Treatment of diuretic resistance

Abstract

The present disclosure provides a method for treating diuretic resistance by administering an IL-6 antagonist to a patient in need of diuresis. In an exemplary embodiment, the patient has heart failure. Optionally, the patient has elevated urinary IL-6 levels, plasma IL-6 levels, or urinary and plasma IL-6 levels. [Selected Figure] Figure 1B

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 453,257, filed February 1, 2017, which is incorporated by reference in its entirety.

[0002] 2. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with Government support under Grants 5R01HL128973 and 4K23HL114868 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0003] 3. Statement Regarding Joint Research Agreement This invention was made under a collaborative research agreement between Yale University, MedImmune Ltd., AstraZeneca Pharmaceuticals LP, and Corvidia Therapeutics, Inc.

[0004] 4.Sequence Listing This application contains a Sequence Listing submitted via EFS-Web, which is incorporated herein by reference in its entirety. The ASCII copy, created in XX, 2018, is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size. [Background technology]

[0005] 5.Background Renal injury is frequently observed in heart failure, and heart failure is frequently observed in renal disease. The term cardiorenal syndrome ("CRS") encompasses a variety of clinical conditions in which dysfunction of the heart, kidneys, or both leads to accelerated dysfunction of both organs. Evidence of this deleterious organ crosstalk represents a high degree of morbidity and mortality. Despite its dire clinical implications, the mechanistic basis of CRS is only now being elucidated. Summary of the Invention [Problem to be solved by the invention]

[0006] New methods are needed to treat renal injury and damage in heart failure patients, as well as new methods to detect cardiorenal syndrome, determine which cardiorenal patients will respond to treatment, and monitor the effectiveness of treatment.

[0007] Diuretics are the mainstay of treatment for heart failure. However, certain patients are or become resistant to diuretics. New methods of treating patients who require diuretics but are resistant to diuretics are needed. [Means for solving the problem]

[0008] 6. Overview As further described below in Example 1, ongoing heart failure ("HF") patients receiving high-dose diuretic therapy at an outpatient treatment center were enrolled in a prospective observational study. Plasma levels of IL-6 were measured to examine the systemic association of this proinflammatory cytokine with various disease parameters, and urinary levels of IL-6 were measured to examine the association of IL-6 with local inflammation and neurohormonal activation at the level of renal tissue.

[0009] Urinary IL-6 and plasma IL-6 levels were found to be only weakly correlated with each other.

[0010] Increased urinary levels of IL-6 correlate significantly with measures of renal damage, such as diuretic resistance, reduced estimated glomerular filtration rate ("eGFR"), and increased tissue-level renin-angiotensin-aldosterone system ("RAAS") activation in these heart failure patients.

[0011] An inverse correlation between diuretic efficiency and plasma IL-6 was also observed, but after adjusting for eGFR, only urinary IL-6 was the condition significantly associated with the risk of poor diuretic efficiency in these patients.Furthermore, when both urinary IL-6 and plasma IL-6 were entered into a logistic regression model, only urinary IL-6 was the condition associated with the risk of poor diuretic efficiency; plasma IL-6 showed no such association.

[0012] These data indicate that urinary IL-6 levels are a useful biomarker of renal inflammation and can be used to determine renal dysfunction in the setting of heart failure (cardiorenal syndrome). The data further suggest that serial measurements of urinary IL-6 can be used to measure the renal benefit of treatments administered to patients with heart failure, particularly those with cardiorenal syndrome.

[0013] The urinary IL-6 data, and to some extent the plasma IL-6 data, also predict that treatment with an IL-6 antagonist should be effective in reducing renal inflammation in heart failure patients and thus treating the renal symptoms of cardiorenal syndrome.

[0014] However, because infections are a frequent cause of acute decompensation in heart failure patients, it is important to limit anti-cytokine and other immunosuppressive therapies to heart failure patients who are more likely to respond to improved renal and / or cardiac function. The cost of chronic IL-6 antagonist therapy also serves to limit treatment to heart failure patients who are more likely to respond to improved renal and / or cardiac function.

[0015] The analysis was expanded to 129 patients and each patient's genotype at the rs855791 single nucleotide polymorphism ("SNP") of transmembrane protease serine 6 ("TMPRSS6") was further evaluated.

[0016] Urinary levels of IL-6 were inversely correlated with diuretic efficiency only in patients with at least one copy of the major allele (AG and GG) of the TMPRSS6 rs855791 SNP. Urinary levels of IL-6 were not significantly correlated with diuretic efficiency in patients homozygous for the minor allele (AA). Plasma levels of IL-6 were inversely correlated with diuretic efficiency only in patients with at least one copy of the major allele of the TMPRSS6 rs855791 SNP. Plasma levels of IL-6 were not significantly correlated with diuretic efficiency in patients homozygous for the minor allele.

[0017] These data suggested that diuretic resistance (low diuretic efficiency) in patients with heart failure could be treated with IL-6 antagonists, but only in patients who carry at least one copy of the TMPRSS6 rs855791 major allele.

[0018] In mouse M1 CCD cells, which are genotypically similar to human cells homozygous for the TMPRSS6 rs855791 major allele, addition of IL-6 correlated with expression of the ion transporters, NKCC2, ENaC-beta, and NCC. Increased expression of these ion transporters provides a putative mechanism for IL-6-mediated diuretic resistance.

[0019] Because IL-6-mediated increased expression of ion transporters is not known to be associated with hepcidin expression, these data suggested that IL-6 antagonism may also be effective in treating diuretic resistance in patients homozygous for the TMPRSS6 rs855791 minor allele.

[0020] Secondary analyses of data from two additional large heart failure clinical trials confirmed the association between diuretic resistance and IL-6 levels (Example 5), independent of TMPRSS6 rs855791 genotype (Example 6), providing evidence that IL-6 antagonism should also be effective in treating diuretic resistance in patients homozygous for the TMPRSS6 rs855791 minor allele.

[0021] Thus, in a first aspect, a method of treating a patient who is in need of, but resistant to, a diuretic is provided, comprising administering to the patient a therapeutically effective amount of an IL-6 antagonist in combination with a diuretic.

[0022] In some embodiments, the patient has elevated pre-treatment plasma IL-6 levels. In certain embodiments, the patient has a pre-treatment plasma IL-6 level greater than 2 pg / mL. In certain embodiments, the patient has a pre-treatment IL-6 level greater than 3 pg / mL. In certain embodiments, the patient has a pre-treatment IL-6 level greater than 5 pg / mL. In certain embodiments, the patient has a pre-treatment IL-6 level greater than 10 pg / mL.

[0023] In some embodiments, the patient has a diuretic efficiency of less than 500. In some embodiments, the patient has a diuretic efficiency of less than 200. In some embodiments, the patient has a diuretic efficiency of less than 150. In some embodiments, the patient has a diuretic efficiency of less than 100.

[0024] In some embodiments, the patient has diuretic-resistant heart failure. In certain embodiments, the patient has acute heart failure. In certain embodiments, the patient has chronic heart failure.

[0025] In some embodiments, the patient has cardiorenal syndrome. In some of these embodiments, the patient has cardiorenal syndrome type 4.

[0026] In some embodiments, the patient has renal disease. In certain embodiments, the patient has hepatorenal syndrome.

[0027] In some embodiments, the patient has at least one copy of the TMPRSS6 rs855791 major allele.

[0028] In certain embodiments, the IL-6 antagonist is an anti-IL-6 antibody or an antigen-binding fragment or derivative thereof. In particular embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative has a K of less than 100 nM, less than 50 nM, less than 10 nM, or even less than 1 nM for binding to human IL-6. D has.

[0029] In certain embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative has an elimination half-life following intravenous administration of at least 7 days, at least 14 days, at least 21 days, or at least 30 days.

[0030] In certain embodiments, the IL-6 antagonist is a full length monoclonal anti-IL-6 antibody. In particular embodiments, the antibody is an IgG1 or IgG4 antibody. In certain embodiments, the antibody is an IgG1 antibody.

[0031] In certain embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative is fully human. In certain embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative is humanized.

[0032] In certain embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable region CDRs of MED5117. In a specific embodiment, the antibody comprises the VH and VL of MED5117. In a specific embodiment, the antibody is MED5117.

[0033] In certain embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable region CDRs of an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, elsilimomab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In certain embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative comprises a heavy chain V region and a light chain V region from an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In certain embodiments, the anti-IL-6 antibody or antigen-binding fragment or derivative is an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza) and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).

[0034] In certain embodiments, the IL-6 antagonist is a single domain antibody, V HH It is a nanobody, Fab, or scFv.

[0035] In certain 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 comprises the six CDRs of tocilizumab or bovalilizumab.

[0036] In certain 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, upadacitinib, baricitinib, filgotinib, lestaurtinib, pacritinib, peficitinib, INCB-039110, ABT-494, INCB-047986, and AC-410.

[0037] In certain embodiments, the IL-6 antagonist is a STAT3 inhibitor.

[0038] In certain embodiments, the IL-6 antagonist is administered parenterally. In a specific embodiment, the IL-6 antagonist is administered subcutaneously.

[0039] In certain embodiments, the IL-6 antagonist is administered orally.

[0040] In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase diuretic efficiency. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase diuretic efficiency to normal levels. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase eGFR. In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase eGFR to normal levels.

[0041] In certain embodiments, the method further comprises a subsequent step of determining the level of IL-6 in urine, determining the level of IL-6 in plasma, or determining the level of IL-6 in urine and plasma. In certain embodiments, the method further comprises a final step of adjusting the dose of the IL-6 antagonist for subsequent administration based on the IL-6 level determined in the immediately preceding step.

[0042] 7. Brief description of the drawings The following detailed description of the preferred embodiments of the invention will be better understood when read in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. [Brief description of the drawings]

[0043] [Figure 1A] FIG. 1 is a bar graph showing diuretic efficiency ("DE") by tertiles of urinary IL-6 in 129 heart failure ("HF") patients receiving high-dose diuretic therapy enrolled in the study described in Example 1 herein. Whiskers extend from the 10th to the 90th percentile. [Figure 1B] FIG. 1B is a bar graph showing diuretic efficiency ("DE") by tertiles of plasma IL-6 in 129 heart failure ("HF") patients whose data are shown in FIG. 1A. Whiskers extend from the 10th to the 90th percentile. [Figure 2A] DE by tertiles of urinary IL-6 in patients reported in Figures 1A and 1B, further stratified by genotype. The left panel shows results for patients homozygous for the TMPRSS6 rs855791 SNP minor allele (2321G→A, A736V), and the right panel shows results for patients with at least one copy of the TMPRSS6 rs855791 SNP major allele (2321G, A736). [Figure 2B]DE by tertile of plasma IL-6 in patients reported in Figures 1A and 1B, further stratified by genotype. The left panel shows results for patients homozygous for the TMPRSS6 rs855791 SNP minor allele (2321G→A, A736V), and the right panel shows results for patients with at least one copy of the TMPRSS6 rs855791 SNP major allele (2321G, A736). [Figure 3A-B] Figure 1 plots the association in a subset of 98 patients out of 129 patients reported in Figures 1 and 2 of (A) urinary IL-6 levels and (B) plasma IL-6 levels with the following clinical decisions: reduced renal function, low diuretic efficiency ("DE"), increased neurohormonal activation, and risk of mortality. Whiskers represent 95% confidence intervals ("CI"). All analyses were adjusted for both urinary and plasma levels of IL-6. Urinary IL-6 levels were indexed to urinary creatinine. Due to skewed distributions of urinary and plasma IL-6 variables, log transformation was applied before performing logistic and Cox regressions. Abbreviations: OR = odds ratio. HR = hazard ratio. IL = interleukin. SD = standard deviation. eGFR = estimated glomerular filtration rate. * = adjusted for use of angiotensin-converting enzyme inhibitors (ACE-I) or angiotensin receptor blockers (ARB). ** = adjusted for baseline characteristics including age, race, amino-terminal pro-B-type natriuretic peptide (NT-proBNP), use of ACE-I or ARB, home loop diuretic dose, and eGFR. [Figure 4A] Figure 4 shows the expression of NKCC2, ENaC-beta, and NCC in M1 CCD cells after treatment with IL-6 and / or ruxolitinib. Figure 4A shows the expression of NKCC2 after treatment with IL-6 and / or ruxolitinib. [Figure 4B] Figure 4B shows the expression of NKCC2, ENaC-beta, and NCC in M1 CCD cells after treatment with IL-6 and / or ruxolitinib.Figure 4B shows the expression of ENaC-beta after treatment with IL-6 and / or ruxolitinib. [Figure 4C] Figure 4C shows the expression of NKCC2, ENaC-beta, and NCC in M1 CCD cells after treatment with IL-6 and / or ruxolitinib. Figure 4C shows the expression of NCC after treatment with IL-6 and / or ruxolitinib. [Figure 5A] Figure 5 shows the expression of NKCC2, ENaC-beta, and NCC in M1 CCD cells after treatment with IL-6 and / or spironolactone. Figure 5A shows the expression of NKCC2 after treatment with IL-6 and / or spironolactone. [Figure 5B] Figure 5B shows the expression of NKCC2, ENaC-beta, and NCC in M1 CCD cells after treatment with IL-6 and / or spironolactone.Figure 5B shows the expression of ENaC-beta after treatment with IL-6 and / or spironolactone. [Figure 5C] Figure 5C shows the expression of NKCC2, ENaC-beta, and NCC in M1 CCD cells after treatment with IL-6 and / or spironolactone. Figure 5C shows the expression of NCC after treatment with IL-6 and / or spironolactone. [Figure 6] FIG. 1 shows the association of higher levels of IL-6 with baseline characteristics in the PROTECT study described in Example 5. [Figure 7A] Figure 7 shows Kaplan-Meier survival curves by tertiles of IL-6.Figure 7A shows all-cause mortality at 180 days. [Figure 7B] Figure 7B shows Kaplan-Meier survival curves by tertiles of IL-6.Figure 7B shows 60-day all-cause mortality or cardiovascular-related readmission. [Figure 8A] Figure 8 shows Kaplan-Meier survival curves for change in IL-6 between baseline and day 7. Figure 8A shows all-cause mortality at 180 days. [Figure 8B] Figure 8B shows Kaplan-Meier survival curves for change in IL-6 between baseline and day 7. Figure 8B shows all-cause mortality or cardiovascular-related readmission at 60 days. [Figure 9]FIG. 1 shows the association of higher levels of IL-6 with baseline characteristics in the BIOSTAT-CHF study described in Example 6. [Figure 10A] Figure 10 shows Kaplan-Meier survival curves by tertiles of IL-6.Figure 10A shows all-cause mortality and / or rehospitalization for heart failure at 2 years. [Figure 10B] Figure 10B shows Kaplan-Meier survival curves by tertiles of IL-6.Figure 10B shows all-cause mortality at 2 years. [Figure 11] FIG. 1 shows tertile analysis of ferritin levels and IL-6. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] 8. Detailed Description 8.1. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It is to be understood that the terminology used herein is for the purpose of the particular embodiments described only and is not intended to be limiting.

[0045] "Transmembrane protease serine 6 (TMPRSS6) polypeptide" refers to a polypeptide or fragment thereof having at least about 85% or more amino acid identity to the amino acid sequence provided in NCBI Accession No. NP_001275929 and having serine proteinase activity. TMPRSS6 polypeptide, also known as matriptase-2 (MT2), cleaves hemojuvelin and inhibits bone morphogenetic protein signaling. An exemplary TMPRSS6 amino acid sequence having an alanine at position 736 (736A) is provided below:

[0046] 1 MPVAEAPQVA GGQGDGGDGE EAEPEGMFKA CEDSKRKARG YLRLVPLFVL LALLVLASAG 61 VLLWYFLGYK AEVMVSQVYS GSLRVLNRHF SQDLTRRESS AFRSETAKAQ KMLKELITST 121 RLGTYYNSSS VYSFGEGPLT CFFWFILQIP EHRRLMLSPE VVQALLVEEL LSTVNSSAAV 181 PYRAEYEVDP EGLVILEASV KDIAALNSTL GCYRYSYVGQ GQVLRLKGPD HLASSCLWHL 241 QGPKDLMLKL RLEWTLAECR DRLAMYDVAG PLEKRLITSV YGCSRQEPVV EVLASGAIMA 301 VVWKKGLHSY YDPFVLSVQP VVFQACEVNL TLDNRLDSQG VLSTPYFPSY YSPQTHCSWH 361 LTVPSLDYGL ALWFDAYALR RQKYDLPCTQ GQWTIQNRRL CGLRILQPYA ERIPVVATAG 421 ITINFTSQIS LTGPGVRVHY GLYNQSDPCP GEFLCSVNGL CVPACDGVKD CPNGLDERNC 481 VCRATFQCKE DSTCISLPKV CDGQPDCLNG SDEEQCQEGV PCGTFTFQCE DRSCVKKPNP 541 QCDGRPDCRD GSDEEHCDCG LQGPSSRIVG GAVSSEGEWP WQASLQVRGR HICGGALIAD 601 RWVITAAHCF QEDSMASTVL WTVFLGKVWQ NSRWPGEVSF KVSRLLLHPY HEEDSHDYDV 661 ALLQLDHPVV RSAAVRPVCL PARSHFFEPG LHCWITGWGA LREGALRADA VALFYGWRNQ 721 GSETCCCPIS NALQKADVQL IPQDLCSEVY RYQVTPRMLC AGYRKGKKDA CQGDSGGPLV 781 CKALSGRWFL AGLVSWGLGC GRPNYFGVYT RITGVISWIQ QVVT (SEQ ID NO: 1)

[0047] An exemplary TMPRSS6 amino acid sequence with a valine at position 736 (736V) is provided below: 1 MPVAEAPQVA GGQGDGGDGE EAEPEGMFKA CEDSKRKARG YLRLVPLFVL LALLVLASAG 61 VLLWYFLGYK AEMVVSQVYS GSLRVLNRHF SQDLTRRESS AFRSETAKAQ KMLKELITST 121 RLGTYYNSSS VYSFGEGPLT CFFWFILQIP EHRRLMLSPE VVQALLVEEL LSTVNSSAAV 181 PYRAEYEVDP EGLVILEASV KDIAALNSTL GCYRYSYVGQ GQVLRLKGPD HLASSCLWHL 24 QGPKDLMLKL RLEWTLAECR DRLAMYDVAG PLEKRLITSV YGCSRQEPVV EVLASGAIMA 30 VVWKKGLHSY YDPFVLSVQP VVFQACEVNL TLDNRLDSQG VLSTPYFPSY YSPQTHCSWH 36 LTVPSLDYGL ALWFDAYALR RQKYDLPCTQ GQWTIQNRRL CGLRILQPYA ERIPVVATAG 42 ITINFTSQIS LTGPGVRVHY GLYNQSDPCP GEFLCSVNGL CVPACDGVKD CPNGLDERNC 48 VCRATFQCKE DSTCISLPKV CDGQPDCLNG SDEEQCQEGV PCGTFTFQCE DRSCVKKPNP 54 QCDGRPDCRD GSDEEHCDCG LQGPSSRIVG GAVSSEGEWP WQASLQVRGR HICGGALIAD 60 RWVITAAHCF QEDSMASTVL WTVFLGKVWQ NSRWPGEVSF KVSRLLLHPY HEEDSHDYDV 66 ALLQLDHPVV RSAAVRPVCL PARSHFFEPG LHCWITGWGA LREGALRADA VALFYGWRNQ 72 GSETCCCPIS NALQKVDVQL IPQDLCSEVY RYQVTPRMLC AGYRKGKKDA CQGDSGGPLV 78 CKALSGRWFL AGLVSWGLGC GRPNYFGVYT RITGVISWIQ QVVT (SEQ ID NO:2)

[0048] By "TMPRSS6 nucleic acid molecule" is meant a polynucleotide encoding a TMPRSS6 polypeptide (matriptase-2, MT2). ​​An exemplary TMPRSS6 nucleic acid molecule sequence is provided in NCBI Accession No. NM_001289000. A TMPRSS6 nucleic acid sequence having a G at nucleotide position 2321 (the "G allele", the "major allele") is provided below:

[0049] 1 GGACAAACAG AGGCTCCTGA GGCCTGTGTG CAGGCCCGGC ACCTATCTGC CGCTCCCAAA 61 GGATGCCCGT GGCCGAGGCC CCCCAGGTGG CTGGCGGGCA GGGGGACGGA GGTGATGGCG 121 AGGAAGCGGA GCCGGAGGGG ATGTTCAAGG CCTGTGAGGA CTCCAAGAGA AAAGCCCGGG 181 GCTACCTCCG CCTGGTGCCC CTGTTTGTGC TGCTGGCCCT GCTCGTGCTG GCTTCGGCGG 241 GGGTGCTACT CTGGTATTTC CTAGGGTACA AGGCGGAGGT GATGGTCAGC CAGGTGTACT 301 CAGGCAGTCT GCGTGTACTC AATCGCCACT TCTCCCAGGA TCTTACCCGC CGGGAATCTA 361 GTGCCTTCCG CAGTGAAACC GCCAAAGCCC AGAAGATGCT CAAGGAGCTC ATCACCAGCA 421 CCCGCCTGGG AACTTACTAC AACTCCAGCT CCGTCTATTC CTTTGGGGAG GGACCCCTCA 481 CCTGCTTCTT CTGGTTCATT CTCCAAATCC CCGAGCACCG CCGGCTGATG CTGAGCCCCG 541 AGGTGGTGCA GGCACTGCTG GTGGAGGAGC TGCTGTCCAC AGTCAACAGC TCGGCTGCCG 601 TCCCCTACAG GGCCGAGTAC GAAGTGGACC CCGAGGGCCT AGTGATCCTG GAAGCCAGTG 661 TGAAAGACAT AGCTGCATTG AATTCCACGC TGGGTTGTTA CCGCTACAGC TACGTGGGCC 721 AGGGCCAGGT CCTCCGGCTG AAGGGGCCTG ACCACCTGGC CTCCAGCTGC CTGTGGCACC 781 TGCAGGGCCC CAAGGACCTC ATGCTCAAAC TCCGGCTGGA GTGGACGCTG GCAGAGTGCC 841 GGGACCGACT GGCCATGTAT GACGTGGCCG GGCCCCTGGA GAAGAGGCTC ATCACCTCGG 901 TGTACGGCTG CAGCCGCCAG GAGCCCGTGG TGGAGGTTCT GGCGTCGGGG GCCATCATGG 961 CGGTCGTCTG GAAGAAGGC CTGCACAGCT ACTACGACCC CTTCGTGCTC TCCGTGCAGC 1021 CGGTGGTCTT CCAGGCCTGT GAAGTGAACC TGACGCTGGA CAACAGGCTC GACTCCCAGG 1081 GCGTCCTCAG CACCCCGTAC TTCCCCAGCT ACTACTCGCC CCAAACCCAC TGCTCCTGGC 1141 ACCTCACGGT GCCCTCTCTG GACTACGGCT TGGCCCTCTG GTTTGATGCC TATGCACTGA 1201 GGAGGCAGAA GTATGATTTG CCGTGCACCC AGGGCCAGTG GACGATCCAG AACAGGAGGC 1261 TGTGTGGCTT GCGCATCCTG CAGCCCTACG CCGAGAGGAT CCCCGTGGTG GCCACGGCCG 1321 GGATCACCAT CAACTTCACC TCCCAGATCT CCCTCACCGG GCCCGGTGTG CGGGTGCACT 1381 ATGGCTTGTA CAACCAGTCG GACCCCTGCC CTGGAGAGTT CCTCTGTTCT GTGAATGGAC 1441 TCTGTGTCCC TGCCTGTGAT GGGGTCAAGG ACTGCCCCAA CGGCCTGGAT GAGAGAAACT 1501 GCGTTTGCAG AGCCACATTC CAGTGCAAAG AGGACAGCAC ATGCATCTCA CTGCCCAAGG 1561 TCTGTGATGG GCAGCCTGAT TGTCTCAACG GCAGCGACGA AGAGCAGTGC CAGGAAGGGG 1621 TGCCATGTGG GACATTCACC TTCCAGTGTG AGGACCGGAG CTGCGTGAAG AAGCCCAACC 1681 CGCAGTGTGA TGGGCGGCCC GACTGCAGGG ACGGCTCGGA TGAGGAGCAC TGTGACTGTG 1741 GCCTCCAGGG CCCCTCCAGC CGCATTGTTG GTGGAGCTGT GTCCTCCGAG GGTGAGTGGC 1801 CATGGCAGGC CAGCCTCCAG GTTCGGGGTC GACACATCTG TGGGGGGGCC CTCATCGCTG 1861 ACCGCTGGGT GATAACAGCT GCCCACTGCT TCCAGGAGGA CAGCATGGCC TCCACGGTGC 1921 TGTGGACCGT GTTCCTGGGC AAGGTGTGGC AGAACTCGCG CTGGCCTGGA GAGGTGTCCT 1981 TCAAGGTGAG CCGCCTGCTC CTGCACCCGT ACCACGAAGA GGACAGCCAT GACTACGACG 2041 TGGCGCTGCT GCAGCTCGAC CACCCGGTGG TGCGCTCGGC CGCCGTGCGC CCCGTCTGCC 2101 TGCCCGCGCG CTCCCACTTC TTCGAGCCCG GCCTGCACTG CTGGATTACG GGCTGGGGCG 2161 CCTTGCGCGA GGGCGCCCTA CGGGCGGATG CTGTGGCCCT ATTTTATGGA TGGAGAAACC 2221 AAGGCTCAGA GACATGTTGC TGCCCCATCA GCAACGCTCT GCAGAAAGTG GATGTGCAGT 2281 TGATCCCACA GGACCTGTGC AGCGAGGTCT ATCGCTACCA 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: 3)

[0050] The TMPRSS6 nucleic acid sequence having an A at nucleotide position 2321 is provided below: 1 GGACAAACAG AGGCTCCTGA GGCCTGTGTG CAGGCCCGGC ACCTATCTGC CGCTCCCAAA 61 GGATGCCCGT GGCCGAGGCC CCCCAGGTGG CTGGCGGGCA GGGGGACGGA GGTGATGGCG 121 AGGAAGCGGA GCCGGAGGGG ATGTTCAAGG CCTGTGAGGA CTCCAAGAGA AAAGCCCGGG 181 GCTACCTCCG CCTGGTGCCC CTGTTTGTGC TGCTGGCCCT GCTCGTGCTG GCTTCGGCGG 241 GGGTGCTACT CTGGTATTTC CTAGGGTACA AGGCGGAGGT GATGGTCAGC CAGGTGTACT 301 CAGGCAGTCT GCGTGTACTC AATCGCCACT TCTCCCAGGA TCTTACCCGC CGGGAATCTA 361 GTGCCTTCCG CAGTGAAACC GCCAAAGCCC AGAAGATGCT CAAGGAGCTC ATCACCAGCA 421 CCCGCCTGGG AACTTACTAC AACTCCAGCT CCGTCTATTC CTTTGGGGAG GGACCCCTCA 481 CCTGCTTCTT CTGGTTCATT CTCCAAATCC CCGAGCACCG CCGGCTGATG CTGAGCCCCG 541 AGGTGGTGCA GGCACTGCTG GTGGAGGAGC TGCTGTCCAC AGTCAACAGC TCGGCTGCCG 601 TCCCCTACAG GGCCGAGTAC GAAGTGGACC CCGAGGGCCT AGTGATCCTG GAAGCCAGTG 661 TGAAAGACAT AGCTGCATTG AATTCCACGC TGGGTTGTTA CCGCTACAGC TACGTGGGCC 721 AGGGCCAGGT CCTCCGGCTG AAGGGGCCTG ACCACCTGGC CTCCAGCTGC CTGTGGCACC 781 TGCAGGGCCC CAAGGACCTC ATGCTCAAAC TCCGGCTGGA GTGGACGCTG GCAGAGTGCC 841 GGGACCGACT GGCCATGTAT GACGTGGCCG GGCCCCTGGA GAAGAGGCTC ATCACCTCGG 901 TGTACGGCTG CAGCCGCCAG GAGCCCGTGG TGGAGGTTCT GGCGTCGGGG GCCATCATGG 961 CGGTCGTCTG GAAGAAGGC CTGCACAGCT ACTACGACCC CTTCGTGCTC TCCGTGCAGC 1021 CGGTGGTCTT CCAGGCCTGT GAAGTGAACC TGACGCTGGA CAACAGGCTC GACTCCCAGG 1081 GCGTCCTCAG CACCCCGTAC TTCCCCAGCT ACTACTCGCC CCAAACCCAC TGCTCCTGGC 1141 ACCTCACGGT GCCCTCTCTG GACTACGGCT TGGCCCTCTG GTTTGATGCC TATGCACTGA 1201 GGAGGCAGAA GTATGATTTG CCGTGCACCC AGGGCCAGTG GACGATCCAG AACAGGAGGC 1261 TGTGTGGCTT GCGCATCCTG CAGCCCTACG CCGAGAGGAT CCCCGTGGTG GCCACGGCCG 1321 GGATCACCAT CAACTTCACC TCCCAGATCT CCCTCACCGG GCCCGGTGTG CGGGTGCACT 1381 ATGGCTTGTA CAACCAGTCG GACCCCTGCC CTGGAGAGTT CCTCTGTTCT GTGAATGGAC 1441 TCTGTGTCCC TGCCTGTGAT GGGGTCAAGG ACTGCCCCAA CGGCCTGGAT GAGAGAAACT 1501 GCGTTTGCAG AGCCACATTC CAGTGCAAAG AGGACAGCAC ATGCATCTCA CTGCCCAAGG 1561 TCTGTGATGG GCAGCCTGAT TGTCTCAACG GCAGCGACGA AGAGCAGTGC CAGGAAGGGG 1621 TGCCATGTGG GACATTCACC TTCCAGTGTG AGGACCGGAG CTGCGTGAAG AAGCCCAACC 1681 CGCAGTGTGA TGGGCGGCCC GACTGCAGGG ACGGCTCGGA TGAGGAGCAC TGTGACTGTG 1741 GCCTCCAGGG CCCCTCCAGC CGCATTGTTG GTGGAGCTGT GTCCTCCGAG GGTGAGTGGC 1801 CATGGCAGGC CAGCCTCCAG GTTCGGGGTC GACACATCTG TGGGGGGGCC CTCATCGCTG 1861 ACCGCTGGGT GATAACAGCT GCCCACTGCT TCCAGGAGGA CAGCATGGCC TCCACGGTGC 1921 TGTGGACCGT GTTCCTGGGC AAGGTGTGGC AGAACTCGCG CTGGCCTGGA GAGGTGTCCT 1981 TCAAGGTGAG CCGCCTGCTC CTGCACCCGT ACCACGAAGA GGACAGCCAT GACTACGACG 2041 TGGCGCTGCT GCAGCTCGAC CACCCGGTGG TGCGCTCGGC CGCCGTGCGC CCCGTCTGCC 2101 TGCCCGCGCG CTCCCACTTC TTCGAGCCCG GCCTGCACTG CTGGATTACG GGCTGGGGCG 2161 CCTTGCGCGA GGGCGCCCTA CGGGCGGATG CTGTGGCCCT ATTTTATGGA TGGAGAAACC 2221 AAGGCTCAGA GACATGTTGC TGCCCCATCA GCAACGCTCT GCAGAAAGTG GATGTGCAGT 2281 TGATCCCACA GGACCTGTGC AGCGAGGTCT ATCGCTACCA AGTGACGCCA CGCATGCTGT 2341 GTGCCGGCTA CCGCAAGGGC AAGAAGGATG CCTGTCAGGG TGACTCAGGT GGTCCGCTGG 2401 TGTGCAAGGC ACTCAGTGGC CGCTGGTTCC TGGCGGGGCT GGTCAGCTGG GGCCTGGGCT 2461 GTGGCCGGCC TAACTACTTC GGCGTCTACA CCCGCATCAC AGGTGTGATC AGCTGGATCC 2521 AGCAAGTGGT GACCTGAGGA ACTGCCCCCC TGCAAAGCAG GGCCCACCTC CTGGACTCAG 2581 AGAGCCCAGG GCAACTGCCA AGCAGGGGGA CAAGTATTCT GGCGGGGGGT GGGGGAGAGA 2641 GCAGGCCCTG TGGTGGCAGG AGGTGGCATC TTGTCTCGTC CCTGATGTCT GCTCCAGTGA 2701 TGGCAGGAGG ATGGAGAAGT GCCAGCAGCT GGGGGTCAAG ACGTCCCCTG AGGACCCAGG 2761 CCCACACCCA GCCCTTCTGC CTCCCAATTC TCTCTCCTCC GTCCCCTTCC TCCACTGCTG 2821 CCTAATGCAA GGCAGTGGCT CAGCAGCAAG AATGCTGGTT CTACATCCCG AGGAGTGTCT 2881 GAGGTGCGCC CCACTCTGTA CAGAGGCTGT TTGGGCAGCC TTGCCTCCAG AGAGCAGATT 2941 CCAGCTTCGG AAGCCCCTGG TCTAACTTGG GATCTGGGAA TGGAAGGTGC TCCCATCGGA 3001 GGGGACCCTC AGAGCCCTGG AGACTGCCAG GTGGGCCTGC TGCCACTGTA AGCCAAAAGG 3061 TGGGGAAGTC CTGACTCCAG GGTCCTTGCC CCACCCCTGC CTGCCACCTG GGCCCTCACA 3121 GCCCAGACCC TCACTGGGAG GTGAGCTCAG CTGCCCTTTG GAATAAAGCT GCCTGATCCA 3181 AAAAAAAAAA AAAAAA (SEQ ID NO:4)

[0051] "Variant" means a polynucleotide or polypeptide sequence that differs from a reference sequence by one or more nucleotides or one or more amino acids. An exemplary TMPRSS6 variant is TMPRSS6(A736V), resulting from SNP rs855791(G→A).

[0052] "Single Nucleotide Polymorphism" or "SNP" refers to a naturally occurring DNA sequence variant in which a single base in a genome differs between members of a species or between paired chromosomes in an individual. SNPs can be used as genetic markers for mutant alleles. In one embodiment, the TMPRSS6 SNP is rs855791.

[0053] "rs855791" refers to a single nucleotide polymorphism (SNP) in the human TMPRSS6 gene, 2321G→A, which results in an alanine to valine substitution (A736V) in the catalytic domain of matriptase-2 (MT2), encoded by the TMPRSS6 gene. The allele with the highest frequency in the human population (major allele) is 2321G, which encodes 736A. The allele with the lowest frequency in the human population (minor allele) is 2321A, which encodes 736V.

[0054] "Heterozygous" means that a chromosomal locus has two different alleles. In one embodiment of the methods described herein, heterozygous refers to a genotype in which one allele has a TMPRSS6 nucleic acid sequence that encodes a TMPRSS6 polypeptide having an alanine at amino acid position 736 (e.g., having a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 major allele) and the other allele comprises a mutant TMPRSS6 nucleic acid sequence that encodes a TMPRSS6 polypeptide that includes a valine at amino acid position 736 (e.g., having an A or T at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 minor allele).

[0055] "Homozygous" means that a chromosomal locus has two identical alleles. In certain embodiments of the methods described herein, homozygous refers to a genotype in which both alleles have a TMPRSS6 nucleic acid sequence that encodes a TMPRSS6 polypeptide that includes an alanine at amino acid position 736 (e.g., has a G or C at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 homozygous major allele). In certain embodiments, homozygous refers to a genotype in which both alleles have a TMPRSS6 nucleic acid sequence that encodes a TMPRSS6 polypeptide that includes a valine at amino acid position 736 (e.g., has an A or T at nucleotide position 2321 of the TMPRSS6 nucleic acid molecule) (rs855791 homozygous minor allele).

[0056] "Determining that a patient has at least one copy of the TMPRSS6 rs855791 major allele" includes, but is not limited to, performing an assay to determine that a patient has at least one copy of the TMPRSS6 rs855791 major allele, prescribing an assay to determine that a patient has at least one copy of the TMPRSS6 rs855791 major allele, otherwise directing or controlling the performance of an assay to determine that a patient has at least one copy of the TMPRSS6 rs855791 major allele, and reviewing TMRSS6 genotype assay data or protein or nucleic acid sequence data to determine that a patient has at least one copy of the TMPRSS6 rs855791 major allele.

[0057] "Interleukin 6 (IL-6)" or "IL-6 polypeptide" refers to a polypeptide or fragment thereof having at least about 85% or more amino acid identity to the amino acid sequence provided in NCBI Accession No. NP_000591 and having IL-6 biological activity. IL-6 is a co-functional cytokine with multiple biological functions. Exemplary IL-6 biological activities include immune stimulatory and proinflammatory activities. An exemplary IL-6 amino acid sequence is provided below:

[0058] 1 MCVGARRLGR GPCAALLLLG LGLSTVTGLH CVGDTYPSND RCCHECRPGN GMVSRCSRSQ 61 NTVCRPCGPG FYNDVVSSKP CKPCTWCNLR SGSERKQLCT ATQDTVCRCR AGTQPLDSYK 121 PGVDCAPCPP GHFSPGDNQA CKPWTNCTLA GKHTLQPASN SSDAICEDRD PPATQPQETQ 181 GPPARPITVQ PTEAWPRTSQ GPSTRPVEVVP GGRAVAAILG LGLVLGLLGP LAILLALYLL 241 RRDQRLPPDA HKPPGGGSFR TPIQEEQADA HSTLAKI (SEQ ID NO:5)

[0059] "Interleukin 6 (IL-6) nucleic acid" refers to a polynucleotide that encodes an Interleukin 6 (IL-6) polypeptide. An exemplary Interleukin 6 (IL-6) nucleic acid sequence is provided at NCBI Accession No. NM_000600. An exemplary sequence at NCBI Accession No. NM_000600 is provided below:

[0060] 1 AATATTAGAG TCTCAACCCC CAATAAATAT AGGACTGGAG ATGTCTGAGG CTCATTCTGC 61 CCTCGAGCCC ACCGGGAACG AAAGAGAAGC TCTATCTCCC CTCCAGGAGC CCAGCTATGA 121 ACTCCTTCTC CACAAGCGCC TTCGGTCCAG TTGCCTTCTC CCTGGGGCTG CTCCTGGTGT 181 TGCCTGCTGC CTTCCCTGCC CCAGTACCCC CAGGAGAAGA TTCCAAAGAT GTAGCCGCCC 241 CACACAGACA GCCACTCACC TCTTCAGAAC GAATTGACAA ACAAATTCGG TACATCCTCG 301 ACGGCATCTC AGCCCTGAGA AAGGAGACAT GTAACAAGAG TAACATGTGT GAAAGCAGCA 361 AAGAGGCACT GGCAGAAAAC AACCTGAACC TTCCAAAGAT GGCTGAAAAA GATGGATGCT 421 TCCAATCTGG ATTCAATGAG GAGACTTGCC TGGTGAAAAT CATCACTGGT CTTTTGGAGT 481 TTGAGGTATA CCTAGAGTAC CTCCAGAACA GATTTGAGAG TAGTGAGGAA CAAGCCAGAG 541 CTGTGCAGAT GAGTACAAAA GTCCTGATCC AGTTCCTGCA GAAAAAGGCA AAGAATCTAG 601 ATGCAATAAC CACCCCTGAC CCAACCACAA ATGCCAGCCT GCTGACGAAG CTGCAGGCAC 661 AGAACCAGTG GCTGCAGGAC ATGACAACTC ATCTCATTCT GCGCAGCTTT AAGGAGTTCC 721 TGCAGTCCAG CCTGAGGGCT CTTCGGCAAA TGTAGCATGG GCACCTCAGA TTGTTGTTGT 781 TAATGGGCAT TCCTTCTTCT GGTCAGAAAC CTGTCCACTG GGCACAGAAC TTATGTTGTT 841 CTCTATGGAG AACTAAAAGT ATGAGCGTTA GGACACTATT TTAATTATTT TTAATTATT 901 AATATTTAAA TATGTGAAGC TGAGTTAATT TATGTAAGTC ATATTTTATAT TTTTAAAGAAG 961 TACCACTTGA AACATTTTAT GTATTAGTTT TGAAATAATA ATGGAAAGTG GCTATGCAGT 1021 TTGAATATCC TTTGTTTCAG AGCCAGATCA TTTCTTGGAA AGTGTAGGCT TACCTCCAAAT 1081 AAATGGCTAA CTTATACATA TTTTTAAAGA AATATTATA TTGTATTTAT ATAATGTATA 1141 AATGGTTTTT ATACCAATAA ATGGCATTTT AAAAAATTCA GCAAAAAAAA AAAAAAAAAA 1201 A (SEQ ID NO:6)

[0061] By "interleukin-6 receptor (IL-6R) complex" is meant a protein complex that includes the IL-6 receptor subunit alpha (IL-6Rα) and interleukin-6 signal transducer glycoprotein 130, also known as interleukin-6 receptor subunit beta (IL-6Rβ).

[0062] "Interleukin-6 receptor subunit alpha (IL-6Rα) polypeptide" refers to a polypeptide or fragment thereof having at least about 85% or more amino acid identity with the amino acid sequence provided in NCBI Accession No. NP_000556 or NP_852004 and having IL-6 receptor biological activity. Exemplary IL-6Rα biological activities include binding to IL-6, binding to glycoprotein 130 (gp130), and regulating cell proliferation and differentiation. An exemplary IL-6R sequence is provided below:

[0063] 1 MLAVGCALLA ALLAAPGAAL APRRCPAQEV ARGVLTSLPG DSVTLTCPGV EPEDNATVHW 61 VLRKPAAGSH PSRWAGMGRR LLLRSVQLHD SGNYSCYRAG RPAGTVHLLV DVPPEEPQLS 121 CFRKSPLSNV VCEWGPRSTP SLTTKAVLLV RKFQNSPAED FQEPCQYSQE SQKFSCQLAV 181 PEGDSSFYIV SMCVASSVGS KFSKTQTFQG CGILQPDPPA NITVTAVARN PRWLSVTWQD 241 PHSWNSSFYR LRFELRYRAE RSKTFTTWMV KDLQHHCVIH DAWSGLRHVV QLRAQEEFGQ 301 GEWSEWSPEA MGTPWTESRS PPAENEVSTP MQALTTNKDD DNILFRDSAN ATSLPVQDSS 361 SVPLPTFLVA GGSLAFGTLL CIAIVLRFKK TWKLRALKEG KTSMHPPYSL GQLVPERPRP 421 TPVLVPLISP PVSPSSLGSD NTSSHNRPDA RDPRSPYDIS NTDYFFPR (SEQ ID NO: 7)

[0064] "Interleukin 6 receptor subunit beta (IL-6Rβ) polypeptide" refers to a polypeptide or fragment thereof having at least about 85% or more amino acid identity with the amino acid sequence provided in NCBI Accession No. NP_002175, NP_786943, or NP_001177910 and having IL-6 receptor biological activity. Exemplary IL-6Rβ biological activities include binding to IL-6Rα, IL-6 receptor signaling activity, and regulation of cell proliferation, differentiation, hepcidin expression, and the like. An exemplary IL-6Rβ sequence is provided below:

[0065] 1 MLTLQTWLVQ ALFIFLTTES TGELLDPCGY ISPESPVVQL HSNFTAVCVL KEKCMDYFHV 61 NANYIVWKTN HFTIPKEQYT IINRTASSVT FTDIASLNIQ LTCNILTFGQ LEQNVYGITI 121 ISGLPPEKPK NLSCIVNEGK KMRCEWDGGR ETHLETNFTL KSEWATHKFA DCKAKRDTPT 181 SCTVDYSTVY FVNIEVWVEA ENALGKVTSD HINFDPVYKV KPNPPHNLSV INSEELSSIL 241 KLTWTNPSIK SVIILKYNIQ YRTKDASTWS QIPPEDTAST RSSFTVQDLK PFTEYVFRIR 301 CMKEDGKGYW SDWSEEASGI TYEDRPSKAP SFWYKIDPSH TQGYRTVQLV WKTLPPFEAN 361 GKILDYEVTL TRWKSHLQNY TVNATKLTVN LTNDRYLATL TVRNLVGKSD AAVLTIPACD 421 FQATHPVMDL KAFPKDNMLW VEWTTPRESV KKYILEWCVL SDKAPCITDW QQEDGTVHRT 481 YLRGNLAESK CYLITVTPVY ADGPGSPESI KAYLKQAPPS KGPTVRTKKV GKNEAVLEWD 541 QLPVDVQNGF IRNYTIFYRT IIGNETAVNV DSSHTEYTLS SLTSDTLYMV RMAAYTDEGG 601 KDGPEFTFTT PKFAQGEIEA IVVPVCLAFL LTTLLGVLFC FNKRDLIKKH IWPNVPDPSK 661 SHIAQWSPHT PPRHNFNSKD QMYSDGNFTD VSVVEIEAND KKPFPEDLKS LDLFKKEKIN 721 TEGHSSGIGG SSCMSSSRPS ISSSDENESS QNTSSTVQYS TVVHSGYRHQ VPSVQVFSRS 781 ESTQPLLDSE ERPEDLQLVD HVDGGDGILP RQQYFKQNCS QHESSPDISH FERSKQVSSV 841 NEEDFVRLKQ QISDHISQSC GSGQMKMFQE VSAADAFGPG TEGQVERFET VGMEAATDEG 901 MPKSYLPQTV RQGGYMPQ (SEQ ID NO:8)

[0066] "IL-6 antagonist" refers to an agent capable of reducing the biological activity of IL-6. IL-6 antagonists include agents that reduce the level of IL-6 polypeptide in serum; agents that reduce the expression of IL-6 polypeptide or nucleic acid, agents that reduce the ability of IL-6 to bind to IL-6R, agents that reduce the expression of IL-6R, and agents that reduce signaling by the IL-6R receptor when bound to IL-6. In a preferred embodiment, the IL-6 antagonist reduces the biological activity of IL-6 by at least about 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95%, or even 100%. As further described in Section 6.3.4 below, IL-6 antagonists include IL-6 binding polypeptides, such as anti-IL-6 antibodies and antigen-binding fragments or derivatives thereof; IL-6R binding polypeptides, such as anti-IL-6R antibodies and antigen-binding fragments or derivatives thereof; and synthetic chemical molecules, such as JAK1 and JAK3 inhibitors.

[0067] By "IL-6 antibody" or "anti-IL-6 antibody" is meant an antibody that specifically binds to IL-6. Anti-IL-6 antibodies include monoclonal and polyclonal antibodies specific for IL-6, as well as antigen-binding fragments or derivatives thereof. IL-6 antibodies are described in more detail below in Section 8.3.6.1.

[0068] As used herein, "diuretic efficiency" is calculated as mmol urinary sodium per two times loop diuretic dose (mmol Na / 2 times loop diuretic dose) according to the method described in Hanberg et al., Circ. Heart Fail. 2016;9:e003180, the disclosure of which is incorporated herein by reference in its entirety.

[0069] "Diuretic-resistant heart failure" means heart failure in which the patient has a diuretic efficiency of less than 100.

[0070] As used herein, the term "biomarker" or "marker" refers to a molecule that can be detected. Thus, biomarkers according to the present invention include, but are not limited to, nucleic acids, polypeptides, carbohydrates, lipids, inorganic molecules, organic molecules, each of which can vary widely in size and nature. A "biomarker" can be a bodily substance that is associated with a bodily condition or disease. A "biomarker" can be detected using any means known in the art, or by previously unknown means that are only apparent to one of skill in the art upon consideration of the marker.

[0071] As used herein, a "biomarker" in the context of the present invention includes, but is not limited to, proteins, nucleic acids, and metabolites, along with their polymorphisms, mutations, variants, modifications, subunits, fragments, protein-ligand complexes, and degradation products, components, associated metabolites, and other analyte- or sample-derived measures. Biomarkers may also include mutant proteins or mutant nucleic acids. Biomarkers also include non-blood-borne or non-analyte physiological markers of health status, such as clinical parameters, as well as traditional laboratory risk factors. As defined by the Food and Drug Administration (FDA), a biomarker is a feature (e.g., measurable DNA and / or RNA or protein) that is "objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic or other interventions." Biomarkers also include mathematically generated calculated indices, or combinations of one or more of the above measurements, including time trends or differences. Biomarkers may be measured at any level of spatial or temporal localization, such as, but not limited to, within a tumor, within a cell, or on the cell membrane.

[0072] "Drug" means any compound or composition suitable for administration in therapy, and expressly includes chemical compounds, proteins, including antibodies or antigen-binding fragments thereof, peptides; and nucleic acid molecules.

[0073] "Subject" or "individual" means a human or non-human mammal, including, but not limited to, bovine, equine, canine, ovine, feline, and rodent subjects such as mice and rats. A "patient" is a human subject.

[0074] As used herein, the terms "treat," "treating," "treatment," and the like refer to the reduction or amelioration of a disorder and / or its associated signs or symptoms, or the slowing or halting of its progression. Although not excluded, it is understood that treating a disorder or condition does not require that the disorder, condition, or symptoms associated therewith be completely eliminated.

[0075] "Pre-treatment" means prior to the first administration of an IL-6 antagonist according to the methods described herein. Pre-treatment does not exclude, and often includes, prior administration of a treatment other than an IL-6 antagonist, such as a diuretic (such as a loop diuretic).

[0076] By "biological sample" is meant any tissue, cell, fluid, or other substance derived from an organism (e.g., a human subject). In certain embodiments, the biological sample is serum, plasma, urine, or whole blood.

[0077] As used herein, "instructional material" includes publications, records, drawings, or any other medium of expression that can be used to communicate the utility of a component of the invention in a kit for detecting a biomarker disclosed herein. The instructional material of the kits of the invention can be, for example, attached to a container that includes a component of the invention or shipped together with a container that contains the component. Alternatively, the instructional material can be shipped separately from the container with the intention that the instructional material and the component be used in concert by the recipient.

[0078] "Level" of one or more biomarkers refers to the absolute or relative amount or concentration of the biomarker in a sample, as determined by measuring mRNA, cDNA, small organic molecules, nucleotides, ions, or proteins, or any portion thereof, such as oligonucleotides or peptides. The level of a biomarker may refer to an overall level, or to the level within a subsection of an organism or within a particular sample, depending on the context; as non-limiting examples, the level may refer to the amount or concentration of the biomarker in a urine sample, or the level may refer to the amount or concentration of the same biomarker in a plasma sample.

[0079] "Measuring" or "measuring", or alternatively "detecting" or "detection", means determining the presence, absence, quantity or amount (which may be an effective amount) of a given substance in a clinical sample or a sample derived from a subject, e.g., deriving a qualitative or quantitative concentration level of such a substance, or otherwise determining the value or classification of a clinical parameter of the subject.

[0080] A "reference level" of a biomarker refers to a level of the biomarker that indicates the presence or absence of a particular phenotype or characteristic. When the level of the biomarker in a subject exceeds the reference level of the biomarker, it indicates the presence, or a relatively elevated level, of a particular phenotype or characteristic. When the level of the biomarker in a subject is below the reference level of the biomarker, it indicates the absence, or a relative absence, of a particular phenotype or characteristic.

[0081] 8.1. Other Interpretive Conventions Unless otherwise specified, residue numbering of antibody constant regions is according to the EU index as in Kabat.

[0082] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. The ranges include the recited endpoints. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have all possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range, e.g., 1-6, should be considered to have specifically disclosed subranges, e.g., 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is true regardless of the breadth of the range.

[0083] Unless otherwise stated or clear from the context, as used herein, the term "or" is understood to be inclusive.

[0084] Unless otherwise specified or clear from the context, as used herein, the terms "a", "an" and "the" are understood to be in the singular or the plural. That is, the articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0085] In this disclosure, the words "comprises," "comprising," "containing," "having," "includes," "including," and variations of these words have the meaning given to them in U.S. patent law and permit the presence of additional elements beyond those expressly recited.

[0086] Unless otherwise specified or clear from the context, as used herein, the term "about" is meant to be understood as within normal tolerances in the art, e.g., within two standard deviations of the mean, and encompasses a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the stated value.

[0087] Antibody equilibrium dissociation constant (K D ) is reported, K D is determined by surface plasmon resonance with an antibody (or antigen-binding fragment thereof) immobilized on the chip surface and a ligand flowed over it.

[0088] 8.2. Experimental Observation Overview As further described below in Example 1, ongoing heart failure ("HF") patients receiving high-dose diuretic therapy at an outpatient treatment center were enrolled in a prospective observational study. Plasma levels of IL-6 were measured to examine the systemic association of this proinflammatory cytokine with various disease parameters, and urinary levels of IL-6 were measured to examine the association of IL-6 with local inflammation and neurohormonal activation at the level of renal tissue.

[0089] Plasma and urinary IL-6 levels were only weakly correlated with each other.

[0090] Increased urinary levels of IL-6 correlated significantly with measures of renal damage, such as diuretic resistance, lower estimated glomerular filtration rate ("eGFR"), and increased tissue levels of renin-angiotensin-aldosterone system ("RAAS") activation in these heart failure patients.

[0091] An inverse correlation between diuretic efficiency and plasma IL-6 was also observed, but after adjusting for eGFR, only urinary IL-6 was the condition significantly associated with the risk of poor diuretic efficiency in these patients.Furthermore, when both urinary IL-6 and plasma IL-6 were entered into a logistic regression model, only urinary IL-6 was the condition associated with the risk of poor diuretic efficiency; plasma IL-6 showed no such association.

[0092] These data indicate that urinary IL-6 levels are a useful biomarker of renal inflammation and can be used to determine renal dysfunction in the setting of heart failure (cardiorenal syndrome). The data further suggest that serial measurements of urinary IL-6 can be used to measure the renal benefit of treatments administered to patients with heart failure, particularly those with cardiorenal syndrome.

[0093] Urinary IL-6 data, and to some extent plasma IL-6 data, predict that IL-6 antagonist treatment should be effective in reducing renal inflammation in heart failure patients, thus treating symptoms of cardiorenal syndrome. However, because infections are often the cause of acute decompensation in heart failure patients, it is important to limit anti-cytokine and other immunosuppressive therapies to heart failure patients who are more likely to respond to improved renal and / or cardiac function. The cost of chronic IL-6 antagonist therapy also works to limit treatment to heart failure patients who are more likely to respond to improved renal and / or cardiac function.

[0094] As detailed in Example 2 below, the analysis performed in Example 1 was extended to 129 patients. Figure 1A is a bar graph showing diuretic efficiency ("DE") by tertiles of urinary IL-6, confirming the inverse correlation between diuretic efficiency and urinary IL-6 levels observed in the 98 patient subset. Figure 1B is a bar graph showing diuretic efficiency ("DE") by tertiles of plasma IL-6 in these 129 patients, confirming the inverse correlation between plasma IL-6 levels and diuretic efficiency.

[0095] The genotype of each of 129 patients at the rs855791 single nucleotide polymorphism ("SNP") of transmembrane protease serine 6 ("TMPRSS6") was evaluated. The TMPRSS6 polypeptide, also known as matriptase-2 (MT2), cleaves hemojuvelin and inhibits bone morphogenetic protein signaling. The rs855791 (G2321A) SNP alters the TMPRSS6 protein sequence. The most frequent allele in the human population (major allele) is 2321G, which codes for 736A. The least frequent allele in the human population (minor allele) is 2321A, which codes for 736V.

[0096] As shown in Figure 2A, urinary levels of IL-6 were inversely correlated with diuretic efficiency only in patients with at least one copy of the major allele of the TMPRSS6 rs855791 SNP (Figure 2A, right panel, "AG+GG"). Urinary levels of IL-6 were not significantly correlated with diuretic efficiency in patients homozygous for the minor allele (Figure 2A, left panel, "AA").

[0097] As shown in Figure 2B, plasma levels of IL-6 were inversely correlated with diuretic efficiency only in patients with at least one copy of the major allele of the TMPRSS6 rs855791 SNP (Figure 2B, right panel, "AG+GG"). Plasma levels of IL-6 were not significantly correlated with diuretic efficiency in patients homozygous for the minor allele (Figure 2B, left panel, "AA").

[0098] These data suggested that diuretic resistance (low diuretic efficiency) in patients with heart failure could be treated with IL-6 antagonists, but only in patients with at least one copy of the TMPRSS6 rs855791 major allele.

[0099] In mouse M1 CCD cells, which are genotypically similar to human cells homozygous for the TMPRSS6 rs855791 major allele, addition of IL-6 correlated with expression of the ion transporters, NKCC2, ENaC-beta, and NCC. Increased expression of these ion transporters provides a putative mechanism for IL-6-mediated diuretic resistance. Also, because increased expression is not known to be related to hepcidin expression, these data suggested that IL-6 antagonism may be effective in treating diuretic resistance even in patients homozygous for the TMPRSS6 rs855791 minor allele.

[0100] Secondary analyses of data from two large clinical trials in different heart failure patient populations confirmed the association between diuretic resistance and IL-6 levels (Example 5), independent of TMPRSS6 rs855791 genotype (Example 6), providing evidence that IL-6 antagonism should also be effective in treating diuretic resistance in patients homozygous for the TMPRSS6 rs855791 minor allele.

[0101] 8.3. Methods of Treating Diuretic Resistance Thus, in a first aspect, a method of treating a patient in need of diuresis but resistant to a diuretic is provided, comprising administering to the patient a therapeutically effective amount of an IL-6 antagonist in combination with a diuretic.

[0102] In certain embodiments, the patient has elevated pre-treatment urinary levels of IL-6. In some embodiments, the patient has elevated pre-treatment plasma IL-6 levels. In certain embodiments, the patient has elevated pre-treatment levels of IL-6 in urine and plasma.

[0103] In some embodiments, the patient has diuretic-resistant heart failure, hi various embodiments, the patient has cardiorenal syndrome.

[0104] In some embodiments, the patient is determined to have at least one copy of the TMPRSS6 rs855791 major allele, hi other embodiments, the patient is homozygous for the TMPRSS6 rs855791 minor allele.

[0105] Diuretic Efficiency In the methods described herein, the patient in need of IL-6 antagonist treatment has a disease or condition requiring diuresis and is diuretic resistant.

[0106] In certain embodiments, the patient is being treated or is being treated with a thiazide diuretic, such as chlorothiazide (Diuril®), chlorthalidone, hydrochlorothiazide (Microzide®), indapamide, or metolazone. In certain embodiments, the patient is being treated or is being treated with a loop diuretic, such as bumetanide (Bumex®), ethacrynic acid (Edecrin®), furosemide (Lasix®), or torsemide (Demadex®). In certain embodiments, the patient is being treated or is being treated with a potassium-sparing diuretic, such as amiloride, eplerenone (Inspra®), spironolactone (Aldactone®), or triamterene (Dyrenium®). In some embodiments, the patient is being treated or is being treated with more than one diuretic. In some embodiments, the patient is being treated or is being treated with multiple different types of diuretics.

[0107] By definition, patients who are resistant to diuretics have low diuretic efficiency. Diuretic efficiency is calculated as the increase in sodium excretion per doubling of loop diuretic dose, centered around a dose of 40 mg IV furosemide equivalent: Diuretic efficiency = (mmol Na excretion) / (log2(administered loop diuretic dose)-4.32). See Hanberg et al., Circ. Heart Fail. 2016, 9:e003180, the disclosure of which is incorporated herein by reference in its entirety.

[0108] In some embodiments, the patient has a diuretic efficiency of less than 500, e.g., less than 450, 400, 350, 300, 250, or 200. In some embodiments, the patient has a diuretic efficiency of less than 200, e.g., less than 195, 190, 185, 180, 175, 170, 165, 160, 155, or 150. In some embodiments, the patient has a diuretic efficiency of less than 150, e.g., less than 145, 140, 135, 130, 125, 120, 110, 105, or 100. In some embodiments, the patient has a diuretic efficiency of less than 100, e.g., less than 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50. In certain embodiments, the patient has a diuretic efficiency of less than 50, e.g., less than 45, 40, 35, 30, or even less than 25, 20, 15, or 10.

[0109] In some embodiments, the diuretic resistant patient requires diuretic therapy of 40 mg or more of furosemide (or equivalent) daily. In some of these embodiments, the patient requires diuretic therapy of 80 mg or more of furosemide (or equivalent) daily. In some of these embodiments, the patient requires diuretic therapy of 120 mg or more of furosemide (or equivalent) daily.

[0110] 8.3.2. Pre-treatment IL-6 levels 8.3.2.1. Pretreatment levels of urinary IL-6 In certain embodiments, the patient has a pretreatment urinary level of IL-6 greater than 5.0 pg IL-6 / g creatinine, 6.0 pg IL-6 / g creatinine, 7.0 pg IL-6 / g creatinine, 8.0 pg IL-6 / g creatinine, 9.0 pg IL-6 / g creatinine, or 10.0 pg IL-6 / g creatinine. In certain embodiments, the patient has a pretreatment urinary level of IL-6 greater than 11.0 pg IL-6 / g creatinine, 12.0 pg IL-6 / g creatinine, 13.0 pg IL-6 / g creatinine, 14.0 pg IL-6 / g creatinine, or 15.0 pg IL-6 / g creatinine. In further embodiments, the patient has a pre-treatment urinary IL-6 level greater than 16.0 pg IL6 / g creatinine, 17.0 pg IL-6 / g creatinine, 18.0 pg IL-6 / g creatinine, 19.0 pg IL-6 / g creatinine, or 20.0 pg IL-6 / g creatinine. In certain embodiments, the patient has a pre-treatment urinary IL-6 level of greater than 21.0 pg IL-6 / g creatinine, 22.0 pg IL-6 / g creatinine, 23.0 pg IL-6 / g creatinine, 24.0 pg IL-6 / g creatinine, 25.0 pg IL-6 / g creatinine, 26.0 pg IL-6 / g creatinine, 27.0 pg IL-6 / g creatinine, 28.0 pg IL-6 / g creatinine, 29.0 pg IL-6 / g creatinine, or even greater than 30.0 pg IL-6 / g creatinine. In certain embodiments, the patient has a pre-treatment urinary IL-6 level of greater than 35.0 pg / g creatinine.

[0111] In certain embodiments, the patient has a pre-treatment urinary level of greater than 14.2 pg of IL-6 / g creatinine ("elevated urinary IL-6 level"). In other embodiments, the patient has a pre-treatment urinary level of less than 14.2 pg of IL-6 / g creatinine.

[0112] In certain embodiments, the patient has a urinary IL-6 level prior to treatment with the IL-6 antagonist and prior to treatment with the loop diuretic of greater than 5.0 pg IL-6 / g creatinine, 6.0 pg IL-6 / g creatinine, 7.0 pg IL-6 / g creatinine, 8.0 pg IL-6 / g creatinine, 9.0 pg IL-6 / g creatinine, or 10.0 pg IL-6 / g creatinine. In some of these embodiments, the patient has an IL-6 urinary level of greater than 11.0 pg IL-6 / g creatinine, 12.0 pg IL-6 / g creatinine, 13.0 pg IL-6 / g creatinine, 14.0 pg IL-6 / g creatinine, or 15.0 pg IL-6 / g creatinine. In a further embodiment, the patient has a urinary IL-6 level prior to treatment with the IL-6 antagonist and prior to treatment with the loop diuretic of greater than 16.0 pg IL6 / g creatinine, 17.0 pg IL-6 / g creatinine, 18.0 pg IL-6 / g creatinine, 19.0 pg IL-6 / g creatinine, or 20.0 pg IL-6 / g creatinine. In certain embodiments, the patient has a urinary IL-6 level of greater than 21.0 pg IL-6 / g creatinine, 22.0 pg IL-6 / g creatinine, 23.0 pg IL-6 / g creatinine, 24.0 pg IL-6 / g creatinine, 25.0 pg IL-6 / g creatinine, 26.0 pg IL-6 / g creatinine, 27.0 pg IL-6 / g creatinine, 28.0 pg IL-6 / g creatinine, 29.0 pg IL-6 / g creatinine, or even greater than 30.0 pg IL-6 / g creatinine. In certain embodiments, the patient has a urinary IL-6 level before treatment with the IL-6 antagonist and before treatment with the loop diuretic of greater than 35.0 pg / g creatinine.

[0113] In certain embodiments, the patient has a urinary IL-6 level prior to treatment with the IL-6 antagonist and prior to treatment with the loop diuretic that is greater than 14.2 pg of IL-6 / g creatinine, while in other embodiments, the patient has a urinary IL-6 level prior to treatment with the IL-6 antagonist and prior to treatment with the loop diuretic that is less than 14.2 pg of IL-6 / g creatinine.

[0114] 8.3.2.2. Pretreatment levels of IL-6 in plasma In various embodiments, the patient has elevated pre-treatment plasma IL-6 levels.

[0115] In certain embodiments, the patient has pre-treatment plasma levels of IL-6 greater than 2.0 pg / mL. In other embodiments, the patient has pre-treatment plasma levels of IL-6 less than 2.0 pg / mL.

[0116] In certain embodiments, the patient has a pretreatment plasma level of IL-6 greater than 1.0 pg / ml, 1.1 pg / ml, 1.2 pg / ml, 1.3 pg / ml, 1.4 pg / ml, 1.5 pg / ml, 1.6 pg / ml, 1.7 pg / ml, 1.8 pg / ml, 1.9 pg / ml, or 2.0 pg / ml. In certain embodiments, the patient has a pretreatment plasma level of IL-6 greater than 2.1 pg / ml, 2.2 pg / ml, 2.3 pg / ml, 2.4 pg / ml, 2.5 pg / ml, 2.6 pg / ml, 2.7 pg / ml, 2.8 pg / ml, 2.9 pg / ml, or 3.0 pg / ml. In certain embodiments, the patient has a pre-treatment plasma level of IL-6 greater than 3.1 pg / ml, 3.2 pg / ml, 3.3 pg / ml, 3.4 pg / ml, 3.5 pg / ml, 3.6 pg / ml, 3.7 pg / ml, 3.8 pg / ml, or 3.9 pg / ml.

[0117] In some embodiments, the patient has a pre-treatment IL-6 level of greater than 2 pg / mL, e.g., greater than 3 pg / mL, 4 pg / mL, 5 pg / mL, 6 pg / mL, 8 pg / mL, 10 pg / mL, 15 pg / mL, or 20 pg / mL. In certain embodiments, the patient has a pre-treatment IL-6 level of greater than 3 pg / mL. In certain embodiments, the patient has a pre-treatment IL-6 level of greater than 5 pg / mL. In certain embodiments, the patient has a pre-treatment IL-6 level of greater than 10 pg / mL.

[0118] Measurement of Pre-Treatment IL-6 Levels Concentrations of IL-6 in urine, plasma, and serum can be determined using any standard assay known in the art. When IL-6 is measured in urine, levels can be indexed or normalized to another biomarker, in certain embodiments, urinary creatinine.

[0119] In certain embodiments, the concentration is measured using the MesoScale Discovery (MSD) platform (Meso Scale Diagnostics, Gaithersburg, MD, USA).

[0120] Heart failure In an exemplary embodiment of the methods described herein, the patient has heart failure.

[0121] In certain embodiments, the patient has NYHA functional class I heart failure. In certain embodiments, the patient has NYHA functional class II heart failure. In certain embodiments, the patient has NYHA functional class III heart failure. In certain embodiments, the patient has NYHA functional class IV heart failure.

[0122] In certain embodiments, the patient has acute heart failure, hi certain embodiments, the patient has chronic heart failure.

[0123] In certain embodiments, the patient has a type of heart failure selected from Table 1 below.

[0124] [Table 1] TIFF0007630906000002.tif38164

[0125] In certain embodiments, the patient has cardiorenal syndrome. In certain embodiments, the patient has cardiorenal syndrome type 1. In certain embodiments, the patient has cardiorenal syndrome type 2. In certain embodiments, the patient has cardiorenal syndrome type 3. In certain embodiments, the patient has cardiorenal syndrome type 4.

[0126] In certain embodiments, the patient has diuretic-resistant heart failure. In some of these embodiments, the heart failure patient has a diuretic efficiency of less than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50. In certain embodiments, the patient has a diuretic efficiency of less than 45, 40, 35, 30, or even less than 25, 20, 15, or 10.

[0127] 8.3.4. Kidney disease In some embodiments of the methods described herein, the patient has renal disease.

[0128] In certain embodiments, the patient has diuretic-resistant renal disease. In some of these embodiments, the renal disease patient has a diuretic efficiency of less than 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, or 50. In certain embodiments, the patient has a diuretic efficiency of less than 45, 40, 35, 30, or even less than 25, 20, 15, or 10.

[0129] In certain embodiments, the patient has hepatorenal syndrome.

[0130] TMPRSS6 rs855791 genotype In certain embodiments, the patient has previously been determined to have at least one copy of the TMPRSS6 rs855791 major allele. In other embodiments, the method further comprises an initial step of determining that the patient has at least one copy of the TMPRSS6 rs855791 major allele.

[0131] Preferably, genotypes are determined at both alleles, thus allowing the identification and differentiation of patients who are homozygous for the TMPRSS6 rs855791 major allele, heterozygous for both the major and minor alleles of TMPRSS6 rs855791, and homozygous for the TMPRSS6 rs855791 minor allele.

[0132] The absence (major allele) or presence (minor allele) of SNP rs855791 (2321G→A) in the TMPRSS6 gene is determined using standard techniques.

[0133] Typically, PCR is used to amplify a biological sample obtained from a patient.

[0134] In certain embodiments, the absence or presence of the polymorphism is detected simultaneously with amplification using real-time PCR (RT-PCR). In certain embodiments, the RT-PCR assay uses 5' nucleases (TaqMan® probes), molecular beacons, and / or FRET hybridization probes. As reviewed in Espy et al., Clin. Microbiol. Rev. January 2006, 19(1):165-256, which is incorporated herein by reference in its entirety. In typical embodiments, commercially available assays are used. In selected embodiments, the commercially available assays are selected from the group consisting of TaqMan™ SNP genotyping assays (ThermoFisher), PCR SNP genotyping assays (Qiagen), Novallele genotyping assays (Canon), and SNP Type™ assays (formerly SNPtype) (Fluidigm).

[0135] In certain embodiments, the absence or presence of the polymorphism is detected by hybridization with a probe specific for SNP rs855791 after amplification, restriction endonuclease digestion, nucleic acid sequencing, primer extension, microarray or gene chip analysis, mass spectrometry, and / or DNAse protection assay. In certain embodiments, the allelic variant is called by sequencing. In certain embodiments, Sanger sequencing is used. In certain embodiments, one of a variety of next-generation sequencing technologies is used, including, for example, a sequencing technology selected from the group consisting of microarray sequencing, Solexa sequencing (Illumina), Ion Torrent (Life Technologies), SOliD (Applied Biosystems), pyrosequencing, single molecule real-time sequencing (Pacific Bio), nanopore sequencing, and tunneling current sequencing.

[0136] In certain embodiments, the absence or presence of a polymorphism is detected using the procedures described in Example 2 below.

[0137] 8.3.6 IL-6 Antagonists The IL-6 antagonists used in the methods described herein are capable of reducing the biological activity of IL-6.

[0138] 8.3.6.1. Anti-IL-6 antibodies In certain embodiments, the IL-6 antagonist is an anti-IL-6 antibody, or an antigen-binding fragment or derivative thereof.

[0139] In certain embodiments, the IL-6 antagonist is a full-length anti-IL-6 monoclonal antibody. In a specific embodiment, the full-length monoclonal antibody is an IgG antibody. In certain embodiments, the full-length monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In certain embodiments, the IL-6 antagonist is a polyclonal composition comprising a plurality of full-length anti-IL-6 antibodies, each of the plurality having a unique CDR. In certain embodiments, the IL-6 antagonist is an antibody fragment selected from Fab, Fab', and F(ab')2 fragments. In certain embodiments, the IL-6 antagonist is a single domain antibody, such as an scFv, a disulfide-linked Fv (dsFv), or a camelid-derived VHH single domain nanobody. In certain embodiments, the IL-6 antagonist is an immunoconjugate or fusion comprising an IL-6 antigen-binding fragment. In certain embodiments, the antibody is bispecific or multispecific, with at least one of the antigen-binding moieties having specificity for IL-6.

[0140] In certain embodiments, the antibody is fully human. In certain embodiments, the antibody is humanized. In certain embodiments, the antibody is chimeric, having non-human V region and human C region domains. In certain embodiments, the antibody is murine.

[0141] In an exemplary embodiment, the anti-IL-6 antibody has a K of less than 100 nM for binding to human IL-6. DFor certain embodiments, the anti-IL-6 antibody has a K of less than 75 nM, 50 nM, 25 nM, 20 nM, 15 nM, or 10 nM for binding to human IL-6. D In specific embodiments, the anti-IL-6 antibody has a K of less than 5 nM, 4 nM, 3 nM, or 2 nM for binding to human IL-6. D In selected embodiments, the anti-IL-6 antibody has a K of less than 1 nM, 750 pM, or 500 pM for binding to human IL-6. D In specific embodiments, the anti-IL-6 antibody has a K of no more than 500 pM, 400 pM, 300 pM, 200 pM, or 100 pM for binding to human IL-6. D has.

[0142] In typical embodiments, the anti-IL-6 antibody neutralizes the biological activity of IL-6, hi certain embodiments, the neutralizing antibody prevents the binding of IL-6 to the IL-6 receptor.

[0143] In typical embodiments, the anti-IL-6 antibody has an elimination half-life following intravenous administration of at least 7 days. In certain embodiments, the anti-IL-6 antibody has an elimination half-life of at least 14 days, at least 21 days, or at least 30 days.

[0144] In certain embodiments, the anti-IL-6 antibody has a human IgG constant region with at least one amino acid substitution that extends serum half-life compared to an unsubstituted human IgG constant domain.

[0145] In certain practical forms, the IgG constant domain comprises substitutions at residues 252, 254, and 256, where the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, the amino acid substitution at amino acid residue 254 is a substitution with threonine, and the amino acid substitution at amino acid residue 256 is a substitution with glutamic acid ("YTE"). See U.S. Patent No. 7,083,784, which is incorporated herein by reference in its entirety. In certain extended half-life embodiments, the IgG constant domain comprises substitutions selected from T250Q / M428L (Hinton et al., J. Immunology 176:346-356 (2006)), N434A (Yeung et al., J. Immunology 182:7663-7671 (2009)), or T307A / E380A / N434A (Petkova et al., International Immunology, 18: 1759-1769 (2006)).

[0146] In certain embodiments, the excretion half-life of anti-IL-6 antibodies is increased by utilizing the FcRN binding properties of human serum albumin. In certain embodiments, the antibodies are conjugated to albumin (Smith et al., Bioconjug. Chem., 12: 750-756 (2001)). In certain embodiments, the anti-IL-6 antibodies are fused to bacterial albumin binding domains (Stork et al., Prot. Eng. Design Science 20: 569-76 (2007)). In certain embodiments, the anti-IL-6 antibodies are fused to albumin binding peptides (Nguygen et al., Prot Eng Design Sel 19: 291-297 (2006)). In certain embodiments, the anti-IL-antibody is bispecific, one specificity directed against IL-6 and one specificity directed against human serum albumin (Ablynx, WO2006 / 122825 (bispecific nanobody)).

[0147] In certain embodiments, the excretion half-life of an anti-IL-6 antibody is increased by PEGylation (Melmed et al., Nature Reviews Drug Discovery 7: 641-642 (2008)), by HPMA copolymer conjugation (Lu et al., Nature Biotechnology 17: 1101-1104 (1999)), by dextran conjugation (Nuclear Medicine Communications, 16: 362-369 (1995)), by conjugation with homoamino acid polymers (HAP, Hap addition) (Schlapschy et al., Prot Eng Design Sel 20: 273-284 (2007)), or by polysialylation (Constantinou et al., Bioconjug. Chem. 20: 924-931 (2009)).

[0148] 8.3.6.1.1. MEDI5117 and derivatives In certain embodiments, the anti-IL-6 antibody, or antigen-binding portion thereof, comprises all six CDRs of MEDI5117. In particular embodiments, the antibody, or antigen-binding portion thereof, comprises the MEDI5117 heavy chain V region and light chain V region. In a specific embodiment, the antibody is a full-length MEDI5117 antibody. The MEDI5117 antibody is described in WO2010 / 088444 and US 2012 / 0034212, the disclosures of which are incorporated herein by reference in their entireties. The MEDI5117 antibody has the following CDRs and heavy and light chain sequences:

[0149] MEDI5117 VH CDR1 SNYMI (SEQ ID NO:9) MEDI5117 VH CDR2 DLYYYAGDTYYADSVKG (SEQ ID NO: 10) MEDI5117 VH CDR3 WADDHPPWIDL (SEQ ID NO:11) MEDI5117 VL CDR1 RASQGISSWLA (SEQ ID NO:12) MEDI5117 VL CDR2 KASTLES (SEQ ID NO:13) MEDI5117 VL CDR3 QQSWLGGS (SEQ ID NO:14)

[0150] MEDI5117 heavy chain EVQLVESGGGLVQPGGSLRLSCAASGFTISSNYMIWVRQAPGKGLEWVSDLYYYAGDTYY ADSVKGRFTMSRDISKNTVYLQMNSLRAEDTAVYYCARWADDHPPWIDLWGRGTLVTVSS ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALAPIEKTISKAKGQPREPQVYTLPSREE MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 15)

[0151] MEDI5117 light chain DIQMTQSPSTLSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKVLIYKASTLESGVPS RFSGSGSGTEFTLTISSLQPDDFATYYCQQSWLGGSFGQGTKLEIKRTVAAPSVFIFPPS DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:16)

[0152] In certain embodiments, the anti-IL-6 antibody is a derivative of MED5117.

[0153] In certain embodiments, MED5117 derivatives contain one or more amino acid substitutions in the MED5117 heavy and / or light chain V regions.

[0154] In certain embodiments, the derivative is a derivative of the original V H and / or V L While retaining specificity for human IL-6, compared to the above, it contains fewer than 25 amino acid substitutions, fewer than 20 amino acid substitutions, fewer than 15 amino acid substitutions, fewer than 10 amino acid substitutions, fewer than 5 amino acid substitutions, fewer than 4 amino acid substitutions, fewer than 3 amino acid substitutions, fewer than 2 amino acid substitutions, or fewer than 1 amino acid substitution.

[0155] In certain embodiments, a MED5117 derivative comprises an amino acid sequence that comprises an amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the VH and VL domains of MEDI5117. Percent sequence identity is determined using the BLAST algorithm using default parameters.

[0156] In certain embodiments, a MED5117 derivative comprises an amino acid sequence whose CDRs are at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to the amino acid sequence of the respective CDR of MEDI5117. Percent sequence identity is determined using the BLAST algorithm using default parameters.

[0157] In certain embodiments, V H and / or V L The CDR derivatives contain conservative amino acid substitutions at one or more predicted non-essential amino acid residues (ie, amino acid residues that are not important for the antibody to specifically bind human IL-6).

[0158] 8.3.6.1.2. Other anti-IL-6 antibodies In certain embodiments, the anti-IL-6 antibody comprises six CDRs from an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, elsilimomab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (Argen-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In certain embodiments, the anti-IL-6 antibody comprises a heavy chain V region and a light chain V region from an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). In specific embodiments, the anti-IL-6 antibody is an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb).

[0159] In certain embodiments, the anti-IL-6 antibodies are described in US 2016 / 0168243, US 2016 / 0130340, 2015 / 0337036, US 2015 / 0203574, US 2015 / 0140011, US 2015 / 0125468, US 2014 / 0302058, US 2014 / 0141013, US 2013 / 0280266, US 2013 / 0017575, US 2010 / 0215654, US 2008 / 0075726, US Patent No. 5,856,135, US 2006 / 0240012, US No. 2006 / 0257407, or U.S. Pat. No. 7,291,721.

[0160] 8.3.6.2. Anti-IL-6 Receptor Antibodies In certain embodiments, the IL-6 antagonist is an anti-IL-6 receptor antibody, or an antigen-binding fragment or derivative thereof.

[0161] In certain embodiments, the IL-6 antagonist is a full length anti-IL-6 receptor monoclonal antibody. In a specific embodiment, the full length monoclonal antibody is an IgG antibody. In certain embodiments, the full length monoclonal antibody is an IgG1, IgG2, IgG3, or IgG4 antibody. In certain embodiments, the IL-6 antagonist is a polyclonal composition comprising a plurality of full length anti-IL-6 receptor antibodies, each of the plurality having a unique CDR. In certain embodiments, the IL-6 antagonist is an antibody fragment selected from Fab and Fab' fragments. In certain embodiments, the IL-6 antagonist is a single domain antibody, an scFv, including a camelid-derived VHH single domain nanobody. In certain embodiments, the antibody is bispecific or multispecific, with at least one of the antigen-binding moieties having specificity for IL-6R.

[0162] In certain embodiments, the antibody is fully human. In certain embodiments, the antibody is humanized. In certain embodiments, the antibody is chimeric, having non-human V region and human C region domains. In certain embodiments, the antibody is murine.

[0163] In an exemplary embodiment, the anti-IL-6 receptor antibody has a K of less than 100 nM for binding to human IL-6R. D In certain embodiments, the anti-IL-6R antibody has a K of less than 75 nM, 50 nM, 25 nM, 20 nM, 15 nM, or 10 nM for binding to human IL-6R. D In specific embodiments, the anti-IL-6 receptor antibody has a K of less than 5 nM, 4 nM, 3 nM, or 2 nM for binding to human IL-6R. D In selected embodiments, the anti-IL-6 receptor antibody has a K of less than 1 nM, 750 pM, or 500 pM for binding to human IL-6R. D In specific embodiments, the anti-IL-6 receptor antibody has a K of 500 pM, 400 pM, 300 pM, 200 pM, or 100 pM or less for binding to human IL-6R. D has.

[0164] In an exemplary embodiment, the anti-IL-6R reduces the biological activity of IL-6.

[0165] In typical embodiments, the anti-IL-6R antibody has an elimination half-life after intravenous administration of at least 7 days. In certain embodiments, the anti-IL-6R antibody has an elimination half-life of at least 14 days, at least 21 days, or at least 30 days.

[0166] In certain embodiments, the anti-IL-6R antibody has a human IgG constant region with at least one amino acid substitution that extends serum half-life compared to the unsubstituted human IgG constant domain.

[0167] In certain practical forms, the IgG constant domain comprises substitutions at residues 252, 254, and 256, where the amino acid substitution at amino acid residue 252 is with tyrosine, the amino acid substitution at amino acid residue 254 is with threonine, and the amino acid substitution at amino acid residue 256 is with glutamic acid ("YTE"). See U.S. Patent No. 7,083,784, which is incorporated herein by reference in its entirety. In certain extended half-life embodiments, the IgG constant domain comprises substitutions selected from T250Q / M428L (Hinton et al., J. Immunology 176:346-356 (2006)), N434A (Yeung et al., J. Immunology 182:7663-7671 (2009)), or T307A / E380A / N434A (Petkova et al., International Immunology, 18: 1759-1769 (2006)).

[0168] In certain embodiments, the excretion half-life of anti-IL-6R antibodies is increased by utilizing the FcRN binding properties of human serum albumin. In certain embodiments, the antibodies are conjugated to albumin (Smith et al., Bioconjug. Chem., 12: 750-756 (2001)). In certain embodiments, the anti-IL-6R antibodies are fused to bacterial albumin binding domains (Stork et al., Prot. Eng. Design Science 20: 569-76 (2007)). In certain embodiments, the anti-IL-6 antibodies are fused to albumin binding peptides (Nguygen et al., Prot Eng Design Sel 19: 291-297 (2006)). In certain embodiments, the anti-IL-antibody is bispecific, with one specificity directed against IL-6R and one specificity directed against human serum albumin (Ablynx, WO2006 / 122825 (bispecific nanobody)).

[0169] In certain embodiments, the excretion half-life of an anti-IL-6R antibody is increased by PEGylation (Melmed et al., Nature Reviews Drug Discovery 7: 641-642 (2008)), by HPMA copolymer conjugation (Lu et al., Nature Biotechnology 17: 1101-1104 (1999)); by dextran conjugation (Nuclear Medicine Communications, 16: 362-369 (1995)), by conjugation with homoamino acid polymers (HAP, Hap addition) (Schlapschy et al., Prot Eng Design Sel 20: 273-284 (2007)), or by polysialylation (Constantinou et al., Bioconjug. Chem. 20: 924-931 (2009)).

[0170] In certain embodiments, the anti-IL-6R antibody or antigen-binding portion thereof comprises all six CDRs of tocilizumab. In particular embodiments, the antibody or antigen-binding portion thereof comprises the heavy chain V region and the light chain V region of tocilizumab. In a specific embodiment, the antibody is a full-length tocilizumab antibody.

[0171] In certain embodiments, the anti-IL-6R antibody or antigen-binding portion thereof comprises all six CDRs of sarilumab. In particular embodiments, the antibody or antigen-binding portion thereof comprises the heavy chain V region and the light chain V region of sarilumab. In a specific embodiment, the antibody is a full-length sarilumab antibody.

[0172] In certain embodiments, the anti-IL-6R antibody, or antigen-binding portion thereof, comprises all six CDRs of VX30 (Vaccinex), ARGX-109 (arGEN-X), FM101 (Formatech), SA237 (Roche), NI-1201 (NovImmune), or an antibody described in US 2012 / 0225060.

[0173] In certain embodiments, the anti-IL-6R antibody or antigen-binding portion thereof is a single domain antibody. In a particular embodiment, the single domain antibody is a camelid VHH single domain antibody. In a specific embodiment, the antibody is bovalilizumab (ALX-0061) (Ablynx NV).

[0174] 8.3.6.3. Anti-IL-6:IL-6R complex antibody In certain embodiments, the IL-6 antagonist is an antibody specific for the complex of IL-6 and IL-6R, hi certain embodiments, the antibody has six CDRs of an antibody selected from those described in US 2011 / 0002936, the entirety of which is incorporated herein by reference.

[0175] JAK and STAT Inhibitors IL-6 is known to signal through the JAK-STAT pathway.

[0176] In certain embodiments, the IL-6 antagonist is an inhibitor of the JAK signaling pathway. In certain embodiments, the JAK inhibitor is a JAK1 specific inhibitor. In certain embodiments, the JAK inhibitor is a JAK3 specific inhibitor. In certain embodiments, the JAK inhibitor is a pan-JAK inhibitor.

[0177] 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.

[0178] In certain embodiments, the IL-6 antagonist is a STAT3 inhibitor. In certain embodiments, the inhibitor is AZD9150 (AstraZeneca, Isis Pharmaceuticals), a STAT3 antisense molecule.

[0179] 8.3.6.5. Additional IL-6 Antagonists In certain embodiments, the IL-6 antagonist is an antagonist peptide.

[0180] In certain embodiments, the IL-6 antagonist is C326 (IL-6 antagonist by Avidia, also known as AMG220), or FE301, a recombinant protein inhibitor of IL-6, (Ferring International Center SA, Conaris Research Institute AG). In certain embodiments, the anti-IL-6 antagonist comprises soluble gp130, FE301 (Conaris / Ferring).

[0181] 8.3.7. Medication regimens 8.3.7.1. Antibodies, antigen-binding fragments, and peptides In an exemplary embodiment, the antibody, antigen-binding fragment, and peptide IL-6 antagonists are administered parenterally.

[0182] In some parenteral embodiments, the IL-6 antagonist is administered intravenously. In certain intravenous embodiments, the IL-6 antagonist is administered as a bolus. In certain intravenous embodiments, the IL-6 antagonist is administered as an infusion. In certain intravenous embodiments, the IL-6 antagonist is administered as a bolus followed by an infusion. In some parenteral embodiments, the IL-6 antagonist is administered subcutaneously.

[0183] In certain embodiments, the antibody, antigen-binding fragment, or peptide IL-6 antagonist is administered at a dose that is independent of the patient's weight or surface area (flat dose).

[0184] In certain embodiments, the intravenous plateau dose is 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, or 10 mg. In certain embodiments, the intravenous plateau dose is 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, or 20 mg. In certain embodiments, the intravenous plateau dose is 25 mg, 30 mg, 40 mg, or 50 mg. In certain embodiments, the intravenous plateau dose is 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg. In certain embodiments, the intravenous plateau dose is 1-10 mg, 10-15 mg, 15-20 mg, 20-30 mg, 30-40 mg, or 40-50 mg. In certain embodiments, the intravenous plateau dose is 1-40 mg or 50-100 mg.

[0185] In certain embodiments, the subcutaneous plateau dose is 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, or 100 mg. In certain embodiments, the subcutaneous plateau dose is 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, or 200 mg. In certain embodiments, the subcutaneous plateau dose is 210 mg, 220 mg, 230 mg, 240 mg, or 250 mg. In certain embodiments, the subcutaneous plateau dose is 10-100 mg, 100-200 mg, or 200-250 mg. In certain embodiments, the subcutaneous flat dose is 10-20 mg, 20-30 mg, 30-40 mg, 40-50 mg, 50-60 mg, 60-70 mg, 70-80 mg, 80-90 mg, or 90-100 mg. In certain embodiments, the subcutaneous flat dose is 100-125 mg, 125-150 mg, 150-175 mg, 175-200 mg, or 200-250 mg.

[0186] In certain embodiments, the antibody, antigen-binding fragment, or peptide IL-6 antagonist is administered as a patient weight-based dose.

[0187] In certain embodiments, the antagonist is administered at an intravenous dose of 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1.0 mg / kg. In certain embodiments, the antagonist is administered at a dose of 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, or 5 mg / kg.

[0188] In certain embodiments, the dose on a subcutaneous weight basis is 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 0.6 mg / kg, 0.7 mg / kg, 0.8 mg / kg, 0.9 mg / kg, or 1.0 mg / kg. In certain embodiments, the antagonist is administered at a dose of 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, or 5 mg / kg.

[0189] In various intravenous embodiments, the IL-6 antagonist is administered once every 7 days, once every 14 days, once every 21 days, once every 28 days, or once a month. In various subcutaneous embodiments, the IL-6 antagonist is administered once every 14 days, once every 28 days, once a month, once every two months (every other month), or once every three months.

[0190] In certain preferred embodiments, the IL-6 antagonist is a MEDI5117 antibody. In certain embodiments, MEDI5117 is administered IV once weekly at a flat dose of 1-30 mg. In certain embodiments, MEDI5117 antibody is administered IV once weekly at a flat dose of 1, 2, 3, 4, 5, 7.5, 10, 15, 20, 25, or 30 mg. In certain embodiments, MEDI5117 antibody is administered sc once monthly to once every three months at a flat dose of 25-250 mg. In particular embodiments, MEDI5117 is administered sc once monthly, every two months, or every three months at a dose of 30 mg, 45 mg, 60 mg, 75 mg, 100 mg, 120 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 240 mg, or 250 mg.

[0191] In certain embodiments, the IL-6 antagonist is tocilizumab. In certain embodiments, tocilizumab is administered sc once a week to patients weighing 100 kg or more at a starting dose of 162 mg. In certain embodiments, tocilizumab is administered intravenously at a dose of 4 mg / kg once every 4 weeks, then increased to 8 mg / kg every 4 weeks based on clinical response.

[0192] JAK and STAT Inhibitors In an exemplary embodiment, the small molecule JAK inhibitor and STAT inhibitor are administered orally.

[0193] In certain embodiments, the inhibitor is administered at an oral dose of 1-10 mg, 10-20 mg, 20-30 mg, 30-40 mg, or 40-50 mg once or twice daily. In certain embodiments, the inhibitor is administered at a dose of 50-60 mg, 60-70 mg, 70-80 mg, 80-90 mg, or 90-100 mg once or twice daily. In certain embodiments, the inhibitor is administered at a dose of 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 mg PO once or twice daily. In certain embodiments, the inhibitor is administered at a dose of 75 mg PO QD or BID, 100 mg PO QD or BID.

[0194] In certain embodiments, the JAK inhibitor is tofacitinib, administered at a dose of 5 mg PO BID or 11 mg PO qDay.

[0195] In certain embodiments, the JAK inhibitor is decernotinib and is administered at a dose of 25 mg, 50 mg, 100 mg, or 150 mg PO BID.

[0196] In certain embodiments, the inhibitor is ruxolitinib and is administered at a dose of 25 mg PO BID, 20 mg PO BID, 15 mg PO BID, 10 mg PO BID, or 5 mg PO BID.

[0197] 8.3.7.3. Addressing goals In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase diuretic efficiency. In some of these embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase diuretic efficiency to normal levels.

[0198] In certain embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase eGFR, and in some of these embodiments, the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase eGFR to normal levels.

[0199] 8.3.7.4. IL-6 Level Monitoring In certain embodiments, the impact of treatment with an IL-6 antagonist on cardiorenal parameters can be monitored by measuring the level of IL-6 in urine or plasma samples from patients. It is specifically contemplated that the methods of the present invention can be used to monitor the effectiveness of treatment of cardiorenal syndrome, for example, to motivate changes in dosage or treatment.

[0200] Thus, in certain embodiments, the methods further comprise a subsequent step of determining the level of IL-6 in urine, plasma, or both urine and plasma, and in some of these embodiments, the methods further comprise a final step of adjusting the dose of at least one subsequent administration of the IL-6 antagonist based on the IL-6 level determined in the immediately preceding step.

[0201] 8.3.8. Additional Therapeutic Drugs In certain embodiments of the methods described herein, the methods further comprise administering at least one therapeutic agent in addition to the IL-6 antagonist, the additional therapeutic agent treating one or more cardiovascular or renal symptoms of heart failure. The particular treatment is determined on a case-by-case basis by the attending physician.

[0202] 8.3.8.1. Standard heart failure medications In certain embodiments, the additional therapeutic agent is a diuretic.

[0203] In certain embodiments, the diuretic is a loop diuretic. In selected embodiments, the loop diuretic is selected from the group consisting of furosemide, torsemide, bumetanide, and ethacrynic acid. In certain embodiments, the loop diuretic is furosemide. In certain embodiments, the furosemide is administered orally. In certain embodiments, the furosemide is administered intravenously. In certain embodiments, the diuretic is a thiazide diuretic. In certain embodiments, the thiazide diuretic is chlorothiazide, hydrochlorothiazide, chlorthalidone, indapamide, or metolazone. In certain embodiments, the diuretic is a potassium-sparing diuretic.

[0204] In certain embodiments, the additional therapeutic agent is an ACE inhibitor, hi certain embodiments, the ACE inhibitor is selected from the group consisting of benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quinapril, ramipril, and trandolapril.

[0205] In certain embodiments, the additional therapeutic agent is an angiotensin receptor blocker ("ARB"). In certain embodiments, the ARB is eprosartan, olmesartan, valsartan, telmisartan, losartan, azilsartan medoxomil, candesartan, or irbesartan.

[0206] In certain embodiments, the additional therapeutic agent is a beta-blocker, a calcium antagonist, or a mineralocorticoid receptor antagonist.

[0207] In certain embodiments, the additional therapeutic agent is a natriuretic peptide, such as B-type natriuretic peptide or N-terminal pro-B-type natriuretic peptide.

[0208] In certain embodiments, the additional therapeutic agent is an adenosine antagonist, such as rolofylline.

[0209] Specific treatment will be determined on a case-by-case basis by the treating physician.

[0210] 8.3.8.2. Nitroxyl donors In certain embodiments, the additional therapeutic agent is a nitroxyl donor, and the method further comprises administering a therapeutically effective amount of the nitroxyl donor.

[0211] In certain embodiments, the nitroxyl donor is selected from compounds described in one or more of U.S. Pat. Nos. 9,499,511, 9,487,498, 9,464,061, 9,458,127, 9,221,780, 9,181,213, 9,156,804, 9,115,064, 9,018,411, 8,987,326, RE45,314, 8,674,132, 8,227,639, and 8,030,356, the disclosures of which are incorporated by reference herein in their entireties.

[0212] In selected embodiments, the nitroxyl donor is selected from the compounds described in U.S. Patent No. RE45,314. In certain embodiments, the nitroxyl donor is selected from the compounds described in U.S. Patent No. 9,156,804.

[0213] 8.3.8.3. Sodium-free chloride supplement In certain embodiments, the additional therapeutic agent is a chloride salt that does not contain a sodium ion, hi certain embodiments, the agent is lysine chloride.

[0214] 8.4. Ways to improve the treatment of heart failure In another aspect, a method is provided for improving the treatment of heart failure by stopping ineffective treatment, thereby reducing side effects and reducing costs without compromising therapeutic efficacy.The method comprises stopping administration of an IL-6 antagonist to a patient with heart failure, the patient being determined to be homozygous for the TMPRSS6 rs855791 minor allele.In one set of embodiments, the patient has previously been determined to be homozygous for the TMPRSS6 rs855791 minor allele.In another set of embodiments, the method further comprises an initial step of determining that the patient is homozygous for the TMPRSS6 rs8557791 minor allele.

[0215] In certain embodiments, the patient has elevated pre-treatment urinary levels of IL-6. In certain embodiments, the patient has elevated pre-treatment plasma levels of IL-6. In certain embodiments, the patient has elevated pre-treatment urinary levels and elevated pre-treatment plasma levels of IL-6.

[0216] In certain embodiments, the patient has cardiorenal syndrome.

[0217] 8.5. Methods of diagnosis, prognosis, and treatment guidance In another aspect, a method is provided for determining whether a subject will benefit from IL-6 antagonist treatment for heart failure. The method includes the steps of measuring the level of IL-6 in a urine sample or plasma sample from a subject, comparing the measured level of IL-6 with a predetermined reference level, and determining whether the measured level of IL-6 is greater than the corresponding reference level, and if the measured level of IL-6 is greater than the corresponding reference level, IL-6 antagonist treatment is recommended.

[0218] IL-6 can be measured in plasma or urine according to the methods described above in section 6.3.2.3. Reference levels of IL-6 in urine and plasma can be determined by measuring IL-6 levels in a reference population. Those skilled in the art can determine reference levels for the levels of the biomarker in a population based on clinical experience and the general levels of the biomarker in samples from the population.

[0219] In a further embodiment, a method is provided for determining whether a subject requires IL-6 antagonist treatment due to glomerular filtration impairment, low diuretic efficiency, high urinary angiotensin, high plasma renin, or is at risk of death due to cardiorenal syndrome using the above method. As shown in Figure 3 and discussed in Example 1 below, high levels of IL-6 in urine or plasma are associated with these parameters.

[0220] 8.6.Kit In a further aspect, kits are provided. Generally, the kits include detection reagents suitable for detecting the presence of a biomarker of interest and include instructions for use according to the methods of the invention. The kits may include antibodies or other immunohistochemical reagents capable of binding to IL-6. The kits may include capture and detection antibodies suitable for performing an ELISA to measure IL-6 in urine or plasma. In certain embodiments, the kits may include tools and reagents for preparing urine and plasma samples for ELISA or for indexing IL-6 in urine against the concentration of another biomarker, in certain embodiments IL-6 is indexed against creatinine.

[0221] 8.7. Further embodiments Further embodiments are provided in the following numbered embodiments. 1. A method of treating heart failure comprising administering a therapeutically effective amount of an IL-6 antagonist to a patient having heart failure, The method, wherein the patient has been determined to have at least one copy of the TMPRSS6 rs855791 major allele. 2. The method of embodiment 1, wherein the patient has previously been determined to have at least one copy of the TMPRSS6 rs855791 major allele. 3. The method of embodiment 1, further comprising the initial step of determining that the patient has at least one copy of the TMPRSS6 rs855791 major allele. 4. The method of any one of embodiments 1-3, wherein the patient has elevated pre-treatment urinary levels of IL-6. 5. The method of any one of embodiments 1-4, wherein the patient has elevated pre-treatment plasma levels of IL-6. 6. The method of any one of embodiments 1-5, wherein the patient has acute heart failure. 7. The method of any one of embodiments 1-5, wherein the patient has chronic heart failure. 8. The method of any one of embodiments 1-7, wherein the patient has cardiorenal syndrome. 9. The method of embodiment 8, wherein the patient has cardiorenal syndrome type 4. 10. The method of any one of embodiments 1-9, wherein the patient has diuretic-resistant heart failure. 11. The method of embodiment 10, wherein the patient has a diuretic efficiency of less than 95. 12. The method of embodiment 11, wherein the patient has a diuretic efficiency of less than 90. 13. The method of embodiment 12, wherein the patient has a diuretic efficiency of less than 85. 14. The method of embodiment 13, wherein the patient has a diuretic efficiency of less than 80. 15. The method of embodiment 14, wherein the patient has a diuretic efficiency of less than 75. 16. The method of embodiment 15, wherein the patient has a diuretic efficiency of less than 70. 17. The method of embodiment 16, wherein the patient has a diuretic efficiency of less than 65. 18. The method of any one of embodiments 1-17, wherein the IL-6 antagonist is an anti-IL-6 antibody, or an antigen-binding fragment or derivative thereof. 19. The anti-IL-6 antibody, or antigen-binding fragment or derivative, has a K of less than 100 nM for binding to human IL-6. D 19. The method of embodiment 18, comprising: 20. The antibody or antigen-binding fragment or derivative has a K of less than 50 nM for binding to human IL-6. D 20. The method of embodiment 19, comprising: 21. The antibody or antigen-binding fragment or derivative has a K of less than 10 nM for binding to human IL-6. D 21. The method of embodiment 20, comprising: 22. The antibody or antigen-binding fragment or derivative has a K of less than 1 nM for binding to human IL-6. D 22. The method of embodiment 21, comprising: 23. The method of any one of embodiments 18-22, wherein the anti-IL-6 antibody, or antigen-binding fragment or derivative, has an elimination half-life following intravenous administration of at least 7 days. 24. The method of embodiment 23, wherein the anti-IL-6 antibody, or antigen-binding fragment or derivative, has an elimination half-life following intravenous administration of at least 14 days. 25. The method of embodiment 24, wherein the anti-IL-6 antibody, or antigen-binding fragment or derivative, has an elimination half-life following intravenous administration of at least 21 days. 26. The method of embodiment 25, wherein the anti-IL-6 antibody, or antigen-binding fragment or derivative, has an elimination half-life following intravenous administration of at least 30 days. 27. The method of any one of embodiments 18-26, wherein the IL-6 antagonist is a full-length monoclonal anti-IL-6 antibody. 28. The method of embodiment 27, wherein the antibody is an IgG1 or IgG4 antibody. 29. The method of embodiment 28, wherein the antibody is an IgG1 antibody. 30. The method of any one of embodiments 18-29, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is fully human. 31. The method of any one of embodiments 18-29, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is humanized. 32. The method of any one of embodiments 18-31, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable region CDRs of MED5117. 33. The method of embodiment 32, wherein the antibody comprises the VH and VL of MED5117. 34. The method of embodiment 33, wherein the antibody is MED5117. 35. The method of any one of embodiments 18-31, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable region CDRs of an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, elsilimomab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza) and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). 36. The method of embodiment 35, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises a heavy chain V region and a light chain V region from an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza) and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). 37. The method of embodiment 36, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza) and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). 38. IL-6 antagonist is a single domain antibody, V HH The method of any one of embodiments 18-26, wherein the antibody is a nanobody, Fab, or scFv. 39. The method of any one of embodiments 1-17, wherein the IL-6 antagonist is an anti-IL-6R antibody, or an antigen-binding fragment or derivative thereof. 40. The method of embodiment 39, wherein the anti-IL-6R antibody, antigen-binding fragment or derivative comprises the six CDRs of tocilizumab. 41. The method of embodiment 39, wherein the anti-IL-6R antibody, antigen-binding fragment or derivative comprises the six CDRs of bovalilizumab. 42. The method of any one of embodiments 1-17, wherein the IL-6 antagonist is a JAK inhibitor. 43. The method of embodiment 42, wherein the JAK inhibitor is selected from the group consisting of tofacitinib (Xeljanz), decernotinib, ruxolitinib, upadacitinib, baricitinib, filgotinib, lestaurtinib, pacritinib, peficitinib, INCB-039110, ABT-494, INCB-047986 and AC-410. 44. The method of any one of embodiments 1-17, wherein the IL-6 antagonist is a STAT3 inhibitor. 45. The method of any one of embodiments 18-41, wherein the IL-6 antagonist is administered parenterally. 46. ​​The method of embodiment 45, wherein the IL-6 antagonist is administered subcutaneously. 47. The method of any one of embodiments 42 or 43, wherein the IL-6 antagonist is administered orally. 48. The method of any one of embodiments 1-47, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase diuretic efficiency. 49. The method of embodiment 48, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase diuretic efficiency to normal levels. 50. The method of any one of embodiments 1-49, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase eGFR. 51. The method of embodiment 50, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase eGFR to normal levels. 52. The method of any one of embodiments 1-51, further comprising a subsequent step of determining the level of IL-6 in the urine. 53. The method of any one of embodiments 1-51, further comprising a subsequent step of determining the level of IL-6 in plasma. 54. The method of any one of embodiments 1-51, further comprising the subsequent step of determining the levels of IL-6 in urine and plasma. 55. The method of any one of embodiments 52-54, further comprising a final step of adjusting the dose of the IL-6 antagonist for subsequent administration based on the IL-6 level determined in the immediately preceding step. 56. A method for determining whether a subject is in need of treatment for cardiorenal syndrome, comprising: (a) measuring the level of IL-6 in a urine or plasma sample from a subject; (b) comparing the measured level of IL-6 to a predetermined reference level; (c) determining whether the measured level of IL-6 is greater than the corresponding reference level, and if the measured level of IL-6 is greater than the corresponding reference level, the subject has cardiorenal syndrome and treatment is recommended. 57. A method for determining whether a subject is in need of treatment for glomerular filtration disorder, comprising: (a) measuring the level of IL-6 in a urine sample from a subject; (b) comparing the measured level of IL-6 to a predetermined reference level; (c) determining whether the measured level of IL-6 is greater than the corresponding reference level, wherein if the measured level of IL-6 is greater than the corresponding reference level, the subject has a glomerular filtration disorder and treatment is recommended. 58. A method for determining whether a subject requires treatment for low diuretic efficiency, comprising: (a) measuring the level of IL-6 in a urine sample from a subject; (b) comparing the measured level of IL-6 to a predetermined reference level; (c) determining whether the measured level of IL-6 is greater than the corresponding reference level, and if the measured level of IL-6 is greater than the corresponding reference level, the subject has low diuretic efficiency and treatment is recommended. 59. A method for determining whether a subject is in need of treatment for hyperuric angiotensin II, comprising the steps of: (a) measuring the level of IL-6 in a urine sample from the subject; (b) comparing the measured level of IL-6 to a predetermined reference level; (c) determining whether the measured level of IL-6 is greater than the corresponding reference level, and if the measured level of IL-6 is greater than the corresponding reference level, the subject has hyperuricemia and treatment is recommended. 60. A method for determining whether a subject is in need of treatment for elevated plasma renin, comprising: (a) measuring the level of IL-6 in a plasma sample from a patient; (b) comparing the measured level of IL-6 to a predetermined reference level; (c) determining whether the measured level of IL-6 is greater than the corresponding reference level, and if the measured level of IL-6 is greater than the corresponding reference level, the subject has high plasma renin and treatment is recommended. 61. A method for determining whether a subject is at risk for death from cardiorenal syndrome and in need of treatment, comprising: (a) measuring the level of IL-6 in a plasma sample from a patient; (b) comparing the measured level of IL-6 to a predetermined reference level; (c) determining whether the measured level of IL-6 is greater than the corresponding reference level, and if the measured level of IL-6 is greater than the corresponding reference level, the subject is at risk of death from cardiorenal syndrome and treatment is recommended. 62. The method of any one of embodiments 56-61, wherein the patient is a heart failure patient. 63. The method of any of embodiments 56-62, wherein the level of IL-6 is measured using an enzyme-linked immunosorbent assay (ELISA). 64. A method for treating cardiorenal syndrome in a patient, comprising: (a) measuring the level of IL-6 in a urine or plasma sample from a patient; (b) comparing the measured level of IL-6 to a predetermined reference level; (c) determining whether the measured level of IL-6 is greater than a corresponding reference level; and if the measured level of IL-6 is greater than the corresponding reference level, treatment of cardiorenal syndrome is performed. 65. The method of embodiment 64, wherein the patient is a heart failure patient. 66. The method of embodiment 64 or embodiment 65, wherein the level of IL-6 is measured using an enzyme-linked immunosorbent assay (ELISA). 67. The method of any one of embodiments 64-66, wherein the treatment administered for cardiorenal syndrome comprises administering one or more of at least one diuretic, angiotensin-converting enzyme inhibitor, angiotensin receptor blocker, natriuretic peptide, adenosine antagonist, IL-6 antagonist, or any combination thereof. 68. The method of embodiment 67, wherein the treatment of cardiorenal syndrome comprises administering at least one anti-IL-6 antibody or anti-IL-6R antibody. 69. A kit comprising reagents and written instructions for an assay for measuring levels of IL-6, the written instructions comprising: (a) measuring the level of IL-6 in a urine or plasma sample from a subject; (b) comparing the measured level of IL-6 to a predetermined reference level; (c) determining whether the measured level of IL-6 is greater than the corresponding reference level, wherein if the measured level of IL-6 is greater than the corresponding reference level, the subject has cardiorenal syndrome and treatment is recommended. 70. The kit of embodiment 69, wherein the instructions instruct the kit to be used in a heart failure patient. 71. The kit of embodiment 69 or 70, wherein the kit comprises reagents for measuring the level of IL-6 using an enzyme-linked immunosorbent assay (ELISA). The present invention also includes the following embodiments: [Embodiment 1] A method for treating a patient who requires but is resistant to a diuretic, comprising administering to the patient a therapeutically effective amount of an IL-6 antagonist in combination with a diuretic. [Embodiment 2] The method of embodiment 1, wherein the patient has elevated pre-treatment plasma IL-6 levels. [Embodiment 3] The method of embodiment 2, wherein the patient has a pre-treatment plasma IL-6 level greater than 2 pg / mL. [Embodiment 4] The method of embodiment 3, wherein the patient has a pre-treatment IL-6 level greater than 3 pg / mL. [Embodiment 5] The method of embodiment 4, wherein the patient has a pre-treatment IL-6 level greater than 5 pg / mL. [Embodiment 6] The method of embodiment 5, wherein the patient has a pre-treatment IL-6 level of greater than 10 pg / mL. [Embodiment 7] The method of any one of embodiments 1 to 6, wherein the patient has a diuretic efficiency of less than 500. [Embodiment 8] The method of embodiment 7, wherein the patient has a diuretic efficiency of less than 200. [Embodiment 9] The method of embodiment 8, wherein the patient has a diuretic efficiency of less than 150. [Embodiment 10] The method of embodiment 9, wherein the patient has a diuretic efficiency of less than 100. [Embodiment 11] The method according to any one of embodiments 1 to 10, wherein the patient has diuretic-resistant heart failure. [Embodiment 12] The method of embodiment 11, wherein the patient has acute heart failure. [Embodiment 13] The method of embodiment 11, wherein the patient has chronic heart failure. [Embodiment 14] The method according to any one of embodiments 1 to 13, wherein the patient has cardiorenal syndrome. [Embodiment 15] The method of embodiment 14, wherein the patient has cardiorenal syndrome type 4. [Embodiment 16] The method according to any one of embodiments 1 to 10, wherein the patient has renal disease. [Embodiment 17] The method of embodiment 16, wherein the patient has hepatorenal syndrome. [Embodiment 18] The method of any one of embodiments 1 to 17, wherein the patient has at least one copy of the TMPRSS6 rs855791 major allele. [Embodiment 19] The method of any one of embodiments 1 to 18, wherein the IL-6 antagonist is an anti-IL-6 antibody, or an antigen-binding fragment or derivative thereof. [Embodiment 20] The anti-IL-6 antibody, or antigen-binding fragment or derivative thereof has a K of less than 100 nM for binding to human IL-6. D 20. The method of embodiment 19, comprising the steps of: [Embodiment 21] The anti-IL-6 antibody or antigen-binding fragment or derivative has a K of less than 50 nM for binding to human IL-6. D 21. The method of embodiment 20, comprising: [Embodiment 22] The anti-IL-6 antibody or antigen-binding fragment or derivative has a K of less than 10 nM for binding to human IL-6. D 22. The method of embodiment 21, comprising: [Embodiment 23] The anti-IL-6 antibody, or antigen-binding fragment or derivative thereof has a K of less than 1 nM for binding to human IL-6. D 23. The method of embodiment 22, comprising: [Embodiment 24] The method of any one of embodiments 19 to 23, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative has an excretion half-life after intravenous administration of at least 7 days. [Embodiment 25] The method of embodiment 24, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative has an excretion half-life after intravenous administration of at least 14 days. [Embodiment 26] The method of embodiment 25, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative has an excretion half-life after intravenous administration of at least 21 days. [Embodiment 27] The method of embodiment 26, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative has an excretion half-life after intravenous administration of at least 30 days. [Embodiment 28] The method of any one of embodiments 19 to 27, wherein the IL-6 antagonist is a full-length monoclonal anti-IL-6 antibody. [Embodiment 29] The method described in embodiment 28, wherein the antibody is an IgG1 or IgG4 antibody. [Embodiment 30] The method described in embodiment 29, wherein the antibody is an IgG1 antibody. [Embodiment 31] The method of any one of embodiments 19 to 30, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is fully human. [Embodiment 32] The method according to any one of embodiments 19 to 30, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is humanized. [Embodiment 33] The method according to any one of embodiments 19 to 32, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable region CDRs of MED5117. [Embodiment 34] The method described in embodiment 33, wherein the antibody comprises the VH and VL of MED5117. [Embodiment 35] The method of embodiment 34, wherein the antibody is MED5117. [Embodiment 36] The method according to any one of embodiments 19 to 32, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises all six variable region CDRs of an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, elsilimomab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza), and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). [Embodiment 37] The method of embodiment 36, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative comprises a heavy chain V region and a light chain V region derived from an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza) and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). [Embodiment 38] The method described in embodiment 37, wherein the anti-IL-6 antibody or antigen-binding fragment or derivative is an antibody selected from the group consisting of siltuximab, gerilimuzumab, sirukumab, clazakizumab, olokizumab, VX30 (VOP-R003, Vaccinex), EB-007 (EBI-029, Eleven Bio), ARGX-109 (ArGEN-X), FM101 (Femta Pharmaceuticals, Lonza) and ALD518 / BMS-945429 (Alder Biopharmaceuticals, Bristol-Myers Squibb). [Embodiment 39] The IL-6 antagonist is a single domain antibody, V HH 28. The method of any one of embodiments 19 to 27, wherein the antibody is a nanobody, a Fab, or a scFv. [Embodiment 40] The method according to any one of embodiments 1 to 18, wherein the IL-6 antagonist is an anti-IL-6R antibody or an antigen-binding fragment or derivative thereof. [Embodiment 41] The method of embodiment 40, wherein the anti-IL-6R antibody, antigen-binding fragment or derivative comprises the six CDRs of tocilizumab. [Embodiment 42] The method of embodiment 40, wherein the anti-IL-6R antibody, antigen-binding fragment or derivative comprises the six CDRs of bovalilizumab. [Embodiment 43] The method of any one of embodiments 1 to 18, wherein the IL-6 antagonist is a JAK inhibitor. [Embodiment 44] The method of embodiment 43, wherein the JAK inhibitor is selected from the group consisting of tofacitinib (Xeljanz), decernotinib, ruxolitinib, upadacitinib, baricitinib, filgotinib, lestaurtinib, pacritinib, peficitinib, INCB-039110, ABT-494, INCB-047986 and AC-410. [Embodiment 45] The method of any one of embodiments 1 to 18, wherein the IL-6 antagonist is a STAT3 inhibitor. [Embodiment 46] The method of any one of embodiments 19 to 42, wherein the IL-6 antagonist is administered parenterally. [Embodiment 47] The method of embodiment 46, wherein the IL-6 antagonist is administered subcutaneously. [Embodiment 48] The method of embodiment 43 or 44, wherein the IL-6 antagonist is administered orally. [Embodiment 49] The method of any one of embodiments 1 to 48, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase diuretic efficiency. [Embodiment 50] The method of embodiment 49, wherein the IL-6 antagonist is administered at a dose, schedule, and duration sufficient to increase diuretic efficiency to normal levels. [Embodiment 51] The method of any one of embodiments 1 to 50, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase eGFR. [Embodiment 52] The method of embodiment 51, wherein the IL-6 antagonist is administered at a dose, on a schedule, and for a duration sufficient to increase eGFR to normal levels. [Embodiment 53] The method described in any one of embodiments 1 to 52, further comprising a subsequent step of determining the level of IL-6 in urine. [Embodiment 54] The method described in any one of embodiments 1 to 52, further comprising a subsequent step of determining the level of IL-6 in plasma. [Embodiment 55] The method described in any one of embodiments 1 to 52, further comprising a subsequent step of determining the levels of IL-6 in urine and plasma. [Embodiment 56] The method described in any one of embodiments 53 to 55, further comprising a final step of adjusting the dose of the IL-6 antagonist for subsequent administration based on the IL-6 level determined in the immediately preceding step.

[0222] 8.8. Experimental Example The present invention will be further described in detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples in any way, but should be construed as embracing any and all variations that become evident as a result of the teachings provided herein.

[0223] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, practice the methods recited in the claims of the present invention. Accordingly, the following examples specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0224] The materials and methods used in the experiments disclosed herein are now described.

[0225] 8.8.1. Example 1: Urinary and Plasma IL-6 Association in Patients with Heart Failure METHODS: Patients with ongoing heart failure ("HF") receiving high-dose diuretic therapy at Yale Transitional Care Clinic (YTCC) were enrolled in a prospective observational study.

[0226] YTCC is an outpatient clinic with an emphasis on diuretic and fluid status management. Patients are administered 4-8 hours of treatment during which they receive 1-3 doses of loop diuretic. The dosing protocol is determined by the patient's fluid status. The choice of oral (PO) torsemide or intravenous (IV) bumetanide is at the discretion of the treating physician. During the treatment period, all urine produced is stored in a cumulative collection container and sent to the clinical laboratory for electrolyte measurement at the end of the visit. Cumulative urine collections are performed during the treatment period. Additional spot urine samples are stored both before and 1 hour after diuretic administration.

[0227] Assays. Urine and serum electrolytes were measured using ion-selective electrodes using a Randox RxDaytona™ automated clinical chemistry analyzer. Urea, creatinine, bicarbonate, and cystatin C were measured using Randox reagents according to the manufacturer's instructions (Randox Laboratories™, UK). Interleukin-10 and IL-6 concentrations were measured using the MesoScale Discovery (MSD) platform (Meso Scale diagnostics™, Gaithersburg, MD, USA). Amino-terminal pro-B-type natriuretic peptide (NT-proBNP) levels were measured at Yale Clinical Laboratories on a Roche Elecsys 120 analyzer (Roche Diagnostics, Indianapolis, USA). Plasma renin activity (PRA), angiotensinogen, and active renin were measured using commercially available competitive ELISA kits from ALPCO™ according to the manufacturer's instructions (ALPCO™, Salem, NH, USA). Total renin was also analyzed using a commercially available ELISA kit (R&D Systems™, Minneapolis, USA). The R&D Systems™ total renin immunoassay kit recognizes both active and prorenin. The mean detection limits of the assay are 4.43 pg / ml for total renin and 0.81 pg / ml for active renin. Urinary bumetanide and torsemide levels were measured using liquid chromatography mass spectrometry. Ultra-performance liquid chromatography was performed on an Agilent Infinity 1290 UPLC System™. Chromatographic separation was achieved on a Zorbax Bonus RP™ 2.1 x 50 mm 1.8μ column at a flow rate of 0.6 ml / min. The mobile phase included 0.1% formic acid (buffer A) and 80% acetonitrile in 0.1% formic acid (buffer B). Mass spectrometry was performed on an Agilent Q-TOF system™ (Agilent™, Santa Clara, Calif., USA) in positive ion mode.

[0228] Calculations and definitions. eGFR was calculated using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula. Loop diuretic doses were converted to furosemide equivalents: 1 mg bumetanide = 20 mg torsemide = 40 mg intravenous furosemide = 80 mg oral furosemide. As previously published, diuretic efficiency was defined as the increase in urinary sodium excretion during the treatment period for each doubling of loop diuretic dose, centered around 40 mg furosemide equivalents. This scale was chosen to account for the sigmoidal dose-response curve of these drugs. Urinary excretion of diuretic was calculated by multiplying the concentration of diuretic in urine by the volume of urine produced during the first 3 hours after diuretic administration. This amount of estimated diuretic was then normalized to the administered diuretic dose in furosemide equivalents, taking into account the published urinary clearance of the particular diuretic (bumetanide or torsemide) the patient received. Fractional excretion of sodium and potassium was calculated using the following formula: Fractional excretion of X (FEX) = [X]urine × [creatinine]serum / ([X]serum or plasma × [creatinine]urine). Urinary proteins such as renin, angiotensinogen and IL-6 were indexed to urinary creatinine. Low urinary IL-6 was defined as a value below the median urinary creatinine of 14.2 pg / g. Low plasma IL-6 was defined as a value below the median of 2.0 pg / mL. High levels of urinary and plasma renin and angiotensinogen were defined as values ​​greater than or equal to the cohort median of these variables.

[0229] Statistical analysis. Values ​​reported are mean + / - SD, median (quartile 1-quartile 3) and percentages. Correlations between continuous variables are Spearman's rho, except for adjusted correlations. Pearson's chi-square test was used to compare categorical variables between groups. Student's t-test or Wilcoxon rank sum test was used to compare continuous variables between groups. Logarithmic transformation was applied to skewed variables including plasma IL-6, urinary IL-6 and NT-pro-BNP and then entered into multivariable models and partial correlation analyses. Logistic regression was used to predict odds of low diuretic efficiency, eGFR < 60 mL / min / 1.73 m at the univariate level and with adjustment for plasma or urinary IL-6 and / or eGFR. 2 Associations of plasma and urinary levels of IL-6 with low or high levels of urinary or plasma neurohormonal parameters were assessed. Cox proportional hazards modeling was used to assess the association of time to event with all-cause mortality. Statistical analyses were performed with IBM SPSS Statistics version 23 (IBM Corp., Armonk, NY) and Stata version 13 (StataCorp™, College Station, TX). Statistical significance was defined as two-sided p<0.05 for all analyses.

[0230] The baseline characteristics of the population we used are described in Table 2 below. In this subset, 98 patients underwent determination of blood and urinary IL-6 levels. The median (IQR) pre-diuretic level of urinary IL-6 was 14.2 pg / g creatinine (5.6-36.2 pg / g), whereas the median plasma IL-6 level was 2.0 pg / mL (1.2-3.9 pg / mL). Plasma and urinary IL-6 levels were only weakly correlated (r=0.40, p<0.001). Notably, individuals with lower than median urinary IL-6 levels were younger, Caucasian, more likely to be on angiotensin receptor blocker (ARB) or angiotensin-converting enzyme (ACE-I) therapy, more likely to have heart failure with reduced ejection fraction (HFrEF), higher GFR and substantially lower plasma levels of NT-proBNP. Patient profiles defined by lower than median plasma IL-6 levels were somewhat different but largely reflected the same trends.

[0231] [Table 2] TIFF0007630906000004.tif253142TIFF0007630906000005.tif25379

[0232] [Table 3]

[0233] Renal function and IL-6 As shown in Table 2, eGFR was lower in patients with high urinary or plasma IL-6, but this association was only significant between eGFR and urinary IL-6 (p=0.01). A correlation was observed between plasma IL-6 and eGFR (r=-0.26, p=0.01), as well as between urinary IL-6 and eGFR (r=-0.38, p<0.001). However, after adjusting for urinary IL-6, there was no longer a significant association between plasma IL-6 and eGFR (p=0.20), whereas after adjusting for plasma IL-6, a significant association remained between urinary IL-6 and eGFR (partial r=-0.32, p=0.002). Similarly, patients with eGFR<60mL / min / 1.73m 2 The risk of a decline in eGFR as defined by IL-6 increased with higher urinary IL-6 levels (OR per SD increase = 1.9, 95% CI = 1.2, 3.1, p = 0.006) but not with higher plasma IL-6 levels (OR per SD increase = 1.3, 95% CI = 0.8 to 2.0, p = 0.25).

[0234] Diuretic response and IL-6 There was an inverse correlation between diuretic efficiency and both urinary IL-6 (r=-0.43, p<0.001) and plasma IL-6 (r=-0.31, p=0.002, Figure 3). The odds of low diuretic efficiency increased with higher levels of either urinary IL-6 (OR=2.3 per SD increase, 95% CI 1.4-3.8, p=0.001) or plasma IL-6 (OR=1.7 per SD increase, 95% CI=1.1-2.7, p=0.02). Adjusting for eGFR, urinary IL-6 was the only condition significantly associated with risk of low diuretic efficiency (adjusted OR=1.8 per SD increase, 95% CI 1.1-3.1, p=0.02, Figure 3). Furthermore, when both urinary IL-6 and plasma IL-6 were entered into a logistic regression model, only urinary IL-6 was the condition associated with the risk of poor diuretic efficiency (adjusted OR per SD increase = 2.1, 95% CI 1.3 to 3.5, p = 0.004), whereas plasma IL-6 showed no such association (OR = 1.4, 95% CI 0.9 to 2.2, p = 0.17).

[0235] Neurohormonal activation Plasma IL-6 was associated with higher levels of plasma renin (Table 3 and Figure 3). Notably, higher levels of plasma IL-6 conferred an additional risk of high plasma total renin (OR=1.9 per SD increase, 95% CI 1.2-3.0, p=0.008), which persisted even after adjusting for ACE-I or ARB use and urinary IL-6 levels (adjusted OR=2.3 per SD increase, 95% CI 1.3-3.9, p=0.003). Urinary IL-6 was not associated with an increased risk of high plasma renin (OR=1.0, 95% CI 0.7-1.5, p=0.98).

[0236] Urinary IL-6 was strongly associated with higher tissue levels of RAAS activation as measured by urinary angiotensinogen (OR per SD increase = 4.2, 95% CI 2.2-7.9, p < 0.001) and urinary renin (OR per SD increase = 2.1, 95% CI 1.3-3.4, p = 0.002, Figure 3). These associations persisted after adjustment for plasma IL-6 levels and ACE-I or ARB use (adjusted OR for higher urinary angiotensinogen per SD increase = 4.2, 95% CI 2.2-8.3, p < 0.001; adjusted OR for higher urinary renin per SD increase = 2.0, 95% CI 1.2-3.3, p = 0.005). Plasma IL-6 showed no univariate association with risk of high urinary renin (OR=1.3 per SD increase, 95% CI 0.9-2.0, p=0.16). A trend for an association with urinary angiotensinogen (OR=1.5 per SD increase, 95% CI 0.98-2.3, p=0.06) was observed but was eliminated with adjustment for urinary IL-6 (adjusted OR=1.1, 95% CI 0.7-1.7, p=0.76).

[0237] 8.8.1.4. Association with survival Over a median follow-up of 713 days, 32 deaths occurred. Consistent with previous reports, increased plasma IL-6 was associated with a higher risk of mortality (univariate HR per SD increase = 2.8, 95% CI 2.0-4.0, p < 0.001). Plasma IL-6 remained associated with mortality after multivariate adjustment for baseline characteristics such as age, race, baseline NT-proBNP level, use of ACE-I or ARB, home loop diuretic dose, and eGFR (HR per SD increase = 2.3, 95% adjusted CI = 1.5-3.7, p < 0.001). In contrast, urinary IL-6 was not associated with risk of mortality (univariate HR per SD increase = 1.3, 95% adjusted CI 0.9-1.8, p = 0.15; adjusted HR = 1.02, 95% adjusted CI = 0.6-1.6, p = 0.93).

[0238] OVERVIEW Plasma and urinary IL-6 levels capture distinct aspects of the role of IL-6 in the pathophysiology of cardiorenal disease. Plasma IL-6 levels are associated with global measures of disease severity, e.g., risk of mortality. In contrast, urinary IL-6, likely a measure of renal inflammation, strongly correlates with multiple measures of the cardiorenal syndrome ("CRS") in these patients with heart failure, such as diuretic resistance, renin-angiotensin-aldosterone and system (RAAS) activation, and reduced estimated glomerular filtration rate (eGFR).

[0239] These data demonstrate that urinary IL-6 levels are a useful biomarker of renal inflammation and are particularly useful for assessing renal symptoms in patients with heart failure. Furthermore, serial urinary IL-6 levels should prove useful for monitoring and evaluating the renal benefit of therapeutic interventions in heart failure.

[0240] 8.8.2. Example 2: TMPRSS6 genotype stratifies heart failure patients whose renal symptoms predict response to IL-6 antagonists The data obtained in Example 1 also predict that treatment with an IL-6 antagonist should be effective in reducing renal inflammation in heart failure patients.

[0241] However, because infections are a frequent cause of acute decompensation in heart failure patients, it is important to limit anti-cytokine and other immunosuppressive therapies to heart failure patients who are more likely to respond to improved renal and / or cardiac function. The cost of chronic IL-6 antagonist therapy also serves to limit treatment to heart failure patients who are more likely to respond to improved renal and / or cardiac function.

[0242] The analysis performed in Example 1 was extended to 129 patients. Figure 1A is a bar graph showing diuretic efficiency ("DE") by tertiles of urinary IL-6, confirming the inverse correlation between urinary IL-6 and diuretic efficiency observed in a subset of 98 patients. Figure 1B is a bar graph showing diuretic efficiency ("DE") by tertiles of plasma IL-6 in these 129 patients, confirming the inverse correlation between plasma IL-6 and diuretic efficiency (uncorrected for urinary IL-6 levels).

[0243] Each patient's genotype at the rs855791 single nucleotide polymorphism ("SNP") of transmembrane protease serine 6 ("TMPRSS6") was further evaluated. The TMPRSS6 polypeptide, also known as matriptase-2 (MT2), cleaves hemojuvelin and inhibits bone morphogenetic protein signaling. The rs855791 (G2321A) SNP alters the TMPRSS6 protein sequence: the most frequent allele in the human population (major allele) is 2321G, which codes for 736A. The least frequent allele in the human population (minor allele) is 2321A, which codes for 736V.

[0244] Genomic DNA was isolated from the buffy coat using the ReliaPrep high-capacity HT gDNA isolation system on an HSM Instrument (Promega, Madison, USA). The purity of the isolated DNA was assessed by Nanodrop. Genotyping was performed at Yale University for genomic analysis. Whole genome genotyping was performed using Illumina's Infinium® Exome-24 v1.0 BeadChip kit using standard protocols recommended by the manufacturer (Illumina, Inc., San Diego, CA). Amplification, fragmentation, precipitation, resuspension and hybridization steps were performed manually. Arrays were scanned with an Illumina Hiscan instrument. The Illumina HiScan or iScan system uses a laser to excite the fluorophores of the single-base extension products on the beads and scans the BeadChip. The scanner records high-resolution images of the light emitted from the fluorophores. The Illumina GenomeStudio genotyping module included in the Illumina Infinium assay system was used to extract genotyping data from intensity data files (*.idat files) collected from the Illumina HiScan system. The Infinium Exome-24 v1.0 BeadChip contains over 240,000 putative functional exonic variants selected from over 12,000 individual human exome and whole genome sequences. The over 240,000 markers represent diverse populations including individuals from Europe, Africa, China, and Spain, as well as a range of common conditions, such as type 2 diabetes, cancer, metabolic disorders, and psychiatric disorders. A detailed Illumina genotyping protocol is available at http: / / support.illumina.com (infinium_hts_assay_protocol_user_guide_15045738_a.pdf). Details of the SNPs on this exome chip are available at http: / / support.illumina.com / downloads / infinium-exome-24-v1-0-product-files.html.

[0245] As shown in Figure 2A, urinary levels of IL-6 were inversely correlated with diuretic efficiency only in patients with at least one copy of the major allele of the TMPRSS6 rs855791 SNP (Figure 2A, right panel, "AG+GG"). Urinary levels of IL-6 were not significantly correlated with diuretic efficiency in patients homozygous for the minor allele (Figure 2A, left panel, "AA").

[0246] As shown in Figure 2B, plasma levels of IL-6 were inversely correlated with diuretic efficiency only in patients with at least one copy of the major allele of the TMPRSS6 rs855791 SNP (Figure 2B, right panel, "AG+GG"). Plasma levels of IL-6 were not significantly correlated with diuretic efficiency in patients homozygous for the minor allele (Figure 2B, left panel, "AA").

[0247] These data indicate that treatment of heart failure with an IL-6 antagonist improves renal symptoms only in heart failure patients who carry at least one copy of the TMPRSS6 rs855791 major allele.

[0248] 8.8.3. Example 3: Correlation of IL-6 with NKCC2, ENaC-beta, and NCC Expression in the Absence or Presence of Ruxolitinib method The mouse M1 CCD cell line (American Type Culture Collection (ATCC), Cat. No. CRL-2038) was maintained in cell culture medium containing equal volumes of DMEM (Sigma-Aldrich, Cat. No. D6046) and Han F12 (Sigma-Aldrich, Cat. No. 11765-047), supplemented with 5% fetal bovine serum (FBS), 1% penicillin-streptomycin (Thermo Fisher Scientific, Cat. No. 15140-122), 1% insulin-transferrin-selenium (Thermo Fisher Scientific, Cat. No. 51500-056) and 100 nM dexamethasone (Sigma-Aldrich, Cat. No. D4902-100MG).

[0249] M1 CCD cells were seeded at 1 million cells / well in 6-well plates in cell culture medium and incubated overnight at 37°C in a 5% CO2 incubator on day 0. On day 1, cell culture medium was changed to DMEM / F12 serum-free medium and cells were incubated overnight at 37°C in a 5% CO2 incubator. On day 2, serum-free medium was removed and cell culture medium was added to each well. Ruxolitinib (Selleckchem, Catalog No. S1378) was added at a final concentration of 1 μM or 100 μM 10 minutes before the addition of IL-6 (Sigma-Aldrich, Catalog No. SRP3096-20UG) at a final concentration of 10 ng / mL, 100 ng / mL, or 500 ng / mL. Control wells without ruxolitinib or IL-6 were included. Cells were treated with IL-6 and / or ruxolitinib for 24 hours. Cells in each well were washed once with 1x PBS and collected in 250 μL of 1x PIPA buffer (10x, Millipore, Cat. No. 20-188) supplemented with 1% protease inhibitor cocktail (100x, Thermo Fisher Scientific, Cat. No. 78430). Samples were analyzed by immunoblotting using anti-NKCC2 antibody (Millipore, Cat. No. AB3562P), anti-ENaC-beta antibody (Millipore, Cat. No. AB3532P), or anti-NCC antibody (Millipore, Cat. No. AB3553) to quantify protein expression. Each experiment was performed in triplicate.

[0250] result We investigated the expression of NKCC2 (Na-K-Cl cotransporter 2), ENaC-beta (epithelial sodium channel, beta subunit), and NCC (sodium chloride cotransporter) proteins in mouse M1 CCD cell lines after treatment with IL-6 in the absence or presence of the JAK inhibitor ruxolitinib. Mouse M1 CCD cells are genotypically similar to human cells that are homozygous for the TMPRSS6 rs855791 major allele. As shown in Figures 4A, 4B, and 4C, treatment with IL-6 increased the expression of NKCC2, ENaC-beta, and NCC. Ruxolitinib blocked the effect of IL-6 on ion transporter proteins.

[0251] Increased expression of these ion transporters provides a putative mechanism for IL-6-mediated diuretic resistance.

[0252] Because increased expression is not known to be associated with expression of hepcidin, these data suggested that IL-6 antagonism may be effective in treating diuretic resistance even in patients who are homozygous for the TMPRSS6 rs855791 minor allele.

[0253] 8.8.4. Example 4: Correlation of IL-6 with NKCC2, ENaC-beta, and NCC Expression in the Absence or Presence of Spironolactone method The mouse M1 CCD cell line (American Type Culture Collection (ATCC), Cat. No. CRL-2038) was maintained in cell culture medium containing equal volumes of DMEM (Sigma-Aldrich, Cat. No. D6046) and Han F12 (Sigma-Aldrich, Cat. No. 11765-047), supplemented with 5% fetal bovine serum (FBS), 1% penicillin-streptomycin (Thermo Fisher Scientific, Cat. No. 15140-122), 1% insulin-transferrin-selenium (Thermo Fisher Scientific, Cat. No. 51500-056) and 100 nM dexamethasone (Sigma-Aldrich, Cat. No. D4902-100MG).

[0254] M1 CCD cells were seeded at 1 million cells / well in 6-well plates in cell culture medium and incubated overnight at 37°C in a 5% CO2 incubator on day 0. On day 1, cell culture medium was changed to DMEM / F12 serum-free medium and cells were incubated overnight at 37°C in a 5% CO2 incubator. On day 2, serum-free medium was removed and cell culture medium was added to each well. Spironolactone (Selleckchem, Cat. No. S4054) was added at a final concentration of 1 μM or 100 μM 10 min before the addition of IL-6 (Sigma-Aldrich, Cat. No. SRP3096-20UG) at a final concentration of 10 ng / mL, 100 ng / mL, or 500 ng / mL. Control wells without spironolactone or IL-6 were included. Cells were treated with IL-6 and / or spironolactone for 24 h. Cells in each well were washed once with 1x PBS and collected in 250 μL of 1x PIPA buffer (10x, Millipore, Cat. No. 20-188) supplemented with 1% protease inhibitor cocktail (100x, Thermo Fisher Scientific, Cat. No. 78430). Samples were analyzed by immunoblotting using anti-NKCC2 antibody (Millipore, Cat. No. AB3562P), anti-ENaC-beta antibody (Millipore, Cat. No. AB3532P), or anti-NCC antibody (Millipore, Cat. No. AB3553) to quantify protein expression. Each experiment was performed in triplicate.

[0255] result We investigated the expression of NKCC2 (Na-K-Cl cotransporter 2), ENaC-beta (epithelial sodium channel, beta subunit), and NCC (sodium chloride cotransporter) proteins in mouse M1 CCD cell lines after treatment with IL-6 in the absence or presence of the potassium-sparing diuretic spironolactone. Mouse M1 CCD cells are genotypically similar to human cells that are homozygous for the TMPRSS6 rs855791 major allele. As shown in Figures 5A, 5B, and 5C, treatment with IL-6 increased the expression of NKCC2, ENaC-beta, and NCC. Spironolactone blocked the effect of IL-6 on ion transporter proteins.

[0256] Increased expression of these ion transporters provides a putative mechanism for IL-6-mediated diuretic resistance.

[0257] Because increased expression is not known to be associated with expression of hepcidin, these data suggested that IL-6 antagonism may be effective in treating diuretic resistance even in patients who are homozygous for the TMPRSS6 rs855791 minor allele.

[0258] 8.8.5. Example 5: Association of IL-6 with Diuretic Response in Patients Hospitalized with Acute Heart Failure (HF) Methods: Data from the PROTECT trial (Weatherley et al., 2010, J. Card. Fail. 16:25-35; Massie et al., 2010, N. Engl. J. Med. 363:1419-1428) were analyzed by tertiles of IL-6. PROTECT was a randomized, placebo-controlled study testing the effect of the adenosine A1 receptor antagonist rolofylline on relief of dyspnea, risk of worsening renal function, and clinical outcomes. Key inclusion and exclusion criteria for the study are listed below.

[0259] TIFF0007630906000007.tif119169

[0260] In total, 2033 patients with ADHF were included in the PROTECT study. Among these patients, IL-6 was measured by Singulex in 1445 patients at admission (baseline), 1462 patients at day 2 (24 hours after baseline), and 1445 patients at day 7. Diuretic response was defined as the change in weight on day 4 per 40 mg of furosemide (or equivalent dose) administered from baseline to day 3. The primary endpoint of the study was all-cause mortality at 180 days.

[0261] Statistical analysis. Baseline characteristics are presented according to tertiles of IL-6. Differences between tertiles of baseline characteristics were tested using one-way analysis of variance (ANOVA), Kruskal Wallis or chi-2-tests, as appropriate. Univariate linear regression was performed using diuretic response as the dependent variable and (log-transformed) IL-6 at baseline as the independent variable adjusting for clinically relevant variables associated with diuretic response. Survival analysis was performed using Cox regression analysis adjusting for clinically relevant variables and the PROTECT risk model (O'Connor et al., 2012, Eur. J. Heart Fail. 14:605-612). The PROTECT risk model includes previous hospitalization for HF, edema, systolic blood pressure, sodium levels, BUN, creatinine and albumin at admission.

[0262] Results. The baseline characteristics of the population are described below in Table 4. Higher levels of IL-6 at baseline are associated with higher levels of BNP, anemia, eGFR<60, and older age (Figure 6).

[0263] [Table 4] TIFF0007630906000009.tif91168

[0264] The association between IL-6 levels and diuretic response is shown below in Table 5. Diuretic response was defined as the change in body weight on day 4 per 40 mg of furosemide (or equivalent dose) administered from baseline to day 3.

[0265] [Table 5]

[0266] Table 6 and Figures 7A and 7B show the association between IL-6 levels and 180-day all-cause mortality, and the association between IL-6 levels and 60-day all-cause mortality and / or cardiovascular-related rehospitalization (CV hospitalization).

[0267] [Table 6]

[0268] Table 7 and Figures 8A and 8B show that increased IL-6 levels on day 7 compared to baseline predict adverse outcome.

[0269] [Table 7]

[0270] 8.8.6. Example 6: Association of IL-6 Levels and Outcomes in a Deteriorating Heart Failure Population Methods. Data from the BIOlogy Study to TAilored Treatment in Chronic Heart Failure (BIOSTAT-CHF) study were analyzed to investigate the association of IL-6 with outcomes in patients with worsening heart failure. Briefly, BIOSTAT-CHF was a multicenter, multinational, prospective, observational study including 2516 patients from 69 centers in 11 European countries (Voors et al., 2016, Eur. J. Heart Fail. 18:716-726). We performed a secondary analysis in the BIOSTAT-CHF study and excluded patients with ferritin <100 from subsequent analyses. Inclusion criteria for the index cohort included patients over 18 years of age with symptoms of new-onset or worsening HF, confirmed by either LVEF ≤ 40% or BNP and / or NT-proBNP plasma levels > 400 pg / ml or > 2,000 pg / ml, respectively. Furthermore, these patients were not previously treated with ACEi / ARBs and / or beta-blockers or were receiving 50% or less of the target dose of these drugs at the time of enrollment and were anticipating initiation or titration of ACEi / ARBs and beta-blockers. All patients were required to be treated with a loop diuretic.

[0271] TIFF0007630906000013.tif55168

[0272] Overall, IL-6 was measured in 2329 patients with worsening HF from the BIOSTAT-CHF study. The primary outcome of this study was a composite outcome of all-cause mortality and hospitalization for HF.

[0273] Statistical analysis. Baseline characteristics are presented according to tertiles of IL-6. Differences between tertiles of baseline characteristics were tested using one-way analysis of variance (ANOVA), Kruskal Wallis or chi-2-tests, as appropriate. Survival analyses were performed using Cox regression analyses adjusting for clinically relevant variables and the BIOSTAT-CHF risk model. The BIOSTAT-CHF risk model of all-cause mortality and / or hospitalization for heart failure included age, N-terminal pro-B-type natriuretic peptide (NT-proBNP), hemoglobin (Hb), beta-blocker use at inclusion, HF hospitalization in the year prior to inclusion, peripheral edema, systolic blood pressure, high-density lipoprotein cholesterol and sodium (Voors et al., 2017, Eur. J. Heart Fail. 19:627-634). To investigate the association of IL-6 with outcomes according to the location of the TMPRS6 SNP (rs855791), we performed an interaction analysis of ferritin levels and IL-6 for the primary outcome.

[0274] Results. The baseline characteristics of the population are described below in Table 8. Higher levels of IL-6 at baseline are associated with higher levels of NTproBNP and anemia (Figure 9).

[0275] [Table 8] TIFF0007630906000015.tif100167

[0276] As shown in Table 9 and Figures 10A and 10B, levels of IL-6 measured at baseline were associated with the combined outcome of all-cause mortality and / or hospitalization for HF, and with all-cause mortality only at 2 years.

[0277] [Table 9]

[0278] As shown in Table 10 and FIG. 11, there is no differential association of IL-6 with outcome depending on ferritin status.

[0279] [Table 10]

[0280] As shown in Table 11, there is no differential association of IL-6 with outcome depending on TMPRSS6 genotype.

[0281] [Table 11]

[0282] 9. INCORPORATION BY REFERENCE The disclosures of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entireties.

[0283] 10. Equivalents While the present invention has been disclosed with reference to specific embodiments, it will be apparent that other embodiments and modifications of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention, and it is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. A pharmaceutical composition for treating a patient with chronic heart failure, comprising a therapeutically effective amount of an anti-IL-6 antibody, wherein the patient is a human subject.

2. The pharmaceutical composition of claim 1, wherein the patient has elevated pre-treatment plasma IL-6 levels.

3. The pharmaceutical composition of claim 2, wherein the patient has a pre-treatment plasma IL-6 level greater than 2 pg / mL.

4. 2. The pharmaceutical composition of claim 1, wherein the patient has a diuretic efficiency of less than 500 mmol Na per twice the loop diuretic dose.

5. 5. The pharmaceutical composition of claim 4, wherein the patient has a diuretic efficiency of less than 200 mmol Na per twice the loop diuretic dose.

6. The pharmaceutical composition of claim 1, wherein the patient has diuretic-resistant heart failure.

7. The pharmaceutical composition of claim 1, wherein the anti-IL-6 antibody comprises all six variable region CDRs of MEDI5117.

8. The pharmaceutical composition of claim 7, wherein the antibody comprises the VH and VL of MEDI5117.

9. 9. The pharmaceutical composition of claim 8, wherein the antibody is MEDI5117.

10. 13. The pharmaceutical composition of claim 1, administered in combination with a diuretic.

11. 2. The pharmaceutical composition of claim 1, wherein the anti-IL-6 antibody is MEDI5117, and MEDI5117 is administered subcutaneously at a flat dose of 10-20 mg.

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