele. In accordance with another series of embodiments, the method further comprises an earlier step of determining that the patient is homozygous for the minor rs855791 TMPRSS6 allele. According to exemplary embodiments, the patient has elevated serum levels of IL-6 prior to treatment. In accordance with various embodiments, the patient has elevated serum levels of CRP prior to treatment.

In accordance with various embodiments, the patient has a hepcidin-mediated disorder selected from the disorders described in section 5.2.1 above. In accordance with certain embodiments, the patient is anemic for a chronic disease.

6.6. Methods for the treatment of IL-6 mediated inflammatory disorders.

From the data presented in Examples 2, 3, and 5 below, it can be seen that IL-6 antagonists are beneficial in subjects who have elevated pre-treatment IL-6 levels and at least one copy of the major TMPRSS6 allele, even in the absence of anemia. . Therefore, in accordance with another aspect, the present invention relates to methods for the treatment of IL-6 mediated inflammatory disorders in patients without anemia with chronic inflammation.

The methods include administering a therapeutically effective amount of an IL-6 antagonist to a subject, typically a human patient, who has an IL-6 mediated inflammatory disease, the patient is not anemic, and the subject has been determined to have at least one copy of the major rs855791 TMPRSS6 allele. In accordance with the first series of embodiments, the subject has previously been determined to have at least one copy of the core rs855791 TMPRSS6 allele. In accordance with another series of embodiments, the method further comprises an earlier step of determining that the subject has at least one copy of the core rs855791 TMPRSS6 allele. Typically, the methods include excluding treatment of subjects if they are positively homozygous for the rs855791 minor allele of TMPRSS6. Usually the patient has elevated serum levels of IL6 before starting treatment.

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

In accordance with specific embodiments, the patient has a DAS28 score greater than 5.1 prior to treatment. In accordance with some embodiments, the patient has a DAS28 score of 3.2 to 5.1 prior to treatment. In accordance with some embodiments, the patient has a DAS28 score of less than 2.6 prior to treatment. According to various embodiments, the patient's RA is highly active prior to treatment. According to some embodiments, the patient's RA is moderately active prior to treatment.

In accordance with certain embodiments, the patient has been treated with methotrexate. According to some embodiments, methotrexate is discontinued when treatment with the IL-6 antagonist is initiated. According to some embodiments, methotrexate is continued at the start of treatment with the IL-6 antagonist.

In accordance with certain embodiments, the patient has been treated with an anti-TNFa agent. In certain embodiments, the anti-TNFa agent is selected from etanercept, adalimumab, infliximab, certolizumab, and golimumab. In certain embodiments, the anti-TNFa agent is discontinued when treatment with the IL-6 antagonist is initiated.

In accordance with certain embodiments, the patient has been treated with an IL-1 receptor antagonist. According to specific embodiments, the IL-1 receptor antagonist is anakinra. According to certain embodiments, the IL-1 receptor antagonist is interrupted when treatment with the IL-6 antagonist is initiated.

In accordance with certain embodiments, the patient has been treated with abatacept. In certain embodiments, abatacept is discontinued when treatment with an IL-6 antagonist is started.

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.

6.7. Serum levels of IL-6 and CRP before starting treatment.

In accordance with typical embodiments of the methods described herein, the patient has elevated serum levels of IL-6 prior to treatment.

In accordance with some embodiments, the patient has a pre-treatment serum IL-6 level greater than 2.5 pg/mL. According to various embodiments, the patient has a pre-treatment serum IL-6 level of 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

In some embodiments, the IL-6 antagonist is administered at a dose, schedule, and for a period sufficient to reduce the patient's serum IL-6 levels below pre-treatment levels. In certain embodiments, the IL-6 antagonist is dosed according to the schedule and for a period sufficient to reduce the patient's serum levels of IL-6 by at least 10%, 20%, 30%, 40%, or 50% compared to the levels before starting treatment.

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

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

6.8. Genotyping rs855791 TMPRSS6.

The methods described herein involve administering a therapeutically effective amount of an IL-6 antagonist to a subject who has been determined to have at least one copy of the core rs855791 TMPRSS6 allele. Preferably, both alleles are detected that correspond to the gene of interest, thus allowing the identification and differentiation of patients who are homozygous for the major rs855791 TMPRSS6 allele, heterozygous for the major and minor alleles of rs855791 TMPRSS6, and homozygous for the minor allele of rs855791 TMPRSS6.

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

As a rule, PCR is used to amplify a biological sample obtained from a patient.

According to some embodiments, the absence or presence of a polymorphism is detected simultaneously with amplification by real-time PCR (RT-PCR). In certain embodiments, the RT-PCR assay uses 5'-nuclease (TaqMan® probes), molecular beacons, and/or FRET hybridization probes. For a review, see Espy et al., Clin. microbiol. Rev. January 2006; 19(1):165-256, incorporated herein by reference in its entirety. In accordance with typical embodiments, a commercially available assay kit is used. According to the selected embodiments, the commercially available assay kit is selected from the group consisting of TaqMan™ SNP Genotyping Assays (ThermoFisher), PCR SNP Genotyping Assay (Qiagen), Novallele Genotyping Assays ( Canon) and SNP Type™ assays (formerly SNPtype) (Fluidigm).

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

6.9. Antagonists of IL-6.

An IL-6 antagonist is used in the methods described herein that can reduce the biological activity of IL-6.

6.9.1. Antibodies to IL-6.

According to various embodiments, the IL-6 antagonist is an anti-IL-6 antibody, or an antigen-binding fragment or derivative thereof.

According to some embodiments, the IL-6 antagonist is a full-length anti-IL-6 monoclonal antibody. In certain embodiments, the full length monoclonal antibody is an IgG antibody. In certain embodiments, the full-length monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the IL-6 antagonist is a polyclonal composition comprising a plurality of full-length anti-IL-6 antibodies, each of such a plurality having unique CDRs. According to 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 an scFv, a disulfide-linked Fv(dsFv) or a single domain antibody such as a camel-backed VHH single domain nanobody. According to some embodiments, the IL-6 antagonist is an immunoconjugate or hybrid containing an IL-6 antigen-binding fragment. In some embodiments, the antibody is bispecific or multispecific, wherein at least one of the antigen-binding moieties is specific for IL-6.

In some embodiments, the antibody is fully human. In accordance with some embodiments, the antibody is humanized. In some embodiments, the antibody is chimeric and has non-human V regions and human C region domains. In accordance with some embodiments, the antibody is murine.

According to exemplary embodiments, an anti-IL-6 antibody has a K _D for human IL-6 binding of less than 100 nM. In some embodiments, an anti-IL-6 antibody has a K _D for human IL-6 binding of less than 75, 50, 25, 20, 15, or 10 nM. In certain embodiments, an anti-IL-6 antibody has a K _D for human IL-6 binding of less than 5, 4, 3, or 2 nM. According to selected embodiments, the anti-IL-6 antibody has a K _D for human IL-6 binding of less than 1 nM, 750 pM, or 500 pM. According to specific embodiments, an anti-IL-6 antibody has a KD for human IL-6 binding of no more than 500, 400, 300, 200, or 100 pM.

According to exemplary embodiments, an anti-IL-6 antibody neutralizes the biological activity of IL-6. In some embodiments, the neutralizing antibody prevents IL-6 from binding to the IL-6 receptor.

According to exemplary embodiments, an anti-IL-6 antibody has a half-life after intravenous administration of at least 7 days. In certain embodiments, the anti-IL-6 antibody has a half-life of at least 14 days, at least 21 days, or at least 30 days.

In some embodiments, an anti-IL-6 antibody has a human IgG constant region with at least one amino acid substitution that prolongs the serum half-life compared to a human IgG constant domain without the substitution.

In certain embodiments, the IgG constant domain contains substitutions at residues 252, 254, and 256, wherein the amino acid substitution at amino acid residue 252 is a tyrosine substitution, the amino acid substitution at amino acid residue 254 is a threonine substitution, and the amino acid substitution at amino acid residue 254 is a threonine substitution, and the amino acid substitution at amino acid residue 252 is residue 256 is a substitution for glutamic acid (YTE). See US Pat. No. 7,083,784, incorporated herein by reference in its entirety. According to specific 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)).

In accordance with some embodiments, the half-life of an anti-IL-6 antibody

- 23 041768 increase using 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, an anti-IL-6 antibody is hybridized to bacterial albumin binding domains (Stork et al., Prot. Eng. Design Science, 20:569-76 (2007)). In some embodiments, an anti-IL-6 antibody is hybridized to an albumin binding peptide (Nguygen et al., Prot. Eng. Design Sel. 19:291297 (2006)). In some embodiments, an anti-IL antibody is bispecific, with one specificity directed to IL-6 and the other specificity directed to human serum albumin (Ablynx, WO 2006/122825 (bispecific nanobody)).

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

6.9.1.1. MED5117 and derivatives.

In certain embodiments, the anti-IL-6 antibody, or antigen-binding portion thereof, contains all six CDRs from MEDI5117. In certain embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain V region and a light chain V region from MEDI5117. In accordance with specific embodiments, the antibody is a full length MEDI5117 antibody. The MEDI5117 antibody is described in WO 2010/088444 and US 2012/0034212, the disclosures of which are incorporated herein by reference in their entirety. The MEDI5117 antibody has the following CDR and heavy and light chain sequences: 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)

MEDI5117 Heavy chain

EVQLVESGGGLVQPGGSLRLSCAASGFTISSNYMIWVRQAPGKGLEWVSDLYYYAGDTYY

ADSVKGRFTMSRDISKNTVYLQMNSLRAEDTAVYYCARWADDHPPWIDLWGRGTLVTVSS

ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLYITREPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:18)

MEDI5117 Light chain

DIQMTQSPSTLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKVLIYKASTLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQSWLGGSFGQGTKLEIKRTVAAPSVFIFPPS

- 24 041768

According to various embodiments, the anti-IL-6 antibody is a derivative of MED5117.

In accordance with some embodiments, a derivative of MED5117 comprises one or more amino acid substitutions in the V regions of the heavy and/or light chain of MED5117.

According to certain embodiments, the derivative contains 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 1 amino acid substitution relative to the original VH and/or V _L of the anti-IL-6 antibody MEDI5117 while maintaining specificity for human IL-6.

In accordance with certain embodiments, a derivative of MED5117 contains an amino acid sequence that is 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% amino acid sequence identical VH and VL domains from MEDI5117. Percent sequence identity is determined using BLAST algorithms using default parameters.

In accordance with certain embodiments, the MED5117 derivative comprises an amino acid sequence wherein the CDRs comprise an amino acid sequence that is 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 at least 99% identical to the amino acid sequence of the corresponding CDRs from MEDI5117. Percent sequence identity is determined using BLAST algorithms using default parameters. In certain embodiments, the VH and/or VL 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 essential for the antibody to specifically bind to human IL6).

6.4.1.2. Other antibodies to IL-6.

According to various embodiments, an anti-IL-6 antibody comprises six CDRs from an antibody selected from the group consisting of siltuximab, gerilimzumab, sirukumab, clasakizumab, 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, an anti-IL-6 antibody comprises a heavy chain V region and a light chain V region from an antibody selected from the group consisting of siltuximab, gerilimzumab, sirukumab, clasakizumab, 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 certain embodiments, the anti-IL-6 antibody is an antibody selected from the group consisting of siltuximab, gerilimzumab, sirukumab, clasakizumab, 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 some embodiments, an anti-IL-6 antibody contains six CDRs from an antibody selected from those described in US 2016/0168243, US 2016/0130340, US 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, US 5856135, US 2006/02400012 or US 2006/0240012 7291721, the disclosure of which is incorporated herein by reference in their entirety.

6.9.2. Antibodies to the IL-6 receptor.

According to various embodiments, the IL-6 antagonist is an anti-IL-6 receptor antibody, or an antigen-binding fragment or derivative thereof.

According to some embodiments, the IL-6 antagonist is a full-length monoclonal antibody to the IL-6 receptor. In certain embodiments, the full length monoclonal antibody is an IgG antibody. In certain embodiments, the full-length monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In some embodiments, the IL-6 antagonist is a polyclonal composition comprising a plurality of full-length IL-6 receptor antibodies, each of such a plurality having unique CDRs. According to some embodiments, the IL-6 antagonist is an antibody fragment selected from Fab, Fab' fragments. In some embodiments, the IL-6 antagonist is an scFv, single domain antibody, including a camel VHH single domain nanobody. In some embodiments, the antibody is bispecific or multispecific, wherein at least one of the antigen-binding moieties is specific for IL-6R.

In some embodiments, the antibody is fully human. In accordance with some embodiments, the antibody is humanized. In some embodiments, the antibody is chimeric and has non-human V regions and human C region domains. In accordance with some embodiments, the antibody is murine.

According to exemplary embodiments, an anti-IL-6 receptor antibody has a K _D for human IL-6R binding of less than 100 nM. In some embodiments, an anti-IL-6R antibody has a K _D for human IL-6R binding of less than 75, 50, 25, 20, 15, or 10 nM. In certain embodiments, the IL-6 receptor antibody has a KD for human IL-6R binding of less than 5, 4, 3, or 2 nM. According to selected embodiments, the IL-6 receptor antibody has a KD for human IL-6R binding of less than 1 nM, 750 pM, or 500 pM. According to specific embodiments, the IL-6 receptor antibody has a K _D for human IL-6R binding of no more than 500, 400, 300, 200, or 100 pM.

According to exemplary embodiments, an anti-IL-6R antibody reduces the biological activity of IL-6.

According to exemplary embodiments, an anti-IL-6R antibody has a half-life after intravenous administration of at least 7 days. In certain embodiments, an anti-IL-6R antibody has a half-life of at least 14 days, at least 21 days, or at least 30 days.

In some embodiments, an anti-IL-6R antibody has a human IgG constant region with at least one amino acid substitution that prolongs the serum half-life compared to a human IgG constant domain without the substitution.

In certain embodiments, the IgG constant domain contains substitutions at residues 252, 254, and 256, wherein the amino acid substitution at amino acid residue 252 is a tyrosine substitution, the amino acid substitution at amino acid residue 254 is a threonine substitution, and the amino acid substitution at amino acid residue 254 is a threonine substitution, and the amino acid substitution at amino acid residue 252 is residue 256 is a substitution for glutamic acid (YTE). See US Pat. No. 7,083,784, incorporated herein by reference in its entirety. According to specific 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)).

In some embodiments, the half-life of an anti-IL-6R antibody is increased by 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, an anti-IL-6R antibody is hybridized to bacterial albumin binding domains (Stork et al., Prot. Eng. Design Science, 20:569-76 (2007)). In some embodiments, an anti-IL-6 antibody is hybridized to an albumin binding peptide (Nguygen et al., Prot. Eng. Design Sel. 19:291297 (2006)). In some embodiments, an anti-IL antibody is bispecific, with one specificity directed to IL-6R and the other specificity directed to human serum albumin (Ablynx, WO 2006/122825 (bispecific nanobody)).

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

In certain embodiments, the anti-IL-6R antibody, or antigen-binding portion thereof, contains all six CDRs from tocilizumab. In certain embodiments, the antibody or antigen-binding portion thereof comprises a heavy chain V region and a light chain V region from tocilizumab. In accordance with specific embodiments, the antibody is a full-length tocilizumab antibody.

In certain embodiments, the anti-IL-6R antibody, or antigen-binding portion thereof, contains all six CDRs from sarilumab. In certain embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain V region and a light chain V region from sarilumab. In accordance with specific embodiments, the antibody is a full length sarilumab antibody.

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

- 26 041768

In some embodiments, the anti-IL-6R antibody, or antigen-binding portion thereof, is a single domain antibody. In certain embodiments, the single domain antibody is a camel VHH single domain antibody. According to specific embodiments, the antibody is vobarilizumab (ALX0061) (Ablynx NV).

6.9.3. Antibodies to the IL-6:IL-6R complex.

According to various embodiments, the IL-6 antagonist is an antibody specific for a complex of IL-6 and IL-6R. In accordance with certain embodiments, the antibody has six CDRs from an antibody selected from those described in US 2011/0002936, which is incorporated herein by reference in its entirety.

6.9.4. JAK and STAT inhibitors.

IL-6 is known to signal through the JAK-STAT signaling pathway.

According to various embodiments, the IL6 antagonist is an inhibitor of the JAK signaling pathway. According to some embodiments, the JAK inhibitor is a JAK1-specific inhibitor. According to some embodiments, the JAK inhibitor is a JAK3-specific inhibitor. According to some embodiments, the JAK inhibitor is a pan-JAK inhibitor.

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.

According to various embodiments, the IL-6 antagonist is a STAT3 inhibitor. According to a specific embodiment, the inhibitor is AZD9150 (AstraZeneca, Isis Pharmaceuticals), a STAT3 antisense molecule.

6.9.5. Additional antagonists of IL-6.

According to various embodiments, the IL-6 antagonist is an antagonist peptide.

In certain embodiments, the IL-6 antagonist is C326 (an IL-6 inhibitor from Avidia, also known as AMG220) or FE301, a recombinant IL-6 inhibitor protein (Ferring International Center S.A., Conaris Research Institute AG). In some embodiments, the IL-6 antagonist antibody comprises soluble gp130, FE301 (Conaris/Ferring).

6.10. Dosing regimens.

6.10.1. Antibodies, antigen-binding fragments, peptides.

According to exemplary embodiments, the antibody, antigen-binding fragments, and IL-6 peptide antagonists are administered parenterally.

In some parenteral embodiments, the IL-6 antagonist is administered intravenously. In some intravenous embodiments, the IL-6 antagonist is administered as a single dose. In some intravenous embodiments, the IL-6 antagonist is administered as an infusion. In some intravenous embodiments, the IL-6 antagonist is administered as a bolus followed by an infusion. In some parenteral embodiments, the IL-6 antagonist is administered subcutaneously.

According to various embodiments, the antibody, antigen-binding fragment, and IL-6 peptide antagonist are administered at a dose that is independent of the patient's weight or surface area (fixed dose).

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

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

- 27 041768 or 90-100 mg. In some embodiments, the subcutaneous fixed dose is 100-125, 125-150, 150-175, 175-200, or 200-250 mg.

According to various embodiments, the antibody, antigen-binding fragment, and IL-6 peptide antagonist are administered in a weight-dependent dose of the patient.

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

In some embodiments, the subcutaneous weight dependent dose is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 .0 mg/kg. In some embodiments, the antagonist is administered at a dose of 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 mg/kg.

According to various intravenous-related 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. According to various subcutaneous embodiments, the IL-6 antagonist is administered every 14 days, every 28 days, once a month, every two months (every other month), or every three months.

According to certain preferred embodiments, the IL-6 antagonist is the MEDI5117 antibody. According to various embodiments, MEDI5117 is administered as a fixed dose of 1-30 mg intravenously once a week. In certain embodiments, the MEDI5117 antibody is administered as a fixed dose of 1, 2, 3, 4, 5, 7.5, 10, 15, 20, 25, or 30 mg intravenously once a week. In accordance with some embodiments, the MEDI5117 antibody is administered in a fixed dose of 25-250 mg subcutaneously at a frequency of once a month - every three months. In certain embodiments, MEDI5117 is administered at a dose of 30, 45, 60, 75, 100, 120, 125, 150, 175, 200, 225, 240, or 250 mg subcutaneously once a month, every two months, or every 3 months .

According to some embodiments, the IL-6 antagonist is tocilizumab. According to various embodiments, tocilizumab is administered subcutaneously to patients <100 kg at an initial dose of 162 mg once a week. In some embodiments, tocilizumab is administered intravenously at 4 mg/kg every 4 weeks followed by an increase to 8 mg/kg every 4 weeks based on clinical response.

6.10.2. JAK and STAT inhibitors.

According to exemplary embodiments, small molecule JAK inhibitors and STAT inhibitors are administered orally.

In various embodiments, the inhibitor is administered once or twice a day in an oral dose of 1-10, 10-20, 20-30, 30-40, or 40-50 mg. In some embodiments, the inhibitor is administered once or twice a day at a dose of 50-60, 60-70, 70-80, 80-90, or 90-100 mg. In some embodiments, the inhibitor is administered at a dose of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg orally once or twice a day. In some embodiments, the inhibitor is administered at a dose of 75 mg orally once a day or twice a day, 100 mg orally once a day or twice a day.

In certain embodiments, the JAK inhibitor is tofacitinib and is administered at a dose of 5 mg orally twice a day or 11 mg orally every day,

In certain embodiments, the JAK inhibitor is decernotinib and is administered at a dose of 25, 50, 100, or 150 mg orally twice a day.

In certain embodiments, the inhibitor is ruxolitinib and is administered at a dose of 25 mg orally twice a day, 20 mg orally twice a day, 15 mg orally twice a day, 10 mg orally twice a day, or 5 mg orally twice a day.

6.11. Additional therapeutic agents.

According to various embodiments of the methods described herein, the method further comprises administering a therapeutic agent in addition to the IL-6 antagonist, wherein the second therapeutic agent is also capable of downregulating hepcidin expression.

In accordance with some embodiments, the second therapeutic agent is a BMP antagonist. In certain embodiments, the BMP antagonist is an anti-BMP6 antibody. In certain embodiments, an anti-BMP6 antibody has six CDRs from the antibody described in US 2016/0176956 or US 2016/0159896, the disclosure of which is incorporated herein by reference in its entirety.

In accordance with certain embodiments, the second therapeutic agent is a hemojewelin antagonist. In certain embodiments, the hemojewelin antagonist is an anti-hemojewelin antibody. According to specific embodiments, the anti-haematolin antibody has six CDRs from the antibodies disclosed in Kovac et al., Haematologica (2016), doi:10.3324/haematol.2015.140772 [ePub ahead of print].

- 28 041768

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

6.12. Sets.

In accordance with another aspect, the present invention relates to kits.

According to exemplary embodiments, the kits provide reagents for determining, from a biological sample obtained from a patient, the patient's genotype at the location of the rs855791 TMPRSS6 SNP.

6.13. Additional Aspects and Embodiments.

6.13.1. Methods for treating inflammation in chronic kidney disease or cardiovascular disease.

In accordance with other aspects and embodiments, the present invention relates to compositions and methods for characterizing and treating inflammation in chronic kidney disease or cardiovascular disease with an IL-6 antagonist, as well as methods for characterizing a patient's responsiveness to treatment.

These aspects and embodiments are based, at least in part, on the finding that inflammation in patients with chronic kidney disease and in patients with cardiovascular disease having one or more TMPRSS6 alleles containing G or C at nucleotide position 2321 ( encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736) increased the risk of death in these patients, and that such subjects can be treated with an IL-6 antagonist to reduce this risk. As discussed in more detail below, patients with chronic kidney disease were subjected to genotyping, and analyzed their levels of IL-6 and CRP in the blood serum, and these diagnostic data compared with the administered dose of EPO and the risk of death. Patients with one or more TMPRSS6 alleles containing G or C at nucleotide position 2321 (encoding the TMPRSS6 polypeptide containing alanine at amino acid position 736) and elevated levels of IL-6 and/or CRP required higher doses of EPO for therapy and had a higher mortality. The nucleotide at this position has been shown to be important in identifying patients with iron deficiency anemia (see Finberg et al., Nat. Genet. 2008; 40(5): 569-571, which is incorporated herein by reference throughout its completeness in relation to all the ideas and sequences, variants, nomenclature, etc. disclosed in it). These data strongly support the identification of a subset of patients, based on the TMPRSS6 genotype, who require a higher dose of EPO and/or have a higher risk of death and are likely to respond to IL-6 inhibition with or without standard therapy during anemia treatment (e.g., associated with chronic kidney disease). By inhibiting inflammation, the dose of EPO can be reduced, thereby avoiding the adverse side effects of EPO (eg, the risk of developing cardiovascular disorders).

These aspects and embodiments are further based on the finding that patients with one or more TMPRSS6 alleles containing G or C at nucleotide position 2321 (encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736) have a higher risk of death associated with with myocardial infarction or cardiovascular disease. These patients are also likely to benefit from IL-6 inhibition, which will reduce inflammation and risk.

Accordingly, the present invention relates to therapeutic methods for treating inflammation associated with cardiovascular disease or chronic kidney disease, including anemia in chronic kidney disease, and/or reducing the risk of death associated with such conditions, by inhibiting the biological activity of IL-6, e.g., in patients selected by TMPRSS6 genotyping at the rs855791 SNP, by blocking either IL-6 or its receptor (gp80) binding to each other or signaling or expressing it (e.g., by anti-IL-6 antibody or anti-IL -6R or by inhibition of JAK1/STAT3). In one embodiment, treatment of chronic kidney disease is administered with or without standard treatment for anemia, as well as methods for characterizing the susceptibility of a patient suffering from chronic kidney disease to treatment of anemia, for example, by genotyping TMPRSS6 at the rs855791 SNP and detecting levels of inflammatory markers (eg, elevated serum levels of IL-6 and/or CRP).

The present invention relates to methods for treating cardiovascular disease or anemia in chronic kidney disease and/or reducing the likelihood of death associated with chronic inflammation in such patients by administering an agent that inhibits the biological activity or expression of IL-6.

In accordance with some aspects and embodiments, the present invention relates to compositions and methods for the treatment of chronic inflammation, which increases mortality in

- 29 041768 subjects with chronic kidney disease or cardiovascular disease, and characteristics of patients' susceptibility to such therapies. In accordance with certain embodiments, the present invention relates to methods for characterizing and treating anemia in chronic inflammation and mortality (e.g., in chronic kidney disease), as well as characterizing a patient's responsiveness to anemia treatment (e.g., administration of erythropoietin or erythropoiesis-stimulating agents). In one aspect, the present invention relates to methods for treating chronic inflammation in a selected subject, which method comprises administering to the subject an IL-6 antagonist, the subject being selected for treatment by the presence of one or more alleles encoding a TMPRSS6 polypeptide containing an alanine in the amino acid position 736.

According to another aspect, the present invention provides methods for treating inflammation or chronic inflammation in a selected subject with cardiovascular disease or chronic kidney disease, the method comprising administering to the subject an IL-6 antagonist (e.g., an anti-IL-6 antibody), wherein the subject is selected for treatment by the presence of one or more alleles encoding the TMPRSS6 polypeptide containing alanine at amino acid position 736. In accordance with one embodiment, the method reduces the risk of mortality in the subject. In accordance with one embodiment, the subject has a history of myocardial infarction or heart failure.

According to another aspect, the present invention relates to methods for reducing inflammation and risk of mortality in a selected subject with cardiovascular or kidney disease, the method comprising administering to the subject an IL-6 antagonist (e.g., an anti-IL-6 antibody), wherein the subject selected for treatment by the presence of one or more alleles encoding the TMPRSS6 polypeptide containing alanine at amino acid position 736 and increased inflammation relative to the reference. In accordance with one embodiment, the subject has a history of myocardial infarction or heart failure.

According to another aspect, the present invention relates to methods for reducing the risk of mortality in a subject with chronic kidney disease or heart failure, the method comprising administering to the subject an IL-6 antagonist, the subject being identified as having one or more alleles encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736, and increased inflammation relative to the reference.

In another aspect, the present invention relates to methods for treating anemia in a subject, the method comprising administering to the subject an IL-6 antagonist alone or in combination with anemia therapy, the subject being identified as having one or more alleles encoding a TMPRSS6 polypeptide (also referred to as matriptase-2; MT2) containing alanine at amino acid position 736 (eg, having G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) and increased inflammation relative to the reference.

According to another aspect, the present invention provides methods for treating anemia in a subject with increased inflammation, the method comprising administering an IL-6 antagonist (e.g., an anti-IL-6 antibody) alone or in combination with an erythropoietic factor in an amount effective to neutralize inflammation in a subject with one or more alleles encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736 (eg, with G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule).

According to yet another aspect, the present invention relates to methods for enhancing EPO susceptibility in a subject identified as in need thereof, the method comprising administering an IL-6 antagonist (e.g., an anti-IL-6 antibody) in an amount effective to neutralize inflammation in a subject with one or more alleles encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736 (eg, with G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule), thereby reducing the dose of EPO.

According to another aspect, the present invention relates to methods for reducing mortality in a subject with increased inflammation, the method comprising administering an IL-6 antagonist in an amount effective to neutralize inflammation in a subject with one or more alleles encoding a TMPRSS6 polypeptide containing an alanine in amino acid position 736 (eg, with G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule).

According to yet another aspect, the present invention relates to methods for selecting therapy for a subject who has been identified as in need thereof, the method comprising characterizing a subject with one or more alleles encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736 (e.g., with G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule); and detecting the level of one or more of the inflammatory markers IL-6 or CRP, such a characteristic indicating that it is necessary to administer the IL-6 antagonist alone or in combination with anemia therapy.

In accordance with another aspect, the present invention relates to methods for increasing the proliferation or survival of an erythrocyte or its precursor (for example, hematopoietic

- 30 041768 stem cell, proerythroblast, erythroblast or reticulocyte) in a subject who is identified as needing it, while the method involves administering to the subject an IL-6 antagonist or erythropoietic factor, and the subject is identified as having one or more alleles encoding the TMPRSS6 polypeptide, containing alanine at amino acid position 736 (eg, having G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule), and increased inflammation relative to the reference.

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

According to various embodiments of any of the aspects described herein, anemia therapy involves the administration of an erythropoietic factor. According to various embodiments, the erythropoietic factor is one or more of an erythropoietin, an erythropoiesis-stimulating agent, a HIF stabilizer, and supplemental iron.

According to various embodiments, increased inflammation is characterized by elevated levels of IL-6 and/or CRP relative to a reference (e.g., as measured by conventional CRP assays or high sensitivity assays (hsCRP), both of which can detect CRP but are different analytical properties). According to various embodiments, increased inflammation is characterized by an IL-6 greater than about 5 pg/mL. According to various embodiments, increased inflammation is characterized by a CRP greater than about 2 mg/l.

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

In accordance with various embodiments of any of the aspects described herein, administration of an IL-6 antagonist or anti-IL-6 antibody reduces the dose of EPO. In certain embodiments, the dose of EPO is reduced by about 40 IU/kg/week, about 50 IU/kg/week, about 80 IU/kg/week, about 100 IU/kg/week, or more. According to various embodiments, administration of an IL-6 antagonist or an anti-IL-6 antibody reduces the side effect of an increased dose of EPO.

According to one embodiment, patients with chronic kidney disease are treated with or without standard anemia management. In particular, an agent that inhibits the biological activity or expression of IL-6 is administered to a subject with anemia associated with chronic kidney disease with or without treatment for the anemia (eg, with EPO, ESA, HIF stabilizers, supplemental iron, or with red blood cell transfusion) . Various treatments for anemia involve stimulating erythropoiesis, or the production of red blood cells. Thus, these are agents that increase the growth or proliferation and/or decrease the death of an erythrocyte or its precursor. To the number

- 31 041768 precursors of erythrocytes include, for example, hematopoietic stem cells, common myeloid precursors, proerythroblasts, erythroblasts, reticulocytes, or any cell capable of differentiating or maturing into an erythrocyte.

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

According to another aspect, the present invention provides assays that are useful in characterizing a subject with anemia associated with chronic inflammation (eg, chronic kidney disease). The inflammatory markers IL-6 and CRP can be detected by any suitable method. The methods described herein can be used alone or in combination to detect an IL-6 or CRP biomarker and/or an inflammatory condition. According to one embodiment, inflammation is characterized by detecting the level of an IL-6 polypeptide and/or CRP in a biological sample (e.g., blood serum) from a subject relative to expression in a reference (e.g., blood serum from a healthy control subject), with an increase in IL expression -6 and/or CRP is indicative of inflammation. In another embodiment, an increase in IL-6 and/or CRP expression indicates that a subject with anemia associated with chronic kidney disease will not be responsive to anemia treatment and/or will be responsive to anemia treatment when administered in combination with an IL antagonist. -6 (for example, an antibody to IL-6).

In accordance with one embodiment, the level of the IL-6 polypeptide and/or CRP is measured using an immunoassay. An immunoassay typically involves the use of an antibody (or other agent that specifically binds a marker) to detect the presence or level of a biomarker in a sample. Antibodies can be obtained by methods well known in the art, for example by immunizing animals with a biomarker or fragments thereof. Biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of the polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies using methods that are well known in the art.

In accordance with various embodiments, traditional immunoassays are used, including, for example, Western blotting, sandwich-type immunoassays, including ELISA, and other enzyme immunoassays, fluorescent immunoassays, and chemiluminescence. Nephelometry is a liquid-phase analysis in which antibodies are in solution. The binding of an antigen to an antibody results in changes in absorbance, which is measured. Other forms of immunoassay include magnetic immunoassay, radioimmunoassay, and real-time immunoquantitative PCR (iqPCR). Other detection methods include liquid chromatography and mass spectrometry.

Immunoassays can be performed on solid substrates (eg, chips, beads, microfluidic platforms, membranes) or any other form that supports binding of the antibody to the marker and subsequent detection. One marker may be detected at a time, or a multiplex format may be used. The multiplex immunoassay may involve the use of flat microarrays (protein chips) or granular microarrays (suspension assays).

Anemia patients with chronic kidney disease who are identified as having elevated levels of IL-6 polypeptides and/or CRP are selected for treatment with an agent that reduces the expression or activity of IL-6 (for example, an antibody to IL-6), in combination with treatment anemia. Patients treated with the method of the present invention can be monitored by detecting changes in hemoglobin, hematocrit, erythropoietin dose, IL-6 and/or CRP expression after treatment. Patients who experience a decrease in IL-6 and/or CRP expression and/or a decrease in inflammation are found to be susceptible to IL-6 inhibition.

Other aspects and embodiments are presented in the following numbered paragraphs.

1. A method of treating chronic inflammation in a selected subject, the method comprising administering to the subject an IL-6 antagonist, the subject being selected for treatment by the presence of one or more alleles encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736.

2. A method of treating inflammation in a selected subject with cardiovascular disease, heart failure, or chronic kidney disease, the method comprising administering an IL-6 antagonist to the subject, the subject being selected for treatment by the presence of one or more alleles encoding an alanine-containing TMPRSS6 polypeptide at amino acid position 736.

3. A method for reducing inflammation and risk of mortality in a selected subject with cardiovascular disease, heart failure and/or chronic kidney disease, the method comprising administering an IL-6 antagonist to the subject, the subject being selected for treatment by the presence of one or more alleles encoding a TMPRSS6 polypeptide , containing alanine at amino acid position 736, and increased inflammation relative to the reference.

4. A method of treating anemia in a subject with chronic kidney disease, the method comprising administering to the subject an IL-6 antagonist, the subject being identified as having one or more alleles encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736 and increased inflammation relative to a reference.

5. A method according to any one of claims 1 to 4, wherein the IL-6 antagonist is administered in an amount effective to neutralize inflammation.

6. A method according to any one of claims 1 to 4, wherein the IL-6 antagonist is an anti-IL-6 antibody.

7. The method of claim 5, wherein the method further comprises administering an erythropoietic factor to the subject.

8. A method according to any one of claims 1 to 4, wherein the method reduces the risk of mortality in the subject.

9. A method for reducing the risk of mortality in a subject with chronic kidney disease or heart failure, the method comprising administering to the subject an IL-6 antagonist, the subject being identified as having one or more alleles encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736, and an increased inflammation relative to the standard.

10. A method of treating anemia in a subject with increased inflammation, the method comprising: administering an erythropoietic factor and an anti-IL-6 antibody in an amount effective to neutralize inflammation in a subject with one or more alleles encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736 .

11. A method according to any one of claims 1 to 10, wherein the increased inflammation is characterized by elevated levels of IL-6 and/or CRP relative to a reference.

12. The method of claim 11, wherein the increased inflammation is characterized by an IL-6 greater than about 5 pg/ml, about 10 pg/ml, or about 15 pg/ml.

13. The method of claim 10, wherein the increased inflammation is characterized by a CRP greater than about 2 mg/l.

14. The method of claim 10, wherein the erythropoietic factor is one or more of an erythropoietin, an erythropoiesis-stimulating agent, a HIF stabilizer, and additional iron.

15. A method of enhancing EPO susceptibility in a subject identified as in need thereof, the method comprising administering an IL-6 antagonist or an anti-IL-6 antibody in an amount effective to neutralize inflammation in a subject with one or more alleles encoding a TMPRSS6 polypeptide , containing alanine at amino acid position 736, thus enhancing EPO susceptibility in the subject.

16. The method of claim 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. ml.

17. The method of claim 16, wherein the amount of the IL-6 antagonist or anti-IL-6 antibody effective to neutralize inflammation reduces the CRP to less than about 2 mg/L.

18. The method of claim 15, wherein administration of the IL-6 antagonist or anti-IL-6 antibody reduces the dose of EPO.

19. The method of claim 17, wherein the dose of EPO is reduced by about 40 IU/kg/week, by about 50 IU/kg/week, by about 80 IU/kg/week, by about 100 IU/kg/week, or more.

- 33 041768

20. The method of claim 15, wherein administration of the IL-6 antagonist or anti-IL-6 antibody reduces the side effect of the increased dose of EPO.

21. A method for selecting therapy for a subject who is identified as in need of it, while the method includes:

a) characterizing the subject as having one or more alleles encoding a TMPRSS6 polypeptide containing an alanine at amino acid position 736; And

b) detecting the level of one or more of the inflammatory markers IL-6 and CRP, such a characteristic indicating that it is necessary to administer an IL-6 antagonist in combination with anemia therapy.

22. The method of claim 21, the method further comprising administering an IL-6 antagonist to the subject and administering therapy for the anemia.

23. The method of claim 21, wherein the treatment of anemia involves the administration of an erythropoietic factor.

24. A method for increasing the proliferation or survival of an erythrocyte or a precursor thereof in a subject identified as in need thereof, the method comprising administering to the subject an IL-6 antagonist and an erythropoietic factor, wherein the subject is identified as having one or more alleles encoding a TMPRSS6 polypeptide containing alanine at amino acid position 736, and moreover, the subject has increased inflammation relative to the reference.

25. The method of claim 24, wherein the method reduces the death of an erythrocyte or its precursor.

26. The method of claim 24, wherein the precursor is a hematopoietic stem cell, proerythroblast, erythroblast, or reticulocyte.

27. The method of any one of claims 15-24, wherein the subject has chronic kidney disease.

28. The method of any one of claims 15-24, wherein the subject is anemic.

29. The method of claim 28, wherein the anemia is cancer anemia, chronic autoimmune disease anemia, chronic inflammatory disease anemia, or metabolic syndrome anemia.

30. The method of any one of claims 15 to 24, wherein the IL-6 antagonist is administered in an amount effective to neutralize inflammation.

31. The method of any one of claims 15-24, wherein the IL-6 antagonist is an anti-IL-6 antibody.

32. The method of any one of claims 15-24, wherein the increased inflammation is characterized by elevated levels of IL-6 and/or CRP relative to a reference.

33. The method of any one of claims 15-24, wherein the increased inflammation is characterized by an IL-6 greater than about 5 pg/ml, about 10 pg/ml, or about 15 pg/ml.

34. The method of any one of claims 15-24, wherein the increased inflammation is characterized by a CRP greater than about 2 mg/l.

35. The method of any one of claims 15-24, wherein the amount effective to neutralize inflammation reduces IL-6 to less than about 10 pg/ml or less than about 5 pg/ml.

36. The method of any one of claims 15-24, wherein the amount effective to neutralize inflammation reduces the CRP to less than about 2 mg/L.

37. The method of any one of claims 15-24, wherein the erythropoietic factor is one or more of an erythropoietin, an erythropoiesis-stimulating agent, a HIF stabilizer, and additional iron.

38. The method of claim 24, wherein administering the IL-6 antagonist reduces the dose of EPO.

39. The method of claim 38, wherein the IL-6 antagonist is an anti-IL-6 antibody.

40. The method of claim 38, wherein the dose of EPO is reduced by about 40 IU/kg/week, by about 50 IU/kg/week, by about 80 IU/kg/week, by about 100 IU/kg/week, or more.

41. The method of claim 23, wherein administration of the IL-6 antagonist reduces a side effect of an increased dose of EPO.

42. The method of any one of claims 1-40, wherein the allele contains a G at position 2321 of the TMPRSS6 polynucleotide.

43. The method according to any one of claims 1 to 42, wherein the IL-6 antagonist is an anti-IL-6 antibody that has one or more CDRs selected from nucleic acid sequences:

- 34 041768

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).

44. The method according to claim 42, providing that antibody to IL-6 has CDR1 heavy chain, containing the sequence SNYMI (SEQ ID NO: 12); heavy chain CDR2, containing the sequence DLYYYAGDTYYADSVKG (SEQ ID NO: 13); CDR3 heavy chain, containing the sequence WADDHPPWIDL (SEQ ID NO: 14); CDR1 light chain, containing the sequence RASQGISSWLA (SEQID NO: 15); CDR2 light chain, containing the sequence KASTLES (SEQ ID NO: containing the sequence QQSWLGGS (SEQ ID NO: 17). 16); and CDR3 light chain,

45. The method of claim 42, wherein the anti-IL-6 antibody has a heavy chain containing the sequence:

EVQLVESGGGLVQPGGSLRLSCAASGFTISSNYMIWVRQAPGKGLEWVSDLYYYAGDTYY

ADSVKGRFTMSRDISKNTVYLQMNSLRAEDTAVYYCARWADDHPPWIDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLYITREPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 18).

46. The method according to p. 42, providing that the antibody to IL-6 has a light chain containing the sequence:

DIQMTQSPSTLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKVLIYKASTLESGVPS

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 19).

47. The method of claim 42, wherein the anti-IL-6 antibody is MEDI5117.

48. A method according to any one of claims 1 to 47, wherein the subject is a human.

6.1 3.2. Methods of treatment of cardiorenal syndrome/

In accordance with other aspects and embodiments, the present invention relates to compositions and methods for the treatment of cardiorenal syndrome.

These aspects and embodiments are based, at least in part, on the finding that treatment with an anti-IL-6 antibody to cardiac injury in a rodent model with cardiorenal syndrome had an equivalent effect as standard prophylactic treatment. As reported in more detail below, rodent models with cardiorenal syndrome were treated with anti-IL-6 antibody or standard prophylactic therapy (ACE inhibitor, perindopril) after myocardial infarction. After treatment, the ejection fraction, the force of cardiac contractility and the percentage of fibrous tissue in the heart tissue were measured. Ejection fraction levels in both groups of subjects that were treated with anti-IL-6 antibody and groups of subjects that were treated with standard prophylactic therapy were increased compared to levels in the group of subjects that were treated with control treatment. Cardiac contractility in both groups that were treated with anti-IL-6 antibody and that were treated with standard prophylactic therapy was increased compared to levels in the group of subjects that were treated with control treatment. The amount of fibrous tissue in both groups that was treated with anti-IL-6 antibody and that was treated with standard prophylactic therapy was reduced compared to the amount in the group of subjects that were treated with control treatment. In addition, ejection fraction levels and the amount of fibrous tissue were similar in the group of subjects that were treated with anti-IL-6 antibody and the group of subjects that were treated with standard prophylactic therapy. From these results, it appears that anti-IL-6 antibody therapy had equivalent efficacy as standard prophylactic therapy in the treatment of cardiorenal syndrome in a rodent model.

These aspects and embodiments are further based, at least in part, on the finding that patients who were identified as having cardiorenal syndrome after myocardial infarction and having elevated levels of IL-6 had a particularly increased risk

- 35 041768 deaths associated with the cardiovascular system, including heart failure. Without being limited by theory, IL-6 may play a causal role in the development and/or progression of cardiorenal syndrome. Thus, patients having elevated IL-6 levels after myocardial infarction or patients with cardiorenal syndrome and elevated IL-6 levels are likely to benefit from IL-6 inhibition.

Accordingly, the present invention provides therapeutic methods for treating heart and/or kidney injury in a subject having cardiorenal syndrome, comprising administering an IL-6 antagonist to the subject. In accordance with some embodiments, treatment of heart and/or kidney injury in a subject having cardiorenal syndrome is performed with or without standard treatment for cardiorenal syndrome. Also, the present invention relates to methods for characterizing the risk of death associated with the cardiovascular system in a patient after myocardial infarction, and the method provides for the detection of elevated levels of IL-6 in a biological sample obtained from the patient.

According to one aspect, the present invention relates to methods for treating heart and/or kidney injury in a subject with cardiorenal syndrome, the method comprising administering an IL-6 antagonist to the subject.

According to another aspect, the present invention relates to methods for improving cardiac function in a subject with cardiorenal syndrome, the method comprising administering an IL-6 antagonist to the subject.

In another aspect, the present invention provides methods for reducing fibrosis in a subject with cardiorenal syndrome, the method comprising administering an IL-6 antagonist to the subject.

In accordance with various embodiments of any of the aspects described herein, the method further comprises administering a standard prophylactic agent to the subject. According to various embodiments, the standard prophylactic agent is an angiotensin converting enzyme (ACE) inhibitor.

In accordance with various embodiments of any of the aspects described herein, an increase in cardiac function is characterized by an increase in a subject's ejection fraction and/or cardiac contractility relative to a reference. In accordance with various embodiments of any of the aspects described herein, fibrosis reduction is characterized by a decrease in the percentage of fibrous tissue in a tissue sample from a subject relative to a reference. According to various embodiments, the fibrosis is in heart tissue.

In accordance with various embodiments of any of the aspects described herein, the subject has heart and/or kidney damage. In accordance with various embodiments of any of the aspects described herein, the subject has a heart injury followed by a kidney injury.

According to another aspect, the present invention relates to a method for detecting an increased risk of cardiovascular-related death (e.g., heart failure) in a subject following a myocardial infarction, the method comprising measuring the level of one or more of an IL- 6 in a sample from a subject relative to a reference, an elevated level of one or more of the IL-6 polynucleotide or polypeptide is indicative of an increased risk of cardiovascular-related death.

According to another aspect, the present invention relates to a method for characterizing the risk of cardiovascular-related death (e.g., heart failure) in a subject after myocardial infarction, the method comprising measuring the level of one or more of an IL-6 polynucleotide or polypeptide in a sample from a subject relative to a reference, wherein an elevated level of one or more of the IL-6 polynucleotide or polypeptide is indicative of an increased risk of cardiovascular-related death.

In accordance with various embodiments of any of the aspects described herein, the subject has cardiorenal syndrome, heart failure, chronic kidney disease, or no cardiorenal pathology. In accordance with various embodiments of any of the aspects described herein, the subject is identified as having cardiorenal syndrome, heart failure, chronic kidney disease, or no cardiorenal disease approximately one month after myocardial infarction.

According to another aspect, the present invention relates to a method of treating heart and/or kidney injury in a selected subject with cardiorenal syndrome, the method comprising administering to the subject an IL-6 antagonist, wherein the subject is selected for treatment by detecting elevated levels of one or more of the polynucleotide or an IL-6 polypeptide in a biological sample from a subject relative to a reference.

In accordance with another aspect, the present invention relates to a method for reducing the risk

- 36 041768 death associated with the cardiovascular system (for example, heart failure) in a selected subject with cardiorenal syndrome, and the method involves administering an IL-6 antagonist to the subject, while the subject is selected by detecting an increased level of one or more of the polynucleotide or IL-6 polypeptide in a biological sample from a subject relative to a reference. In accordance with various embodiments of any of the aspects described herein, the subject has already suffered a myocardial infarction.

According to various embodiments of any of the aspects described herein, the IL-6 antagonist is an anti-IL-6 antibody. According to various embodiments, the anti-IL-6 antibody is MEDI5117.

In accordance with various embodiments of any of the aspects described herein, the biological sample is a plasma sample or a blood serum sample. In accordance with various embodiments of any of the aspects described herein, the subject is a human.

According to another aspect, the present invention relates to methods for treating cardiorenal syndrome in patients and/or reducing the risk of death or heart failure in such patients by administering an agent that inhibits the biological activity or expression of IL-6. In accordance with one embodiment, patients with cardiorenal syndrome are treated with or without standard treatment for cardiorenal syndrome (eg, angiotensin converting enzyme (ACE) inhibitors). In particular, a subject with a cardiovascular syndrome is administered an agent that inhibits the biological activity or expression of IL-6 (eg, administration of an anti-IL-6 antibody).

In accordance with another aspect, the present invention relates to methods for improving cardiac function and methods for reducing fibrosis in subjects with cardiorenal syndrome. The methods involve administering to the subject an agent that inhibits the biological activity or expression of IL-6. According to some embodiments, an increase in cardiac function is characterized by an increase in the subject's ejection fraction relative to a reference (e.g., ejection fraction in a healthy control subject) or cardiac contractility (e.g., dP/dt _max ) relative to a reference (e.g., heart rate in a healthy control subject). ). According to some embodiments, fibrosis reduction is characterized by a decrease in the percentage of fibrous tissue in a tissue sample from a subject relative to a reference (eg, in a tissue sample obtained from a healthy control subject). According to one embodiment, the fibrosis is in the tissue of the heart.

An agent that inhibits the biological activity of IL-6 by blocking either the binding of IL-6 or its receptor (gp80) to each other, or its signaling or expression, can be administered to a subject with cardiorenal syndrome, in a pharmaceutical composition, wherein the pharmaceutical composition contains an effective amount of said agent; and a suitable excipient. In one embodiment, the agent is an IL-6 antagonist or anti-IL-6 antibody that reduces the level or activity of an IL-6 polypeptide or polynucleotide in a subject or inhibits intracellular signaling triggered by IL-6 receptor activation. You can enter an antibody to IL-6 (for example, MEDI5117). Methods of treatment of cardiorenal syndrome may vary depending on the stage of cardiorenal syndrome, age of the patient, health and physical condition.

In accordance with various embodiments, subjects with cardiorenal syndrome are treated with an IL6 antagonist. In addition, subjects at increased risk of cardiovascular death and/or heart failure following myocardial infarction can be identified by characterizing the subject's plasma IL-6 level. Subjects with elevated levels of IL-6 have an increased risk of cardiovascular-related death and/or heart failure. Such subjects can be selected for treatment with an IL-6 antagonist. Additionally, subjects having a cardiovascular syndrome and elevated levels of IL-6, including those who have suffered a myocardial infarction, may be selected for treatment. Once selected for treatment, such subjects may be administered essentially any IL-6 antagonist known in the art. Suitable IL-6 antagonists include, for example, known IL-6 antagonists, commercially available IL-6 antagonists, IL-6 antagonists developed using methods well known in the art, and antagonists of intracellular signaling systems associated with IL-6R.

In accordance with another aspect, the present invention relates to analyzes for characterizing the risk of death associated with the cardiovascular system, heart failure and/or mortality in a subject after myocardial infarction. The assays detect IL-6 in a biological sample obtained from a subject. IL-6 can be detected by any suitable method. In one embodiment, the risk of cardiovascular-related death or heart failure is characterized by detecting the level of an IL-6 polypeptide in a biological sample (eg, serum or plasma) of a subject relative to expression in a reference ( for example, serum or plasma from a healthy control subject or from a control subject who does not have cardiorenal pathology), while an increase in IL-6 indicates an increased risk of death associated with the cardiovascular system, or heart failure. Subjects who have been identified as having an increased risk of cardiovascular-related death, heart failure, or mortality may be selected for treatment. According to another embodiment, a subject with cardiorenal syndrome and elevated levels of IL-6 is selected for treatment with an IL-6 antagonist (eg, an anti-IL-6 antibody).

According to one embodiment, the level of an IL-6 polynucleotide is measured. IL-6 polynucleotide levels can be measured by standard methods such as quantitative PCR, northern blotting, microarray, mass spectrometry, and in situ hybridization.

According to one embodiment, the level of the IL-6 polypeptide is measured. Levels of IL-6 polypeptides can be measured by standard methods such as immunoassay. An immunoassay typically involves the use of an antibody (or other agent that specifically binds a marker) to detect the presence or level of a biomarker in a sample. Antibodies can be obtained by methods well known in the art, for example, by immunizing animals with a biomarker or fragments thereof. Biomarkers can be isolated from samples based on their binding characteristics. Alternatively, if the amino acid sequence of the polypeptide biomarker is known, the polypeptide can be synthesized and used to generate antibodies using methods that are well known in the art.

In accordance with various embodiments, the assays involve the use of conventional immunoassays, including, for example, Western blotting, sandwich type immunoassays, including ELISA, and other enzyme immunoassays, fluorescent immunoassays, and chemiluminescence. Nephelometry is a liquid-phase analysis in which antibodies are in solution. The binding of an antigen to an antibody results in changes in absorbance, which is measured. Other forms of immunoassay include magnetic immunoassay, radioimmunoassay, and real-time immunoquantitative PCR (iqPCR). Other detection methods include liquid chromatography and mass spectrometry.

Immunoassays can be performed on solid substrates (eg, chips, beads, microfluidic platforms, membranes) or any other form that supports binding of the antibody to the marker and subsequent detection. One marker may be detected at a time, or a multiplex format may be used. The multiplex immunoassay may involve the use of flat microarrays (protein chips) or granular microarrays (suspension assays).

Patients with cardiorenal syndrome who are identified as having elevated levels of IL-6 polypeptides are selected for treatment with an agent that reduces IL-6 expression or activity (eg, an anti-IL-6 antibody). Treatment can be done in combination with standard treatment for cardiorenal syndrome (eg, an ACE inhibitor). Patients treated with the method of the present invention can be monitored by detecting changes in IL-6 after treatment.

Other aspects and embodiments are presented in the following numbered paragraphs.

1. A method of treating heart and/or kidney injury in a subject with cardiorenal syndrome, the method comprising administering an IL-6 antagonist to the subject.

2. A method for improving cardiac function in a subject with cardiorenal syndrome, the method comprising administering an IL-6 antagonist to the subject.

3. A method for reducing fibrosis in a subject with cardiorenal syndrome, the method comprising administering an IL-6 antagonist to the subject.

4. The method of claim 2, wherein the increase in cardiac function is characterized by an increase in the subject's ejection fraction relative to the reference.

5. The method according to claim 3, providing that the fibrosis is in the tissue of the heart.

6. The method of claim 3 or 5, wherein the reduction in fibrosis is characterized by a reduction in the percentage of fibrous tissue in the tissue sample from the subject relative to the reference.

7. A method according to any one of claims 1 to 6, wherein the subject has heart and/or kidney damage.

8. A method according to any one of claims 1 to 7, wherein the subject has a heart injury followed by a kidney injury.

9. The method of any one of claims 1 to 8, further comprising administering a standard prophylactic agent to the subject.

10. The method of claim 1-9, wherein the standard prophylactic agent is an angiotensin converting enzyme (ACE) inhibitor.

11. A method for detecting an increased risk of cardiovascular-related death in a subject after myocardial infarction, the method comprising measuring the level of one or more of an IL-6 polynucleotide or polypeptide in a sample from the subject relative to a reference,

- 38 041768 and an increased level of one or more of the polynucleotide or polypeptide of IL-6 indicates an increased risk of death associated with the cardiovascular system.

12. A method for characterizing the risk of cardiovascular-related death in a subject after myocardial infarction, the method comprising measuring the level of one or more of an IL-6 polynucleotide or polypeptide in a sample from the subject relative to a reference, wherein an elevated level of one or more of polynucleotide or polypeptide IL-6 indicates an increased risk of death associated with the cardiovascular system.

13. The method of claim 11 or 12, wherein the subject has cardiorenal syndrome, heart failure, chronic kidney disease, or no cardiorenal pathology.

14. The method of any one of claims 11-13, wherein the subject is identified as having cardiorenal syndrome, heart failure, chronic kidney disease, or no cardiorenal pathology approximately one month after myocardial infarction.

15. A method of treating heart and/or kidney injury in a selected subject with cardiorenal syndrome, the method comprising administering to the subject an IL-6 antagonist, wherein the subject is selected for treatment by detecting elevated levels of one or more of an IL-6 polynucleotide or polypeptide in a biological sample from the subject relative to the standard.

16. A method for reducing the risk of cardiovascular-related death in a selected subject with cardiorenal syndrome, the method comprising administering an IL-6 antagonist to the subject, wherein the subject is selected by detecting elevated levels of one or more of an IL-6 polynucleotide or polypeptide in a biological sample from a subject relative to a reference.

17. The method of claim 15 or 16, wherein the subject has already suffered a myocardial infarction.

18. The method of any one of claims 1-10 or 15-17, wherein the IL-6 antagonist is an anti-IL-6 antibody.

19. The method of claim 18, wherein the anti-IL-6 antibody is MEDI5117.

20. The method according to any one of claims 11 to 19, wherein the biological sample is a blood plasma sample.

21. A method according to any one of claims 1 to 20, wherein the subject is a human.

6.14. Examples.

The following examples are provided by way of illustration and not limitation.

6.14.1. Example 1

EPO dosage and overall survival in patients with chronic kidney disease correlate with serum IL-6 and CRP levels only in patients with at least one copy of the major rs855791 TMPRSS6 SNP allele.

The peptide hormone, hepcidin, plays a central role in systemic iron homeostasis. Hentze et al., Cell, 142:24-38 (2010). Hepcidin expression is known to be influenced by the product of the TMPRSS6 gene, matriptase-2, a type II transmembrane serine protease. Common variants of the TMPRSS6 gene have been shown to correlate with iron status, Benyamin et al., Nature Genetics, 41(11):1173-1175 (2009), and certain mutations in the TMPRSS6 gene have been shown to cause iron refractory and iron deficiency anemia (IRIDA ), Finberg et al., Nature Genetics, 40(5):569-571 (2008). SNP rs855791 (2321G^A; A736V) is a natural variant of the TMPRSS6 gene that is associated with natural fluctuations in hepcidin expression and blood hemoglobin levels.

To determine whether the rs855791 TMPRSS6 SNP genotype could predict the degree of anemia in end-stage kidney disease, data previously collected from clinical trials in patients with chronic kidney disease 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):el003421 (2014), data were also analyzed to determine whether serum IL-6 levels could predict the degree of anemia in end-stage kidney disease.

Ways.

We reviewed data from N=257 patients in the MIMICK1, MIMICK2 (Mapping of Inflammatory Markers in Chronic Kidney Disease) and MIA (malnutrition, inflammation and atherosclerosis) cohorts recruited since October 2003 to September 2004 in six dialysis units in the region of Stockholm - Uppsala (Sweden), bringing down to N=208 according to the criteria of the well-known dialysis method: ferritin >100 ng/ml and Hb >10 mg/dl, for patient selection , which were stable on hemodialysis without iron deficiency and without significant anemia, thus excluding patients with factors that could separate iron regulation and hemoglobin levels.

All patient clinical data, including erythropoietin (EPO) dose in IU/kg/week, serum IL-6 level in pg/ml, serum CRP level in mg/l, survival in months and TMPRSS6 SNP genotype rs855791 were compared and analyzed with statistical analysis software (SPSS Statistics Desktop; IBM). The studied TMPRSS6 alleles and their nucleotide and amino acid are listed in the table.

- 39 041768

TMPRSS6 alleles

Allele TMPRSS6 Nucleotide in position 2321 Amino acid at position 736 Basic G or C Ala Minor A or T Vai

The cohort was divided into rs855791 subgroups (homozygous AA, heterozygous AG, and homozygous GG) and each genotype group was divided into tertiles or quartiles by 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 (CRP <2 mg/L vs >2 mg/L). Comparisons were made between EPO dose in upper and lower tertiles and quartiles. Conducted statistical analysis in groups of genotypes using Student's T-test and between groups using analysis of variance.

Results.

Because the dose of EPO in each patient was adjusted by the attending physicians to achieve normal hemoglobin levels, the dose of EPO could be used as an indicator for the underlying degree of anemia. It was found that the dose of EPO in subjects homozygous for the minor allele (A/A) was relatively insensitive to fluctuations in IL-6 (Fig. 1A; left panel). However, the dose of EPO in subjects with at least one copy of the major allele, i.e. patients heterozygous (A/G) or homozygous (G/G) for the major allele (G) was sensitive to their IL-6 level (Fig. 1B; right panel). In these latter subjects, elevated serum levels of IL-6 (eg, >5 pg/ml) were associated with an increased dose of EPO.

Without being bound by a particular theory, homozygosity for the minor allele abolished the effect of IL-6 on iron regulation. Thus, the dose of EPO in these patients (A/A) was approximately the same, regardless of the level of IL-6.

Subjects homozygous for the minor allele rs855791 TMPRSS6 (A) had similar mortality regardless of IL-6 levels (FIG. 2A). However, survival in subjects with at least one copy of the major allele, i. patients heterozygous or homozygous for the major allele (G) varied depending on the level of IL-6 (Fig. 2B). In fact, the G allele of TMPRSS6 conferred higher overall mortality in response to elevated levels of IL-6 in dialysis subjects with stage 5 chronic kidney disease. In subjects with at least one copy of the major allele (G), IL-6 levels <5 pg/mL (i.e., middle and upper tertile IL-6) were associated with increased mortality compared to IL-6 levels <5 pg /ml (ie the lower tertile of IL-6) (Fig. 2B).

The level of the acute phase reagent, CRP, an inflammatory marker, also correlated with elevated EPO dosage in subjects heterozygous or homozygous for the major allele (G), but not in patients homozygous for the minor allele (FIG. 3).

Discussion.

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

Similarly, overall survival correlated with IL-6 levels only in patients with at least one copy of the major allele with the rs855791 TMPRSS6 SNP. In subjects with at least one copy of the major rs855791 TMPRSS6 allele, survival was inversely correlated with serum IL-6 levels, with patients in the highest tertile of serum IL-6 levels having statistically significantly worse survival than those in the lowest tertile of IL levels. -6 (Fig. 2B). In contrast, overall survival of patients homozygous for the minor allele in rs855791 was independent of IL-6 levels (Fig. 2A).

Without wishing to be bound by any theory, in patients with at least one copy of the major TMPRSS6 allele, an increase in serum IL-6 may lead to an increase in hepcidin expression, thereby increasing anemia. The increased risk of mortality is due to impaired iron metabolism resulting from anemia and/or an increased dose of an erythropoiesis-stimulating agent such as EPO. If these correlations reflect causation, they raise the possibility that lowering IL-6 levels or IL-6 signaling can reduce anemia, reduce the required EPO dose, and increase survival in patients with chronic kidney disease, but only in those patients that have at least one copy of the core rs855791 TMPRSS6 allele, and are most effective in patients with elevated serum IL-6 levels.

6.14.2. Example 2

Risk of death and risk of heart failure after acute myocardial infarction correlate with serum IL-6 levels only in patients with at least one copy

-40041768 of the main allele of SNP rs855791 TMPRSS6.

To determine whether the rs855791 TMPRSS6 genotype affects IL-6 susceptibility in patients with acute but not chronic disease, data previously collected in clinical trials of patients hospitalized with acute coronary syndrome were analyzed in combination with newly determined SNP genotyping.

Ways.

Data were analyzed from subjects previously enrolled in the multicenter Platelet Inhibition and Patient Outcomes PLATO study. Patients were eligible for enrollment in PLATO if they were hospitalized for an acute coronary syndrome with onset of symptoms within the previous 24 hours. Mortality and the presence of heart failure were measured in these subjects starting 30 days after myocardial infarction.

Results.

Mortality of subjects homozygous for the minor allele (A) of the rs855791 TMPRSS6 SNP did not correlate with fluctuations in IL-6 (Fig. 4A). However, one or two copies of the major allele (G) gave higher overall mortality in response to elevated levels of IL-6 in subjects after myocardial infarction (Fig. 4B). Thus, TMPRSS6 modulated the IL-6 mediated risk of death after myocardial infarction.

The effect of TMPRSS6 genotype on IL-6 mediated risk of heart failure was also measured in subjects participating in PLATO starting at 30 days post myocardial infarction. Heart failure in subjects homozygous for the minor allele (A) did not correlate with fluctuations in IL-6 (FIG. 5A). However, the G allele of TMPRSS6 conferred a higher incidence of heart failure in response to elevated levels of IL-6 in subjects after myocardial infarction (Fig. 5B). Thus, TMPRSS6 modulated the IL-6 mediated risk of heart failure after myocardial infarction.

Discussion.

From these data, it appears that the correlation between TMPRSS6 genotype, IL-6 levels, and adverse clinical outcomes is not limited to patients with chronic kidney disease. Without wishing to be bound by any theory, in patients with at least one copy of the major TMPRSS6 allele, an increase in serum IL-6 may lead to an increase in hepcidin expression, followed by an increase in iron sequestration in cardiomyocytes, with subsequent iron-mediated cellular toxicity. If such correlations reflect causation, they raise the possibility that lowering IL-6 levels or IL-6 signaling may reduce heart failure and mortality in patients with acute coronary syndrome, but only in those patients who have at least at least one copy of the core rs855791 TMPRSS6 allele, and with the greatest effect in patients with elevated serum IL-6 levels.

6.14.3. Example 3

In vitro studies on iPS-derived human cardiomyocytes support a causal relationship between the TMPRSS6 genotype and IL-6 mediated cytotoxicity.

Although the correlations observed in Examples 1 and 2 suggest that a reduction in IL-6 mediated signaling should be clinically beneficial in patients with at least one copy of the major rs855791 TMPRSS6 allele, elevated levels of IL-6, and either anemia or hepcidin-mediated cellular toxicity, the observed correlations did not support a causal relationship. Accordingly, experiments were performed on induced human pluripotent cell derived cardiomyocytes (iPS-CM) transfected with TMPRSS6 variants to test the effect of BMP and BMP together with IL-6 on hepcidin expression and cellular susceptibility to ischemic injury.

6.14.3.1. Ways.

Cultivation of human iPS-derived cardiomyocytes. iCell cardiomyocytes (Cellular Dynamics International, CDI Inc.) were plated in a 0.1% gelatin coated 6-well or 96-well culture plate with iCell cardiomyocyte medium plate (CDI Inc.). 48 hours after inoculation, the plate medium was replaced with maintenance medium (CDI Inc.). The support medium was changed every two days until the day of the experiment.

Simulated ischemia/reoxygenation protocol: iPS cardiomyocytes were subjected to simulated ischemia (SI) for 90 min by replacing the cell medium with ischemic buffer that contained 118 mM NaCl, 24 mM NaHCO ₃ , 1.0 mM NaH ₂ PO ₄ , 2.5 mM CaCl ₂ 2H ₂ O, 1.2 mM MgCl ₂ , 20 mM sodium lactate, 16 mM KCl, 10 mM 2-deoxyglucose (pH adjusted to 6.2) as previously reported (Das, A., Xi, L., and Kukreja, KC (2005), J Biol Chem 280:12944-12955 Das A, Smolenski A, Lohmann SM, Kukreja RC (2006), J Biol Chem 281(50):38644-52 ). Cells were incubated at 37°C in a trigas controlled incubator, controlling 1-2% O2 and 5% CO2 throughout the entire SI period. Reoxygenation (RO) was performed by replacing the ischemic buffer with a normal cellular environment under normal oxygen conditions. Cell necrosis was observed after 2 or 18 hours, respectively.

- 41 041768 reoxygenation. iCells were subjected to 4 h SI and 24 h RO as described above.

Assessment of cell viability and apoptosis. A trypan blue exclusion assay was performed to assess cell necrosis as previously described (Das, A., Xi, L., and Kukreja, K.C. (2005), J. Biol. Chem. 280,

12944-12955; Das A., Smolenski A., Lohmann S.M., Kukreja R.C. (2006), J. Biol. Chem. 281(50):38644-52).

Transfection of iCell cardiomyocytes. On day 8 post-plating, medium was replaced with fresh maintenance medium and cells were incubated for 4 h. Cells were transfected with pCMV6-XL5 TMPRSS6 (K523) or pCMV6-XL5 TMPRSS6 (K523) V763A using ViaFect™ Transfection Reagent as instructed. manufacturer (Promega Corp., Madison, Wisconsin). 48 hours after transfection, further experiments were performed on the cells.

Western blotting. Western blotting was performed as previously described (Das, A., Xi, L., and Kukreja, K.C. (2005), J. Biol. Chem. 280, 12944-12955; Das A, Smolenski A., Lohmann S. M., Kukreja R. C. (2006), J Biol Chem 281(50):38644-52). Total soluble protein was extracted from cells with lysis buffer (Cell Signaling, MA). The homogenate was centrifuged at 10000xg for 5 min at 4°C and the supernatant was isolated. Protein (50 µg from each sample) was separated using 12% acrylamide gels and transferred to a nitrocellulose membrane, and then blocked with 5% skimmed milk powder in TBST (10 mM Tris-HCl, pH 7.4, 100 mM NaCl and 0.1 % Tween 20) for 1 hour. The membrane was then incubated overnight with rabbit monoclonal/polyclonal primary antibody or goat polyclonal primary antibody at a dilution of 1:1000 for each of the respective proteins, i.e. rabbit mAb phospho-beclin 1 (Ser93) (D9A5G), beclin-1, SQSTM1/p62, rabbit mAb LC3A/B (D3U4C) XP®, rabbit mAb phospho-Akt (Ser473) (D9E) XP®, rabbit mAb Akt ( pan) (C67E7), rabbit mAb to ribosomal protein phospho-S6 (Ser240/244) (D68F8) XP®, rabbit mAb to ribosomal protein S6 (5G10) from Cell Signaling, Massachusetts, antibody to matriptase 2 (TMPRSS6) and antibody to SLC40A1 (ferroportin) from Abcam Company, Massachusetts, and HRP-conjugated goat anti-actin polyclonal antibody (Santa Cruz Biotechnology, Texas). The membranes were then incubated with a horseradish peroxidase conjugated secondary antibody to rabbit antibodies (1:2000 dilution, Amersham Biosciences) for 2 h. The blots were developed using a chemiluminescent system, and the bands were scanned and quantified by densitometric analysis.

Taqman real-time PCR analysis. Total RNA, including small RNA, 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 was measured using a Nanodrop ND-1000 spectrophotometer (Agilent technologies, California, USA). Briefly, 1 μg of total RNA was converted to random hexamer cDNA using the High Efficiency cDNA Synthesis Kit (Applied Biosystems, CA, USA). The reverse transcription reaction was carried out using the following PCR conditions: 25°C for 10 min; 37°C for 120 min and 85°C for 5 min. Real-time PCR was performed using Taqman (Applied Biosystems, CA, USA) Hamp amplicon-specific probes (CGGCTCTGCAGCCTTG) (SEQ ID NO: 20) under the following PCR cycle conditions: 95° C. for 10 min; 95°C for 15 s and 60°C for 60 s. Hamp expression was normalized to the constitutive GAPDH gene (CTTCCAGGAGCGAGATCCCGCTAA) (SEQ ID NO: 21).

Relative gene expression was analyzed using the 2-AACt method.

Mutagenesis of TMPRSS6 and transfection of iPS cells. pCMV6-XL5 TMPRSS6 was purchased from Origene Technologies (Rockville, Maryland), catalog number SC306623, corresponding to GenBank accession number NM 153609. This clone contained a mutation resulting in the K253A amino acid substitution. Site directed mutagenesis was performed to convert the amino acid at position 253 to canonical lysine (K). After the conversion 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); semantic (s) TMPRSS6 E253K CTGCAGGGCCCCAAGGACCTCATGC (SEQ ID NO: 23); antisense (as) TMPRSS6 V736A CCTGGTAGCGATAGGCCTCGCTGCACAGG (SEQ ID NO: 24); semantic (s) TMPRSS6 V736A CCTGTGCAGCGAGGCCTATCGCTACCAGG (SEQ ID NO: 2).

6.14.3.2. Results.

Human iPS-CMs only minimally expressed matriptase-2 at baseline. Cells were transfected with a construct driving constitutive expression of matriptase-2 736A encoded by the major allele of the rs855791 TMPRSS6 SNP or matriptase-2 736V encoded by the minor allele, mimicking, respectively, cardiomyocytes with a homozygous major allele and a homozygous minor allele.

Hepcidin expression is regulated by both BMP6/SMAD and IL-6/STAT signaling pathways, with BMP and IL-6 acting through their respective receptors and driving hepcidin expression.

- 42 041768

Casanovas et al., PLOS Comp. Biol. 10(1):e1003421 (2014). Major and minor allele iPS cardiomyocytes were treated in vitro with agonists of both signaling pathways: recombinant BMP2 and IL-6 or BMP2 alone to model clinical interventions in which IL-6 levels (or signaling) are reduced. iPS control cells were not treated with any agonist at all. Cell death was measured at normal oxygen pressure (normal oxygen conditions) as well as under conditions that mimic hypoxia followed by reoxygenation (reperfusion).

In FIG. 6A shows results when cells were treated at normal oxygen levels. iPS cardiomyocytes expressing only the minor rs855791 TMPRSS6 allele (minor allele 736V) were not significantly affected (n.s.) by abolishing IL-6 signaling: cell death, measured as the percentage of trypan blue positive cells, was not significantly reduced when cells were treated with BMP2 alone compared to BMP2+IL-6 processing. In contrast, iPS cardiomyocytes expressing the major rs855791 TMPRSS6 allele showed statistically significant lower cell death when IL-6 signaling was abolished.

In FIG. 6B shows the results when cells were subjected to hypoxia followed by reoxygenation. Compared to normal oxygen conditions, hypoxia/reoxygenation is significantly toxic to iPS cardiomyocytes, with approximately 40 percent of major and minor allele control cells dying compared to approximately 20% of control cells under normal oxygen conditions (cf. FIG. 6B - Fig. 6A). Relative to this increased background toxicity, minor allele iPS cardiomyocytes were not significantly affected by abolishing IL-6 signaling: cell death was not significantly reduced when cells were treated with BMP2 alone compared to treatment with BMP2 + IL-6. In contrast, iPS cardiomyocytes expressing the major rs855791 TMPRSS6 allele showed statistically significant lower cell death when IL-6 signaling was abolished.

6.14.3.3. Discussion.

These data provide strong support for the conclusions drawn from the post hoc analysis of clinical trial data in Examples 1 and 2: reduction of IL-6 signaling may be effective in reducing IL-6 mediated toxicity in cardiomyocytes expressing the major rs855791 TMPRSS6 allele, but not in cardiomyocytes. expressing only the minor allele. Without wishing to be bound by any theory, the IL-6-driven increase in toxicity in iPS cardiomyocytes with the major allele may be the result of an IL-6-mediated increase in hepcidin expression, followed by an increase in cellular iron sequestration, and subsequent iron-mediated cellular toxicity.

6.14.4. Example 4

Anti-IL-6 antibody therapy is as effective as standard prophylactic therapy in a model of cardiorenal syndrome in rats genotypically similar to human homozygotes for the major rs855791 TMPRSS6 allele

Patients with chronic kidney disease, such as those who participated in the MIMICK studies analyzed in example 1, often develop impaired cardiac function, which is a major factor influencing mortality rates. This secondary myocardial injury after primary chronic kidney disease is called cardiorenal syndrome type 4 (CRS type 4).

To test whether anti-IL-6 antibody therapy is effective as a treatment in CRS4 patients having at least one copy of the major rs855791 TMPRSS6 allele, as suggested by the data in Examples 1 and 3, we used the CRS4 rat model , which are genotypically similar to people homozygous for the main rs855791 TMPRSS6 allele. In FIG. 7 shows the scheme of the study.

At week 0, myocardial infarction was induced in animals with CRS. On the 2nd week, a nephrectomy was performed. The control group was given sham operations instead. Various evaluations of subjects were performed prior to nephrectomy. Evaluations included measurements of serum creatinine, glomerular filtration rate, 24-hour urinary protein levels, echocardiography, tail cuff blood pressure, and biomarkers in plasma and urine.

Treatment was started on the 1st day after nephrectomy. Animals were divided into three groups:

(i) control processing;

(ii) anti-IL-6 antibody therapy;

(iii) standard preventive therapy.

As anti-IL-6 antibody therapy, an anti-IL-6 antibody suitable for use in rodents was used. The standard means of prophylactic therapy was the administration of perindopril, an ACE (angiotensin-converting enzyme) inhibitor. At the start of treatment, the subjects in all groups were evaluated. Assessments included measurement of serum creatinine, glomerular filtration rate, 24-hour protein levels, and plasma biomarkers.

On the 3rd and 7th days after nephrectomy, evaluations of subjects in all groups were performed. Estimates included

- 43 041768 measurement of serum creatinine and plasma biomarkers on day 3 and measurement of serum creatinine, glomerular filtration rate, 24-hour protein levels, echocardiography, blood pressure and plasma biomarkers on day 7.

At week 6, subjects were sacrificed. Prior to sacrifice, various assessments were made of subjects in all groups. Assessments included measurements of serum creatinine, glomerular filtration rate, 24-hour protein levels, blood pressure, plasma biomarkers, echocardiography, and pressure-volume loop analysis. After sacrifice, tissues were also taken from subjects in all groups for histological evaluation (ie, staining of cardiac tissue with Sirius red dye).

In FIG. 8A-8D show cardiac ejection fraction in non-CRS rats (blank), in CRS animals treated with a pharmacologically irrelevant isotype control antibody (isotype), in CRS animals treated with an anti-IL-6 antibody (anti-IL-6 Ab), and in animals with CRS treated with standard prophylactic therapy, ie. ACE inhibitor (Peri) in the cardiorenal syndrome model shown in FIG. 7.

In FIG. 8A shows baseline ejection fraction levels for all groups two weeks after myocardial infarction, but before nephrectomy and before treatment, showing that experimentally induced myocardial infarction caused a significant decrease in cardiac ejection fraction. In FIG. 8B is a graph showing ejection fraction levels for all groups one week after nephrectomy, after 1 week of treatment. In FIG. 8C is a graph showing ejection fraction levels for all groups two weeks after nephrectomy, after 2 weeks of treatment. In FIG. 8D is a graph showing ejection fraction levels for all groups four weeks after nephrectomy, after 4 weeks of treatment. Results are expressed as mean ± SEM.

After 4 weeks of treatment, both treatment groups, ie. the anti-IL-6 antibody-treated group and the standard ACE inhibitor prophylactic treatment group had statistically significantly elevated ejection fraction levels compared to the isotype control group (FIG. 8D) (p<0.001). Similar ejection fraction levels in the anti-IL-6 antibody and standard prophylactic therapy groups, measured after week 4 of treatment, indicated that anti-IL-6 antibody therapy had equivalent efficacy to that of the ACE inhibitor perindopril (standard prophylactic therapy), demonstrating that anti-IL-6 antibody therapy had therapeutic efficacy in preserving cardiac function in a model of cardiorenal syndrome equivalent to standard prophylactic therapy as measured by changes in cardiac ejection fraction.

From the results of the measurement of cardiac contractility (Fig. 9) it is seen that the therapy with antibody to IL-6 also had an effect equivalent to the standard prophylactic therapy, i. an ACE inhibitor. After 4 weeks of treatment, cardiac contractility in the anti-IL-6 antibody and standard prophylactic treatment groups significantly increased relative to that of the isotype control group. The similar contractility in the anti-IL-6 antibody and standard prophylactic groups indicated that anti-IL-6 antibody therapy had an efficacy in preserving cardiac function in a model of cardiorenal syndrome equivalent to that of the ACE inhibitor perindopril (standard prophylaxis) as measured by by contraction.

Measurement of fibrosis in heart tissue taken from animals in all groups also showed that anti-IL-6 antibody therapy had an effect equivalent to that of standard prophylactic therapy (FIGS. 10A-10C). Fibrosis in heart tissue was quantified by measuring the percentage area of fibrous tissue in two sites: a normal site and a fibrous margin site. An example of a normal site is indicated by the outlined portion of the tissue section shown in the photomicrograph of FIG. 10A. The micrograph inset shows an enlarged view of the normal area showing that small portions of the normal area had fibrous tissue. The fibrous margin site was an area of tissue in the normal area that was peripheral to the fibrous tissue.

On the graphs in Fig. 10B and 10C show that heart tissue from subjects in the anti-IL-6 antibody or standard prophylactic treatment groups had a significantly reduced percentage of fibrous tissue area compared to the isotype control group, in both cases measurements were made in the normal region (Fig. .10B) or in the area of the fibrous margin (Fig. 10C). In addition, the percentage areas of fibrous tissue measured in the anti-IL-6 antibody and conventional prophylactic groups were similar (both in the normal area and in the fibrous margin area), indicating that the anti-IL-6 antibody had antifibrotic effect equivalent to the ACE inhibitor perindopril (standard prophylactic agent).

From these data, it appears that anti-IL-6 antibody treatment is effective in reducing myocardial injury and restoring function in an in vivo model of cardiorenal syndrome in animals that are genotypically similar to humans homozygous for the major rs855791 TMPRSS6 allele.

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6.14.5. Example 5

Anti-IL-6 antibody therapy is effective in maintaining cardiac function in a model of acute myocardial infarction in mice genotypically similar to human homozygotes for the major allele rs855791 TMPRSS6/

The data in Examples 2 and 3 suggest that reducing IL-6 levels or IL-6 signaling may reduce heart failure and mortality in patients with acute coronary syndrome, but only in those patients who have at least one copy of the major allele. rs855791 TMPRSS6, and with the greatest effect in patients with elevated serum IL-6 levels.

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

In FIG. 11A and 11B show data from an in vivo model in which myocardial infarction was induced in mice genotypically similar to humans homozygous for the major rs855791 TMPRSS6 allele. The control group received no therapy. The experimental group was treated with anti-mouse IL-6 antibody. In FIG. 11A shows that anti-IL-6 antibody treatment provided a statistically significant improvement in ejection fraction. In FIG. 11B shows that anti-IL-6 antibody treatment provided a statistically significant improvement in contractility, measured as a shortening of the cardiac fraction. The data show that anti-IL-6 antibody therapy given immediately after myocardial infarction improved functional recovery of the left ventricle in rodents that are genetically similar to human patients with the major rs855791 TMPRSS6 allele.

7. Inclusion with a link.

All publications, patents, patent applications and other documents cited herein 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 document were identified separately as incorporated by reference for all purposes.

8. Equivalents.

While various specific embodiments have been illustrated and described, the above description is not intended to be limiting. It is understood that various changes can be made without deviating from the spirit and scope of the present invention(s). Many variations will become apparent to those skilled in the art upon review of the present disclosure.

Claims (11)

CLAIM 1. A method of treating cardiovascular disease in a subject, comprising administering to the subject a therapeutically effective amount of an anti-IL-6 antibody, wherein the patient has at least one copy of the major rs855791 TMPRSS6 allele, and wherein the anti-IL-6 antibody is MED5117. 2. The method of claim 1 wherein the cardiovascular disease is atherosclerosis. 3. The method of claim 2 wherein the cardiovascular disease is inflammation. 4. The method of claim 1, wherein the subject additionally has chronic kidney disease. 5. The method of claim 4, wherein the subject further has chronic kidney disease stage 1 KDOQI. 6. The method of claim 4, wherein the subject further has chronic kidney disease stage 2 KDOQI. 7. The method of claim 4, wherein the subject further has chronic kidney disease stage 3 KDOQI. 8. The method of claim 4, wherein the subject further has chronic kidney disease stage 4 KDOQI. 9. The method of claim 4, wherein the subject further has chronic kidney disease stage 5 KDOQI. 10. The method of claim 3 wherein the anti-IL-6 antibody is administered in an amount effective to neutralize inflammation. 11. The method of claim 3, wherein the subject has a C-reactive protein level greater than 2 mg/mL prior to treatment.